role of citrate toxicity on cardiac excitation … · "kulk: uf ci'i'kate toxicity...
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ROLE OF CITRATE TOXICITY ON CARDIAC EXCITATION-CONTRACTION
COUPLING MECHANISMS
Samuel J. Yoon
ROLE OF CITRATE TOXICITY ON CARDIAC EXCITATION- CONTMCTION COUPLING MECHANISMS
Samuel J. Yoon
A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology
University of Toronto
@ Copyright by Samuel J. Y o m (1 997)
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"KULk: UF CI'I'KATE TOXICITY ON CARDIAC EXCITATION- CONTRACTION COUPLING MECHANISMS ."
Master's of Science, 1997 Samuel J. Yoon
Department of Physiology, University of Toronto
ABSTRACT
It has been proposed that citrate inhibits contractile force by reducing L-type Ica,
independent of ca2+-buffering (Hryshko & Bers, 1992). Using whole-ce11 techniques in
rat ventricular myocytes, we found significant Ica-rundown contamination in our
recordings, despite attempts to minimize it. Based on perforated patch recordings,
however, Ica did not change significantly with citrate (Ica = -5.2k1.1 to -6.1*0.7 pA/pF;
p>0.5, n=3). Action potentials and single myocyte [ca2+]i transients data were consistent
with these findings. The mechanism of force inhibition was then studied using h a -
loaded rat trabeculae, where a decrease in peak force (pX0.05, n-4) was accompanied
without changes in [ca2']i @>0.1). Inhibition in force due to intracellular acidosis was
ruled out. Using force-[ca2+]i loops from CPA-treated trabeculae, citrate decreased Fm,
(pc0.05, n=6) without changes in MFT ca2+ sensitivity (p0.1). In conclusion, citrate
inhibits force by reducing Fm,, without affecting cardiac Ica, [ca2'li transients, or MFT
ca2' sensitivity.
ACKNOWLEGEMENTS
1 would like to thank the following people:
Dr. Peter H. Backx, whose incredible expertise, guidance and supervision allowed me to understand and appreciate the intricacies of cardiac research. His support and continua1 challenges contributed to the realization of my potential and the successful completion of my master's, for which I am grateful.
Dr. Ivan M. Rebeyka and Dr. Uwe Ackerrnann, who were always there to encourage me in my research, and provide vaiuable insights-both c1inicaI and physiological-into my work. Their contributions were both intriguing and enriching.
Dr. Carin Wittnich, who at short notice agreed to step in my thesis committee, despite her busy schedule. Her wil work were paramount in the completion of my thesis.
Dr. Robert Tsushima, who provided new perspectives i
and become another member of ingness and contributions to my
ito my work, and especially for helping me maintain my sanity and focus during my research.
Zarnaneh Kassiri, whose work on the muscle preparations were an invaluable component of my study. Her superb expertise and assistance helped me gain an important perspective on the nature of citrate toxicity. Throughout this collaboration, she has become both fiiend and confidarit, and 1 wish her success in her .future research aspirations.
John L. Yoon and Moon Yoon, whose loving support and dedication to my continuing education has culminated in the completion of my thesis. Their etemal patience and understanding, and their continuing love, has become a source of strength and compassion. Love, always.
Daniel H. Yoon and Michael J. Yoon, whose humour and wit have provided me with an outlet for al1 of my moments of anguish and suffering. Thanks for everything! O
RESEARCH QUOTE
"But it is the grandeur of all truth which can occupy a very high place in human interests that it is never absolutely novel to the meanest of rninds: it exists eternally, by way of germ or latent principle, in the Zowest as in the highest, needing to be developed but never to be planted. "
-- Thomas De Quincey (1 785 - 1 859)
DEDICATION
To my grandfather, Byong-Moorz Choi, who provided for me a genuine inspiration in overcoming all obstacles with immortal dedication and compassion.
To my grandrnother, Hyun-Sook Choi, whose rnernory serves as a legacy for my own life.
- Love, SJY
Page
... ABSTRACT ....................................................................................................... lii
ACKNO WLEDGEMENTS ......................................................................... iv DEDICATION .................................................................................................... v . . TABLE OF CONTENTS .................................................................................. vil
LIST OF TABLES ............................................................................................. xi . . LIST OF FIGURES ........................................................................................... xi1
Chapter 1: INTRODUCTION ..................................................................... 1
1.1 Purpose .................................................................................................... 1 1.2 Statement of the Problem ........................................................................ 1 1.3 Hypotheses .............................................................................................. 2 1.4 Specific Objectives ................................................................................. 2
........................................................ Chapter 2: LITERATURE REVIEW 4
2.1 CITRATE: AN INTRODUCTION .................................................... 4 2.1.1 Metabolic nature of citrate ...................................................... 4
...................................... 2.1.2 Clinical toxicity associated with citrate 5 2.1.3 L-type Ca channel study: citrate interaction
directly with the channel ............................................................ 7 2.1.4 Mechanism for citrate uptake/transport:
intracellular role of citrate .......................................................... 7
CITRATE: ROLE IN CARDIAC E-C COUPLWG MECHANISMS .. 9 ................................. 2.2.1 Role of citrate on cardiac ca2+-homestasis 9
................. 2.2.2 Role of citrate on cardiac contractile myofilarnents 10
ROLE OF CITRATE ON CARDIAC ca2+-HOMEOSTASIS ............. 12 2.3.1 Calcium cycling in heart: potential implications for citrate ... 12 2.3 .2.i Sarcolemmal calcium channels: influx ........................... 14 2.3.2.ii Biochemical characterization of L-type Ca channels:
structural significance ............................................................ 14 2.3.2.iii Regulation of L-type Ca channels .......................................... 1 6 2.1.2.i~ Calcium current: # channels, channel gating, open
................................................. probability. and regulation 1 7 2.1.2.v The sarcolemmal pump: calcium extrusion .......................... 18 2.1.2.vi NaKa exchanger .................................................................... 19
2.1.2.vii The sarcoplasmic reticulum (SR): Ca-uptake. contents. and .................................................................................. release 20
................................................................. The SR Ca-pump 20 The SR Ca-release channel/ryanodine receptor ................. 20
2.1.2.viii Mitochondrial calcium transport ......................................... - 2 1
2.4 ROLE OF CITRATE ON CARDIAC CONTRACTILE ........................................................................... MYOFILAMENTS 2 3
2.4.1 The machinery of the cardiac contractile mechanism: ...................................................................... the myofilarnents 23
2.4.2 Mechanism of calcium-activated contractile force: cooperative mode1 of thin filament activation ......................... 25
2.4.3 Factors affecting the force-calcium relationships in .......................................................................... cardiac muscle 28
2.5 OUR APPROACH IN EXAMINING THE ROLE OF CITRATE IN E-C COUPLING MECHANISMS ....................................................... 29
Chapter 3: METHODOLOGY ................................................................. 31
3.1 ELECTROPHY SIOLOGICAL STUDIES AT THE SINGLE-CELL LEVEL .................................................................................................. 31 3.1.1 Isolation of rat ventricular myocytes ......................................... 31 3.1.2 Voltage-clamp experiments
3.1 .2.i Voltage-clamp technique ........................................ 3 2 3.1.2.ii Experimental Rationale ............................................. 3 2 3.1.2.iii Experimental Method and Protocols .......................... 33 3.1.2.i~ Determination of free [ca2'], using association
constants .................................................................... 35
3.2 SINGLE-CELL [ca2+li TRANSIENTS MEASUREMENTS .............. 37 3.2.1 Fluorescent Measurements using Fura-2 salt; calibration of
............................................................................. fùra-2 signals 37 3 2.2 Experimental Protocols ............................................................. 37
3.3 STUDIES IN MULTI-CELLULAR PREPARATIONS ....................... 39 3.3.1 Isolation of right ventricular trabeculae in rats ......................... 39 3.3.2 Fluorescence Measurements using Fura-2 ............................. -39 3.3.3 Experimental Rationale ......................................................... 4 0 3.3.4 pHi experiments ........................................................................ 43
.................... 3.3.4.i pHi measurements in cardiac trabeculae 43 3.3 .4.ii Loading of carboxy-SNARF- 1 and calibration .......... 43 3.3 .4.iii Experimental Protocols .............................................. 44
3.4 Statistics ................................................................................................ 44
Chapter 4: RESULTS ....................... .. ................................................... 45
4.1 ROLE OF CITRATE ON CARDIAC CA^+-HOMEOSTASIS ............ 45 4.1.1 L-type Ca charnel study: problems with Ica-rundown ............ 45 4.1.2 Other methods for reducing cardiac Ica rundown ..................... 53 4.1.3 Chronic study: effects of citrate versus EGTA on Ica. ............ 62
......... 4.1.4 Conclusions on the role of citrate on L-type Ca channels 65 4.1.5 Effects of citrate on action potential profile: other
............................................................. modes of citrate action 66 4.1.6 Effects of citrate on cardiac [ca2+li transients in
rnyocytes: other possible targets responsibIe for ..................................................... disrupted ca2+-homeostasis 70
4.1.7 Conclusions on the role of citrate in cardiac 2+ Ca -homeostasis ..................................................................... 75
4.2 ROLE OF CITRATE ON THE CARDIAC CONTRACTILE MECHANISM ...................................................................................... 76 4.2.1 Effects of citrate on force and [ca2+]i in rat
............................................................... ventricular trabeculae 76 4.2.2 Role of citrate on MFT ~a~'-sensitivity intracellular
.................................................................. acidosis (pHi) study 81 4.2.3 Role of citrate on MFT ca2+ -sensitivity: CPA study ............... 86 4.2.5 Control experiments on auto fluorescence ............................... 100 4.2.6 Conclusions on the role of citrate in the cardiac
........................................................... contractile mechanism 102
........................................................................ Chapter 5: DISCUSSION 103
5.1 INTRODUCTION .............................................................................. 103
5.2 ROLE OF CITRATE ON L-TY PE Ca CHANNELS ......................... 104
5.3 ROLE OF CITRATE ON CARDIAC ca2+ -HOMEOSTASIS ........... 106
5.4 ROLE OF CITRATE ON FORCE AND [ca2+]i IN CARDIAC MUSCLE ............................................................................................ 106
Chapter 6: SUMMARY ........................................................................... 110
Chapter 7: FUTURE DIRECTIONS ...................................................... 111
\ . . . . / = .. bV
Chapter 8: REFERENCES .............................................................. 1 12
APPENDIX 1 ................................................................................................. 145
APPENDIX 2 ................................................................................................ 146
LIST OF TABLES
Page
Table 1
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
Effects of acute exposure (< 2 hours) of rat ventricular myocytes to I O mM citrate .................................................................. 49 Effects of 10 mM citrate on rabbit ventricular myocytes using perforated patch-clamp: arnphotericin B ........................................... 6 1 Effects of chronic exposure (> 2 hours) of rat ventricular
................................................................ myocytes to 10 rnM citrate ..64 Effects of 10 mM citrate on resting membrane potential, &,,-plateau
.................................................. and action potential duration (n = 1 1 69 Effects of 10 mM citrate on various parameters of calcium transients in rat ventricular myocytes: relaxation phase (T0.5), diastolid
......... systolic calcium transients, and calcium transients magnitude. .74 Effects of acute exposure (1 0 minutes) to 10 mM citrate on right ventricular trabeculae ................................................................ -79 Effects of 10 mM citrate and 100 pM CPA in right ventricular trabeculae: changes in peak force and KD .......................................... 98
Page
Figure 4.1
Figure 4.2
Figure 4.3
Figure 4.4
Figure 4.5
Figure 4.6
Figure 4.7
Figure 4.8
Figure 4.9
Figure 4.10
Figure 4.1 1 Figure 4.1 2
Figure 4.13
Figure 4.1 4
Figure 4.1 5
Figure 4.16
Figure 4.17
Figure 4.18
Figure 4.19
Cwrent-voltage (1-V) relationship showing 10 rnM citrate exposure in rat ventricular myocytes: 5 mM [EGTAIi ...................... 48 1-V relationship with 10 mM citrate exposure in rat ventricular myocytes: 1 O mM [BAPTAIi.. ........................................ -50 Time-course of Ica with 10 mM citrate: Evidence of rundown without leupeptin in rat ventricular myocytes .................................... S2 Evidence of Ica rundown in rat ventricular myocytes with proteolytic inhibition ........................................................................... 54 Tirne-course of Ica with 10 mM citrate: evidence of rundown prior to citrate application despite proteolytic inhibition. .......,........... 56 Inhibition of Ica rundown via phosphorylation of L-type Ca channels .............................................................................. 59 Perforated patch-clamp recordings showing effects of 10 mM citrate on Ica in rabbit ventricular myocytes ...............,........... 60
Population study showing effects of chronic exposure ( > 2 hours) to 10 mM citrate and 10 mM EGTA in rat ventricular myocytes ...... .63 Effects of 10 mM citrate on action potential profile in rat ventricular myocytes ..................................................................... .67 Effects of 10 mM citrate on resting membrane potential (r.m.p.) in rat ventricular myocytes .................................................................. 68 Control intracellula [ca2+] over time in rat ventricular myocytes ..... .7 1 Effects of 10 mM citrate on [ca2+li transients in rat ventricular myocytes .......................................................................... ,72 Effects of citrate wash-out on [Ch2+]* transients in rat ventricular myocytes ...................................................................... ..73 Effects of 10 mM citrate on force and [ca2+li traces (raw) in rat ventricular trabeculae ................................................................ 77 Effects of 10 mM citrate on peak force and twitch duration (TD) in rat ventricular trabeculae ................................................................. 78 Effects of 10 mM citrate on peak force, twitch duration, and pHi in rat ventricular trabecuIae .................................................. 83 Effects of 10 mM citrate and NH&l on peak force, TD, and
.......................................................... pHi in rat ventricular trabeculae 83 Effects of increasing [ca2'], in the presence of 100 pM CPA
................................................................. in rat ventricular trabeculae 87 Force and [ca2']i traces (uaw) showing effects of high-frequency stimulation (7 Hz) in rat ventricular trabeculae in the presence
................................................................................... of 100 pM CPA 89
Figure 4.20 Effects of 10 mM citrate on force-[ca2+]i relationships in rat ventricular trabeculae in the presence of 100 FM CPA: maximal loaded with hra-2 ................................................................ 90
Figure 4.21 Effects of 10 mM citrate on force and [ca2+li traces ( r m ) in rat ventricular trabeculae with 100 pM CPA: maximal loading with fura-2 ......................................................................................... ..92
Figure 4.22 F O ~ C ~ - [ C ~ ~ + ] ~ relationships derived fiom twitches slowed with 100 pM CPA in the presence of citrate ............................................... 94
Figure 4.23 Normalized f o r c e - [ ~ a ~ + ] ~ relationships derived fiom twitches slowed with 100 pM CPA in the presence of citrate .......................... 95
Figure 4.24 ~ o r c e - [ ~ a ~ + ] i relationships denved h m twitches slowed with .................................................... 100 pM CPA after citrate wash-out 96
Figure 4.25 Normalized force-[ca2+li relationships derived fiom twitches slowed with 100 pM CPA after citrate wash-out ................................ 97
Figure 4.26 Effects of 10 mM citrate on peak force, autofluorescence and fura-2 340 and 3 80 signals (raw) in rat ventricular trabeculae ........ .10 1
1.1 PURPOSE
The purpose of this study was to elucidate the mechanism by which citrate alters
the contractile properties of the heart. The study involved using voltage- and current-
clamp techniques on cardiac myocytes, force/[~a~~]~/sarcomerecoere recordings on cardiac
trabeculae, and calcium transients recordings in cardiac myocytes.
1.2 STATEMENT OF THE PROBLEM
The use of sodium citrate as a blood anticoagulant for transfusion purposes is a
well-accepted standard practice. The three ionized carboxyl groups are responsible for
the major pharmacological properties of citrate: the binding of divalent cations, including
ca2+ (Dzik & Kirkley, 1988). Citrate is highly soluble in aqueous media even when
bound or unbound to divalent cations, and this c m be attributed to the presence of the
third ionized carboxyl group, creating a strongly polar molecule.
Toxicity of citrate is also well-known. With the advent of surgical techniques of
increasing cornplexity requiring rapid transfùsion of larger volumes of blood, several
cases of citrate intoxication following transfusion have been recorded (Bunker et al.,
1955). Since then, nurnerous cases have been reported and considerable controversy has
developed regarding the management of citrate toxicity during massive transfusion.
Recognition and therapy of citrate toxicity was enhanced by the widespread application in
the 1970s of ion-selective calcium electrodes. With the development of advanced trauma
care, liver transplantation, and prolonged extensive surgical procedures in pediatrics,
renewed interest has been developed in the role of citrate toxicity in the setting of
ultramassive transfusion.
In recent years, nurnerous studies have demonstrated that depression of
myocardial force occurs during massive blood transfusion, and this effect is presumed to
be secondary to the chelating effect of citrate on serum ionized calcium levels (Ludbrock
et al., 1958; Denlinger et al., 1976; Abbott, 1983; Rebeyka et al., 1990). However,
previous studies have suggested that citrate's effects on contractile function might also
involve action independent of ca2+-chelation (Hryshko & Bers, 1992). This correlation
between infusion of citrated blood products and depression of sem-ionized calcium
levels is dependent on the balance between the citrate dose versus clearance. A number
of factors affect this balance, including rate and duration of citrate administration and
circulating blood volume as well as hepatic and renal clearance (Dzik & Kirkley, 1988).
1.3 HYPOTHESES
Two major hypotheses that were investigated in this study:
(1) Citrate depresses force generation in cardiac muscle by decreasing [ca2+]i transients
as a result of altered L-type Ca channel magnitude and selectivity.
(2) Citrate alters the properties of the contractile proteins, thereby reducing force
generation.
1.4 SPECIFIC OBJECTIVES
The object of this study was to elucidate the mechanism of citrate on the
contractile properties of the heart. Evidence has suggested that the role of citrate as an
anticoagulant in blood-transfusion products and surgical procedures may in fact have
more deleterious consequences (Denlinger et al., 1976; Abbott, 1983). A recent study
hypothesized that citrate disnipted ca2+-homeostasis (Hryshko & Bers, 1992) as a result
of altered Ca channel seIectivity and permeation, which inhibited cardiac contractile
force. The objectives for this study were to examine the nature by which citrate inhibits
contractile force. In this thesis were studied: (1) the effects of citrate on
transsarcolemmal calcium fluxes in single cardiac myocytes; the effects of citrate on, (2)
[cazf]i transients and, (3) action potential profile in myocytes to determine changes in
calcium homeostasis and handling; (4) citrate's effects on the contractile proteins. The
methodology consisted of electrophysiological studies-voltage-clamp, current-clamp-
on rat ventricular myocytes which allowed direct measurements of the effect of citrate on
L-type Ca channels and calcium handling in response to citrate. A parallel study
involving fluorescence measurement of intracellular [ca2+] i and contractile force in right
ventricular trabeculae preparations allowed the rneasurement of [CaL']i and its
relationship to force in order to provide a mechanistic explanation of the varying
inetabolic interventions at the muscle level.
2.1 CITFtATE: AN INTRODUCTION
In order to establish the mechanism of citrate toxicity, a number of features of this
ubiquitous organic compound mrist be identified. Citrate has three ionizable carboxyl
groups (chernical structure, C6H5O7; M.Wt., 192 daltons) and binds to divalent cations. The
normal adult plasma concentration of citrate is from 0.9 to 2.5 mM (Gordon & Craigie,
1960). Citrate is found in al1 human cells and is an intermediary in the Kreb's citric acid
cycle. The metabolic role of this compound is thus an important feature of citrate, and could
explain citrate's toxicity. Questions arise on how citrate is transported, however, since it
could provide clues on whether the effects of citrate are directly involved with the contractile
mechanism, and reflect its extracellular action. An exarnple of this latter effect is the change
in magnitude and selectivity of L-type Ca channels observed by Hryshko and Bers in 1992
after exposure to citrate. The following sections deal with these important aspects of citrate:
(1) whether citrate could act as a metabolic regulator; (2) the clinical effects in using citrate;
(3) the L-type Ca channel study, which establishes that citrate may have a direct effect on the
cardiac contractile mechanism, independent of its calcium-chelating abilities; and (4) the
mechanism by which citrate is transported intracellularly.
2.1.1 Metabolic nature of citrate
In liver, heart and kidney, there exists a mitochondrial tricarboxylate carrier which
exchanges citrate and malate, thereby making acetylCoA available in the cytoplasm for fatty
acid synthesis (Gamble, 1965; LaNoue & Schoolwerth, 1979). Biosynthesis of fatty acids
occurs with a liver and adipose enzyme that cleaves citrate to form acetylCoA and OAA.
.Thus, increased cytoplasmic citrate as a result of massive transfusion might be expected to
temporarily stimulate fatty acid synthesis (Passoneau & Lowry, 1963). However, it was later
shown that both fatty acid and cholesterol synthesis were significantly inhibited (Sullivan et
al., 1972) by citrate, possibly via feedback inhibition, which would lead to decreased ATP-
production. Citrate c m also directly enter the Kreb's TCA cycle to become completely
- - - - - . - . - - -i --- - - A -, --- --- -.- ----. ----- -- b'-'--' -'- Ua------- O--------
Exogenous citrate is actively transported across the mitochondrial membrane by carrier
molecules. Once inside, two carbons of citrate are used to generate two molecules of CO2,
leaving succinate, which is converted to oxaloacetate (OAA). With additional tums of the
Kreb's cycle, the original carbons of citrate are converted to CO2, without either changes in
the citrate concentration or the acidhase balance. Evidence suggests that metabolism of
exogenous citrate results in net transport of malate, the immediate precursor of OAA,
outside the mitochondria. Cytoplasmic malate is then converted into oxaloacetate, which is
then converted to either glucose or pynivate during conditions of ATP-depletion (Dzik &
Kirkley, 1988). Citrate, however, also inhibits phosphofiuctokinase (PFK) (Mansour, 1 972),
a glycolytic enzyme that directs glucose residues into either glycogen or production of
pyruvate. With increased citrate thus inhibiting PFK, the heart then preferentially utilizes
the alburnin-bound fi-ee fatty acids and complex lipids from the blood, and converts them
into acetylCoA for ATP-production.
The rate-limiting step of citrate metabolism following massive transfusion has not
yet been determined. Evidence fiom liver transplantation and fiom studies of renal excretion
of citrate suggests that alkalemia slows the metabolism of citrate presumabIy by retarding
movement of citrate and malate across the mitochondrial membrane (Simpson, 1983).
Whether there are other factors that influence the activity of citrate-cleaving enzymes, and
exert an overall effect on metabolism of exogenous citrate, have not been fully explored. As
will be shown in this study, however, citrate exerts a rapid effect on both voltage-clamp and
trabeculae recordings, suggesting that the effects of citrate are not dependent on processes.
It may be that the effects of citrate on contractility occur as a result of direct actions.
2.1.2 Clinical toxicity associated with citrate
Citrate is known as an effective calcium chelator; a standard clinical use of citrate is
citrate-phosphate-dextrose (CPD) solution, used for its anticoagulant properties in blood-
product transfusions (Abbott, 1983). There have been numerous clinical cases documenting
various symptoms associated with administering citrate. Depression of the ionized [ca2+]
due to citrate, for example, prolongs the QT interval by 80 msec (Olson et al., 1977), which
rerurnea ro normal roiiowing cltrate-Iree solutions. Liver transplant patients ITequently
develop other metabolic abnorrnalities known to exacerbate citrate toxicity: hypothemia,
hyperkalemia from cellular release by the grafied liver, acidemia fiom decreased tissue
perfusion and inability to metabolize lactic acid, and a decreased ability to mobilize calcium
from bony stores (Wu et al., 1987), while neuromuscular signs and symptoms attributed to
hypocalcemia were comrnon as well (Olson et al., 1977). Finally, patients also develop
metabolic alkalosis, as a result of movement of plasma potassium into transfused red cells or
as a result of excessive urinary losses (Pearl & Rosenthal, 1985).
Numerous studies have also investigated the effect of transfusion on serum citrate
levels. In one study, average peak citrate levels during dinical transfusion were 62 mg/dL
and rneasurable cardiovascular depression was detected in patients when citrate levels
exceeded 30 mg/dL (Bunker et al., 1955). Another clinical study docurnented a 26%
reduction in ionized calcium Ievels with an elevation of serum citrate from 1.9 to 6.3 mg/dL
during routine intraoperative transfusions. The hazards of prolonged citrate administration
combined with inadequate removal have become clearly evident during liver transplantation
when median citrate levels of 1 13 mg/dL have been docurnented during the anhepatic period
(Marquez et al., 1986). Finally, an investigation was conducted in which it was found that a
concentration of 42 mg/dL citrate induces a significantly greater depression of contractility
in the neonatal heart; developed pressured recovered partially during the period of citrate
perfusion and subsequently returned toward baseline when citrate was discontinued
(Rebeyka et al., 1990).
The neonatal study showed a significant reduction in end-diastolic ventricular
pressure which was attributed to the Ca-buffering action of citrate (Ginsburg & Shimoni,
1989). It was proposed that citrate dissipated the local elevation of calcium near the outer
sarcolemmal surface, because citrate had a greater affinity for calcium than the extracellular
sites surrounding the pore of the calcium channels, and resulting in decreased serum [~a"],
due to citrate's buffering abilities (Ginsburg & Shimoni, 1989). However, the neonatal
studies suggested that this depression of myocardial contractility may not simply be due to a
reduction in ionized calcium concentration (Rebeyka et al., 1990), and that citrate had a
more complex role than previously shown.
L.1.5 L-type Ca channel study: citrate interaction directIy with the channel
Recent evidence has pointed to effects of citrate that are independent of its action as
a low-affinity Ca buffer (Hryshko & Bers, 1992). Using a 10 mM [citrateIo, and maintaining
free extracellular [cazf] of 2 mM, an alteration in the voltage-dependence of channel gating
and changes in the selectivity of the L-type Ca channel were observed. The conclusion that
arose fi-om the study was that citrate decreased contractile force by direct interaction with the
sarcolemmal Ca channel; the changes in Ica would reduce Ca entry during the action
potential plateau and, consequently, decrease contractile force.
It was postulated that citrate binds at a site near the outer mouth of the calcium
channel, and thus, provides an additional site whereby the passage of calcium ions could be
reduced (Hryshko & Bers, 1992). However, these authors did not demonstrate that citrate's
effects were reversible upon wash-out, making it possible that the effects resulted fiom Ica
rundown, and thus unrelated to a direct action of citrate on the channel, as shown in this
thesis (see Results). Furthemore, it was s h o w in both cell-shortening and in muscle
preparations that force was inhibited in the presence of citrate followed by a complete
recovery with wash-out, and that this was related to the reversible changes in Ca channel
selectivity. This would not be consistent with rundown induced by citrate, as a continual
decrease in Ica would indicate that peak force would also exhibit some f o m of rundown.
2.1.4 Mechanism for citrate uptakeltransport: intracellular role of citrate
There exists the possibility that citrate is transported intracellularly, which could
directly interact with the interna1 cellular processes inherent in cardiac E-C coupling. An
oIder study (thesis; Lawford, 197 1) suggested that citrate could be transported intracelIularIy
within certain strains of bacteria via a citrate-malate CO-transporter. The evidence leads to
the speculation that despite a more highly evolved and selective membrane surrounding
eukaryotic-like cells, such as the specialized cardiac myocytes, a type of transporter,
analogous to the sodium-glucose CO-transporter, exists that would allow citrate to be
transported into the cell. It is already known that the synthesis, uptake, release and oxidative
metabolism of citrate occurs in neurons and astrocytes, and that citrate regulates the
extracellular concentrations of ca2+ by chelation, thereby modulating neuronal excitability
U - -
7 - - - .,. - --- r- ------- -- - ---------'--- '- J ---- ----- - - - - - - - -- - - - - - --r----- in rat cardiac myocytes, allowing for essential oxidation of important respiratory fùels such
as lactate, pyruvate, acetate, acetoacetate, and 8-hydroxybutyrate, as well as citrate (Wang et
al., 1994; Wang et al., 1996).
The transport of citrate into the ce11 could hypothetically alter cellular metabolism.
Citrate entry into the mitochondria could increase the production of NADH and H'
intracellular, via the Krebs' cycle, and thus creating acidosis. Alternatively, citrate could
enter the ce11 directly in its protonated forrn without being metabolized, resulting in
intracellular acidosis. Thus, as evident fiom this, citrate's actions are für more complex than
simply calcium buffering; there may be a direct influence of citrate on the E-C coupling
mechanism itself.
There are two major features of cardiac E-C coupling that serve as the primary focus
for this study: (1) the dynarnic process involving cardiac calcium cycling; and (2) the
cardiac contractile myofilarnents. Since citrate is a well-known clinical ca2+ chelator,
alterations in ca2+-cycling are expected. Alternatively, as suggested by the L-type Ca
channel study (Hryshko & Bers, 1992), it is possible that citrate directly affects cellular
proteins involved in calcium handling, force generation, or metabolism. Finally, the effects
of citrate on cellular metabolism and pHi might also represent a mechanism of action of
citrate, as discussed above.
2.2.1 Role of citrate on cardiac ca2+-homeostasis
It is postulated in this thesis that citrate's calcium chelating properties may be
secondary to a more direct effect on the contractile mechanism (Hryshko & Bers, 1992).
Numerous cellular structures are involved in calcium intrusionlextnision and handling in E-
C coupling, and each represent a potential target for citrate's action. For exarnple, the highly
regulated L-type Ca channel is controlled by nwnerous factors-V,, Cai, channel
agonists/antagonists, phosphorylation, acidosis, allosteric and metabolic regulation-that c m
be linked to some aspect of citrate's multi-faceted nature: calcium chelator, metabolic
regulator, force inhibitor (via some unknown mechanism), TCA metabolite.
Another major structure that may have altered functional aspects is the SR. A recent
study using intracellular citrate showed that calcium-induced calcium release (CICR)
occurred in a regenerative fashion such that the SR would become calcium-overloaded.
causing a spontaneous release of calcium (Callewaert et al., 1994), which could in principle
increase NdCa-exchanger activity in order to extrude the excess calcium. The involvement
of the mitochondria in reguiating calcium transport rnay be another potential target of citrate,
especially in its own regulation of intrarnitochondrial dehydrogenases. Citrate's close
involvement with energy metabolism and the TCA cycle rnay serve to alter mitochondrial
function by driving the TCA cycle fonvard, resulting in an excess of NADH + H' (i.e.
acidosis) and ATP production, which may indirectly contribute to affecting other cellular
processes. The many potential mechanisms by which citrate cm act reinforces the notion
ultimateIy affecting cellular responses.
2.2.2 Role of citrate on cardiac contractile myofilaments
The effects of citrate on calcium handling were initially considered to underlie its
effects on force generation. However, as shown in Our L-type Ca channel studies, action
potential profiles, and measured intracellular [ca2+]-transients (see Results), it is highly
doubtful that citrate significantly affects calcium handling to the extent that could explain
the significant drop in force. The other possibility is a direct effect of citrate on the
contractile proteins resulting in decreased myofilarnent responsiveness, causing force
inhibition. Some possible mechanisms that underlie the citrate-induced force inhibition: (i)
the interference with calcium-binding to troponin C (TnC); (ii) the prevention or inhibition
of ATP-binding to the actin-myosin cornplex, resulting in fewer cross-bridge formations;
(iii) changes in the phosphorylation state of the contractile proteins; (iv) citrate metabolism
may be responsible in creating an intracellular acidotic environment as a result of increased
NADH + H' production via the TCA cycle or direct transport in its protonated form, which
reduces the ca2'-sensitivity of the contractile proteins; and (v) direct alterations in the level
of ATP or other metabolites, which in turn influence contractile proteins.
It is evident fiom the discussion that there is a multi-array of targets that citrate could
act upon. The possibility that there rnay be changes in cardiac ca2+-homeostasis as a result
of disrupted calcium handling was shown with the changes in L-type Ca channel selectivity
that would alter intracellular [ca2+] transients, and indeed as demonstrated above, there are a
number of mechanisms that citrate could act upon. The other possibility lies with the notion
that if citrate is not significantly affecting ca2+-homeostasis, there rnay be another
mechanism downstrearn fiom the various calcium handling targets: narnely, the contractile
proteins. The next sections are concerned with the theory and background of the hypotheses
that were examined in this thesis: (1) the first is Section 2.3: "Role of citrate on cardiac
ca2'-homeostasis", which is a treatment of the different calcium handling cellular proteins,
their regulatory properties, and their potential interactions with citrate; and (2) Section 2.4:
"Role of citrate on cardiac contractile myofilaments", is a detailed report on the
mechanism(s) involved in the contractile machtnery, and the possibility of citrate action on
cardiac contractility.
2.3.1 Calcium cycling in heart: potential implications for citrate.
The two major sources of cal+-ions for cardiac muscle contraction are: (1) voltage-
dependent calcium entry across the sarcolemmal membrane; and (2) calcium release fi-om
the SR. The first source of external calcium depends mainly on calcium movement through
voltage-gated calcium channels. The contractile force mechanism depends on external
calcium sources that permeate the voltage-dependent L-type Ca channels. The second major
source of calcium is the sarcoplasrnic reticulum (SR) which releases calcium from its
intracellular cornpartments using a mechanism known as the calcium-induced calcium
release (CICR) process. The SR is an entirely intracellular, membrane-bounded
cornpartment which is not continuous with the sarcolemma. The main function of this
organelle appears to be sequestration and calcium-release to the myoplasm, removing
calcium from the cytoplasm via mainly the SR Ca-ATPase pump protein (Stewart &
MacLennan, 1974; Katz et al., 1986). The SR junctions with the sarcolemma are highly
specialized and consist of spanning proteins, or "feet" (Franzini-Armstrong, 1970), and
identified as the Ca-release channel of the SR (Lai et al., 1988). These feet (or ryanodine
receptors) are situated on the SR and are found opposite to the T-tubule membrane protein,
identified as the sarcolemmal Ca channel protein, or dihydropyridine receptor (Block et al.,
1988), which is ultimately responsible for the calcium triggering of SR Ca release.
Physiological contractions of striated muscle cells are triggered by action potentials
that elicit transient increases in [ca2+]i at the myofilarnents. In cardiac tissue,
transsarcolemrnal cal+-influx is essential for the rise of [caz'li which activates contraction
in the heart. During the cardiac action potential, caZC may enter the ce11 mainly via the
sarcolernmal L-type Ca channels. While the transsarcolemmal calcium entry rnay contribute
directly to the activation of the myofilaments (discussed later), it is primarily involved in the
activation of SR Ca-re1ease (called "Ca-induced Ca-release process", or CICR; Fabiato,
1985). Calcium channel activation is not sufficient for triggering of phasic cardiac
contractions, however, and so a linkage exists between the L-type Ca channel as the initiator
for a large SR Ca-release. This causes a rapid rise in intracellular [ca2+] which activates
contraction. The amount of SR calcium released is graded with the amount of "trigger"
U L J 8
resulting in a refractory period which prevents a second calcium release. The channel also
replenish SR stores, in which alterations in the charge carried by the L-type Ca channel have
a predictabfe effect on calcium transients and contractility triggered by subsequent
depolarizations.
In order to understand CICR, one must address the question of how such a small
[ca2'li-transient fiom Ica-influx via the sarcolemmal L-type Ca channels c m regulate the
larger transient due to release of SR calcium. Recent evidence has shown that cardiac
myocytes develop upon depolarization a number of discrete regions of concentration [ca2+].
These regions, or "Ca-sparks", are thought to be the main trigger mechanism of CICR
(Canne11 et al., 1995), known as the "local response theory". The ryanodine receptor, which
is believed to make up the SR Ca release channel, has a sufficiently lower conductance
which would potentially prevent spontaneous release of SR calcium. This allows tighter
regulation of SR Ca release and, ultimately, of the global calcium transients.
During reIaxation, calcium is both removed fiom the cytoplasm and dissociates fiom
TnC. Three transport systems are largely responsible for ca2+ removal: the SR Ca-purnp,
the sarcolemmal L-type Ca channels and the NaJCa exchanger. Al1 three influence
intraceilular [ca2+] to within specific concentration ranges in order to prevent overloading or
depletion of the ce11 and SR of calcium. Conversely, the L-type Ca channel and NdCa
exchanger are both influenced by membrane potential and -:dous intracellular constituents,
such as pHi (Philipson et al., 1982), ATP (Caroni & Carafoli, 1983), and Ca chelators (e.g.
EGTA, EDTA; Trosper & Philipson, 1984). These factors make the L-type Ca channel and
NaKa exchanger possible targets for citrate regulation, by virtue of citrate's metabolic and
chelating nature. The proceeding sections will discuss potential interaction between citrate
and structures responsible for calcium homeostasis: the L-type Ca channel, the sarcolemmal
Ca pump, the NaKa exchanger, the SR Ca-ATPasehelease channel, and mitochondrial Ca
transport.
L.5.L.1 Sarcolemmal calcium channels: influx
One major focus of this study was to examine how citrate affected contractile force.
A logical candidate would be the voltage-dependent Ca channels which initiate E-C
coupling. The importance of calcium influx through calcium channels c m be appreciated by
the cellular dependence on intracellular [ ~ a * ~ ] i , which includes examples such as excitation-
contraction and excitation-secretion coupling mechanisms, formation of the action potential
plateau, apoptosis of cells, and acting as the central role in pacemaker activity. In cardiac
muscle, this also determines the level of myoplasmic calcium transients as well as
stimulation of SR Ca-release (Bers, 1985). The resultant calcium influx is manifested in the
strength of cardiac contractility that closely parallels that of the magnitude of Ica
(Beuckelmann & Weir, 1 988).
Of special interest for our studies are the voltage- and time-dependent cardiac L-type
Ca channels, which are localized to the transverse tubules, allowing the movement of ca2*-
ions into the cytosol and triggering the release of stored ca2+ fiorn SR into the myoplasm.
Hryshko and Bers (1992) previously showed that citrate has a direct action on L-type Ca
channe!. The magnitude of Ca-influx through the L-type Ca channels is a key determinant of
the magnitude of the [ca2']i-transients, thereby determining contractile strength of the heart
(Sperelakis et al., 1994). Therefore, we hypothesized that the reduction in Ica reduced
[ca2+li transients which in turn, reduced the twitch force magnitude.
2.3.2.ii Biochemical characterization of L-type Ca channels: structural significance
L-type Ca channels are often characterized by their sensitivity to dihydropyridines
(DHPs). Most DHPs act as calcium channel blockers or antagonists (e.g. nifedipine,
nitrendipine, verapamil) while others act as calcium channel agonists (e.g. (-)Bay K8644).
These blockers act on various states of channel opening; for example, nitrendipine acts on
the closed state of the calcium channel, while veraparnil causes channel blockade while the
channel is in the open state. The DHP-sensitivity of L-type Ca channels and high density of
DHP receptors in T-tubules have also helped in characterizing the L-type Ca channel.
L l l b L - L J ~ ua ~ L I C U U I C ~ I I J u I I ~ L C ~ I U ~ ~ L I L U L I L ~ L V I L ~ V I L L ~ A k u u 8 p r i o w u U r ri r v v u v r u i i b u -1,
w, 6, p, y (Varadi et al., 1995)-a11 of which could be likely targets of citrate. For example,
the endogenous enzyme, calpain, digests the carboxy-terminal domain of the al-subunit of
the rabbit skeletal muscle L-type Ca channel, which results in proteolytic rundown of the Ca
channels (De Jongh et al., 1994). As shown later in this thesis, citrate has been found to
accelerate the rate of Ica rundown, which lead to the speculation that there may be the
involvement of proteolysis of the regulatory ai-subunit. The structural importance of the al-
subunit also becomes apparent when considering that expression of the subunit alone is
sufficient to form a functional voltage-sensitive ion channel (Perez-Reyes et al., 1989). The
ai-subunit bears receptor sites for three classes of calcium channel antagonists (Varadi et al.,
1995), including dihydropyridine (DHP) (Kalasz et al., 1993; Tang et al., 1993). This
makes the al-subunit a likely candidate for modulating the flow of calcium ions through the
calcium channel.
Both the ai- and P-subunits are targets for phosphorylation by CAMP-dependent
protein kinase (Hosey et al., 1986), and it has been well-documented that Ica-influx through
the channel is extensively regulated by phosphorylation (Hyrnel et al., 1988; Sculptoreanu et
al., 1993). The interface that exists between the adjacent al- and P-subunits results in an
allosteric-like control of voltage-sensing, gating and the access of some Ca channel agonists
(Varadi et al., 1995).
The relevance of these subunits and their formation of a functional Ca channel
becomes apparent when the phenomenon of channel rundown is discussed. In experimental
situations where cellular dialysis occurs upon whole-ce11 voltage clamp, Ca channels would
experience rundown after only a brief period of recording, suggesting that small
molecules/proteins required for modulating or maintaining hctioning channels were
diffusing into the pipette solution of soluble cytoplasinic constituents. Two major factors
that have been associated with this rundown phenomenon are: (1) proteolytic degradation of
the L-type Ca channel al-subunit via a calcium-activated protease, confirming that the
subunit may be involved in the regulation of channel function (Chad & Eckert, 1986); (2) a
phosphorylation-dephosphorylation process (e.g. Ca-dependent phosphatase activity) that
can accelerate the rapid rundown of Ca channels were shown to be slowed or even reversed
d - - - , - - - - -- ---- ----- J --- ------- -- r- - ----a ---*A-- - a - \- ---- J --- -- - - - 7
198 1 ; Doroshenko et al., 1982; Chad et al., 1987; Byerly & Hagiwara, 1988; Schouten &
Morad, 1989). Other factors that may also be involved in Ca current rundown are: (3) the
loss of an intracellular source of energy, such as ATP (Belles et al., 1988); (4) the increase
of intracellular calcium (Belles et al., 1988); and finally, ( 5 ) voltage-dependent rundown
(Hockberger & Nam, 1994; Schouten & Morad, 1989). The findings fiom this thesis that
citrate seems to accelerate the rate of rundown (see Results) corroborate with the idea that
citrate may be interacting with one or more of the factors involved in mdown (as evident
from the Results).
2.3.2.iii Regulation of L-type Ca channels
A number of properties have been extensively detailed concerning the different
regulatory mechanisms that exist on the channel. The regulatory pathways that are known to
modulate the L-type Ca channel are as follows: (1) P-adrenergic stimulation of adenylyl
cyclase, which activates the CAMP-dependent phosphorylation pathway via protein kinase
A, resulting in an increased Ica (Hescheler et al., 1986); (2) muscarinic receptor activation,
causing increased cGMP levels which activates protein kinase G, resulting in
phosphodiesterase (PDE) activity; this will stimulate a direct inhibitory action, via GdGi
proteins, which directly inhibits adenylate cyclase activity, resulting in a decrease in CAMP;
both result in the reduction of phosphorylation or enhancement of phosphatase activity,
resulting in the depressed activity of the calcium channel (Hescheler et al., 1986; Sperelakis
et al., 1994); (3) allosteric regulation of the calcium channel, whereby a "coupling factor",
such as glucose or lactate metabolism, cm directly modulate the channel (Cook et al., 1988;
Meats et al., 1989), resulting in increased [ca2+]i transients due to direct Ca channel
activation; (4) ATP-activated Pz-purinoceptors, in which ATP increases the L-type calcium
current amplitude in rat, as well as increasing the [ca2+li transients of electrically stirnulated
and quiescent cells, via direct allosteric regulation or as a substrate for direct
phosphorylation (Danziger et al., 1988; Scamps & Vassort, 1994; Zheng et al., 1993), via
G-protein coupled receptor activation.
of the L-type Ca channel are examples that could involve citrate. Occupation of the P- adrenergic receptor by an agonist leads to the activation of a GTP-binding protein known as
Gs, which activates adenylyl cyclase, hence producing CAMP. This increase in cAMP leads
to the dissociation of the regulatory and catalytic subunits of the CAMP-dependent protein
kinase (PKA). The catalytic subunit of PKA phosphorylates several proteins including the
L-type Ca channel. This activity is terrninated once GTP is hydrolyzed and reassociation of
the al (GDP) and P-y subunits begin. G, has also been s h o w to be directly stimulated via
Gs, thus bypassing the slower CAMP-dependent pathway (Yatani & Brown, 1989).
In Gpmediated inhibition, the process is not clearly defined. There may be direct
actions of an inhibitory a-subunit on the adenylyl cyclase (Wong et al., 1991), or the
activation of protein kinase G, which elevates cGMP and causes the dephosphorylation of
the regulatory site on the Ca channel (MacLeod et al., 1986; Ahmad et al., 1989).
Stimulation of phosphodiesterase (PDE) from muscarinic stimulation by acetylcholine,
which then causes increased conversion of cAMP to AMP, thereby decreasing channel
phosphorylation, may also contribute to the inhibition of the calcium channel.
It is evident that there are numerous modes of regulatory action on the calcium
channel. Stimulation of any of the previously described recept0rs-e.g. isoproterenol on P- adrenergic receptors-can potentiate or modulate calcium influx through the channels.
Although a pharmacological role has not yet been elucidated for citrate, any kind of
interactions with the receptors or downstream proteins could in principle affect the ICaVL
influx, and ultimately intracellular [~a* ' ]~ transients. Citrate may also serve to inhibit
glucose metabolism, whereby its metabolic regulatory properties may result in decreased Ca
channel activation. It c m be concluded that any disruption of these pathways will tamper the
mechanism by which ca2'-influx is regulated, with potentially serious consequences.
2.3.2.i~ Calcium current: # channels, channel gating, open probability and regulation
L-type Ca current (1~~1, ) in cells is detemined by three parameters: N (# of Ca
channels), y (single channel conductance), Po (voltage- and time-dependent open probability;
L ' a/ - . . -. - - - - - - - - -byb - . ,-J - . -, ..----- - -- ---- a-- ----"*
current, and N is calcium channel density, based on whole ce11 Ica and ce11 capacitance. N
depends critically on the open channel probability (Po, assumed from single channel records
= 0.03; Lew et al., 1991). Calcium channels are rapidly activated by depolarization;
channel activation is modulated by temperature and membrane potential, V,, and surface
potential, V, (Wilson et al., 1983; Hille, 1984). Inactivation of calcium channels is time-
dependent, and also depends on both E, and [ca2']i (Haley & Hume, 1987). The calcium-
dependent inactivation of Ica appears to depend on [ca2']i, and rnay provide a feedback
control to limit fùrther calcium entry. This is readily apparent, even with heavy buffering by
EGTA or BAPTA, suggesting that calcium entering the calcium channel must be exerting
this inactivating effect locally, and perhaps directly at the channel.
The importance of understanding how ICqL is characterized is quite clear: citrate rnay
act on any one of the three controlling features of calcium channel activity. For example,
citrate rnay be responsible for causing a massive rundown of the channel (N), resulting in a
decreased number of fùnctional channels available for voltage-dependent activation. Citrate
rnay also be acting at the single-channel level (i), although this isn't likely, since a recent
study (Hryshko & Bers, 1992) deterrnined that citrate did not alter the unitary current
magnitude or conductance of the cardiac Ca channel. Changes in membrane potential (V,) ,
or calcium-dependent inactivation of the L-type Ca channel rnay also occur because of
citrate exposure. Finally, as described above, changes in phosphorylation can significantly
modulate the open probability (Po) of the channel, resulting in altered current magnitudes.
Citrate rnay serve as a potential regulator of the channel by interfering with the
phosphorylation pathways inherent in the heart.
2.3.2.v The sarcolemmal pump: calcium extrusion
The cardiac sarcolemmal calcium pump was first described in vesicle studies (Caroni
& Carafoli, 1980), in which one calcium ion is transported for every ATP that is hydrolyzed
(Rega & Garrahan, 1986), and appears to be coupled to proton influx (1 Ca:l H;
Kuwayama, 1988). A stimulatory effect of CAMP-dependent phosphorylation was also seen
(Caroni & CarafoIi, 1980). This would make the sarcolemmal pump a likely target for
u u a L c , UWWGVGI, SI~UU~U mat: ut: a reaucrion in eirner A I r levels or [La- Ji transients üue to
citrate, a delayed relaxation in force would result, which was clearly not s h o w in our
results. This makes the pump an unlikely target in its importance in contributing to the
effects observed during citrate toxicity.
2.3.2.vi The NaKa exchanger
The cardiac NdCa-exchanger has been recently cloned (Nicholl et al., 1990) and is
found to be structurally similar to the NaK-ATPase. Its greatest activity has been found to
be located in cardiac tissue, compared to smooth and skeletal (Reeves & Philipson, 1989;
Slaughter et al., 1989; Donoso & Hidalgo, 1989). A stoichiometry of 3 Na:l Ca was
reported in a study done which measured the sodium concentrations required to prevent net
calcium transport at various membrane potentials (Reeves & Hale, 1984; Reeves &
Philipson, 1989). An inward-directed exchange current (and calcium extrusion) occurs at
potentials negative to the equilibrium given by the equation E ~ a - c a = EN^ - 2Eca, and an
outward-directed current positive to the equilibrium potential (Mullins, 1979). ENa-Ca
usually exists near -40 mV to ensure calcium extrusion at rest.
It was found that when sarcolemrnal calcium channels and SR Ca release are
inhibited, contractions and [ca2'li transients can be elicited by membrane depolarizations
(Eisner et al., 1983; Canne11 et al., 1986; Bers et al., f 988). While calcium extrusion is
comrnon during diastole, the action potential upstroke carries the membrane potential above
and shifts the exchanger into the calcium influx mode. This, coupled with the rapid
opening of sarcolemmal calcium channels duringlafier the action potential upstroke can
trigger the release of calcium from the SR. What this suggests is that calcium can enter the
ce11 via the sodiurn/calcium exchanger. Therefore the NdCa exchanger could in principle be
a target of citrate. The fact that the NaKa exchanger is also a highly regulated protein only
increases the possibility that citrate may be acting on exchanger activity. For exarnple,
various calcium chelators, such as EGTA, enhance NaKa exchange (Trosper & Philipson,
1984), resulting in increased calcium influx, while lowering pHi inhibits NafCa exchanger
activity (Philipson et al., 1982). These two findings become relevant after consideration that
upon citrate transportluptake intracellularly, it may act as a calcium chelator. Cellular
-
acidosis, and thus resulting in depressed NaIca exchanger activity.
2.3.2.vii The sarcoplasmic reticulum (SR): Ca-uptake, contents, and release
The SR Ca-purnp
An ATPase purnp is found to lower cytoplasmic [ca2+li (Schatmann, 1989) by
transporting in two ca2+ ions for each ATP hydrolyzed (Tada et al., 1982), although lower
stoichiometries are often reported for in vitro cardiac Ca-pumps. The SR Ca-pump is
regulated by phosphorylation fiom the protein phospholamban following P-adrenergic
stimulation (Lindemann et al., 1983). Stimulation of SR Ca-ATPase activity by
phospholamban is a means by which P-adrenergic agonists accelerate relaxation in the heart
(McIvor et al., 1988), favouring ca2+ uptake by the SR Ca-pump at the expense of the NaKa
exchanger; this ultimately increases SR Ca content available for release.
The SR Ca-purnp is also regulated by Ca, pH, ATP, Mg, and potentially citrate as
well. ATP binds to two sites: a high affinity site and a second, regulatory site. When
cellular ATP levels fa11 during ischemia, there may be some decline in SR Ca-purnping and
slowing of relaxation due to allosteric effects, but [ATPIi would have to be very Iow to
prevent ATP binding to the substrate site. Acidosis was shown to depress the rate of SR Ca-
pumping and thus slow relaxation as is observed during acidosis. However, as shown in Our
results, this prolongation in relaxation was not observed, suggesting that citrate may not be
acting via an acidotic mechanism.
The SR Ca-release channeUryanodine receptor
It was demonstrated that the calcium-release from heavy SR vesicles depends on a
high-conductance calcium channel (Rousseau et al., 1987). Ryanodine, a neutral plant
alkaloid, appears to "lock " the Ca-release channel into an open, but lower than normal,
conducting state (Sutko & Willerson 1980).
The SR Ca-release channel was activated by calcium at submicromolar
concentrations. Caffeine also activates the release channel (Rousseau & Meissner, 1989).
nir ana orner aaenine nucieotiaes also activate the cardiac SK Ca release channel at mM
levels, but only if [ca2+] is high enough to partially activate the channel (Rousseau et al.,
1986). The Ca release channei can be blocked by decreasing pH (Rousseau & Pinkos,
1990). Finally, citrate could in principle interfere with SR ca2+-release by interacting with
the receptor itself. For exarnple, since ATP is a direct allosteric regulator of the SR Ca-
release channel (Rousseau et ai., 1986), then there may be implications of a metabolic
regulatory mechanism of citrate. If citrate is responsible for inhibiting ATP production, the
direct ramifications of this would result in inhibition of calcium release fiom the SR.
2.3.2.viii Mitochondrial calcium transport
In the calcium cycle of the inner mitochondrial membrane, calcium enters via a
uniport system d o m a large electrochemical gradient set up by proton extrusion linked to
the passage of electrons down the respiratory chain. This uniporter is blocked competitively
by physiological [ M ~ ~ ' ] ~ (Nicholls & Ackerman, 1982). Calcium entry via the uniport
pathway exhibits a sigmoid dependence on [ca2+]i; at the [ca2+]i associated with the cardiac
cycle (0.1-1 PM) the influx pathway will be at a relatively low level. The ability of
rnitochondria to accumulate calcium led to speculations that rnitochondria may be involved
in removing calcium from the cytoplasm during cardiac relaxation (Lehninger, 1974).
However, in intact cardiac muscle, when both the SR Ca uptake and sarcolemmal NdCa
exchanger activity were inhibited, relaxation was slowed by more than an order of
magnitude and was often incompIete (Bers & Bridge, 1989). Thus, it seems unlikely that
mitochondria can compete effectively with the SR Ca-pwrnp and sarcolemmal NdCa
exchanger activity under physiological conditions and probably do not contribute
quantitatively to normal cardiac relaxation.
Mitochondrial calcium fluxes may be working to regulate intrarnitochondrial
processes. Three mitochondrial matrix enzymes-pyruvate dehydrogenase, a-oxoglutarate
dehydrogenase, and the NAD-dependent isocitrate dehydrogenase-are activated by
calcium, in the low pM range (Denton & McCorrnack 1980, 1985, 1990). Thus increases in
mitochondrial calcium via the above mechanisms would occur when cytosolic [ca2'li is
relatively high and the energy demands are also high (e.g. contractile activation and calcium-
r r , . ̂ *' ""V "..J, C A I V A A U V 111 ",C"yAU.,LLLIv \ W A U A L L l b u ~ I l V L l u L L u l j LLU J1 C l a l lIILIlCIQ3L)
oxidative metabolism and thereby increase ATP production to rneet increased demands. In
cases where cellular calcium load is high, the mitochondria c m temporarily compensate by
taking up large arnounts of calcium, preventing permanent ce11 darnage. But at the same
time, calcium accumulation by the mitochondria diminishes ATP production and eventually
compromises the mitochondria. Thus the survival of the ce11 might depend on whether the
mitochondria can survive a certain level of calcium loading.
The relevance of the mitochondria to the normal contraction-relaxation cycle is not
clear. It rnay be that the mitochondria play a very minor role in calcium movements during
contraction and relaxation, despite its importance in regulation of intramitochondrial
dehydrogenases and in coping with cellular calcium overload. However, with slower
increases in "rnean" cytoplasmic [ca2+] the mitochondrial calcium transport may play a
critical role in increasing metabolisrn to meet increased metabolic demands or in severe
cellular calcium-overload.
It is evident fiom the preceding discussion that the role of calcium is important in
generating the contractile mechanism, and that [ca2'li handling is highly regulated. Even
more significant are the changes that occur as a result of a disruption in the mechanisms
regulating calcium handling, and by nurnerous factors such as pH, Cai, and various
phosphorylating agents. A calcium-chelating agent, such as citrate, could thus result in a
number of consequences pertaining to calcium availability.
Given the complexities of E-C coupling, however, citrate rnay interfere or modulate
ca2+-homeostasis at very different levels, thus decreasing Ica,L influx, as previously
explained. There is the possibility that citrate may be affecting other intracellular processes
not involved in calcium handling. For example, citrate may affect calcium-binding to
troponin C (TnC), thus influencing the removal of the steric hindrance needed for
crossbridge formation. Another possibility is that citrate may be directly intefiering with
force generation by either ATP-inhibition or changing ATP-affinity to the actin-myosin
cornplex. The next sections deals first with the basic machinery of the contractile
mechanism: the myofilarnents. Following that is a review on the mechanisrn of contractile
force, and the possible role of citrate in altering this mechanism.
2.4.1 The machinery of the cardiac contractile mechanism: the myofilaments
The myofilaments are composed of the thick (or myosin) and thin (or actin) filaments
as well as associated contractile and cytoskeletal cornponents. The myofilarnents make up
the basic machinery frorn which contraction occurs, as they represent the end-effector
responsible for transducing chernical energy fiom energy sources such as ATP to mechanical
energy and work. In simple terms, as cytoplasmic [ca2+] rises, the myofilaments are
activated in a [ca2+]-dependent rnanner. Any active mechanical changes in the sarcomere
are due to the subsequent interaction that takes place between the thin and thick
myofilarnents. The resultant is the translation of the thin filaments toward the center of the
sarcomere, via what is generally known as the "sliding filament theory" (Husley &
, - - - - - -, ---- - - - - - - - - ------- ----- -- . - --- -------a J -J ----- O-- --- --- r--- [ca2']i reached during systole.
The thick filament is composed largely of myosin plus additional smaller proteins,
such as the C-protein. Myosin c m act as an enzyme, since it is capable of hydrolyzing ATP;
myosin itseIf consists of two heavy chains and four light chains. Each of these heavy chains
has a long a-helical tail and a globular head, and foms the main mis of the thick filament.
The two tails are intertwined, forming a two-headed structure. The heads contain the
ATPase and actin-binding site, and form the crossbridges which interact with the thin
filaments. Tropornyosin is also a double-stranded protein, connected by a disulfide bridge
(Flicker et al., 1982). At every seventh actin there is a troponin complex attached to
tropomyosin called the troponin complex. This complex is made up of three fiuictionally
distinctive peptides, or subunits: troponin T (the tropomyosin binding subunit), troponin C
(the calcium-binding subunit), and troponin 1 (the inhibitory subunit). TnT controls the
positioning of tropomyosin. TnI binds to both TnT and actin, thus inhibiting myosin fiom
interacting with actin. The interaction between TnC and TnI is strongly affected by calcium-
binding to the lower affinity calcium-binding site and this effect is important in the
physiologic regulation of contraction.
Cardiac TnC has only one calcium-specific binding site. The calcium-specific site
has a KD of about 500 nM (Pan & Solaro, 1987), and thus responds to physiologically
changes in [cazf]i. TnC c m also bind to other di- and trivalent cations (Fuchs, 1974), such
that cardiac myofilarnents can be activated almost as well with Sr as Ca (Kerrick et al.,
1980).
The main conclusion from this brief treatment of the myofilaments is that contractile
force is controlled by ca2+ ions. For exarnple, the interaction between TnC and TnI is
strongly affected by calcium-binding to the lower affinity calcium-binding site, which is
important for contractile force-regulation. The sensitivity of calcium binding to the calciurn-
specific site is only about 500 nM, which is in the physiological range for contractile
regulation (as described earlier). Thus, any significant changes in calcium levels, such as
depressed intracellular [ca2'] transients due to inhibition of rnay result in decreased
calcium binding to the contractile proteins, which could then be related to citrate-induced L-
typc La cnannei innimion. nowever, in the proceeding sections on the mechanisms 01
calcium-activated contractile force and myofilament ca2+ sensitivity, there remains the
possibility that citrate may be inhibiting contractile force independent of calcium-binding.
The significance of this is that it relegates its calcium-chelating ability to that of a secondary
effect, with a vastly different and far more potent mechanism by which force inhibition
occurs with citrate exposure.
2.4.2 Meehanism of calcium-activated contractile force and MFT ca2+ sensitivity: cooperative mode1 of thin filament activation
It has been shown that the rise in cytoplasmic [ca2+] is the trigger for myofilament
activation. During the diastolic phase of contractility, the calcium-specific sites of TnC are
unoccupied, resulting in a weakened interaction between TnI and TnC, and thus allowing a
certain region on the Tnl protein to interact favourably with actin. When [ca2']i rises,
calcium binds to the calcium-specific site of TnC, allowing TnC to interact more forcefully
with TnI, thus weakening the bonds between TnI and actin. The stenc hindrance to myosin
interaction with actin is removed, allowing for the crossbridge (the myosin head) to interact
with the thin filament. The myosin head then rotates, causing relative filament movement
which generates force production. It is apparent that the number of force-generating
crossbridges reacting with the thin filament is related to the force-generating capability of
the myofilarnents, while the rate of crossbridge cycling is related to the shortening velocity.
Chemically, myosin is complexed with ATP at rest (M.ATP or M.ADP.Pi). As
[ca2+li rises, M.ADP.Pi interacts with actin and phosphate is rapidly released. The affinity
of myosin for actin increases along this series of steps and is strongest after ADP has
dissociated. However, at normal [ATPIi, actin-myosin binds to ATP rapidly, and this
induces dissociation of actin from myosin-ATP. The cycle continues until [~a* ' ]~ declines
or until ATP is depleted. (Goldman, 1987; Brenner, 1987).
The approaches towards accurately measuring rnyofilament cazt sensitivity have
been complicated since large differences have been reported to exist in steady-state
relationships between skinned and intact muscle studies (Yue et al., 1986; Gao et al., 1994).
Various quantitative models have been used to assess the different mechanisms that can
-I.vu.riu.v u.uuuJ .,buru A v i v u - L b ~ J~ ~ U L V ~ D V V L L L L ~ i ~ i i b i piicuiiiabviusrbai cuiu yriy3iurugi~ai
interventions. Previous kinetic models of thin filament activation have used the proposa1
that seven actin monomers associated with a single tropomyosin molecule act as a
cooperative unit (Hill et al., 1980; McKillop & Geeves, 1991). However, a recent study
proposed a new cooperative model of thin filament activation, in which twitch relaxation is
dramatically slowed to such a great extent that both force and [ca2']i reach steady state
dwing relaxation independent of the history of contraction (Dobrunz et al., 1995). The
resultant force-[ca2+li relationship yields an in vivo steady-state relationship that provides
evidence for myofilarnent ca2+ sensitivity. The relaxation phases of the force-[~a~+]i curves
were fitted with the Hill equation (Hill et al., 1980) to provide an approxirnate quantitation
in order to facilitate numerical cornparison of the curves and to enable statistical andysis.
The proposed model (Dobrunz et al., 1995) can be used to approximate changes in
myofilament ca2+ sensitivity in response to citrate intervention. This would provide
evidence in determining whether there are changes in cooperativity amongst the regulatory
units on the thin filament with or without changes in calcium-binding, or changes in the
affinity of strongly-binding crossbridges independent of changes in the affinity of TnC for
ca2+.
Myofilarnent ca2+-sensitivity in cardiac muscle is determined primarily by the
number of attached cross-bridges and ca2'-binding to TnC (Allen & Kentish, 1984).
Depression of maximal ca2+-activated force suggests a decrease in the maximal number of
force-generating cross-bridges, while a decrease in myofilarnent ca2+ sensitivity (indicated
by a rightward shift in the steady-state force-ca2+ relationship) suggests a decrease in the
ca2'-binding affinity of the myofibrils and cross-bridge attachment. The relationship of
force to [ca2+]i during physiological twitch contractions cannot be used to predict the steady-
state relationship, and thus cannot address changes in myofilarnent calcium responsiveness
(Backx et al., 1995). The underlying concept for this is that relaxation is limited by the off-
rate of ca2' from troponin or cross-bridge detachment, and not by ca2+ removal fiorn the
cytosol. The slower the changes in intracellular [ca2'] accompanying twitch contraction, the
more closely the relationship between force and [ca2']i approaches the çteady-state [ca2+li
relationship, and this was found to be true in a physiological setting in skinned fibres
, - ---- - ---, -- -'- J J - , " "/' * **Y UUUbY " A l J L U l V U l l A V \ A U V U L UI., I /UV, U V V b &ALI& k L UA.)
1995; Backx et al., 1995) and CPA (Dobrunz et al., 1995; Backx et al., 1995), both of
which increase duration of contraction and associated intracellular [ca2']i transients without
affecting rnyofilament ca2' sensitivity, confirrns that the peak force-peak [ca2']i relationship
should not be used as an index of myofilarnent sensitivity to calcium, since it was found that
drug-induced shifts were caused by changes in the duration of the calcium transient (Backx
et al., 1995). Furthermore, it was also found in an extension of the ryanodine study that the
peak force-peak calcium relationship could be artifactually shifted and should not be used as
an index of myofilament calcium sensitivity (Gwathrney & Hajjar, 1990).
Thus, there are four major possibilities that have been postulated for depression of
contractile as mediated by citrate: (i) citrate may interfere with calcium-binding (via its
calcium chelation ability, as detailed before) to the calcium-specific site of TnC, which
allows TnC-TnI interaction, and resulting in the rotating of the myosin head with the thin
filament, and generating force; this could also occur as a result of citrate's effects on cellular
pHi; (ii) citrate may be directly interfering with force generation by preventing or inhibiting
ATP-binding to the actin-myosin complex; (iii) citrate may also be regulating force above
30% maximal activation by re-distributing the cross-bridges between force-generating
(strongly bound) and non-force-generating (weakly bound) States, resulting in an altered
conformational or kinetic transitional state; (iv) citrate metabolism may also be responsible
force creating an intracellular acidotic environment by driving the TCA cycle forward,
resulting in increased NADH + H+ production, or direct transport in its protonated form,
again resulting in [H']~ build-up, resulting in steric hindrance via H+-ions. The second
possibility may be due to a number of reasons, such as decreased ATP-production due to
some inhibitory action of citrate on various sites of glycolysis, or even a direct inhibition of
ATP-binding. An accurate assessrnent of changes in myofilament ca2' sensitivity, for
exarnpk, may elucidate whether citrate directly affects the contractile mechanism
independent of its calcium-chelating ability.
Numerous factors exist that c m modify the relationship between [ca2+]i and the force
generated by cardiac myofilaments. By understanding how these factors affect this
relationship, a comparative relationship can be deduced with the role of citrate. Soth
cooling and shorter sarcomere length decreases calcium-sensitivity and the maximum force
generated by the myofilaments (Hibberd & Jewell, 1982; Harrison & Bers, 1989).
Similarly, acidosis and accumulation of phosphates (Pi) also decrease both ca2+ sensitivity
and maximum force generated by the myofilaments (Fabiato & Fabiato, 1978). This may be
important during pathological conditions such as hypoxia or ischemia where intracellular pH
is known to decline (Jacobus et al., 1982). Citrate metabolism may also be responsible for
creating intracellular acidosis in two ways: (i) NADH + H+ production via the TCA cycle;
(ii) direct transport in its protonated form, resulting in [ ~ + ] i buildup. An explanation of the
inhibition in contractile force may be due to steric hindrance by an accumulation of H+-ions
due to citrate exposure.
Decreasing [ATP] increases myofilament ~a'+-sensitivit~, while decreasing
maximum force (Best et al., 1977). Increasing free [ M ~ ~ + ] will decrease myofilament
calcium-sensitivity (Fabiato & Fabiato, 1975), while increasing ionic strength decreases both
calcium-sensitivity and maximum force of the myofilaments (Kentish, 1984). Imidazoles,
such as carnosine, and caffeine, a chemically-related compound, increases myofilarnent
calcium-sensitivity. This, however, is offset by caffeine's other ability to deplete the
intracellular calcium stores from the SR.
The CAMP-dependent phosphorylation of cardiac Sn1 in response to P-adrenergic
stimulation induces a decrease in myofilament sensitivity Ca-sensitivity in intact ventricular
muscle (Solaro et al., 1976; Okazaki et al., 1990). Phosphorylation of TnI c m be reversed
by cGMP or cholinergic agonists (Horowits & Winegrad, 1983). In order for P-adrenergic
stimulation to produce its inotropic effect, the amplitude of the intracellular calcium-
transients must more than compensate for reduced MFT ca2+-sensitivity (via enhanced Ica,
SR Ca release); the decline in myofilament calcium sensitivity induced by P-adrenergic
stimulation is accompanied by an increased off-rate of calcium fiom TnC, thus inducing a
faster rate of relaxation of contractions observed in the presence of P-agonists.
Based on the preceding sections, it is obvious that the nature of citrate toxicity could
be both multi-faceted and cornplex. This thesis consisted of two major hypotheses: (1)
citrate depresses force generation in cardiac muscle by decreasing [ca2+]i transients as a
result of altered L-type Ca channel magnitude and selectivity; and (2) citrate alters the
properties of the contractile proteins, thereby reducing force generation. Our study utilized
both electrophysiological analyses of citrate on cardiac L-type Ca channels in rat and rabbit
ventricular myocytes, intracellular calcium transients in myocytes using fluorescent
indicators, curent-clamp studies in order to study the changes in ionic profile due to citrate,
and fluorescent studies on both right ventricular trabeculae, examining the steady-state
force-[~a~+]i relationships with and without citrate.
We were initially motivated by the findings by Hryshko and Bers, suggesting that
citrate altered L-type Ca channel selectivity. The experiments for this thesis duplicated the
methodology previously reported, using citrate concentrations of 10 mM while maintaining
extracellular fiee [ca2+], of 2 mM. Various methods were used to prevent Ica rundown
contamination in our recordings. Intracellular calcium chelators were used to isolate
changes in the L-type Ca channel: both EGTA and BAPTA were used as cornparison. Ica
rundown was also accounted for (Belles et al., 1988), as three techniques were used to
bypass this confounding problem: (i) proteolytic inhibition, using leupeptin; (ii) maximal
phosphorylation of channels, using isoproterenol; and (iii) perforated patch-clamp technique
using amphotericin B in rabbit ventricular myocytes. Observations for the study included a
similar reduction in Ica and similar leftward shift in reversa1 potentials observed during the
Bers study. Whole-ce11 recordings were then used in constructing current-voltage and
current-time relationships. The evidence that there was some change in the channel
selectivity was not strong enough, as there was no citrate-washout trace, while the possibility
of run-down in Ica was not accounted for in their solutions or protocols. Our studies also
incorporated fluorescent measurements of [ca2+],-transients using fura-loaded myocytes to
examine whether citrate ultimately affected intracellular [ca2+li in order to ascertain whether
citrate affected calcium homeostasis via the voltage-dependent L-type Ca channels. Both
- - - - -----.- - - - - - - - - - - --- - -----
ionic currents, and to observe possible alterations in action potential profile attributed to
alteration in the different ionic current profiles, as well as reflected changes in intracellular
[ca2+] transients. The possibility of citrate transport into the ce11 was also examined, using
intracellular citrate as the calcium chelator, and washing in extracellular citrate. Finally, a
chronic versus acute exposure study exarnined the possibility that citrate may be acting long-
term; voltage-clamp recordings from both citrate-suspended ceIls (treatment group) and
EGTA-suspended cells (sharn group) were taken and used to ascertain whether citrate altered
calcium homeostasis.
The second stage of our studies explored the direct effects of citrate on the contractile
proteins by affecting myofilament ca2+-sensitivity and maximal force. This consisted of
using fluorescent-loaded trabeculae preparations (Backx et al., 1993; Backx et al., 1995;
Dobninz et al., 1995), in which the effects of citrate on force and calcium transients were
initially studied. Our studies had initially pointed towards the possibilities of intracellular
pH, SR-involvement and direct effects on the contractile mechanism itself. Our studies
concerning the role of citrate on MFT ca2+-sensitivity entailed the usage of caffeine and
CPA in order to study changes in steady-state force-[ca2+li relationships in order to examine
whether citrate directly affected myofilament sensitivity, or interacts with the contractile
mechanism; caffeine was found to be unreliable, since it already heightens MFT ca2+-
sensitivity. Using the mode1 of cooperativity in thin filament activation (Dobrunz et al.,
1995), and fitting the force-[ca2']i relationships with a modified Hill equation (Hill et al.,
1980; Backx et al., 1995), quantitative comparisons were made regarding changes in MFT
ca2+-sensitivity The possibility of acidosis was ruled out using carboxy-SNARF-AM as a
pH indicator, loaded into trabeculae.
As previously mentioned, electrophysiological analyses of the effects of citrate were
used to determine influences on cardiac L-type Ca channels. Whole-ce11 recordings using
both voltage- and current-clamp will be made on rat ventricular myocytes (Hamill et al.,
1981) in order to ascertain changes in peak Ica, activatiodinactivation properties, as well as
possible alterations in the reversa1 potential. Force, sarcomere length, and calcium was
detemined in rat cardiac trabeculae by microinjecting k a - 2 saIt in the trabeculae. The
temporal relationship between force and the calcium transients can be assessed using
ratiometric measurements in fluorescent levels (Backx & ter Keurs, 1993). Finally, calcium
transients and currents was recorded at the single ce11 level (Berlin & Konish, 1993).
Influence of citrate on al1 of these responses will also be deterrnined.
3.1 ELECTROPHYSIOLOGICAL STUDIES AT THE SINGLE-CELL LEVEL
3.1.1 Isolation of rat ventricular myocytes
250-300 g male Sprague-Dawley rats (Charles River) were used as the mode1 for al1
experiments. The rats were housed in the animal care facility in the basement of Toronto
Hospital, General Division, and were fed using standard rat chow. Ln brief, rats were
pretreated with heparin (Hepalean 1,000 IUkg i.p.) for ten minutes. The rats were then
injected with sodium pentobarbitol (50 mgkg ~omnotol" i.p.) and the hearis were quickly
excised ( 4 0 seconds) by making a midsternal incision and rapidly severing any vascular
attachrnents, and inserted via the aorta ont0 a cannula, where they were retrogradely perfused
initially with pre-bubbled 1 mM [ca2+l0-bicarbonate solution (in mM: NaCl 123, KCl 5.4,
MgCl* 1.2, NaH2P04 1.2, NaHCO, 20) at 35OC. Al1 solutions were pre-bubbled with
carbogen (5%-C02/95%-Oz) in order to maintain a pH of 7.4. Perfusion with 1 mM [ca2+],
at 35OC caused the heart to contract vigorously at 180-200 beatslminute. After the heart was
sufficiently flushed of blood, the solutions were switched over to O mM [~a~+]~-bicarbonate
solution for 5-6 minutes, followed by 7-8 minutes of enzymatic perfusion with collagenase
(0.5 mglml, Boehringer-Mannheim, type B) and protease (0.033 mg/ml, Sigma type XIV) in
l lALvl Lbu J O - u ~ ~ ~ u u ~ ~ a L ~ SUIULIUIL. 1 IIC I I C ~ L W ~ S ~ 1 1 ~ 1 1 GUL JUSL u a u w LIIE: aiilil LU a
potassium-rich (high-~') solution (in mM: K+-glutamate 120, KCl 20, N-2-
hydroxyethylpiperazine-ZV-2-ethanesulfonic acid [HEPES] 10, MgC12 1, glucose 10, K-
EGTA 200 PM, 1% penicillin/streptomyocin, pH 7.4 wl KOH) where the ventricles were
minced (1 mm x 1 mm chunks) and swirled in glass beakers. The solutions were then
filtered and collected into 50cc falcon tubes, afier which the pellets were placed into fiesh
h i g h - ~ + solutions with lmglml bovine serum albumin (Sigma) and diluted. The cells were
then placed in the dark for later use (1 -2 days).
3.1.2 Voltage-clamp experiments
3.1.2.i Voltage-clamp technique
Whole-ce11 voltage-clamp experiments were conducted using an Axopatch 200A
patch-clamp amplifier (Axon Instruments, Inc.). Patch pipettes (thin-wall, single-barrel,
borosilicate glass tubing; 1.5 mm ODl1.12 mm ID, 4") were made using a Flaming/Brown
micropipette puller (Sutter Instruments), and firepolished to a pipette resistance of 1-3 MC2
(the resistance is measured by applying a 2.5 mV voltage pulse in the pipette and monitoring
the resulting current flow on the cornputer screen), The isolated myocytes were placed onto
a perfusion apparatus located on the stage of an inverted microscope. After gigaseal
formation using suction onto the myocyte membrane, and rupture of the membrane patch
(via mechanical andor electrical stimulus), series-resistance (Rs) compensation was then
performed in order to eliminate the R, of the membrane. Access into the ce11 was done at a
holding potential of -40 mV. The membrane potential was then changed to -90 or -80 mV.
Extracellular solutions were then flushed through at various intervals, while recording Ica.
3.1.2.ii Experimental Rationale
It has been shown that the addition of 10 mM citrate at constant free extracellular
[ca2'] of 2 mM reduced contractions in ventricular muscle and cardiac myocytes by 1540%
(Hryshko & Bers, 1992). Also, peak Ica was reduced to 53 + 4 % of control in the presence
of 10 mM citrate (same study; Bers et al., 1991). A shift in both the peak of the current-
- . r - - -. - - . - - - -- - - -------- \-iC*/ -- ..--- --a-.--
1 "'b-" ' - potentials was also a cornmon occurrence. It was also reported in the sarne study that there
was a restoration in the peak Ica of the 1-V curve upon citrate washout (indicating a
reversible effect of citrate), and a temporary alteration of the selectivity of the charnel. We
sought to confirm these results ant to determine the underlying mechanism for these
observations.
3.1.2.G Experimental Method and Protocols
Voltage-clamp measuring techniques were used to assess the effects of citrate on
both Ica and the force-ca2+ relationship. Rat ventricular myocytes will be isolated as
described earlier. The extracellular solutions during the whole-ce11 voltage-clamp
experiments were as follows (in mM): Control = NaCl 140, HEPES 10, MgC12 1, glucose
10, CaC12 2, mannitol 40; Citrate-containing = NaCl 1 10, HEPES 10, Na3-citrate 10,
glucose 10, CaClz 9.9 (see Appendix 1 for calculations), mannitol 25. The pipette solution
as follows (in mM): CsCl 125, BAPTA 10 (or EGTA 5) , HEPES 20, MgC12 1, MgATP 5,
pH 7.1-7.2 wl CsOH. 200 pM leupeptin were added in some experiments to eliminate
proteolytic rundown of Ica (Chad & Eckert, 1986; De Jongh et ai., 1994). Both BAPTA and
EGTA were added as high- to low-affinity calcium buffers in order to prevent calciurn-
dependent inactivation of calcium channels (Hadley & Lederer, 199 1). Mannitol was
initially used in the extracellular solutions during the [EGTAIi study to compensate for
osmotic differences between extracellular and intracellular solutions. Subsequent
experiments used lower [CsClIi at 110 mM which eliminated the usage of mannitol. 200-
400 pM [CdC12] was added at the end of experiments and a 1-V recording was taken. CdC12
eliminates any extraneous currents that may interfere with the Ica-recording, giving an
accurate representation of calcium currents via L-type Ca channels. Al1 Ica-V traces were
cadmium-subtracted.
Ica was recorded before/during/afier 10 m M citrate exposure. ExtracelluIar free
[ca2'] will be rnaintained at a constant concentration of 2 mM. The constant fiee calcium
concentrations was kept constant to establish the effects of citrate independent of its effects
on the [ca2'], as a result of ca2'-buffering. An 1-V recording was taken beforelduringlafter
ULLCLLC Cnpu~ulC IUI LUIII~SUQLIV~: purpuses. ne nolaing porenrial was -YU mv to m~nimize
voltage-dependent calcium current run-dom. A current-time (1-t) relationship was also
recorded (2 minutes, control; 3 minutes, citrate wash-in until steady-state is reached,
followed by 1-V recording; 10 minutes, citrate washout) to detail the progression of the
citrate phenomenon over time. En order to minimize Ica rundown via proteolytic enzymes,
200 pM leupeptin (a general protease inhibitor; Sigma-Aldrich Co.) was added in some
experiments. Isoproterenol experiments were conducted to examine a possible role of citrate
on the phosphorylation mechanisms associated with the L-type Ca channel. Isoproterenol
(200 nM) was used to phosphorylate the channel. Should there arise no changes in the
magnitude of Ica, then citrate works independently of second-messenger pathways. Finally,
M h e r experiments were also conducted in order to examine whether citrate was transported
into the cardiac myocytes. BAPTA was exchanged for 10 mM citrate in the intracellular
pipette solutions. The experimental protocols for 1-t and 1-V were repeated, washing in and
out 10 mM extracellular citrate, in an effort to examine whether citrate is also transported
into myocytes, and acting on an interna1 site.
Current-clamp (1-t) experiments were also be conducted to determine the effects of
citrate on the action potential profile of cardiac myocytes. In these experiments, a small
depolarizing current is injected into the ce11 to trigger an action potential. Any changes in
four pararneters-resting membrane potential (r.m.p.), action potential duration at 50%/90%
(APD T50%/T90%), and profile-may result from citrate's actions on channels other than
L-type calcium channels, or the indirect result of changes to the calcium channels. The
current-time recording protocol for studying action potentials will mirror that in the voltage-
clamp study: 2 minutes, control; 3 minutes, citrate wash-in followed by action potential
recording in citrate; 10 minutes, afier citrate washout, followed by an action potential
recording in citrate washout. Extracellular solutions are (in mM): Control = NaCl 140, KCl
4, MgC12 1, HEPES 10, glucose 10, CaC12 2, pH 7.4 w/ NaOH; Citrate = NaCl 110, KC14,
MgC12 1, HEPES 10, Na3-citrate 10, glucose 10, CaC12 9.9, pH 7.4 w/ NaOH. Tntracellular
solutions are (in mM): K+-glutamate 1 15, KC1 4, EGTA 0.2, HEPES 10, MgATP 5, pH 7.1
w/ KOH.
1 LI l u r a i c u pa~ui-uairlp iznpcr I I I I ~ . I ~ L S wert: aiso prererrea Io reaucea runaoWn 01 Ica.
Both nystatin and arnphotericin B were used for this method, in which there is minimal
dialysis of endogenous cellular proteins and calcium-buffering mechanisms (Horn & Marty,
1988; Bassani et al., 1995). The method ako allows voltage-control of myocytes and has
been reported to eliminate rundown of INa and Ica for up to two hours (three times longer
than the duration of normal experiments). A stock solution of nystatin was made (50 mg
nystatin, Sigma Chernical Co., St. Louis, MO/lmL dimethyl sulfoxide, DMSO), sonicated
for 5 minutes, and refiigerated at -70°C before use. A concentration of 100-150 pglml
nystatin was made with cesium-based intracellular solution (see voltage-clamp recordings).
The pipette tip was filled with regular pipette solution, while the nystatin solution was used
to backfill the rest of the pipette. About 5-10 minutes was the time expected for capacitance
transients to develop. However, after 10 experimental days of changing variables such as
higher temperatures, usage of nystatin was completely unsuccessfid. Arnphotericin B was
used in place of nystatin, as a result. A stock solution of arnphotericin B was also made in
DMSO, sonicated for approximately one minute, and kept in the dark at O°C. The pipette
solution had a concentration of 240 pg/ml arnphotencin B, with 50 pM [ca2']i in the pipette.
About 5-10 minutes is needed for the capacitance transients to develop. Following this, the
normal 1-t and 1-V recordings can be made subsequent to compensation. Rabbits were used
with amphotericin B at higher temperatures of 35"C, since both nystatidamphotericin were
ineffective in rat ventricular rnyocytes.
3.1.2.i~ Determination of free [ca2+], using association constants
In order to calculate the free calcium concentrations of the extracellular solutions, the
association constants of citrate at varying temperatures rnust be taken into account. The
apparent association constant for each metal-ligand interaction must be evaluated, either
from direct measurement (e.g. titration using murexide; Hryshko & Bers, 1992) via
calculations of free ion and ligand concentration. (Harrison & Bers, 1989). Appendix 1 lists
the equations and calculations of the total [ca2'la required with 10 mM citrate. As per the
statement, "...and Ca was titrated to a free Ca concentration of 2.0 mM using murexide
absorbance ...[ which] required the addition of ~ 7 . 9 mM Ca" (Hryshko & Bers, 1992), and
ilulll L l l b urlbbr L b u JO L L L k U L - > C U U A A A U A A L U U d I A A b V U A V I U I A . Y I - i V l - i 7 - -a-------- \- - - - - - - - - - z
General Hospital, Clinical Biochemistry Labs), a total [ca2'], of 9.9 mM was used in citrate-
containing solutions for voltage-clamp experiments, as well as for subsequent trabeculae
experiments.
3.2.1 Fluorescent Measurements using Fura-2 salt; calibration of fura-2 signals
Fura-2 salt has an affinity for ca2' with an in vitro KD close to 200 n M (Grynkiewicz
et al., 1985). In this study, a patch-electrode solution containing the K+-salt form of h a - 2
salt at 100 p M was used to dialyze clarnped cardiac myocytes (see Sections 3.1.1 and
3.1 -2.i). Pipette resistances were fiorn 2-3 MS2 to accelerate the rate of k a - 2 loading in the
cells. The pipettes were coated with an opaque paint to prevent photobleaching and
eliminate the background fluorescence (M-Coat D, Measurernents Group, Raleigh, N.
Carolina; O'Rourke et al., 1993). Intracellular solutions were cesiurn-based (see Section
3.1.2.i); extracellular solutions were potassium-fiee and contained fiee [ ~ a ~ ' ] , = 2 mM.
This allowed the current tracings to be relatively fiee of K+-currents. The [ca2+]i transients
were elicited with depolarizations of 150 msec fiom -80 to + 10 mV, delivered at 0.2 Hz (i.e.
every 5 s).
Fura-2 fluorescence signals fiom the myocytes were calibrated in terms of [ca2'li
with the ratiometric calibration procedure (Grynkiewicz et al., 1 985):
[ca2'] i = KD x p x (R-Rmin)l(Rma-R)
The 3401380 fluorescence ratios were converted into [ca2+li using the in vitro calibration
curve, where R,, = 1 1.1 PM, Rmin = 0.168 PM, Kto = 3.97 PM, and the p-value was 15.6
(see Appendix 2) and used to measure the calcium concentrations (from millivolts to
micromolar). The signals were collected using the program CIampex from the software P-
clamp 6.0 (Axon Instruments) which allowed for simultaneous current and transients
recordings, using multiple channels. Al1 the data was collected via the PMT where the
signals were converted via an A/D converter where they were stored for later analysis.
3.2.2 Experimental Protocols
Rat cardiac myocytes were voltage-clamped in normal Tyrode's solution containing
0.1 pM CaC12 to prevent possible calcium overload. Intracellular solutions were cesium-
based, with 100 p M fura-2 salt and 5-8 mM NaCl. After breaking in, the cells were pulsed
at 1 Hz to ensure rapid loading. The voltage protocols were as follows: Vhold = -90 mV; V I
- s I v 111 v iur I UV- 1 3u rnsec; v 2 = -au m v ror LU msec. rroper ioaaing rooK Perween 4-3
minutes; the duration between pulses were at 0.2 Hz. Fluorescent oscillations were
observed with every pulse; this ensured that the cells were fura-2 loaded and contracting
normally .
Control calcium transients and currents were taken at O", 3", and 6" in order to fully
maximize the magnitude of the fluorescent signals. A solution containing 10 mM citrate + 9.9 mM CaC12 was then washed in for 3-4 minutes, and both current and calcium transients
were recorded. Finally, citrate was washed out, and currentltransient signals were recorded.
The signals were filtered at 300 Hz using the N D acquisition board. For analysis purposes,
the signals were further filtered using the DC-filter on the Clampfit program fiom the P-
clamp 6.0 software in order to eliminate any extraneous noise, and were compared.
3.3.1 Isolation of right ventricular trabeculae in rat
Hearts were excised fiom 250-3008 rats (LBN-FI strain, Harlan, Indianapolis,
Indiana), after injection with sodium pentobarbitol (50mgkg i.p.) and making a midsternal
incision. The h a r t was removed from the chest cavity afier severhg vascular attachrnents
and the aorta was quickly cannulated. The heart was arrested after perfusion with a high-K+
modified Krebs-Henseleit solution (in mM: NaCl 120, KCl 5, MgC12 1.2, NaH2P04 I .2,
NaHC03 19, CaC12 1, glucose 10). The solution was continuously bubbled with 5%-
Co2/%%-o2 carbogen mixture to maintain a pH of 7.4. Long, thin trabecuIae were
dissected fiom the right ventricle, using a dissection solution (modified K-H solution w/ 140
mM KCl) and mounted between a micromanipulator and a stationary hook in a perfùsion
bath located on the stage of an inverted microscope. The trabeculae were stimulated using
an electrical pulse stimulator and platinum electrodes m i n g alongside the bath.
Sarcomere length was measured using laser diffraction by illuminating the muscle
with a 5-mW He-Ne diode laser. The diffiaction pattern was transmitted through the
objective to the fiont optical port of the microscope and projected ont0 a video carnera using
a telephoto lens focused ont0 the back plane of the objective. The first-order difiaction
pattern was calibrated using a diffraction grating and was used to set the end-diastolic
sarcomere length to 2.2-2.3 Fm.
3.3.2 Fluorescence Measurements using Fura-2
Excitation ultra-violet (U-V) light from a 75-W mercury I m p (Oriel Corp, Stratford,
CT) was passed through bandpass filters (Omega Optical, Brattleboro, VT) centered at 340,
360, or 380 nm , located in a filter wheel. The filtered light was projected ont0 the muscle
via a 10X objective (10X Fluor, Nikon, Tokyo, Japan) in the inverted microscope using a
dichroic mirror (4OODPLC, Nikon, Tokyo, Japan). The use of a large field of illumination
minimized ca2+-independent changes in the fluorescent signal associated with movement of
the preparation during a twitch, and reduces the amount of excitation light required to
measure ca2'-dependent changes in fluorescence with a high signal-to-noise ratio.
*A.- -..A... -- "e"' " "' """""- ' J ---- - -J"-- . - --- ------------- - - 0- 1
filter at 5 10 nm to a photomultiplier (PMT) (Hamamatsu, Bridgeport, NJ), whose output was
filtered at 100 Hz (3 dB), and recorded via an A/D data acquisition board (Data Translation,
Marlboro, MA) and stored in the computer for later analysis. The preparations were only
illurninated with the excitation Iight for short periods of time with the use of a filter wheel.
Brief illumination of the preparation reduces the effects of photobleaching, a problem that
interferes with the accurate calibration of the fluorescence signals.
A micropipette containing 1 mM fura-2 salt and backfilled with 140 mM KCl was
used to load the trabeculae. The pipette resistance averaged fiom 150 to 250 Ml2 (< 0.2-pn
tip diameter) when placed in the modified Krebs-Henseleit (K-H) solution. Larger tips
would result in significant damage to the trabeculae, causing local spontaneous contractures
in the region of impalement. After impalement of an unstimulated muscle, the muscle was
injected with 4-6 nA of hyperpolarizing current (depending on dimensions of trabeculae
muscle used) for 30-40 minutes, and the k a - 2 was allowed to spread throughout the muscle
via gap junctions.
Dual excitation of h a - 2 allows the determination of [ca2+li using the ratiometric
technique independent of the arnount of dye being used. The ratio method allows for
accurate measurernents to be made throughout the time-course of experiments (see Backx et
al., 1995). In al1 preparations, the autofluorescence was recorded before loading with k a - 2
and at the end in order to achieve the intensity and contribution of background fluorescence
to the subsequent recordings.
The measured ratio (see Section 3.2.1) was used to estimate [ca2'li by the equation
given before (Grynkiewicz et al., 1985). The accurate determination of these constants is
essential to determine [ca2']i. Calibration of fura-2 signals are discussed at length in
Appendix 2.
3.3.3 ExperimentaI Rationale
The use of fluorescence in a multi-cellular preparation, such as rat cardiac trabeculae,
is a powerful tool in accurately measuring both ca2+ and force for the analysis of regulation
V I Y V . . . i I . . V C . I W * U . I V C I " * I . * Il-"' ""r"""""- -"- .. '--- - - - - - - - - - - - -
and force, thereby providing central insights into its mode of action.
Initial studies showed the effects of I O mM citrate on a trabeculae preparation. The
time-course was compared to current-time recordings in Our voltage-clamp study. We
analyzed the peak systolic and diastolic force and [ca2'li transients. We also analyzed the
tirne-to-peak and the time-to-50% of relaxation of the force and [ca2']i transients.
Autofluorescence before h a - 2 loading and after quenching of k a - 2 with 0.7 mM MnC12
were recorded at wavelengths of 380 and 340 nm to allow accurate calibration of the [ca2+]i
from the fluorescence recordings. In some cases we also measured 360 nm to assess
whether changes in autofluorescence or background fluorescence (Backx et al., 1995)
occurred with citrate application. Sarcomere lengths were maintained at 2.3 pm to ensure
maximal force (F,,) generation.
Both caffeine and cyclopiazonic acid (CPA) will then be used to examine the role of
citrate on the sensitivity of the contractile mechanism. It is possible that citrate may be
interfering with the contractile mechanisms governing the functioning of the heart, as
documented in previous clinical cases. In examining the citrate effects on cardiac
contraction, 10 mM caffeine in modified K-H solution and 2 mM CaC12 was added to the
muscle, and the muscle was allowed to equilibrate with the solution. For the purpose of this
study, the concentration of caffeine is sufficient to deplete the muscle of SR calcium (Bers et
al., 1991). Once steady-state was reached, force and fluorescent measurements at 380 and
340 nrn were then made. A solution of 10 mM caffeine, 10 mM citrate, and 9.9 mM CaC12
was then added to the perfusate tubings, allowing rapid perfusion of the muscle.
Force/fluorescent measurements were again made after achieving steady-state. The citrate
solution was then washed out with 10 mM caffeine plus 2 mM CaC12, subsequently followed
by caffeine washout.
For the CPA experiments, 100 p M CPA was added to modified K-H solution + 8
mM CaC12. 8 mM was a sufficiently high enough calcium concentration in order to reach
Fm,. For citrate solutions, 10 mM citrate + 100 pM CPA + 17.2 mM CaC12 was added to
the K-H solution. The protocol followed the caffeine study. except without the washout of
LYA. CYA 1s Iound to have rrreversible eIIects witn mountea traoecuiae, wirn aeveiopmenr
of spontaneous after-contractures and the quenching of fluorescent signals.
From the CPA studies, phase loops can be constructed using force vs. calcium
values. The insights that can be provided fiom these loops can reveal changes in the state of
the contractile mechanism, such as sensitization, affinity constants, and the relaxation phase.
This can be determined by exarnining the pattern provided by the force-calcium phase loops.
Analyzing the half-maximal tirne from point of stimulation to peak force between control
and citrate using a modified Hi11 equation (Hill et al., 1980) in order to quanti@ the data c m
verie this. The data provided fiom this study, coupled to that previously discovered, will
determine the exact nature by which citrate acts on the cardiac contractile system.
3.3.4.i pHi measurements in cardiac trabeculae
The possibility exists that citrate is acting on the contractile mechanism as the result
of citrate-induced intracellular acidosis. Measurement of pHi therefore provided insights
into the effects seen with citrate wash-in. Ratiometric techniques would be used to improve
the accuracy of pHi recording. The determination of the ratio of two fluorescent signals
originating from an intracellular fluoroprobe at different wavelengths provides a
measurements of pHi independent of dye concentration, path length and excitation intensity
(Grynluewicz et al., 1985). The use of caroxy-seminaphthorhodafluor-1 (carboxy-SNARF-
1; Molecular Probes, Eugene, Oregon, USA) provides two relatively strong pH-sensitive
signals for ratioing, as opposed to the one strong and one weak signal from 2',7'-bis-(2-
carboxyethy1)-5(and-6) carboxyfluorescein (BCECF-AM) (Buckler & Vaughan-Jones,
1990).
3.3.4.ii Loading of carboxy-SNARF-1 and calibration
SNARF-1-AM was initially dissolved in dimethylsulfoxide (DMSO) at a
concentration of 1 mg/mL and stored at -70°C in 50 PL aliquots, and subsequently diluted in
modified K-H solution to a final concentration of 10 PM, where the muscle was perfused
continuously for about 1 hour. The outputs from the photomultiplier tubes (PMT) were fed
to a W converter and filtered at 10 Hz. The signals were passed to an AD converter, and
digitized. The cornputer program automatically calculates the 590 nm/640 nm ratio.
The emission ratio was converted into a linear pH scale, where the following
equation was used to calculate [H~] :
RIHtI = [ H ' I ~ r n a x + (Rrnin-R) (KdF640minlrnax)
3.3.4.iii Experimental Protocols
The pHi measurements documented the effects of 10 mM citrate in O mM [cazf], K-
H solution on mounted cardiac trabeculae. Current-tirne relationships charted the acidosis
-a"" U ' L V A - , --*'-' 0, '"a" ""VA VU'"" ""Y" 1s.. * * L * A I I I V A - 4 " - - - w---V---- 1- ------ J -. - - - - - - - - - -J
washed in to veri@ the drop in pH. Afier steady-state control levels have been reached,
citrate was washed in, while recording force and emission signals at 580 and 640 nm.
Citrate was then washed out, after which point, once steady-state has been reached, force and
emission signals was again be recorded. Finally, NH4Cl was washed in and out to venfy
acidosis. Autofluorescence at 590 and 640 MI were taken before and after the experimental
protocol to eliminate background fluorescence.
3.4 STATISTICS
Statistical values are expressed as mean + S.D. (or * S.E.M. if stated) for myocytes
and muscles. Treatments were analyzed for significant differences using paired t-test for
data under similar experimental conditions. Values of p < 0.05 were considered
significantly different.
The Marquardt-Levenberg algorithm in a non-linear least-squares procedures was
used to fit the force-[cazcli relationships from the ha-loaded rat trabeculae, as defined by
the modified Hill equation. The X2-values that were calculated fiom the fitted curve were
used to assess the accuracy of the approximated curve of best fit, and have been tabulated
(see Table 7).
As described earlier, the approach used for our study was characterized by two
hypotheses in order to establish the mechanism of citrate in cardiac E-C coupling. The first
was related to selective alteration of the L-type Ca channels, causing a depression in
contractile force due to decreased SR Ca-release activity. The second hypothesis proposed
that citrate could somehow interact with the contractile proteins, resulting in force inhibition.
The study was initially inspired by a recent publication (Hryshko & Bers, 1992), which
proposed that citrate altered L-type Ca channel selectivity. The Bers study reported a decrease
in ~a~+-~e rmea t i on during citrate exposure, which could in principle explain the inhibition in
cardiac contractile force. Our study duplicated the experimental conditions and protocols
used by the experimenters, but we could not replicate their results. There were discrepancies
in their statements that may have attributed to experimental error on their part. However, as
shown below, their conclusions were based on fûndamentally incorrect findings, and are
ultirnately insufficient in providing a mechanism for o u . reported observations.
4.1 ROLE OF CITRATE ON CARDIAC CALCIUM HOMEOSTASIS
4.1.1 L-type Ca channels study: problems with Ica rundown
Hryshko and Bers in 1992 reported that in the presence of 10 mM extracellular citrate
and a pipette solution containing 10 mM [EGTAIi, peak Ica was reduced to 53 * 4% of
control, despite maintenance of fiee [ca2'], at 2 mM. In addition, both the peak of the
current-voltage (1-V) relationship and the apparent reversa1 potential (Er,=) were shifted
toward more negative potentials by more than 30 mV. The effects were more pronounced
using lower [~a*+],'s. They suggested that the basis for this phenornenon was due to
alteration in permeation and selectivity of the L-type Ca channel, regardless of surface
membrane potential influence (Ginsburg & Shimoni, 1989).
Our original approach exarnined the possibility that citrate was acting directly on the
L-type Ca channel. One proposed mechanism that could explain the changes observed in Ica,L
was that citrate was somehow metabolically regulating the channel. We therefore conducted a
series of experiments that duplicated the prevlous study's expenmental ~0nditi0nS in rat
ventricular myocytes, and to explore the mechanism for the reduction in peak Ica. One
curiosity from the previous study (Hryshko & Bers, 1992) was the omission of the temporal
dependence of the effects of citrate. In that study, the myocytes were exposed to citrate for
relatively short periods of time (QO minutes). Ica was recorded before/during/after 10 mM
citrate exposure, while extracellular free [ca2'] was 9.9 mM. The rationale in maintaining
constant free [ca2+], was to eliminate the buffering effects of citrate, thereby examining the
direct effects of citrate. The explanation for the initial increase in inward ICaL in the 1-t
relationships probably reflects a slight increase iii the f ke extracellular [ca2'] (Le. for
solutions containing 10 mM citrate, the free [ca2'], was calculated using binding constants, to
be 10.264 rnM; see Appendix 1). Further analysis revealed that this was 0.364 mM more
than necessary, and this was verified using ca2+-sensitive electrodes (Toronto Hospital,
General Division; CIinical Biochemistry Labs; Dr. Allen). Subsequent experiments were
done using 9.9 mM [ca2+],.
Current-voltage (peak Ica-V) relationships were taken before, during, and after citrate
exposure for comparative purposes. Voltage protocols for family recordings were as follows:
Vhold (VH) = -90 mV; VI = -40 mV for 40 msec; Vh = -50 to +60 mV; Vj = -80 mV for 20
msec. The holding potential was at -90 mV, instead of at -80 mV, since more negative
potentials have been shown to prevent voltage-dependent ICaL rundown (Schouten & Morad,
1989; Hockberger & Nam, 1994). A current-time (1-t) relationship was also taken (2 minutes
after initial dialysis, control; 3-5 min., following citrate application until steady-state is
reached, followed by a I-V recording; 8-10 minutes, after citrate washout followed by a 1-V
recording) in order to examine the tirne-course of the effects of citrate. The explanation for
the time-dependent recordings is straightforward: (1) it provides insights into the mechanism
for citrate-induced inhibition; (2) it is also a standard method in assessing for rundown.
Voltage protocols for the current-time relationships were as follows: VH = -90 mV; VI = -40
mV for 40 msec; V2 = +10 mV for 501200 msec (pending on calcium-overload conditions);
V3 = -80 mV for 20 msec. 1-t relationships were sampled every 5 seconds to prevent calcium
overload as well.
I lEU1b T . 1 J l 1 u V v ~ ï ~11b UIXCIULJ V A I V L I A L V A w x r n u v v i i u i - r r r r - - - - r i - - - ir ------ ----- L-- J U
on Ica. The intracellular pipette solution contained 5 mM [EGTAIi, which was lower than the
concentrations used in the previous study. Figure 4.1A shows typical raw traces before citrate
wash-in (control; lep pane2); during citrate exposure (after 3 min., citrate wash-in; middle
panel); and after citrate washout (10 min., citrate wash-out; right panel). Average
capacitance for al1 cells used were 37 & 14 pF. The current-voltage (1-V) relationship in
Figure 4.1B shows the changes in both peak Ica and E,, while the raw traces were taken at
+10 mV of the 1-V relationship, at various phases of each experiment (Figure 4.1A). After 3
minutes of exposure to citrate, peak Ica is reduced by approximately 35 + 2% (n = 7). After
washing out citrate for 5 minutes, Ica is reduced further to a decrease of 44 A 2% of the current
prior to citrate application (n = 7). A leftward shift in ER, of about 15 mV was also observed
with citrate exposure; however, again, the shift persisted even after citrate wash-out. In these
experiments, the initial rate of Ica rundown was -3.1 f 0.3 pA/pF/min during the control
period. Upon citrate wash-in, there is an apparent acceleration in rate of rundown to -4.1
0.4 pA/pF/min (p < 0.05, n = 7) over a time-course of 5 minutes of total citrate exposure.
Upon wash-out, the rate of rundown fell to -1.4 f 0.7 pA/pF/min, which is an almost two-fold
drop between the control and washout rates. The values are listed in Table 1.
The complications introduced by rundown severely limited our ability to characterize
the effects of citrate on Ica. We therefore sought to modie our experimental conditions to
minimize ICqL rundown. A recent study found that the rate of rundown of Ica was accelerated
at higher interna1 [ca2+li, suggesting that rundown was ca2'-dependent. Based on these
findings, we replaced EGTA with BAPTA, a higher-affinity ca2'-chelator capable of rapidly
chelating ca2+, thereby reducing the [ca2'],-localization at the inner rnouth of the pore (Bers
& Peskoff, 199 1). This property would be expected to reduce ca2+-dependent rundown of b. Figure 4.2A shows the raw traces in control (2 minutes after gaining access into the
cell), 3 minutes following citrate application and 8-10 minutes after citrate washout. Notice
that for this cell, the current is actually increased following citrate and returns to control
values after citrate washout. The average peak Ica-V relationships for control, citrate and
following citrate washout recorded at the same time points as in Figure 4.2A are summarized
in Figure 4.2B for seven cells. On average there is no reduction in the peak Ica-voltage
Washout
-F
Figure 4.1: Current-voltage (1-V) relationship showing 10 m M citrate exposure in rat ventncular myocytes: 5 mM [EGTA],. A: Raw traces correspond to peak Ica at +10 mV. B: Constructed 1-V relationship showing effects of 10 mM citrate (mean k SEM; n = 7).
Table 1 : Effects of acute exposure (< 2 hours) of rat ventncular myocytes to 10 m M cityate.
*Exposure to Citrate (pA/pF) Rate of Run-down (p AlpFlmin) % increase / decrease with
Citrate washint Description (I)Iwi (ii)I, after (iii)l, %Inhibition (i)Before (ii)Dunng (iii)After washout
Before 3"citrate afier 10" (Ic Jmin) (IJmin) (IJmin) exposure washout
5 mM EGTA -16.8 -1 1.0 -9.3 -44.4 -3.1 -4.1 -1.4 +32.2%/ ( n = 7 ) k3.6 *2.0 *2.0 *2.3% M.3 M.4 *0.4 -54.8%
10 mM B APTA -15.3 -15.3 -8.6 -43.8 - 1 . 1 -1.4 -0.7 +27.3%/ (n=7) *2.3 s2.5 hl ,6 *1.3% *O. 1 M.2 *O. 1 -36.4%
IO rnM BAPTA -16.9 -16.2 -8.8 -47.9 -1.2 -1.5 -0.4 +25.0%/ + 200 UM &5.5 *5.3 A2.2 *2.6% kO.2 M.2 M.2 -67.0% leupeptin ( n = 5 )
200 nM ISO + -19.4 -20.7 -19.9 +2.6 œq.02 -0.6 1 -0.12 +2,950%/ 10 m M BAPTA, *1.9 *3.7 *4.6 *2.5% kO.01 I0.0 1 M.04 +500% (n=4)
Citrate I I
Figure 4.2: Current-voltage (1-V) relationship with 10 mM citrate exposure in rat ventricular myocytes: 10 mM [BAPTAIi. A: Raw traces correspond to peak Tc, at +10 mV. B: Consmicted 1-V relationship showing effects of 10 mM citrate (mean k SEM; n = 7).
* Y 1 1 - for the changes in peak Ica in response to depolarizations to +10 mV fiom a holding potential
of -90 mV. The rnyocytes were depolarized every 5 seconds. Figure 4.3A shows typical raw
traces at time points A, B. C, and D, corresponding to those labeled in Figure 4.3B. From
phases A-C (initial recording, 2 min.), peak Ica appears to reach steady-state. The
corresponding rau trace at phase A was taken 2 minutes after ce11 dialysis. Following the
application of citrate (as indicated by the arrow) after 2 minutes of control, Ica quickly
increases by about 20% in the cell, corresponding to phase B in Figure 4.3AIB. This is
followed by a continual rundown that does not stabilize for more than 10 minutes. Phase C
indicates time at citrate washout (raw trace C in Figure 4.3A; Figure 4.3B); phase D was
taken 8 minutes following citrate wash-out (raw trace D in Figure 4.3A; Figure 4.3B). What
the figure shows is that citrate actually increases peak Ica, and is followed by a continued
rundown of Ica without ever reaching steady-state. As a result, the relative magnitudes of the
peak Ica depended on the time at which the 1-V recordings were taken. Therefore, for this
study and for al1 subsequent experiments (to be shown below), we took al1 the 1-V recordings
at the same tirne points as those for the EGTA study in Figure 4.1 in order to allow for
accurate cornparisons between the different studies. Phase D of Figure 4.3B shows that Ica
continues to rundown after citrate wash-out at a lower rate (see Table 1, Rates of Rundown),
suggesting again that there was an enhanced rate of rundown caused by citrate.
A summary of these results are shown in Table 1, where there is a comparatively
marked reduction in rate of rundown with 10 mM [BAPTAIi compared to [EGTAIi. The
initial control rate of rundown with 10 mM BAPTA was - 1.1 f O. 1 pA/pF/min (p < 0.05, n =
7). Following citrate wash-in, there was again an acceleration of rundown to -1.4 + 0.2
pA/pF/min. After wash-out, the rate of rundown fell to -0.7 f 0.1 pA/pF/min. These results
establish that BAPTA clearly reduces but does not eliminate the rate of rundown compared to
EGTA. However, our results dernonstrate that the direct extracellular application of citrate
does not significantly reduce ICa,L (Figure 4.3B), as expected from the previous report
(Hryshko & Bers 1992).
Figure 4.3: Tirne-course of ka with 10 m M citrate: evidence of rundown without leupeptin in rat ventricular myocytes. A: Raw traces correspond to peak Ica at +10 mV (A: Control; B: Citrate wash-in; C: Citrate wash-out; D: Wash-out, 10"). B: Current-time relationship (1-t) shows the progression of Ica over time with citrate exposure (B to D). htracellular pipette solutions, and al1 subsequent experiments, contain 10 rnM BAPTA.
Our results suggest that citrate does not decrease Ica irnmediately, but rather, it seems
to accelerate the rate of rundown, despite replacing EGTA with BAPTA in the pipette
solution. Proteolytic digestion by endogenous proteases has been suggested to cause rundown
(Elhamdani et al., 1994; Seydl et al., 1995), as it is directly related to the [ca2']i, since ca2'-
dependent proteases, like calpain, are present in cardiac myocytes (Belles et al., 1988; Seydl
et al., 1995). It has also been shown that nindown depends on the degree of phosphorylation
of the channel (Ono & Fozzard, 1992). Other studies have shown that the holding potentid
can greatly influence the rate of channel rundown possibly by elevating local [ca2']i which
would in turn activate ca2'-dependent proteases (Schouten & Morad, 1989); holding
potentials were changed to -90 mV for the remainder of our ICaL studies. We also used three
methods in an attempt to further minimize rundown: (1) use of leupeptin, a general protease
inhibitor; (2) maximal phosphorylation of the channels using isoproterenol; (3) perforated
patch-clamp technique, using nystatin and arnphotericin B.
In order to control for Ica rundown due to proteolytic digestion, 200 p M leupeptin, a
general protease inhibitor, was added to the pipette solutions, as represented in Figure 4.4.
Raw traces in Figure 4.4A were taken at identical tirne points as in Figures 4.1A/4.2A/4.3A,
taken 2 minutes afier initial cellular dialysis in control solutions, after 3 minutes of citrate
application, and 8-10 minutes following citrate washout. The time-course of Ica for this celi is
shown in Figure 4.4B and shows that prior to citrate application, Ica remains at steady state for
2 minutes fiom phases A to B (raw trace A of Figure 4.4A). 10 mM BAPTA was used
intracellularly over EGTA for reasons discussed previously. The figure shows that citrate
initially causes an immediate decrease in peak Ica before steady-state is reached at phase C
(raw trace C of Figure 4.4A), which corresponds to the second citrate wash-in, in which Ica
slightly increased to a new steady-state level at phases F - G. Upon wash-out, Ica decreased
even further to a new steady-state value at phases D - E, and H - I, which corresponds to
almost half the original Ica magnitude (-24.5 pNpF during control versus -14.0 pA/pF after
the second citrate intervention). This is represented in the 1-V relationships which we
recorded at the same time points as in the EGTA study, in which citrate does not significantly
(p > 0.5, n = 7) change the peak Ic, magnitudes, but upon wash-out, peak Ica is decreased
I 1: Washout #2
\ Ge. Citrate #2
E= Control#2
I l I W I ! l . *O" m . . . . .. --
Figure 4.4: Evidence of ?-, rundown in rat ventricular myocytes with proteolytic inhibition.. A: Raw traces correspond to peak Ica at + 10 mV. B: 1-t relationship showing effects of ka rundown with 200 p M leupeptin (B, E: Citrate wash-in; C, G: citrate wash-out). C: 1-V relationship showing effects of 10 mM citrate with 200 PM leupeptin, showing 1-V plots before/during/after citrate exposure (mean k SEM; n = 7).
. . I I . I V Y C .w I L ..II ~ W I I L - V I .CI. U I \ *".' - "' """" .- a--- -.- - - --- - - - , - -- - - - - - - - - - - -
wash-out; p < 0.05, n = 7).
While the ce11 depicted in Figure 4.4 showed little evidence of rundown (except in the
presence of citrate) this was not consistent in al1 the myocytes studied. In fact, proteolytic
inhibition does not generally eliminate the problems of Ica rundown. Figure 4.5 displays
results fiom a typical ce11 showing progressive rundown. The raw traces (Figure 4.5A) and
the continuous time-course profile of Ica with 10 mM citrate exposure (Figure 4.5B), with a
pipette solution with 200 p M leupeptin, are s h o w in Figure 4.5, illustrating rundown despite
proteolytic inhibition after 5 minutes of control (traces A, B; Fig. 4.5A), followed by citrate
wash-in after 3 minutes of citrate application (trace D), and 10 minutes of citrate wash-out
(trace F). In Figure 4SB, calcium current recordings at phase A already exhibits a persistent
rundown without ever achieving steady-state levels. Citrate addition at phase B, and eventual
removal at phase D, does not seem to affect the rate at which rundown does not occur.
Indeed, upon lürther analysis of the rates of rundown fiom the pooled sample data (n = 7), it
appears that the rate of rundown was not significantly change$ by including leupeptin in the
pipette (peak Ica with [BAPTAIi = -1.1 * 0.1 pA/pF versus -1.2 * 0.2 pMpF with [BAPTAIi + 200 pM leupeptin; p > 0.1, n = 7). Again, with leupeptin, citrate seems to accelerate
rundown (peak Ica = -1 -5 * 0.2 pNpF with citrate), as seen without leupeptin. However, upon
wash-out, the rate of rundown was also reduced (peak Ica = -0.4 h 0.2 pA/pF) in the presence
of leupeptin. Nevertheless, leupeptin did not significantly change the rate of rundown,
suggesting that proteolytic digestion is not responsible for rundown of current in our
experiments.
Phosphorylation of calcium channels is another means by which inhibition of rundown
has been reported (Ono & Fozzard, 1992). Raw traces correspond to set time points 2 minutes
of control, 3 minutes after citrate exposure, and afier 10 minutes following citrate washout, as
shown in Figure 4.6A. Figure 4.6B shows a continuous time-course of Ica with 10 mM citrate
and 200 nM isoproterenol, a P-adrenergic agonist. Intracellular solutions contained 10 mM
BAPTA and 200 pM leupeptin. The current-time relationship shows an initial wash-in of 200
nM isoproterenol. From phase A to B, there is a x2.5-fold increase in peak Ica, developed over
five minutes before reaching steady state (phase C). Important observations can be made fiom
- B: Controi #2
A: Initial conttoi -
Con trol
T
F: Washout
1 - Calcium currents @,.J 1
Tirne (min)
Figure 4.5: Time-course of Ica with 10 mM citrate: evidence of nindown pnor to citrate application despite proteolytic inhibition. A: Raw traces correspond to peak Ica at +10 mV. B: 1-t relationship indicates the time at which citrate wash-inIout occurs (B,D); effects of rundown apparent despite presence of 200 pM leupeptin intracellularly.
----- - -- -me----- - -- = - p K - \ = - - V U
in = -16.0 pA/pF; see Table 1) as shown by the figure is representative of this. Secondly, at
phase C there is a decay in calcium currents after reaching maximal leveis, before eventual
stabilizing, which may be related to activation of phosphatases (e.g. Ca-calmodulin-dependent
phosphatase; Chad & Eckert, 1986). This dual response is characteristic of the response to
syrnpathetic stimulation by isoproterenol before steady state is achieved. Figure 4.6C shows
the peak Ica-V relationship with and without 10 mM citrate in the presence of 200 nM
isoproterenol was not significantly different. For exarnple, at -10 mV, the average current
density was -1 9.4 f 1.9 pA/pF before citrate, and was changed to -20.7 t 3.7 pNpF after
citrate application (see Table 1, under 200 nM ISO-stimulation). The rates of rundown were
significantly reduced, however, as surnmarized in Table 1. The initial control rate was only - 0.024 0.005 pA/pF/min with isoproterenol stimulation, and was accelerated almost 300x-
fold during citrate wash-in to -0.61 h 0.01 pA/pF/min. The rate of rundown after wash-out
then decreased back to -0.080 =t 0.04 pA/pF/min, confirming again that citrate seems to
accelerate rundown, only to be reversed after washout. In the presence of isoproterenol, very
little channel rundown occurs, which is in agreement with the previous studies which
concluded that channel rundown involved dephosphorylation (Belles et al., 1988; Schouten &
Morad, 1989). Furthemore, these results demonstrate that in the presence of isoproterenol
when rundown is minimized, citrate has M e or no effect on ICqL.
In the above experiments using isoproterenol, it is conceivable that the lack of effect of
citrate resulted fiom the phosphorylation of the Ca charnels. We therefore sought to establish
if the observations were generally true in the absence of isoproterenol. As a result, we used
the perforated patch-clamp technique to minimize ce11 dialysis and therefore Ica rundown. The
underlying basis for using the perforated patch-clamp technique relies in the ability of
amphotericin B andlor nystatin to slowly diffuse through the intracellular pipette solution and
to create large conductance transmembrane pores, allowing access to the intraceIIular
cornpartments without ce11 dialysis. Only certain molecules-such as electrolytic salts-are
small enough to pass through these pores. The technique was used to determine the effects of
citrate on ventricular myocytes isolated from New Zealand male rabbits (average wt.=4 kg.),
since the Bers study used rabbits as their experimental model; also, arnphotericin B has been
T h e (min)
5 4
O
-5 - -10 - -15 -
-0- Citrub -20 - -v- wuhout
-25 -
Figure 4.6: Inhibition of Ica rundown via phosphorylation of L-type Ca charnels. A: Raw traces correspond to peak Ica at +10 mV. B: Constructed I-t relationship shows progression in peak Ica: A to B: control; B: 200 nM isoproterenol wash-in; D: Iso- baseline; E ro G: Citrate wash-in; G: Citrate wash-out. C: 1-V relationship showing 1-V plots before/during/afier citrate exposure with isoproterenol stimulation.
--r----- -- . . - - m m "'-"' ""-̂ -*^ * - - Y - - "^J --, "' ‘̂ 'U' ^" ^-'" ' ' . -"-a - -- ----a- - - . . - - - - a a - - -
in rat ventricular myocytes, but as has been reported previously, the technique does not work
in rat.
Figure 4.7 shows perforated patch-clamp recordings, showing the effects of 10 mM
citrate on both current-time (1-t) and curent-voltage (1-V) in rabbit ventricular myocytes.
Raw current traces are shown in Figure 4.7A during control, citrate application, and after
citrate washout; the time-points at which the raw traces were taken correspond to previous
time-dependent experiments. In Figure 4.7B, from phase A to B, the control rate of rundown
is -0.3 * 0.1 pA/pF/min, which is significantly less than that reported for rat ventricular
myocytes with EGTA (rate of decay = -3.1 * 0.3 pNpFIrnin, p < 0.05; see Table 1) or
BAPTA (rate of decay = - 1.1 & 0.1 pA/pF/min, p < 0.05). Tt is significantly improved over
BAPTA and leupeptin (rate of decay = -1.2 * 0.2 pA/pF/min, p < 0.05). However, the
rundown with the perforated patch is still somewhat greater than the rundown with
isoproterenol stimulation (rate of decay = -0.024 * 0.005 pNpF/min, p > 0.1). Nevertheless,
the figure shows that citrate wash-in results in a slight increase in Ica followed by an
accelerated rate of decay (rate = -0.72 k 0.08 pA/pF/min). With citrate wash-out, Ica
continues to decrease at a more slowed rate (rate = -0.4 * 0.1 pA/pF/min); however, it is
apparent as in the isoproterenol experiments that citrate does not seem to reduce ICaL at al1 (p
> 0.1). This is shown more clearly in constructed peak Ica-voltage relationships in Figure
4.7C, where the voltage-dependent Ica,L is actually enhanced in the presence of citrate (see
Table 2). With citrate wash-in, peak Ica increased insignificantly from -5.3 f 0.7 pA/pF to -
6.1 I 0.4 pNpF (paired t-test; p > 0.1). With citrate wash-out, peak Ica was decreased
insignificantly to -5.1 + 1.5 pNpF (p > 0.1). Results are summarized in Table 2.
The perforated patch-clamp experiments, which rninimizes rundown, demonstrate that
citrate slightly increases cardiac Ica. Figure 4.7C shows that with citrate wash-in and wash-
out, there is no significant difference in peak Ica magnitude. The slight increase in Ica
following citrate wash-in may reflect a higher-than-expected free [ca2'],, which was
measured using a ca2+-sensitive electrode, possibly reflecting inaccuracies using this
approach. It is possible, for exarnple, tliat citrate interferes with the selectivity properties of
the ca2'-sensitive electrode used. Regardless, it is clear that citrate does not inhibit Ica when
C. Citrate
- i o - / / . , . , . . . , 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 . 5.0
T h e (min)
-2 - fz ,QI < -4- 3 Y
-6 - L W -
-8 , i I i
-60 I
-40 1
-20 € 1
O I
20 40 60
Figure 4.7: Perforated patch-clamp recordings showing effects of 10 mM citrate on Ica in rabbit ventricular myocytes. A: Raw traces correspond to peak 1, at +10 mV. B: 1-t relationship values correspond to peak Ica usinç the perforated patch-clamp technique in the presence of citrate (B to D). Break at 2.5 minutes of 1-t recording corresponds to 1-V relationshp in the presence of citrate. C: 1-V relationships taken beforelduringlafter citrate exposure (mean k SEM, n = 3).
Table 2: Effects of 10 mM citrate on rabbi t ventricular myocytes using perforated patch-clamp: amphotericin B.
Nature of Study *Exposure to Citrate @AlpF) Rate of Run-down @ AlpFlmin) % Change
( d h g (i) Control (ii) Citrate (iii) Washout citrak/weshout) (i) Control (ii) Citrate (iii) W~shout
wl Amphotericin B (n = 3)
*. ..Correspondhg values are the mean of the capacitancecorrectd magnitudes of rabbit I,, taken at voltages of + 10 mV h m tûe cmt-voltage (1-V) relationships, as detennined by family voltage-protocols (-50 mV to +60 mV, every + 10 mV); the values are given as meanf S.E. Ratcs of mdown were calculated ushg avemged linear regression analyses at al1 time frames along the cwrent-time relationships (a= 1). Description of the study includes the type of study (htracellular amphotericin B, O EGTAIBAPTA); the usage of ihis dnig corresponds to the usage of a perforated patchclamp technique to eliminate effects of 1, nui-down. Rates of xundown show that in presence of citrate, mndown is acceleraied.
rnethod.
4.1.3 Chronic study: effects of citrate versus EGTA on Ic~,L
A study was done to examine whether citrate had any chronic effects on Ica. Clinical
studies showed that liver and heart transplants were suspended in citrate-phosphate-dextrose
solutions (CPD) for preservation. The possibility exists that some of the effects of citrate
were long-terrn, and that experimental protocols only allowed an acute response (< 20 min.) to
be measured.
Figure 4.8 represents a population study in which two ce11 groups (control, n = 10;
citrate, n = 12) were suspended in their respective solutions for a period exceeding 2 hours. A
cadmium-sensitive current-voltage (14) relationship was then recorded and used as a
comparison arnongst each experimental study. EGTA was used for comparison with citrate,
since EGTA was used in a clinical trial as a calcium chelator (communication; Dr. Rebeyka).
It was found that prior chronic exposure to both EGTA and citrate depressed I C 4 ~ by
approximately 20 - 23 % (control peak Ica = -15.0 k 5.8 pA/pF, after 2 hours of chronic
control, n = 12; peak Ica after chronic citrate exposure = -1 1.9 * 2.7 pA/pF, n = 10; peak Ica
after chronic EGTA exposure = -10.5 k 2.4 pA/pF, n = 7), and that there was no significant
difference between EGTA and citrate exposure @ > 0.1). The effects of chronic exposure to
10 mM citrate are sumrnarized in Table 3. This shows that the chronic effects of citrate on
IcaL are identical with those of EGTA, suggesting the effects might be related to ca2+
chelation. However, the effects seen with chronic exposure to both citrate and EGTA indicate
that citrate does not cause significant rundown, and that the acceleration of rundown observed
during citrate exposure is related to the whole-ce11 patch-clamp method of recording in
myocytes. The underlying mechanism for this intriguing observation was not investigated
M e r . Citrate has minimal effect afier chronic exposure, which is relative to the control 1-V
with EGTA.
-i- Control Tymdt's ( n = 12) -4- 10 mM Citrate ( n = 10)
Figure 4.8: Population study showing effects of chronic exposure ( > 2 hours) to 10 mM citrate and 10 m M EGTA in rat ventricular rnyocytes. 1-V relationships show plots in three different solutions taken after chronic exposure to the following solutions: conrroi tyrode 's ( n = 12); lOmMcitrate(n= 10); 10mMEGTA ( n = 7).
Table 3: Effects of chronic exposure (> 2 hours) of rat ventricular myocytes to 10 mM citrate.
*Exposure to citrate @A/pF) Compdson between citrate and EGTA exposure (919)
% Depression % Depression Description O mM Citrate 10 mM Citrate 10 mM EGTA w/ citrate exposure wl EGTA exposui
(n = 12) (n = 10) ( n = a
W/ 10 mM BAPTA -15.5 -11.9 -10.5 -23.2 % -32.3 % O rnM leupeptin f 5.8 f 2.7 f 2.4
*. ..Corresponding values are the mean values of the capacitance-corrected magnitudes of Ica, taken at voltages of + 10 mV fron thc current-voltage' relationships, as determined by fmily voltage-protocols (-50 to +60 mV, every + 10 mV); the values are gi~en a: mcansf SD. Description of each study includes the intracellular contents for the study. The % depression was calculated for bot1 ciirate- and EGTA-suspended cells; it was found that EGTA exerts a much greater, yet ipsignificant @ > 0. l), inhibitory effect thar citrate, suggesting bat the decpise in L due 10 c@te may only'be dve to its cakiuli-bufrcrring I i , l I . l v . capabilities.
, i l .
To conclude, citrate does not affect the L-type Ca channel after inhibition of rundown.
Using various methods in controlling for mdown-replacement of BAPTA with EGTA,
holding at -90 instead of -80 mV, inhibition of proteolytic digestion with leupeptin, channel
phosphorylation using isoproterenol, perforated patch-clamp technique using amphotericin
B-several conclusions can be made. First of all, under our conditions the mechanism of
rundown seems to be dependent on a phosphorylation-dephosphorylation process, since
leupeptin was ineffectual in preventing mndown, while the usage of BAPTA over EGTA also
seemed to prolong the duration of the experiments. Furthemore, it is evident fiom our results
that citrate does not seem to &ect either the magnitude or selectivity of IC4L; in fact, as
demonstrated by the chronic studies using preincubation with EGTA where peak Ica was
reduced by the same magnitude as with citrate, the observations seen are related to the ca2+-
chelating abilities of the two buffers, and not a direct effect of citrate on the channel. The
hypothesis in which citrate may be disrupting ca2' homeostasis via changes in L-type Ca
channel activity has thus proven to be false.
The effects of citrate on the ionic profile of the cardiac myocytes is important, since it
can elucidate the exact nature by which citrate acts upon the cells electrophysiologically. Any
changes in action potential profile could reflect any number of sites of action of citrate on the
sarcolemrna. The previous study showed that citrate did not directly affect ICqL. However,
the amount of catf entering the ce11 through the L-type Ca channel cm also be profoundly
changed by alterations in the action potential profile. Indeed, a recent study showed that
action potential prolongation caused a large increase in the [ca2+li transients by increasing the
arnount of ca2+ entering the channel during the prolonged depolarkation phase (Bouchard et
al., 1995). We therefore studied the effects of citrate on action potentials, with the
anticipation that if changes in action potential underlie the negative inotropic effects of citrate,
then exposure to citrate should cause the action potential to be shortened. Using the current-
clamp technique, fou. parameters were studied: APD T50%/T90% (T50% reflects state of L-
type Ca channel activity; T90% corresponds to the time of the repolarization phase), resting
membrane potential, and the Itcplateau.
Figure 4.9A shows a representative trace of an action potential. As can be seen, citrate
does not aiter the action potential profiles significantly (sample size, n = 11). Figure 4.9B
shows the effects of 10 mM citrate on action potential duration at T50% and T90%. The
figure plots the relative sweep nurnbers and their corresponding duration. Citrate seems to
prolong the two action potential duration parameters slightly, but as shown in Table 4, there
was no significant change. The sole fact that there was no significant changes in action
potential profile is consistent with the lack of changes on peak Ica. Citrate, however, does
have a significant effect on the resting membrane potential (r.m.p.). As shown in Figure 4.10,
the r,m.p. is shown to hyperpolarize by 2-3 mV in the presence of citrate wash-in. After
citrate wash-out, the r.m.p. rernains hyperpolarized, as though there has been a permanent
change in the membrane properties.
-100 ' . 1 l I I i
40 80 120 160 200 Action Potential Duration (mss)
6
Time (min)
Figure 4.9: Effects of 10 rnM citrate on action potential profile in rat ventricular myocytes. A: Raw traces correspond to action potential profiles elicited using the current-clamp technique during three phases of the experimental protocol: control tyrode 's, citrate, wash- out. Action potential traces are given as averaged sweeps during each phase (x = 5; sample size, n = 11) . B: Effects of citrate on A.P. parameters (APD T50%/T90%), showing very little change in both time parameters.
Time (min)
Figure 4.10: Effects of 10 mM citrate on resting membrane potential (r.m.p.) in rat ventncular myocytes. The values from the above temporal relationship were taken by measuring the resting membrane potential of the action potential during various phases of the protocol: control @ode 'S. citrate, wash-out (sample size, n = 1 1).
v . 1 . ~ f i l l C U 3 VI CICI a~t: u11 carumc 1La Ji irdnsrenrs in myocyres: orner pOSSli3le targets responsible for disrupted ca2+-homeostasis
The L-type Ca channel has been described as the main trigger mechanism for
activating the process known as "calcium-induced calcium-release, CICR. Ultimately, it is
the Ic~cinflux that is responsible for triggering intracellular [ca2+li transients, which is
transduced into calcium-activated contractility. The purpose for these experiments was to
determine if citrate affected [ca2+li transients, since it was determined that the effects of
citrate on the L-type Ca channel study were only minimal. If IcKL is unaltered, then
intracellular [ca2+li transients should also be unaffected, unless processes downstream fiom
ICqL are affected by citrate.
In Figure 4.1 1, the 340/380 fluorescence ratio was calculated over a single time sweep
of 1200 msec, following depolarization of a ce11 to +10 mV fiom a holding potentiai of -90
mV. Control calcium transients were taken 2 minutes (solid lines) and 4 minutes (dashed
lines) after initial 2 mM CaC12 wash-in over the clamped myocyte (Figure 4.1 1A). The
current traces (Figure 4.1 1B) also exhibit current magnitudes (PA; not normalized) after 2 and
4 minutes of control Tyrode's solution + 2 mM CaC12. Calibration of h a - 2 signals were
performed in order to convert raw fluorescence ratio recordings to [ca2+li (see Methodology).
Clearly, a slight reduction in the peak ratio is observed 4 minutes post-washin compared to
that recorded at 2 minutes. The reduction could result fiom a rundown of I,, which cannot be
readily identified in Figure 4.1 1B.
Figure 4.12 shows the effects of a fura-loaded cardiac myocyte in response to 10 mM
citrate wash-in. The solid line in Figure 4.12A corresponds to calcium signals taken fiom the
clarnped myocyte afier 2 minutes of control solution; the dashed line is the calcium signal
taken after 1 minute initial citrate exposure. As shown in the 340/380 fluorescence ratio
measurements (Figure 4.12A), diastolic calcium transients are slightly elevated by about 0.10
PM, as in the peak of the ratio transient. However, this change in the peak systolic [ca2+li was
not observed in al1 cells (Table 5). This corresponds to the effects seen in trabeculae
preparations (see Section 4.4.1 Preliminary findings of 10 m M citrate on trabeculae). In
Figure 4.12B, current traces were taken at 2 minutes control (solid line) and during citrate
exposure (dashed line), exhibiting an increased rate of inactivation with citrate wash-in.
Time (msec)
Figure 4.1 1: Control intracellukir [ca2'] over time in rat ventncular myocytes. A: Raw [ca27, transients traces show effects of time (2 to 4 minutes) in control tyrode's solution. B: Current traces show onIy inward currents, exhibiting M e or no time-dependent changes.
Figure 4.12: Effects of 10 rnM citrate on [CLI~']~ transients in rat ventricular rnyocytes. A: Raw [ca2+], transients show enhanced systolic and diastolic calcium levels with 10 mM citrate wash-in (dotred). B: Inward current traces show increased rate of inactivation with citrate wash-in (dotted).
Figure 4.13: Effects of citrate wash-out on [caz'li transients in rat ventncular myocytes. A: Raw [ca2+], transients showing relative decrease in systolic and diastolic calcium magnitude with citrate wash-out (dashed). B: Inward current traces continue to exbibir increased rate of inactivation after wash-out.
Table 5: Table showing effects of lQ mM.citrate on various paramrters of calcium transients in rat ventricular myocytes: relaxation phase (T,,), diastoli~lsystolic calcium tratisients, and calclum ttaansients magnitude.
Relaxation phase
Diastolic Calcium Transients (PM)
Systolic Calcium Transients (PM)
[ ~ a " ] ~ - ~ r a n ~ i e n t s Magnitude (PM)
Perfusion solutions SlGNlFlCANCE TEST ' (y< O. 0.5)
Control + 2 mM CaClz 10 mM Citrate + 9.9 mM CaCI2 Control V.Y. W u
247i9 msec
O. 1 WO.0 1
0.52k0.07
0.32*0.05
347k 14 msec
0.28*0.01
0.55~0.07
0.33~0.06
229*9 msec
O. 19AO.O 1
0.46&0.08
0.2a0.08
sign't. @<O. 00002)
sign't. @<O. 00022)
n.s.
n.s.
*... Significance tests were conducted for Control and Citrate values using a paired t-!est (n -- 9). Significance was detennined if p<0.05; n.s. denotes that then was no signiflmt difference between values at p<O.OS. Values givem are reported as value I S.E.M. Duration for relaxation phase was calculated as being the differences between the time for the transients to decline to 50% of the dc ium signal magnitude h m the tirne- to-peak calcium levels. T0,3 was used as the time-indicaror; values for TD,tr had a similar innease in relaxation duration (conircl - 552 1 l ln~sec; citraie = 6564-1 7msec; wash-out = 521msec; sample size of n = 3).
- - - - - - - - - - - - - - - - - - O -' "" '- '"b.'U'Y.
The values corresponding to diastolic and systolic ca2+-transients measurements and
[caz+li transients magnitude were calculated and sumrnarized in Table 5. The duration for the
relaxation phase was calculated as being the differences between the time for the transients to
decline to 50% of the calcium signal magnitude fiom the time-to-peak calcium levels (T0.5 and
T0.75 were used as the time indicators). As shown in Table 5 there is a significant rise in
diastolic calcium transients following citrate application, indicating the possibility of inhibited
SR Ca-uptake due to citrate. Systolic calcium transients rernain unchanged (p > 0.1); indeed,
there is no significant change in the overall magnitude of the calcium signal. However, there
was a slight prolongation in the relaxation phase by about 100 msec, suggesting that citrate
may modestly inhibit the uptake and sequestration of calcium into the SR. This may result
fiom additional intracellular calcium binding sites (Berlin et al., 1994) made available by
citrate. Another possibility is that citrate may somehow be inhibiting the SR Ca-ATPase
purnp via a metabolic regulatory mechanism. This last scenario rnay be unlikely, since our
experimental conditions included the presence of 5 mM MgATP intracellularly. What these
results demonstrate is that citrate is exerting its effects downstream fiom ca2+-handling at the
level of the contractile proteins. The results establish that citrate application does not affect
[ca2+],-transients which are entirely consistent with the lack of effect observed on ICqL
4.1.7 Conclusions on the role of citrate in cardiac ca2' homeostasis
As shown by the L-type Ca channel study, action potentials, and intracellular [ca2+li
transients study, it can be concluded that the role of citrate on cardiac ca2' homeostasis is only
minimal, and that the action of citrate's toxicity appears to be downstream fiom the cardiac
ca2+-handling processes. The next stage of our study deals with the assumption that if citrate
is indeed being transported intracellularly, then there is the possibility that citrate rnay be
acting at the level of the contractile proteins in order to explain the force inhibition due to
citrate exposure.
4.2.1 Effects of 10 mM citrate on force and [ca2+li in ventricular trabeculae.
Numerous studies have documented citrate-induced inhibition of cardiac contractile
force (Bunker et al., 1955; Olson et al., 1977; Rebeyka et al., 1990). The delicate interplay
that exists between force-generation and intracellular [ca2+] during citrate exposure, however,
has never been previously elucidated, partly because of prior limitations in measuring force
and [ca2+li simultaneously. However, a recent experimental technique (Backx et al., 1993;
Backx et al., 1995) involving microinjection of cardiac muscle with fluorescent indicators has
allowed for accurate measurements of the development in force and calcium levels over tirne.
The technique was used to study citrate interventions on rat ventricular trabeculae, and record
the progression in development of force and [ca2+]i during the experimental protocol.
A preliminary study was performed in order to examine the changes in peak force and
[ca2+li before, during and after 10 m M citrate exposure during constant fiee extracellular
[ca2+] of 2 mM. It was previously demonstrated that force was inhibited by 53% in rabbit
trabeculae (Hryshko & Bers, 1992), and this inhibition was attributed to altered L-type Ca
channel selectivity. Since our studies have s h o w a minimal role of citrate on L-type Ca
channels, and in ca2'-homeostasis in myocytes, we therefore expect reductions in peak force
due to citrate without significant changes in systolic [ca2+li levels. Indeed, Figure 4.14 shows
the effects of 10 mM citrate and 9.9 mM CaC12 on right ventricular trabeculae. Time-traces
(top) show both force (dashed) and [ca2']i transients development: ( A ) before, (B) during,
and (C) after citrate exposure. The strip-chart recording (bottom) corresponds to the raw
recorded data, showing both systolic and diastolic force and fluorescent readings. The
calcium transients (inward/outward deflections on the strip-chart) are measured using 340/380
nm fluorescent measurements.
Figure 4.14 shows that with citrate wash-in, there is a dramatic decrease in systolic
force along with a slight rise in diastolic tone. However, Iittle change in systolic calcium
transients were recorded, which is consistent with Our single cardiac myocyte studies (see
Figures 4.1 1 to 4.13). The rise in diastolic tone is associated with an increase in diastolic
calcium, as shown in diagram (B) of Figure 4.14. During the citrate washout phase, some
(îi) 10 mM Cinate
Figure 4.14: Effects of 10 mM citrate on force and [ca2+li traces (rcnv) in rat ventncular trabeculae. Both force (solid) and calcium signals (dotred) show the progression of citrate- induced changes: A: before, B: during, and C: afier citrate. Time-traces @op) show force and [ca2'li transients development. Strip chart (below) shows corresponding traces at each time point.
Time (msec)
Figure 4.15: Effects of 10 rnM citrate on peal; forcc and twitch duration (TD) in rat ventricular trabeculae. A: Peak force, and B: TD. both represent a time-dependent progression with citrate exposure.
Table 6: Effects of acute exposure (1.0 - 20 minutes) to 10 mM citrate on rat ventricular trabeculae.
*Exposure to 1 O mM Citrate
% Change after % Change afler Trabeculae Parameter Before During Citrate Citrate Wash-in Citrate Wash-out
Wash-in exposure Wash-out
Systolic Force (mWmm2)
Diastol ic Force ( r n ~ ~ r n m ' )
Diastolic [ca2+li (PM)
*... Corresponding values are the mean of each respective parameter (mean i SD), taken at a constant stimulation rate of 0.1 Hz and a room temperature ranging from 19 to 23OC (n = 4). Sarcomere length was kept constant fiom 2.2 to 2.3pm. Bicarbonate solutions were constantly bubbled with carbogen (5% Co2/%% 02), to give a pH of 7.4.
- * - - which are normally associated with ca2+-overload. The development of "aftercontractures"
may be due to an alkalotic effect, causing spontaneous SR Ca-release to occur. This is
probably directly related to the rise in diastolic [ca2']i observed. It should be pointed out that
not al1 trabeculae studied showed aftercontractures (only 3 of 9 trabeculae preparations). A
summary of the effects of acute exposure to citrate on trabeculae are seen in Table 6, in which
there is a significant reduction in systolic force, and increase in both diastolic force and
calcium transients. These results are also charted in Figure 4.15, which shows the effects of
citrate on the magnitudes of peak force (top) and twitch duration (bottom) over t h e . Figure
4.15 shows that with citrate wash-in there is a very small increase in twitch duration. This is
followed by a recovery to original twitch duration values d e r citrate wash-out, indicating that
these effects are reversible.
These results suggest that citrate acts at the level of the contractile proteins, since
citrate does not affect [ca2+]i transients. There are three possibilities that could explain the
force inhibition by citrate without changes in [ca2+]i transients: (1) a reduction in peak
force/maximally generated force; (2) a decrease in the myofilament (MFT) ca2+-sensitivity;
or (3) a combination of (1) and (2). One possible mechanism explaining the results of citrate
may be intracellular acidosis (pHi), due to citrate metabolism, or via direct transport of
protonated citrate. This would inhibit force primarily by reducing the cal+-sensitivity of the
contractile proteins since H+ ions cornpete with ca2+ ions for binding to troponin C (TnC;
Blanchard & Solaro, 1984). Secondly, there may be altered [ATP], by virtue of citrate's
endogenous role as a metabolic regulator. A third explanation involves the possibility that
citrate directly affects the contractile proteins. Citrate may be acting at the level of contractile
proteins by interfering with ca2+-binding to the myofilaments, or altering various steps in the
cross-bridge cycle.
In order to assess the cellular mechanism by which citrate may be acting at the level of
the myofilarnents, we used two methods. Initially, we used carboxy-SNAW-AM to assess for
pH, changes to explain the changes in MFT ca2+-sensitivity,. We then used CPA in order to
study whether there was a change in Fm, (maximally generated force), KD ([ca2+li at half-
maximal activation), or n~ (the Hill coefficient) of the contractile mechanism. The methods
rnar were usea to assess Mr i La- -sensirivny were as roiiows: (1) venuicuiar uaoecuiae,
loaded with the pHi-indicator, carboxy-SNARF-AM, a dual-emission fluoroprobe; (ii) CPA,
slowing down twitch relaxation to the extent that both force and [ca2']i reach steady-state, for
constmcting force-calcium phase loops to rneasure MFT ca2+-sensitivity. CPA eliminates the
contribution of calcium fiom the SR by inhibiting the SR Ca-ATPase, thereby preventing Ca-
uptake back into the SR and elevating [ca2+li. At saturating intracellular calcium, the
contribution of the SR to the cycling of calcium in E-C coupling is minimized. Since the role
of SR to the E-C coupling rnechanism is eliminated, any observations due to citrate can be
attributed to its action on the contractile mechanism of the muscle. Thus, CPA is the ideal
choice for this portion of the study, since it does not itself directly affect the contractile
proteins (Dobrunz et al., 1995; Backx et al., 1995). The application of f O0 p M CPA resuIts
in a marked slowing of the rate of relaxation of the twitch force, and allows high stimulation
rates close to tetanization of the cardiac muscle (Backx et al., 1995). The next sections deal
with the interpretation of the experimental recordings, and the insights on whether citrate is
acting at the level of the contractile proteins.
4.2.2 Role of citrate on MFT ca2'-sensitivity: intracellular acidosis (pHi) study
It has been shown that acidosis decreases both myofilament calcium sensitivity and
maximum force production (Donaldson & Hermansen, 1978; Fabiato & Fabiato, 1 978a;
Blanchard & Solaro, 1984). The shifi in myofilarnent calcium sensitivity can be attributed to
a decrease in the affinity of calcium binding to cardiac troponin C due to the presence of
elevated intracellular [H'] (Blanchard & Solaro, 1984). Other findings have reported a
decrease in cross-bridge formation, increasing intracellular calcium transients (Blanchard &
Solaro, 1984), stimulation of proton extrusion via NaIH-exchanger activity (Vaughan-Jones,
1982), and the inhibition of the sodium pump (Sperelakis & Lee, 1971) and the NaIca-
exchanger (Philipson et al., l982), resulting in cellular (and SR) ca1ciu.m overload.
It seems feasible that citrate may be creating an acidic intracellular environment,
which would result in the aforementioned effects. For example, citrate might enter the ce11 in
a neutral form, and subsequently releasing H'-ions, creating an acidic environment. Another
possibility is that citrate may be metabolized in the mitochondria of the cardiac myocytes via
me i LA-cycle Ior me proauction or A 1 r. 1 ne metaboiism or citrate wouia arive tne i LA-
cycle forward, increasing the production of NADH (a metabolic by-product of the TCA-cycle)
and H+-ions, also creating an acidic intracellular environment, which would result in a
decrease in systolic force without changes in intracellular calcium availability, and a
decreased rate of relaxation, resulting in an increase in twitch duration. Calcium overload
may also be another possibility, since an excess of FI+-ions would overloaci the intracellular
buffering capacity of the muscle, resulting in increased activity of the Na/H exchanger. This
would result an elevation in [ca2+]i via the NdCa exchanger. Small pHi changes due to citrate
can be picked up using a pH-indicator, such as carboxy-SNARF-AM or BCECF-AM. In this
study, SNARF-AM uses a 640/580 nm ratio in fluorescent measurements.
Figures 4.16 and 4.17 show the effects of 10 m M citrate on a mounted rat ventricular
trabeculae loaded with carboxy-SNARF-AM. The muscle is excited with a 540 nm Iight,
whereby two emitted wavelengths at 640 and 580 nm are collected in a photomultiplier tube
(PMT). The two wavelengths correspond to two points along the spectrum with greater
ernission intensity. The ratiometric calculation (640680 nm) allows for greater accuracy in
determining the extent of citrate on intracellular pH.
Figure 4.16 shows a time-course of the effects of citrate on peak force (Figure 4.16A),
twitch duration (Figure 4.16B), and intracellular pH (Figure 4.16C). With citrate wash-in,
peak force decreases fiom 44 to 3 1 m ~ / r n r n ~ , while twitch duration increases fiom 280 to 300
msec at steady state. Intracellular pH (pHi) increases from 0.45 to 0.47 units, which
corresponds to an increase in pHi, or towards a more basic environment. There was no
significant change in duration of the force and calcium transients; however, a rise in pHi
suggests that the mechanism by which citrate is acting on the contractile system may be
independent of the acidosis story, which proposed that H+ ions were potentially competing
with ca2+ ions for binding sites. With citrate wash-out, there is an immediate recovery of
peak force to almost original values. Twitch duration also reverts to original values, but over
the course of 12 minutes of recovery, twitch duration increases slowly a new steady-state
value of 3 10 msec (or an overall increase of 15-20 msec). This may reflect a possible
permanent change in the state of the muscle after citrate exposure. Finally, upon citrate wash-
Coplnoi K-W 1 O mM Citrate C h e Wahour
Figure 4.16: Effects of 10 rnM citrate on peak force, twitch duration, and pHi in rat ventricutar trabeculae. A: Peak force, B: twitch duration, and C: pHi, show effects of citrate on carboxy-SNARF-AM loaded trabeculae.
vu%, r i i u r u i~ u s i w u u u i i r a r ~ r ~ ~ ~ l w iii AIALILIWUIIUICY "A -rr.wfiuA.-.-aJ - ------ ------ r--1
reverts back to previous control values.
In order to veri@ that the change in SNARF fluorescence tmly reflect changes in pH,,
we applied and washed out NH4CI. Typical results of such an experiment are shown in Figure
4.17. Following NH4C1 wash-in, a rise in pHi and intracellular force were seen with 10 and 20
mM NH&l wash-in, as expected. The increases in pHi and force following the application of
NhC1 are the opposite to those observed with citrate application. As s h o w in Figure 4.17,
citrate wash-in decreased peak force, while increasing pHi, suggesting that the reduced force
occurs independent of acidosis.
LEGEND: A...ConPol f 2 mM Ca%
B...10 mM Ci- warh-i C...Ci- Warbaut D...10 mM NH,CI
I E...2O mM N X p F.. .NH,CI -ut
Time (sec)
Figure 4.17: Effects of 10 mM citrate and W C 1 on peak force, TD, and pHi in rat ventncular trabeculae. A: Peak force, B: TD, and C: pHi, show the time-progression of citrate exposure on carboxy-SNARF-AM loaded trabeculae. W C 1 wash-in's of 10 and 20 rnM show pH-dependent deflections as a pH reference.
Results fiom the pHi study suggestecî that citrate may be directly affecting the
contractile parameters of cardiac muscle. The study had shown that citrate was causing
alkalosis, rather than acidosis. Hence, desensitization of the myofilarnents due to acidosis was
ruled out, indicating that citrate was directly affecting the contractile system via another
mechanism. The usage of CPA allows the elimination of th(: SR, prolonging the relaxation
phases of force and [ca2+li and allowing them to reach steady-state (Dobrunz et al., 1995;
Backx et al., 1995). The steady-state fo rce - [~a~+]~ relationships allow assessrnent of changes
in MFT ca2+-sensitivity upon fitting with the Hill equation.
We therefore exarnined the effects of citrate on the contractile mechanism by
measuring changes in MFT ca2+-sensitivity, via the contractile protein's affinity for calcium
(KD), maximal force generation (F,,), and cross-bridge CO-operativity (nH). Since the
application of CPA allows a mounted muscle to be stimulated at high frequenciesltetanus in
order to achieve Fm,, we needed to determine the maximal [ca2+], required to evoke the
highest possible Fm, values. Figure 4.18 shows the relation between peak force and
increasing [ca2+],. Part A shows control peak force activated by 1 mM [ca2+],. With the
addition of 100 p M CPA in part B in the presence of 1 mM [ca2+], and equilibration for 5-7
minutes, the recorded systolic force was s h o w to reduce fiom 19.5 m ~ l r n m ~ to 13.6
mN/mm2, indicating that a higher [ca2+], is needed to evoke similar magnitude in force in
control solutions. Twitch duration also increased fiom less than 800 msec to 2.7 seconds, an
increase of approximately 3.4-fold, thus reflecting the changes in f o r c e - [ ~ a ~ ~ ] i kinetics with
the addition of CPA. At part C, [ca2+], was elevated to 2 mM, resulting in an increase to 21.8
r n ~ / m m ~ in peak force. Finally, in part D, peak force reached a value of 33.0 m ~ l r n r n ~ in the
presence of 8 mM [ ~ a ~ ~ ] , . At higher [ca2+],, peak force did not increase any higher than 33.0
mN/mm2. In order to ensure maximal [ ~ a ~ + ] , ' s required for Fm, generation, the CPA-treated
muscles were stimulated using tetani at 10 Hz in the presence of 2 mM, 8 mM, and 16 mM
[ca2+], (not s h o w here). From these experiments, we established that 8 rnM [ca2'], was
sufficient to produce Fm, in response to 10 Hz stimulation of the muscle. The total [ca2+], to
achieve free 8 mM [ca2+]i was 17.2 mM in citrate-containing solutions, and this was used for
subsequent CPA experiments.
I i !
I sec
'-..,. CI-
CPA + 2 mM [CalO CrPA + 8 mM [Ca],
rat ventricular Figure 4.18: Effects of increasing [ca2+], in the presence of 100 pM CPA in trabeculae. Raw force (solid) and calcium (dorted) traces revealed that in the presence of CPA, 8 m CaClz with tetani stimulus (10 Hz) was needed to elicit maximum peak force (botrom-righr).
- - Both single-twitch stimuli at 0.1 Hz and high-fiequency stimulation pulses of 7-10 Hz were
used to depolarize the trabeculae, causing surnmation of contractions to occur. ~orce-[~a'+]i
relationships were then constructed by exarnining the relaxation phase twitches, and fitted
using the Hill equation: 2.e .n F = F,,[C~~~]"I([C~ 1, + &")
where F,, is the maximal force (determined by tetani stimulation of 7-10 Hz), KJ- is the
dissociation constant and n is the Hill coefficient which is a measure of the arnount of
cooperativity. This determines the force-[~a~+]i relationships in order to examine the
dependence of force on the [ca2+]i of the contractile system, upon achieving steady-state
levels of activation (Backx et al., 1995). [ca2']i transients measurements were made by using
the ratiometric measurement of the 340 and 380 nm fluorescent signals.
In order to fit the force-[ca2+]i relationships with the Hill equation as indicated before,
a measurable value of the Fm, was required in defining the maximal constraints for force-
generation. A high-fiequency stimulus pulse (Le. fiom 7- 10 Hz, for 10 seconds) was used to
measwe Fm, to allow for norrnalization and to facilitate comparison between preparations
(Dobrunz et al., 1995). Figure 4.19 also show force and [ca2']i traces in a CPA-treated
trabeculae, where a high-fiequency stimulation of 7 Hz for 10 seconds, in the presence of
citrate (rniddle panel), caused force to decrease fiom 51 .O to 46.0 m~/rnrn*, and following
citrate washout (right panel) recovered back to 51.5 m ~ / m m ~ . The observation that the
generated force plateau (solid) remains relatively flat while the [ca2']i tnuisients (dotted)
continue to rise indicates that Fm, had been achieved using the high-frequency stimulus
(Backx et al., 1995). Tetmi stimulation (not shown here) also evoked similar changes in Fm,
during the control/citrate/washout phases, in which Fm, reversibly decreased fiom 5 1 .O to
45.0 rn~ l rn rn~ with 10 mM citrate application. This indicates that there does not seem to be a
difference between the usage of a 7 or 10 Hz stimulus in achieving Fm,.
In Figure 4.20, phase loops were constructed fiom the relaxation phases of the force
and [ca2+]i measurements, showing changes before, during and after citrate exposure. The
first diagram (top-le8 pane[) represents a control loop, in the presence of 100 p M CPA and 8
mM [ca2+],. With 10 mM citrate wash-in, peak force is shown to decrease fiom 64.0 to 33.0
(if) Connol + (ii) Citrate + (iii) Citrute W a h u t . . 60] 8 mM [Ca]* 8 mMfiee [Calo
i t - . - , - , - . 4
O 2 4 6 8 i00 2 4 6 8 100 2 4 6 8 - 1 C
T i (seconds)
Figure 4.19: Force and [ca2+Ii traces (raw) showing effects of high-frequency stimulation (7 Hz) in rat venti-icular trabeculae in the presence of 100 FM CPA. Raw traces correspond to three phases of the solutions protocol: control (le@), citrate (middle), and wash-out (right). At high stimulus rates (7 Hz), figure shows that cinate depresses force reversibly (solid). However, there is a progressive enhancement in systolic [caz+Ii that is irreversible (dmIze6) with citrate and after wash-out.
(i) ControI (iî) Ciirate (IV Washout
K,, = 0.59 UM KD = 0.59 UM = 0.65 uM 70 t
w*
T-mm E .
E Y
O 2 3a4 O IL.,
20 - 104
O 1 i
16 os0 0.' 0 4 l2 lgr.
Figure 4.20: Effects of 10 mM citrate on foice-[~a~+]i relationships in rat ventricular trabeculae in the presence of 100 FM CPA: maximal loaded w/ k a - 2 . Constructed phase loops were taken from the relaxation phase of the force and calcium traces during control, citrate and wash-out stages (top). Phase loops show the KD's during each phase, revealing that MFT ~a~+-sensitivity is shified slightly to the right, indicating decreased sensitivity. Phase loops showing control and citrate force-[ca2*], relationships (bottorn-left) reveal a &op in Fm,, which upon wash-out (bottom-righl) recovers almost to original control values.
---- . .----- ,..-- ---- y-..-./. - -Y".- y- ivvrr I T V V ~ ~ UAV L .IV LIYWWU, IIW TT- T VI, 0 1 1 ~ *T LIIUL L I I Y ~ ~
seems to be no immediate effect on the relaxation phase (leftmost portion, the force-calcium
trace) which reflects the possibility that citrate does not directly alter MFT ca2+-sensitivity.
This was subject to some variability, since in some trabeculae, the rightward shift was
observed in the fo rce - [~a~+]~ relationships. Citrate also decreased Fm, significantly,
decreasing by almost 30-40% of the original value, suggesting that citrate may be sterkally
hindering the cross-bridge formation between actin and myosin. With citrate wash-out (right
panel), the prominent rightward shifl in KD and Fm, is shown to be reversible.
The phase loops were constructed fiom the relaxation phases of the force and calcium
traces shown in Figure 4.21 derived fiom twitches slowed with CPA. The first graph (lefr
panel) shows raw traces in control K-H solution, with free 8 mM [Ca],. An initial peak force
(dotted line) of about 64.0 m~lrnrn* was recorded, with a twitch duration of 2.7 seconds.
Calcium traces (solid Zine) show systolic [ca2'li values of approximately 1.6 PM. Diastolic
[ca2+], is shown to be elevated above 0.2 PM, which reflects the rise in diastolic calcium
levels due to CPA. The next diagram (middle panel) represents a wash-in of 10 mM citrate
and 17.2 mM [ca2+], while recording force and calcium signals. A reduction in systolic force
h m 64.0 to 43.0 r n ~ l r n m ~ was observed after steady-state had been achieved, while diastolic
tone was unaffected. Systolic calcium transients were also largely unaffected, while diastolic
calcium levels were elevated slightly from 0.1 to 0.2 FM. Recovery in diastolic tone and
calcium transients occurred, however, with citrate wash-out (right panel), while a recovery
with a slight overshoot in systolic force fùrther characterized the wash-out effects fiom citrate.
The modified Hill equation was then used to fit the relaxation phase of the force-
[ca2+]i relationships in order to determine citrate-induced changes in MFT caz+-sensitivity
(KD), Fm,, or cross-bridge cooperativity (nd. Figure 4.22 shows fitting curves for force-
[ ~ a ~ + ] i relationships, showing effects of 10 mM citrate on rat ventricular trabeculae. The
curves were fitted to Fm, values (determined fiom high-fiequency stimuli). The diastolic
tautness (Fdias) were subtracted fiom ail force measurements were subtracted of the muscle in
its completely relaxed form (i.e. unstretched), and were plotted against the instantaneous
[ca2+]i measurements from the relaxation phase. From the fined curves, it was found that
T
(Yp 1 2 - 3 - 4
Time (sec)
Figure 4.21: Effects of 10 mM citrate on force and [câ2'li traces ( r a v ) in rat ventricular trabeculae with 100 FM CPA: maximal loading w/ fura-2. Both force (solid) and calcium sjgnals (dorred) show the progression of effects due to citrate exposure in trabeculae. The finding that peak force is depressed in the presence of citrate despite constant systolic calcium levels has been consistent. Pre-tetanus (at 10 Hz) was done before the protocol was started to estabhh that Fm, had been achieved.
- - - - . - - - - - - 1 \ . J A . .. , , -
(rniddle panel; n~ = 6.3), in addition to the reduction in Fm,. Figure 4.23 shows the
normalized force values (with Fm, fiom the high-fiequency stimulus) versus [ca2']i, showing
the same trends as in Figure 4.22, in which there is a reduction in F/Fmm fiom 1.0 to 0.82
(ratio values), without changes in the KD at 0.59 PM in both control and citrate phases ( n ~ =
6.3 in controVcitrate).
The wash-out stage of the phase loops proved to be more difficult to interpret. As
shown in Figure 4.20, there seemed to be a continued rightward shifi in KD after citrate wash-
out, suggesting that perhaps citrate was not being completely washed out of the perfusion
system. As described before, an indicator for citrate wash-out was the recovery in peak force
values during single-twitch measurements; the rightward shifis in MFT ca2+-sensitivity
suggests that citrate may be internalized, resulting in a permanent state of change. As shown
in Figures 4.24 (F-Fdi, vs. [ca2+]i) and 4.25 (normalized FEm, VS. [ca2+]i), the wmh-out KD
was found to be shifted to the right fiom 0.59 to 0.65 PM, indicating an incomplete
reversibility. The magnitude of this change was subject to some variability, but it was
conclusive that there seems to be a permanent effect even after citrate wash-out. The results
have been tabulated in Table 7, in which changes in KD due to citrate were not significant (KD
= 0.45 h 0.16 FM control vs. 0.44 k 0.13 pM citrate; p > 0.1, n = 6). Chi-square values ( X 2 )
using least-squares fit were detemined as follows: control = 9.1 * 4.6; citrate = 6.7 k 6.5;
washout = 12.1 * 4.8. After citrate wash-out, KD was shown to shifted to the right (washout
KD = 0.45 * 0.17 PM; p > 0.1, n = 6). The insignificance o f these results, however, do not
reflect the actual changes fiom muscle to muscle due to variability. Individual muscle
preparations continually show a slight rightward shift in MFT ca2+-sensitivity with citrate that
persists even after wash-out, suggesting that MFT ca2+-sensitivity may be affected.
The conclusions fiom this study suggest a number of possibilities. First of all, there
may be incomplete wash-out of citrate despite a recovery of force. It is possible that the ca2+-
binding affnities of the myofilaments are slower to recover than force, indicating that for
citrate wash-out, a longer wash-out time is actually needed. Another possibility may be that
citrate is changing the phosphorylation state of the contractile proteins, leaving them in a
greater state of dephosphorylation. This is unlikely, since Our experiments are using higher
[ca2+li (UM)
Figure 4.22: ~ o r c e - [ ~ a ~ + ] ~ relationships derived from twitches slowed with 100 pM CPA in the presence of citrate. Steady-state relationships are fitted with a modified Hill equation to the data, with KE = 0.59 FM, and n~ = 6.3 in control (solid-square). Kin was not changed in citrate at 0.59 FM, and n~ = 6.3 in citrate (dotted-diarnond). FM was detemined by using tetani stimulation of 10 Hz for 10 seconds in trabeculae in the presence of CPA; Fm,, decreased with citrate wash-in from 75.0 to 63.2 m ~ i m m ' , while diastolic tone rose from 6.8 rnWrnrn2 in control solution to 7.3 rn~ / rn rn? with citrate wash-in.
Coniml K-H: FE,", = 1 .O
F = 55.0 mWrnm7 K, = 0.59 uM; n, = 8.3
Figure 4.23: Normalized force-[ca2+], relationships derived fiom twitches slowed with 100 FM CPA in the presence of citrate. Steady-state relationships are fitted with a modified Hill equation to the data, with Kg = 0.59 pM and ~ Z H = 6.3 in control (solid-squure). Klx did noi change in citrate at 0.59 pM and n~ = 6.3 (doued-diamond). Peak force values were norrnalized by Fm,, achieved fiom tetani stimulation ( I O Hz) before and afier citrate wash-in. FEms decreased from 1.0 control to 0.82 in citrate.
Figure 4.24: ~orce-[~a'+]~ relationships derived from twitches slowed with 100 FM CPA after citrate wash-out. Steady-state relationships are fitted with a modified Hill equation to the data, with KK = 0.59 PM, and r n ~ = 6.3 in control (solid-square). K% changed siightly after citrate wash-out to 0.65 PM, and n~ = 6 7 in wash-out (dofted-triangle). Fm, was deterrnined by using tetani stimulation of 10 Hz for 10 seconds in trabeculae in the presence of CPA; Fm, reîumed to control values of 75.0 m ~ l m m ' , while diastolic tone also returned to 6.8 m ~ / m m ' upon wash-out .
Figure 4.25: Normalized force-[~a~']~ relationships denved from twitches slowed with 100 pM CPA after citrate wash-out. Steady-state relationships are fitted with a rnodified Hill equation to the data, with K% = 0.59 pM and n~ = 6.3 in control (solid-square). KI/, shified to the right after citrate wash-out to a value of 0.65 pM and n~ = 6.7 (doited-diamond). Peak force values were nomalized by Fm, achieved from tetani stimulation (10 Hz) before and after citrate exposure. FE,, reverted back to 1 .O upon citrate wash-out.
Table 7: Effects of 10 mM citrate and 100 PM CPA in ventricular trabeculae: changes in Fm, and KD.
*Changes in Fm, ( m ~ l m m * ) Sift. test: *KI, (ha1 f-maximal) (PM) Sig't. test: corm-01 v.9. cori?ro/ V.Y.
Description of cilrote / cilraie /
Studv (i) before ( i i ) during ( i i i ) aRer corttrol vs. (i) before (ii) dunng (iii) afier cntrtrol V.S. --J
citrate citrate citrate ~vusho~~t citrate citrate citrate washoir r
100 1iM CPA sig't 1 n.s.
*... Corresponding valucs are given as the means SD (sample size, n = 6). Sarcomere leiigth was kept constant at 2.3 Fm. Bicarbonate solution were constantly bubbled with carbogen (5% Co2/95% 02) to give a constant pH of 7.4. Significance was taken at p 4 0.05 using paired sample i iest. Peak force were the single-twitch peak forces measured at a constant stimulation rate (0.1 Hz, 10 seconds) before, duting and afier chat exposure; the values correspond to the peak value just before initiation of ihe relaxation phase. The KD's were calculated fiom the relaxatio phases of the constmcted force-calcium relationships taken before/during/after citrate exposure, and after fitting the relationships using the Hi1 equation. Parameters for the Hill equation were as follows: Fm,, (which were the maximal generated peak forces at tetani stimulation of 7-10 Hz: Kn ([ca2'li at half-maximal activation of force). The Hill coefiicients were detemined (means I SD): n~ (confrol) = 6.28 * 1.3; ni, (citrate) : 6.42 * 1.6; nll (ivashotrr) = 6.28 * 1 .O; sarnple six, n = 6. Vie data points were also fitted using least-squares anaiysis based on the Marquardi Levenberg algorithm, and the chi2-values that were calculated are as follows (means * SD): control= 9.06 A 4.55; citrate = 6.66 A 6.54; u m l m = 12.135 * 4-81.
r u i i v u i r b o vr LLU JO VVAUVU IAILU~WO LL L ~ I %&IV U ~ ~ V I L L U A ~ V "1 VULVI-SI YVCI .-LI- LWIII VI LWZII
[cazfli relationships. Also, force recovers to nearly 90% of the original control value for
maximal force, indicating that there may not be an effect on the phosphorylation states of the
contractile proteins. However, there still remains the possibility that citrate may be
permanently altering the binding affinity of ca2' to troponin-C, resulting in a decrease in MFT
ca2'-sensitivity. The amount of calcium required to generate an equal magnitude in force
may be a compensatory mechanism by the cardiac tissue in order to achieve the same levels of
contractile force, despite a decreased [ ~ a ~ + ] i - a f i n i t ~ of the contractile proteins. This constant
elevation in systolic [ca2'li may, in the long run, result in muscle fatigue, since MgATP is
required for the actual conformation change in the contractile proteins after activator calcium
is bound to troponin-C. Finally, it is feasible that changes in autofluorescence of the muscle
itself or of citrate itself, rnay have contributed to the continued rightward shifts in KD. There
is a likelihood that the observed trends in KD were due to phase shifts of the h a - 2
measurements, resulting in interference of h a - 2 binding to caz+-ions with citrate. Further
experiments were also conducted to eliminate the contribution of changes in autofluorescence
to the observations: whether citrate displayed its own autofluorescence, or potential changes
in autofluorescence of the muscle without fura-loading. The next section concerns the
possible contribution of autofluorescence changes to our observations.
The purpose of this study was to isolate whether changes in [ca2']i transients were not
related to changes in autofluorescence. Since citrate itself does not exhibit any fluorescence at
340 and 380 nrn (not shown here), any effects observed on [ca2+]i transients cm be solely
attributed to citrate's action on the intrinsic muscle properties in cardiac trabeculae, This is
documented in Figure 4.26, which shows both force and raw fluorescent and auto traces (in
millivolts) during control/citrate/washout phases of the experiment; both muscles used had
similar dimensions, allowing for closer comparisons. As shown in the control (le#), citrate
(middle), and washout (right) phases, both the absolute auto- and h a - 2 fluorescence values
exhibit minimal changes in the presence of citrate. However, the 380 k a - 2 signal is actually
shifted towards a more negative voltage value, as well as decreasing in magnitude. This
cannot be explained by the upward shift in the 380 autofluorescence signal. Indeed, there is
no correspondence between the two signals as it would be expected that the 380
autofluorescence signal would also be shifted downwards, in parallel with that seen with the
380 ha-signal. It cari be concluded, then that changes in autofluorescence may not be a
significant contribution, and that this ensures that the changes seen in [ca2'li are not
attributable to changes in autofluorescence, but to citrate-induced changes in [ca2+li and that
the changes are unlikely to account for prior observations. This is consistent with the notion
that citrate is directly acting on the contractile myofilarnents.
(mi 10 mM Ci- 380 = dcuhad 340 = donGd
Time (sec)
Figure 4.26: Effects of 10 mM citrate on peak force, autofluorescence and fura-2 340 and 380 signals (raw) in rat ventricular trabeculae. Both peak force (solid) and raw autofluorescence signals (380: dashed; 340: dotted) show effects of citrate exposure, in which the autofluorescent 380-signal is elevated during citrate exposure. However, the fluorescent 380-signal is decreased in citrate, indicating that changes in autofluorescence do not contribute to changes in the fluorescent signals.
4.L.b Lonc1usions on the roie oi citrate In tne caraiac conrracrrie mecnanism.
As observed in the trabeculae experiments. the role of citrate is far more complicated
than previously imagined. It was clear that citrate did not interfere with the ca2+-homeostasis
required to maintain cardiac E-C coupling mechanisms, which led to the next obvious
hypothesis that citrate may be somehow interacting with the contractile mechanism. Based on
the force and [caW2+li measurements with and without citrate exposure, it was then postulated
that citrate decreased myofilament ca2+-sensitivity, resulting in force inhibition. Using CPA
as an effective means of studying steady-state force-[~ii~+]~ relationships, it was found that
there was a reduction in systolic force without changes in systolic [~a~*]~-tr~msients; a
reduction in Fm, in the presence of citrate using high-fiequency stimulation may have been
fiuther evidence that citrate may be interacting with the contractile mechanism. A slight
rightward shift in citrate, which was more pronounced with wash-out, suggested also that
citrate may be decreasing myofilament ca2+-sensitivity Possible contributions from
intracellular acidosis and autofluorescence have been ruled out; however, it is apparent that
there seems to be an alternative influence that may be accompanying the trabeculae
recordings. Another explanation is that citrate may be permanently altering the state of the
contractile mechanism, resulting in a decreased responsiveness of cardiac muscle from citrate
exposure. Further experiments are required to validate the observations associated with
exposure to citrate.
5.1 INTRODUCTION
Clinical toxicity of citrate was previously shown to result fiom: hypocalcemic-
induced QT interval, depressed systolic/diastolic blood pressure, abnormal coagulation,
hypokalemic metabolic alkalosis, and neuromuscular manifestations due to hypocalcemia
such as a loss of Chvostek's sign (Dzik & Kirkley, 1988). It was hypothesized that citrate
was interfering with myocardial contractility by decreasing the availability of
extracellular calcium, thus reducing the stimulus for SR Ca-release. It was also
postulated that intracellular citrate rnay also bind to intracellular calcium, reducing
calcium-binding to the contractile mechanism and reducing actin-myosin interaction.
A more recent clinical study reported on the myocardial contractile response to
citrate infusion in neonatai and adult hearts (Rebeyka et al., 1990). These studies
demonstrated that neonatal hearts are far more sensitive to citrate than adult hearts,
possibly because neonatal myocytes rely more on ICaL for contraction (Bers et al., 1981).
In addition, Rebeyka et al. proposed that since citrate c m traverse the cellular membrane
in the nondissociated form (de Hemptine et al., 1983), a decrease in pHi is expected.
However, Rebeyka et al. concluded that this progressive metabolic acidotic derangement
camot adequately explain the effects of citrate, owing to cellular buffering mechanisms
that protect against acidosis. Indeed, our data confirmed this conclusion, since we found
that pHi increased, rather than decreased, with citrate application.
The attribution of the negative inotropic effects of citrate to the L-type Ca channel
was originally proposed by Hryshko and Bers in 1992. Using various buffer controls
versus citrate, they had shown that both whole-ce11 and single-channel recordings
revealed that citrate depressed peak Ica rapidly and shifted both the peak Ica and the
apparent reversa1 potential (E,,) to more negative potentials (Hryshko & Bers, 1992).
Their conclusion was that citrate somehow altered L-type Ca channel gating and
selectivity, rather than a single effect in buffering calcium as had previously been
suggested (Ginsburg & Shimoni, 1989). This ~narked an important step in understanding
A 1
contraction coupling in the heart and other physiological systems. It is understood that a
reduction in Ica via L-type Ca channels would reduce the calcium transients and thus the
contractile force (Bers, 1985). However, our results clearly show that the effects on L-
type Ca channels are in fact minimal, and cannot fully explain the reduction in contractile
force observed in with citrate application. Our results further establish that citrate has
little effect on the [ca2+li and works directly on the contractile system. These results are
consistent with previous results, showing that citrate readily enters the ce11 through the
monocarboxylate transporter (Wang et al., 1 996).
5.2 ROLE OF CITRATE ON L-TYPE Ca CHANNELS
The previous study by Hryshko and Bers (1992) concluded that the effects of
citrate on L-type Ca channels appeared to occw extracellularly, since the inclusion of 10
mM citrate in the pipette did not alter the control 1-V relationship or the response to
extracellular citrate. Ica was found to decrease by 54%, which was comparable to our
results shown in Figures 4.2 and 4.2, and was accompanied by a prominent leftward shift
in the apparent E,,. However, in our studies, we persistently observed rundown of IC%b a
phenornenon which has been reported previously in many studies and has been attributed
to elevated [ca2+li, the resting membrane potential, activation of proteases and
dephosphorylation of the channels. Initially, we used 5 mM EGTA in the pipette and
found that Ica was reduced at a rate of -3.1 fi 0.3 pNpF/min in the absence of citrate; the
rate was increased further to -4.1 It 0.5 pA/pF/min with the application of citrate. We
therefore replaced EGTA in the pipette with BAPTA, a higher-affinity ca2+-chelator.
Since BAPTA chelates ca2+ with very rapid kinetics, in comparison to EGTA, we
expected that BAPTA would reduce or eliminate Ica rundown if the rundown was the
result of elevated [ca2+]i. Inclusion of BAPTA in the pipette did slow the rate of
rundown but did not eliminate it. Moreover, the acceleration of rundown with citrate
application was still observed. These results suggest that elevated [ca2+li was not the
primary cause of channel rundown in oui- experiments.
We then attempted to minimize rundown by preventing proteolytic digestion,
channel dephosphorylation and by eliminating cellular dialysis using the perforated patch-
method. Inclusion of the protease inhibitor, leupeptin (200 PM), in the pipette did not
significantly slowed the rate of rundown of Ica. On the other hand, in the presence of
isoprotemol in order to maintain L-type Ca channel phosphorylation or in the perforated
patch experiments, Ica rundown was minimal. Under these conditions where Ica rundown
was minimized significantly, the application of citrate did not seem to significantly
reduce Ica. In fact, there appeared to be a slight increase in Ica in the presence of citrate,
which may be the result of an increase in pHi observed following citrate application (see
Figure 4.16). This phenomenon was reported recently in which increases in pHi have
been associated with increases in Ica (Schuhmann et al., 1997). Alternatively, the fiee
extracellular [ca2+] rnay have been slightly above 2 mM, despite verification with the
calcium-sensitive electrodes. Clearly, our results are at odds with those of Hryshko and
Bers, who concluded that citrate directly affects L-type Ca channels, and we are uncertain
of the basis for this discrepancy. It is clear, however, that when we used EGTA in the
pipette, the effects of citrate could not be accurately assessed due to rundown. One
potential explanation for this discrepancy between our results and those of Hryshko and
Bers is the fact that these authors had 10 mM EGTA in the pipette while we had only 5
mM. On the other hand, we still observed considerable rundown even when 10 mM
BAPTA was included in the pipette. Unlike Hryshko and Bers, we fully assessed the
effects of citrate on channel rundown by plotting and analyzing the time-dependent
changes in peak Ica before, during, and after the application of citrate. Remarkably, in al1
the experimental conditions studies in which we observed rundown (EGTA, BAPTA,
leupeptin), citrate actually accelerated Ica rundown. The basis for this observation is
unclear, but might be related to citrate's ca2+-chelating properties. Indeed, in our chronic
studies using EGTA or citrate, we observed a reduction in mean Ica-voltage relationship,
compared to control. In any event, it is clear that when Ica rundown is minimized, there is
little or no effect of citrate on Therefore, the proposed changes in L-type Ca
channel activity cannot explain the reduction in contractile force with citrate exposure,
suggesting another mechanism responsible for the observed changes in contractile force.
- - - , - - . - ------------ ---- ------- ---- ---- -""-', - ""' Y" Y
type Ca channel, despite previous assertions made concerning citrate's ability to decrease
Ica-influx.
5.3 ROLE OF CITRATE ON CARDIAC ca2+-HOMEOSTASIS
It was clear that the L-type Ca channels are not affected by citrate, which led us to
examine other targets normally responsible for maintaining cardiac ca2+-homeostasis.
Consistent with these observations, action potential profiles in single cardiac myocytes
were not altered by citrate. The lack of effect of citrate on action potentials demonstrate
that citrate does not affect the arnount of ca2+-entry through L-type Ca channels,
indirectly by changing the action potential profile. These conclusions are entirely
consistent with the absence of significant effects of citrate on [ca2+]i transients recorded
in our single myocytes (Figure 4.13). The absence of changes in the single-myocyte
[ca2+li transients following citrate application might initially be seen to contradict the
observed nindown of Ica. Indeed, in the whole-ce11 voltage-clamp studies designed to
measure Ica, the calcium current was reduced by 44 k 2.3% (n = 7) during the time-course
of a typicaI experirnent, as shown in Figure 4.1 However, in the Ica experiments, 5 mM
EGTA or 10 mM BAPTA was included in the pipette (Figures 4.1, 4.2), while in the
[ca2'] transients experiments, only 1 00 yM fura-2 was included as the ca2'-buffer. It is
conceivable that under the latter conditions, the extent of rundown was reduced.
Regardless, the lack of effect of citrate on the single myocyte [ca2+li transients
magnitude and twitch force amplitude after citrate washout are identical to those recorded
prior to citrate application, suggesting that the effects of citrate are completely reversible.
5.4 ROLE OF CITRATE ON FORCE AND [ca2+]i IN CARDIAC MUSCLE
The results from the L-type Ca channel study, the intracellular [ca2+] transients
recordings, as well as the action potential measurements, suggest that citrate's actions are
downstrearn of ca2+-handling. Therefore citrate action must be at the level of the
contractile proteins. Indeed, Our trabeculae data showed a substantial decrease in force
myofilarnents' responsiveness to generate force. Theoretically, these findings could be
attributed to three major features of contractile force generation: (1) changes in the ca2+-
sensitivity of the myofilarnents; (2) a reduction in the maximal ca2+-activated force
(F,,); (3) altered cross-bridge CO-operativity (nH, or the Hill CO-efficient). It was clear
that a mode1 was required to establish the nature of citrate's direct influence on the
contractile myofilaments.
It has been shown in previous studies that citrate depresses systolic force by 40-
50% (Rebeyka et al., 1990; Hryshko & Bers, 1992), as well as a rise in diastolic tone
(Rebeyka et al., 1990). The reduction in force without changes in [ca2+li transients could
involve a decrease in pHi since H+ and ca2+ ions compete for the binding sites at the level
of the contractile proteins (Blanchard & Solaro, 1984). Intracellular acidosis had been
shown to decrease both MFT ca2+-sensitivity and maximum force production (Donaldson
& Herrnansen, 1978; Fabiato & Fabiato, 1978; Blanchard & Soho , 1984). However,
the results fiom the trabeculae study, using SNARF-AM as the pHi indicator, showed
conclusively that intracellular acidosis does not occur during citrate exposure. In fact, a
slight increase in pHi was observed, rather than the hypothesized decrease in, or acidic,
pHi. A more basic environment would in principle be expected to increase ca2+-
sensitivity of the myofilarnents and increase the peak force magnitude (Godt & Nosek,
1989). In these SNARF-AM experiments, we also applied ammonium chloride (N&CI)
after citrate exposure in order to veri@ the changes in pHi recorded. Indeed, the decrease
in peak force with NH4Cl wash-in compared to that observed with citrate was quite small,
despite the decrease in pHi. On the basis of these observations, we conclude that the
effects of citrate on force are not due to reduced pHi inducing a decrease in MFT ca2'-
sensitivity.
Another possible explanation for the reduction in force is a direct action of citrate
on the contractile proteins. CPA studies were utilized to construct steady-state force-
[ca2+]i relationships in order to examine the changes in rnyofilarnent ca2+-sensitivity,
maximal force, and contractile CO-operativity (Dobrunz et al., 1995; Backx et al., 1995).
These studies have established that citrate reversibly reduced F,,,, by almost 27%.
data in the absence of CPA. These changes in Fm, occurred without significant changes
in MFT cazf-sensitivity or the coefficient for CO-operativity (nH) (Table 7). Therefore, it
appears that citrate intrinsically interferes with cross-bridges, and decreases the force-
generating properties of the myofilaments. The nature of this mechanism, however, is
unclear, and will require further investigation (see below).
One possible mechanism for the inhibition in force generated by citrate may be
related to citrate's regulatory role in the metabolic pathways of the heart. Since citrate
inhibits phosphofructokinase (PFK), this could cause the whole flux through glycolysis to
decrease significantly, resulting in diminished conversion of glucose to ATP. Thus, the
presence of a huge load of extraneous citrate would force the heart to switch over its
energy dependence from that of carbohydrate stores (i.e. glycogen) and FFA (fiee fatty
acids) to sole usage of circulating FFA for ATP production. Furthemore, FFA are the
preferred substrates for the heart to generate ATP (Shipp et al., 1961). Indeed, in our
experiments, we did not supply FFA or acetate to substitute for glucose following the
application of citrate, making it conceivable that citrate causes a large reduction in ATP.
The magnitude of this change in [ATPIi can influence a number of ATP-
dependent processes within the myocyte. Upon considering the relative affinities (KD) of
the various ATP-dependent processes in the heart, a drop in ATP production would
significantly impact ATP-binding to myosin (KD = 6 x IO-' ' M; Cardon & Boyer, 1978),
as well as various calcium handling targets such as the SR Ca-ATPase (Ko = 18.5 x 1 o - ~ M; Vianna, 1979, the SL Ca-ATPase (KD = 0.3 x 10" M; Caroni & Carafoli, 198l),
and the ATP-sensitive K'-channels (KD = 105 x 1 o ' ~ M; Deutsch et al., 1994). However,
no significant changes in the amplitude or relaxation of the [ca2'li transients were
observed following citrate wash-in (Figure 4.17), suggesting that a drop in ATP levels
due to metabolic inhibition by citrate may not serve as an explanation for citrate toxicity.
This conclusion is further bolstered by the lack of change in twitch duration and force
following the application of citrate. A slight prolongation in the relaxation phase of the
single myocyte [CEL~']~ transients was observed, as shown in Figure 4.12 (see Table S) ,
which may suggest a possible decrease in intracellular [ATP]. However, this would be
UIIILKGIY UUG LU ~nt: inclusion or 3 mivi ivlgw ir inro our pipene solutions. rurcnermore,
reductions in ATP (when large enough) actually increase the maximal force by increasing
the number of attached bridges.
It may very well be that the toxicity associated with citrate as a clinical compound
is a direct result of its ability to decrease muscle responsiveness to stimulation at
physiologica1 calcium concentrations. The changes in Fm, as well as a possible
decreased MFT ca2+ sensitivity may to explain the depressed systolic and elevated
diastolic force in transplanted hearts. Removal of citrate in blood transfusion products
and transplant solutions might be considered as a method for reducing force depression in
transplanted hearts. The use of other calcium chelators, such as EGTA, might serve as an
alternative for citrate in the clinic.
The L-type Ca channel studies in the absence of Ica rundown establish that citrate
does not inhibit force by decreasing Ica. Thus, the reduction in peak force without
significant changes in calcium transients in trabeculae experiments appear to be related to
reduction in Fm, without significant changes in the sensitivity (KD) or the Hill co-
efficient (nn). These results are consistent with the pHi measurements which showed an
increase rather than a decrease in pHi. Our results show for the first time that citrate
directly affects the contractile proteins and that citrate does not affect the [ca2+]i.
The underlying question remains: how does citrate alter Fm,? From a more
fundamental perspective, what is the exact role that citrate play in cardiac excitation-
contraction coupling? It is known that citrate plays a detrimental role in its clinical usage
as an anticoagulant in cardioplegic solutions by permanently reducing the strength of
cardiac contractility. Future experiments should explore the possibility that citrate is
directiy affecting various contractile parameters that contribute to the overail function of
the heart. Experiments using skinned muscle fibres, for example, would aliow direct
intervention of citrate on the myofilarnent contractile system, independent of changes in
pHi or ATP availability. Also, since it has now been established that citrate acts upon the
contractile mechanism, experiments using an inhibitor of the monocarboxylate transporter
(MCTI) could be conducted to allow for confirmation of intracellular citrate uptake, and
serve as a protective mechanism for the clinical usage of citrate. Finally, one could assess
ATP-availability and utilization from different energy sources by using a fatty acid (e.g.
acetate, palmitate) instead of glucose, and compare whether the observed results c m be
duplicated while in the presence of citrate; the usage of different substrates cm help
determine how energy utilization in the heart may be affected upon citrate exposure. The
mechanism in which citrate reduces Fm, would provide some insights into the nature of
the cardiotoxicity of citrate.
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AYYEN UIX 1 : Determination of free [ ~ a " ] , using association constants.
In this study, total citrate, ca2', and klg2' were caiculated using the equilibrium constants (Martell & Smith) for citrate bound to calcium, magnesium, and hydrogen ions. The equation that was used to derive the equations in determining total concentrations is as follows:
[CaR] = [Rt]/(l+Kd[Ca])
where [CaR] = amount of calcium bound to the buffer and [Rt] = total buffer concentration
KD = dissociation constant (mM)
The following equations were used in deriving the total volume needed to maintain fiee extracellular 2 mM [ca2+] :
K(I) * H * L ~ - = H L ~ - K(2) * K(I) * H * H * L ~ - = H2L- K(3) * K(2) * K(1) * H * H * H * L ~ - = H3L K(4) * Ca * L'- = CaL K(5) * K(1) * H * Ca * L3' = CaHL K(6) * K(2) * K(I) * H * H * Ca * L ~ - = C ~ H ~ L + K(7) * Mg* L3- = MgL' K(8) * K(I) * Mg * H * L~~ = MgHL K(9) * K(2) * K(1) * Mg * H * H * L ~ ' = M ~ H ~ L +
then, Total Citrate = L3- [l + K(I) *H + K(2) *K(I) *H*H + K(3) *K(2) *K(I) *H*H*H +
K(4) *Ca + K(5) *K(I) *H*Ca + K(6) *K(2) *K(1) *H*H*Ca + K(7) *Mg + K(8) *K(I) *H*Mg+K(9) *K(2) *K(l) *H*H*Mg]
Total [ca2+] = Ca [ 1 + K(4) *L3- + K(5) *K(/) *H*L)- + K(6) *K(2) *K(I) *H*H*HL3-]
Total [M~"] = Mg [l + K(7) *L" + K(8) *K(I) *H*L~- + K(9) *K(2) *K(I) *H*H*L~-]
Using the dissociation constants listed in Martell and Smith for citrate bound to H', ca2', and M ~ ~ ' , the equations were integrated into a cornputer program, where the values for total citrate, ca2', and M ~ ~ ' were calculated, giving the total [calcium] needed to rnaintain a free 2 mM [~a~'] , . The calculated [ca2'], was 10.264 mM in 10 rnM citrate; however, further verification at the clinical biochemistry labs at the Toronto General Hospital revealed a 0.364 mM excess in [C~~'],I added. It was found that using calcium-sensitive electrodes, only 9.9 mM CaC12 was needed to maintain a free extracellular 2 mM CaC12.
Fura-2 salt binds to [cazf] and fluoresces. The difference in fluorescence is due to different wavelengths, and gives the actual [ca2+] inside the muscle. Therefore, an equation is required to calculate the concentrations:
[ca2'] = K'L> + (R - RmiJRmax-R) The procedure for calibration is as follows: (1) Two solutions were made with the following compositions.
200 mL EGTA w/ (in rnM): KC1 100, MgCl2 0.6, HEPES 10, EGTA 10 200 mL Ca-EGTA w/ CaCI2 10
pH 7.1 w/ KOH (2) 20 mL of the EGTA solution, and another 20 mL of the Ca-EGTA solution, are
placed into two conical tubes. Then, 0.2 p M Fura-2 salt (Molecular Probes, tetrapotassiurn salt, KD = 2.3 FM) is added to both aliquots.
(3) The EGTA and Ca-EGTA solutions are then mixed into the 1.5 mL Eppendorf microtubes:
sol' n#: Vol. EGTA (mL) Vol. Ca-EGTA (mL1 1ca2'i, (PM) 1 1 .O 0.0 O 2 0.9 0.1 0.023 3 0.8 0.2 0.052 4 0.7 0.3 0.089 5 0.6 0.4 O. 139 6 0.5 0.5 0.209 7 0.4 0.6 0.3 13 8 0.3 0.7 0.480 9 0.2 0.8 0.836 10 0.15 0.85 1.185 11 O. 1 0.9 1.882 12 0.05 0.95 3.974 13 O 1 .O ---
(4) The solutions were well-rnixed; 150 pL to the perfusion bath on the microscope. (5) The 380, 340 and 360 nm fluorescence signals for each solution were recorded and
tabulated. (6 ) The background fluorescence of EGTA and Ca-EGTA were recorded and used to
subtract from fluorescent recordings. (7) From the above equation, the following can be derived:
where R is the ratio of fluorescence at a known [ca2+]i at 340 and 380 wavelengths. The data points are ploned and fitted using the Hill equation, where Rm,, Rmin, and KD are calculated.
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