the role of na dysregulation in cardiac disease and how it impacts electrophysiology

DRUG DISCOVERY

TODAY

DISEASEMODELS

The role of Na dysregulation incardiac disease and how itimpacts electrophysiologyBrian O’Rourke1,*, Christoph Maack2

1The Johns Hopkins University, Institute of Molecular Cardiobiology, Division of Cardiology, Baltimore, MD, USA2Klinik fur Innere Medizin III, Universitatsklinikum des Saarlandes, Homburg/Saar, Germany

Drug Discovery Today: Disease Models Vol. 4, No. 4 2007

Editors-in-Chief

Jan Tornell – AstraZeneca, Sweden

Andrew McCulloch – University of California, SanDiego, USA

Cardiovascular diseases

Ca2+ is well known as the central player in cardiac cell

physiology, mediating Ca2+ activation of myosin

ATPase and contraction, the stimulation of Ca2+-acti-

vated signaling pathways and modulation of mitochon-

drial energy production. Abnormalities of Ca2+ handling

are a well-studied mechanism of decompensation in

heart failure. Less appreciated is the role of cytosolic

Na+ (Nai+), which can dramatically influence the trans-

fer rates and distribution of Ca2+ among the intracel-

lular compartments of the myocyte. Since Nai+ can vary

widely under different physiological and pathological

conditions, and its effects depend on multiple ion gra-

dients and membrane electrical potentials, unraveling

the global influence of Nai+ on cell function is complex,

requiring an integrative view of cardiomyocyte physiol-

ogy. Here, we discuss how abnormal Nai+ regulation not

only influences the cytosolic Ca2+ transient and the

cellular action potential but also alters mitochondrial

Ca2+ uptake and the balance of energy supply and

demand of the cardiomyocyte, which may contribute

to oxidative stress and cardiac decompensation. The

implications for sudden cardiac death and the potential

for novel therapeutic interventions are discussed.

*Corresponding author: B. O’Rourke ([email protected])

1740-6757/$ � 2007 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddmod.2007.11.003

Section Editors:Rahul Kakkar and Richard T. Lee – Harvard Medical School,Brigham and Women’s Hospital, Cambridge, MA, USA

pensated by NKA action. Uniquely, under physiological con-

ditions, the sarcolemmal Na+/Ca2+ exchanger (NCX) operates

Nai+ balance: the players

Nai+ balance in the heart cell is maintained by a variety of ion

channels, pumps and exchangers whose function is strongly

influenced by the transmembrane gradients of various ions,

and in some cases, by the electrical potential across the

sarcolemmal or organelle membranes (Fig. 1; see [1] for a

review). At the sarcolemma, two well-known processes con-

tribute to unidirectional Na+ fluxes during the cardiac cycle;

voltage-dependent Na+ channels mediate the rapid inward

Na+ current (INa) during the upstroke of the cellular action

potential and may also contribute to persistent Na+ influx,

while the Na+/K+ ATPase (NKA) utilizes the energy released by

ATP hydrolysis to pump Na+ out of the cell against a con-

centration gradient in exchange for K+ in an electrogenic

process (3Na+:2K+). A variety of electroneutral exchangers

also influence Nai+ when ion homeostasis is disturbed under

pathological conditions; these include the Na+/H+ exchanger

(NHE), the Na+/HCO3� cotransporter (NBC), the Na+/K+/2Cl�

cotransporter (NKCC) and the Na+/Mg2+ exchanger (NMgX).

Influx of Na+ through these pathways is generally well com-

in both the forward-mode (Ca2+extrusion/Na+ entry) and

207

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http://dx.doi.org/10.1016/j.ddmod.2007.11.003

Drug Discovery Today: Disease Models | Cardiovascular diseases Vol. 4, No. 4 2007

Figure 1. Factors affecting cytosolic [Na+]i in cardiac myocytes. A variety of electrogenic and nonelectrogenic antiporters, symporters and channels are

involved in cytosolic Na+ homeostasis including the voltage-gated Na+ channel (Na), sarcolemmal Na+/Ca2+ exchanger (NCX), plasmalemmal Ca2+ ATPase

(PCMCA), Na+/H+ exchanger (NHE), Na+/K+ ATPase (NKA), Na+/K+/Cl� cotransporter (NKCC), Na+/HCO3� cotransporter (NBC), Na+/Mg2+

exchanger (NMgX), mitochondrial Na+/Ca2+ exchanger (mNCE) and mitochondrial Na+/H+ exchanger (mNHE).

reverse-mode (Ca2+ entry/Na+ extrusion) during different

phases of the cardiac cycle, depending on the dynamically

changing membrane potential and gradients of Na+ and Ca2+

across the sarcolemma. Its sensitivity to membrane voltage is

conferred by its electrogenic transport properties (predomi-

nantly 3Na+:1Ca2+ stoichiometry, but see [2] for evidence of

other transport modes). The complex dependence of NCX on

the transmembrane gradients of Na+ and Ca2+, as well as

membrane voltage, makes it difficult to make simple assump-

tions about its role in cardiac function in healthy and dis-

eased states. Value judgments about the positive or negative

effects of NCX on cell function can only be made by applying

an integrative approach that takes into account all of the

factors contributing to the thermodynamic driving forces for

this exchanger. In this light, the role of Nai+ is particularly

important.

In addition to the complex interplay between the Na+

transporters, channels and exchangers on the sarcolemma,

208 www.drugdiscoverytoday.com

an important, but underappreciated role for Na+ transport

across the mitochondrial inner membrane has also been

established. In the heart, the mitochondrial Na+/Ca2+

exchanger (mNCE) is the primary mechanism for extruding

Ca2+ that has accumulated in mitochondrial matrix via the

mitochondrial Ca2+ uniporter (MCU) [3]. The stoichiometry

of the mNCE has been debated, but later evidence suggests

that it may be electrogenic [4]. Na+ entering the mitochon-

dria is extruded by H+/Na+ exchange (mNHE) [5–7]. Na+

transport across other intracellular organelles (e.g. the sar-

coplasmic reticulum (SR) or nucleus) has not been well

studied.

Relationship between Nai+ and Ca2+ handling

Physiology

Driven by the steep (�10,000:1) electrochemical gradient for

Ca2+ across the sarcolemma (Cao2+ � 1 mM:Cai

2+ � 100 nM),

excitation–contraction coupling in cardiac muscle employs

Vol. 4, No. 4 2007 Drug Discovery Today: Disease Models | Cardiovascular diseases

voltage-gated Ca2+ influx to trigger the release of Ca2+ stored

in the SR into the cytoplasm. Roughly a tenfold increase, and

a tenfold decrease, in Cai2+ is achieved during the cardiac

cycle (occurring over several hundred milliseconds), high-

lighting the efficiency and speed of both the Ca2+ release and

reuptake processes. Fast Ca2+ release is accomplished by the

local action of Ca2+ entering the space just below the sarco-

lemmal L-type Ca2+ channel to activate the high-conduc-

tance SR Ca2+ release channel (ryanodine receptor). Within

tens of milliseconds, Ca2+ diffuses out of this junctional space

and makes its way to the myofilaments to initiate contrac-

tion. Two main processes contribute to rapid Ca2+ removal

from the cytosol, the SR Ca2+ ATPase (SERCA), which

recharges the intracellular releasable Ca2+ pool, and NCX,

which removes an amount of Ca2+ (�10 mM) equal to that

entering during each beat at steady-state stimulation [8].

Perturbation of any of these fluxes, or a change in the

stimulation frequency, can shift the dynamics of the system

such that a new balance is achieved. This typically results in a

change in the SR Ca2+ load and/or a change in the amplitude

of the cytosolic Ca2+ transient. A notable example would be

the effect of b-adrenergic receptor stimulation, which

strongly enhances Ca2+ entry through the L-type Ca2+ chan-

nel and SR Ca2+ uptake, and may also increase the open

probability of the SR Ca2+ release channel [9] and stimulate

the NCX [10].

The Na+ gradient, on the other hand, is typically on the

order of 10–20:1 in cardiomyocytes at rest (Nao+ � 140 mM:-

mM:Nai+ � 8 mM), and varies much less during stimulation.

Because the Na+ current activates and inactivates rapidly

during the upstroke of the action potential, only a small

change in Nai+ is effected by this mechanism (8–25 mM per

beat [1,11]), though Na+ current may significantly contribute

to an increase in Nai+ during rapid stimulation [12].

Nai+ has been reported to vary over a range from �5 to

20 mM in myocytes from normal and diseased hearts [13].

Changes in Nai+ within this range will have large effects on

excitation–contraction coupling, primarily as a consequence

of the effects of Nai+ on NCX. In simple terms, ignoring

allosteric regulatory effects on the exchanger, current

flow through NCX (INCX) depends on the membrane con-

ductance of NCX and the difference between the membrane

potential (Em) and the equilibrium potential for NCX (ENCX),

that is

INCX ¼ gNCXðEm � ENCXÞ

ENCX is the potential at which the inward and outward

unidirectional charge transport rates are equal and there is

no net driving force for ion transport. It is defined by both the

Na+ and Ca2+ equilibrium potentials by the equation:

ENCX ¼ 3ENa � 2ECa

assuming a 3:1 stoichiometry for Na+:Ca2+ exchange.

ENa and ECa vary as a function of the transmembrane Na+

and Ca2+ gradients, respectively, according to the Nernst

equation, that is

Eion ¼RT

zionFln½ion�out

½ion�in

� �

where R, T, zion and F are the gas constant, temperature,

charge of the ion and Faraday’s constant, respectively. A

change in Nai+ from 5 mM (ENa � +90 mV) to 15 mM

(ENa � +60 mV) would therefore shift ENCX from approxi-

mately +20 to �70 mV, resulting in dramatic changes in

the amplitude and direction of INCX and Ca2+ transport,

depending on membrane potential. NCX in the cardiac cell

at rest, having an Em close to�80 mV, will be operating in the

forward-mode to extrude Ca2+. During the action potential,

the dynamically changing ion gradients and membrane

potential influence the direction of current flow in a rapidly

changing relationship. NCX will influence the shape of both

the action potential and the Ca2+ transient, but will also be

influenced by the action potential and Ca2+ transient.

In terms of the effects on contraction, a rise in Nai+ will

result in reduced Ca2+ efflux via forward-mode NCX and/or

enhanced Ca2+ influx at the plateau of the action potential

via reverse-mode exchange. The net effect will be to increase

the size of the SR Ca2+ store. This is the basis of the positive

inotropic effect of cardiac glycosides like digoxin [14],

inhibitors of the NKA that have been used to treat heart

disease for almost 200 years. The effects of increasing Nai+

on contraction can be quite pronounced – in some

studies, tension increased as the cube of Na+ activity [15],

indicating that even a millimolar increase in Nai+ induced

by rapid pacing could significantly alter excitation–contrac-

tion coupling [12]. However, it has to be considered that the

effect of Na+ on the cytosolic Ca2+ transient is most pro-

nounced at very low stimulation rates and diminishes at

higher rates [16]. Furthermore, digitalis can elevate cytosolic

Ca2+ transients in the absence of Na+, indicating that at least

part of the inotropic effect of digitalis is Na+-independent

[17,18].

In cardiac muscle under physiological conditions, the

main parameters that vary extensively during a heartbeat

are Em and [Ca2+]i. Changes in [Na+]i can be important when

the stimulation rate is changed or in pathological conditions

such as ischemia-reperfusion injury and heart failure.

Pathophysiology – ischemia

At the onset of ischemia, anaerobic metabolism is activated

within 8 s of flow cessation [19] and contributes to intracel-

lular acidification. The decrease in pH contributes to

enhanced Na+ influx through the NHE, and along with other

changes in Na+ flux occurring with prolonged ischemia, such

as an increase in late Na+ current and a decrease in NKA

activity, results in a very large (up to fivefold) increase in Nai+

www.drugdiscoverytoday.com 209


[20,21]. If Nai+ rises to just 30 mM, for example, ENa will

decrease to �+40 mV and ENCX will shift to ��125 mV. This

means that outward NCX current (i.e. Ca2+ entry) will be

strongly favored over the normal membrane potential range

and reverse-mode NCX action will cause the cytoplasmic and

mitochondrial compartments to load with Ca2+ [22]. Cellular

Ca2+ overload can then lead to postischemic electrical and

contractile dysfunction, as well as cell death. In the context of

ischemia and reperfusion then, the normally beneficial

effects of raising intracellular Nai+ are taken to excess, leading

to a disastrous outcome. Rational therapeutic strategies have

therefore included inhibition of NHE (a source of increased

Nai+) [23–25] and inhibition of Ca2+ influx through NCX [26].

High Nai+ might also be expected to alter mitochondrial Ca2+

accumulation and the control of oxidative phosphorylation.

This aspect of intracellular Nai+ homeostasis has not been

extensively investigated, but could have important conse-

quences which we will discuss below.

Pathophysiology – heart failure

An elevation of Nai+ has been reported in both human and

animal models of hypertrophy and heart failure (for a recent

review, see [13]). The increase in Nai+ is typically on the

order of 3–6 mM. The mechanism underlying the increase is

still under investigation; however, several hypotheses have

been explored, including a decrease in NKA activity,

enhanced NHE activity or an increase in persistent Na+

current. A decrease in the expression or isoform distribution

of NKA has been reported in several models of hypertrophy

and heart failure [13,27,28]. Several recent studies have

examined NKA function directly in ventricular myocytes

from failing hearts in different animal models and have

come to disparate conclusions. In a canine model of chronic

AV block and hypertrophy, maximal Na+ pump current was

found to be unchanged, but Nai+ affinity was decreased [29],

while in a rabbit heart failure model of mitral insufficiency

and pressure overload, no change in NKA Vmax or Nai+

affinity was detected [30]. In heart failure induced by myo-

cardial infarction, decreased NKA expression is typically

associated with depressed Na+ pump function [27,31]. Inhi-

bition of NHE activity has recently been shown to improve

cardiac function in several models of heart failure [23,32–

34] and this effect could be related to inhibition of increased

Na+ influx through NHE [25]. In the rabbit heart failure

model in which NKA was unchanged, elevated Nai+ was

attributed to increased Na+ influx through a pathway sen-

sitive to the Na+ channel blocker tetrodotoxin. The proper-

ties of this channel have not yet been characterized, but it

could involve a slowly inactivating component of INa. A

persistent late Na+ current has been shown to increase in an

animal model of heart failure [35,36], but no change in this

current could be found in myocytes from failing human

hearts [37].


The reported increase in Nai+ in these models of heart failure

would cause a marked change in the thermodynamic driving

force for INCX, favoring enhanced reverse-mode Ca2+ entry

during depolarizations and diminished forward-mode Ca2+

extrusion through NCX. This could theoretically improve

contractility in the failing heart in the face of depressed SR

Ca2+ uptake [38]; however, other factors must be considered to

gage the overall effect (Fig. 2). First, it should be noted that

myocytes from failing hearts are much more dependent on

forward-mode NCX rather than SERCA to support Ca2+

removal [39], so the positive effect of higher Nai+ on SR

Ca2+ release is paralleled by slowed Ca2+ removal via NCX as

it switches to forward-mode during diastole. Thus, there is

the danger that diastolic function may be impaired as the

stimulation frequency increases. This has been demon-

strated in human failing cardiac muscle [40].

Another consideration is that the density of NCX has been

shown to be increased in several human and animal models

of heart failure [39,41–43]. The increase in total NCX mem-

brane conductance could offset the effect of higher Nai+ and

contribute to depletion of the SR Ca2+ store by competing

more effectively with SERCA, resulting in enhanced Ca2+

extrusion from the cell during diastole. In the rapid

pacing-induced canine model that we have studied, myo-

cytes from failing hearts display depressed Ca2+ transients

and severely depleted SR Ca2+ loads [44]. Inhibition of NCX

by �30% using the exchange inhibitor peptide XIP restored

the SR Ca2+ load to normal levels and improved the kinetics

and amplitude of the Ca2+ transient (Fig. 3) [45]. These

experiments were performed using whole-cell patch clamp

at equivalent Nai+s in the control and failing groups (20 mM)

in order to eliminate differences related to the changes in Nai+

noted above. Thus, inhibition of forward-mode NCX could

improve Ca2+ handling in the failing heart without the

potential detrimental effects of elevating Nai+ (such as with

digoxin), which could include impairment of mitochondrial

Ca2+ uptake (see below), suppression of H+ efflux by the NHE

or a decrease in other Na+ cotransport processes that depend

on the transmembrane Na+ gradient.

Role of Nai+ in modulating the action potential

The electrogenic processes that depend directly on Nai+ will

have strong effects on the cellular action potential and in turn

will be most affected by the waveform of depolarization. The

3Na+:2K+ stoichiometry of NKA, along with its unidirectional

transport behavior, ensures that the direction of current flow

will always be outward, which would facilitate repolarization.

Therefore, the overall effect of NKA inhibition should be to

prolong the action potential, but this is complicated by the fact

that Nai+ will rise concomitantly, making it difficult to accu-

rately determine how much NKA current contributes to nor-

mal electrical function. NKA current density at physiological

Nai+ (e.g. 10 mM, see [46]) is smaller than that of NCX, the


Figure 2. Influence of Nai+ on Ca2+ fluxes during the action potential in myocytes from normal and failing hearts. In the failing heart, decreased activity of

the sarcoplasmic reticulum (SR) Ca2+ pump and enhanced leak of Ca2+ from the SR contribute to a decrease in SR Ca2+ content. Nai+ is elevated

and SR Ca2+ release is blunted, contributing to more reverse-mode Ca2+ entry (net outward NCX current) during the early and late phases of the AP

plateau, as compared to normal myocytes. The impairment of SR Ca2+ uptake shifts the failing myocyte toward a greater dependence on NCX to remove

Ca2+ during diastole.

other major Nai+-sensitive electrogenic transporter, but inhibi-

tion of NKA wouldbe expected to prolong the action potential.

Understanding the impact of INCX on the action potential is

an even more difficult problem, because both the membrane

potential and ion gradients driving flux are rapidly changing.

In animal models with a short action potential, such as the

mouse and rat, NCX might only operate in the reverse-mode

for a very brief time during the action potential overshoot, and

early studies confirmed that the predominant direction of INCX

was inward during the AP plateau of the rat [47]. We have

previously examined the net effect of changing Nai+ on action

potentials in myocytes from normal and failing canine hearts

[48] (Fig. 4). Like human cardiac cells, canine APs show a

pronounced notch-and-dome morphology and an extended

action potential that could favor outward INCX during the

plateau. In both groups, the action potential duration was

markedly decreased when Nai+ was increased from 5 to

15 mM. This was consistent with the calculated shift of the

thermodynamic driving force for NCX toward more outward

current at high Nai+ during the plateau of the action potential.

Moreover, the driving force for reverse-mode NCX current

was increased in myocytes from failing hearts compared to

controls at any equivalent Nai+. This was because first, the

amplitude of the Ca2+ transient was depressed due to

impaired SR Ca2+ loading in these cells (i.e. the Ca2+

gradient favored less forward-mode exchange during sys-

tole) and second, the early phases of the action potential

plateau occurred at more depolarized potentials as a result

of the diminution of the notch phase of the AP in the

failing group. These differences between groups would be



Figure 3. Effects of partial inhibition of NCX on Ca2+ handling in heart failure. Treatment with 10 or 30 mM sarcolemmal Na+/Ca2+ exchange inhibitor

peptide (XIP) in the cytosolic compartment of myocytes from failing canine hearts (solid symbols) increases the amplitude of the Ca2+ transient (Cai),

restores SR Ca2+ load (CaSR) and improves the rate of rise of the Ca2+ transient DCai/Dt without affecting Ca2+ current (ICa) as compared to controls (open

symbols). From Hobai et al. [45] with permission.

expected to be exacerbated under more physiological cir-

cumstances, considering that Nai+ is also elevated with the

development of heart failure. This view is consistent with

other recent studies in which reverse-mode Ca2+ entry via

NCX during the AP plateau is thought to significantly

contribute to contraction in cells from failing hearts [49].

In the same study, to assess whether the effects of Nai+ on the

AP were indeed mediated by changes in NCX driving force, we

examined the short-term effect of partially inhibiting NCX at

different Nai+. At 5 mM Nai

+, intracellular application of XIP

caused action potential shortening, consistent with NCX con-

tributing inward current (depolarizing influence) during the

AP plateau, while at 10 or 15 mM Nai+, XIP-induced AP pro-

longation, indicating that NCX was predominantly contribut-

ing outward current (repolarizing influence). With more

prolonged inhibition of NCX, the effects of XIP to increase

SR Ca2+ load usually overrode the short-term effects on the AP;

that is, an increase in the amplitude of the Ca2+ transient

tended to shorten the AP at physiological Nai+. These compet-

ing influences tend to offset each other, resulting in very little

effect on the APD overall (e.g. see Fig. 7 of Ref. [45]).

An importantquestionhasbeenraisedbythestudyofWeber

etal. [50],whoconcludedthatNCXsensesasubmembraneCa2+

domain that has higher Ca2+ concentrations than the average

cytosolic Ca2+. High Ca2+ persisted in this microdomain well

into the plateau period, thus resulting in a calculated driving


force for NCX strongly favoring the forward-mode throughout

mostoftheAPatphysiologicalNai+.TheexpectedeffectofNCX

inhibition on the action potential of normal cells should then

always be to shorten the action potential, contradictory to the

findings discussed above. Further studies and perhaps more

selective NCX inhibitors could help to resolve the discrepancy

between these viewpoints in the future.

Contribution of altered INCX to arrhythmogenesis

Given the multiple direct and indirect effects of Nai+ on Ca2+

handling and ion transport, as discussed above, it is difficult

to predict what effect a change in Nai+ will have on the

propensity for arrhythmias in the setting of cardiac hyper-

trophy or failure. From first principles, the direct effects of

elevated Nai+ on the electrogenic processes described above

would be expected to facilitate repolarization of the action

potential. An increase in Nai+ would stimulate the Nai

+ pump

to increase outward current, and the shift in ENCX would

enhance outward INCX during the action potential plateau.

These action potential shortening effects may be superceded

by other factors during the development of cardiac disease.

Prolongation of the action potential in the failing heart may

be conferred by first, a decrease in repolarizing K+ currents

[51]; second, a decrease in Ca2+-dependent inactivation of ICa

as a result of reduced SR Ca2+ release [52]; third, an increase in

the density of NCX and the relative dependence on Na+/Ca2+


Figure 4. Effects of heart failure and Nai+ on NCX driving force during the action potential. (Top) Representative APs (black traces) and Ca2+ transients

(red traces) from myocytes isolated from normal hearts dialyzed with 5, 10 or 15 mmol/L [Na+]i. Left panel illustrates the method for calculating

the NCX driving force (Em � ENCX, magenta trace) from ENCX (blue trace). (Bottom) Analogous records for myocytes from failing hearts with 5, 10 or 15

[Na+]i. RP is the potential at which Em � ENCX = 0 (indicated with a dashed line in all panels). Em: membrane potential and ENCX: equilibrium potential for

NCX. From Armoundas et al. [48] with permission.

exchange as a Ca2+ extrusion mechanism, which could con-

tribute larger inward currents during late repolarization and

diastole. Increased inward INCX occurring late in the action

potential could increase the likelihood for the generation of

an early afterdepolarization.

Delayed afterdepolarizations (DADs), occurring during dia-

stole, are another potential source of triggered arrhythmias.

Pogwizd et al. [53] have proposed that increased NCX activity,

combined with a reduction in the background inward recti-

fier K+ current, decreases the threshold for triggering DADs in

a rabbit model of heart failure. These arrhythmias would be

most likely to occur during sympathetic activation, when a

spontaneous Ca2+ release from an overloaded SR would have

a higher probability of triggering a DAD.

Mitochondrial Na–Ca exchange

An integrative view of the effects of Nai+ on cardiac cell

function must include the important role played by Nai+

on mitochondrial Ca2+ homeostasis. Recent studies have

spurred renewed interest in the participation of mitochon-

dria in excitation–contraction coupling. In many tissues, and

especially in heart and brain, the mNCE is the primary path-

way for Ca2+ efflux from the matrix. This antiporter has yet to

be identified at the molecular level, but evidence indicates

that it catalyzes a 3Na+:1Ca2+ electrogenic exchange [4,54]

with a Km for Nai+ of�8 mM [55]. An electroneutral transport

mode was also reported in the earliest studies identifying

mNCE [3,56]. An increase in mitochondrial Ca2+ concentra-

tion plays a major role in the stimulation of mitochondrial

ATP production in response to a change in cardiac work, both

by stimulation of Ca2+-sensitive dehydrogenases [57] and by

activation of other sites in the respiratory chain [58].

The Km for Na+ activation of mNCE lies close to the physio-

logical setpoint for Nai+, so it follows that changes inNai

+ in the

range we have discussed above could have a significant impact

on the bioenergetic response to a change in the inotropic state

of the myocyte. This hypothesis is supported by a previous

study on the effects of Nai+ and the mNCE inhibitor CGP-


�

�

�


37157 on the oxidative phosphorylation rates of isolated

mitochondria. Increasing extramitochondrial Na+ decreased

matrix Ca2+ accumulation, NADH production and the oxida-

tive phosphorylation rate, while inhibition of mNCE elimi-

nated the Na+ sensitivity of these parameters [59]. We have

addressed these issues in a recent study in our own laboratory.

In patch-clamped cardiac myocytes, mitochondrial Ca2+

dynamics during excitation–contraction coupling were found

to have fast uptake kinetics and a slow rate of Ca2+ removal as

compared to the rate of decline of Ca2+ in the cytoplasm [60].

The rate of mitochondrial Ca2+ removal was sensitive to Nai+ in

the range of 5–15 mM. Moreover, the altered kinetics of mito-

chondrial Ca2+ accumulation in the presence of high Nai+ were

associated with an altered compensatory response of NADH

when work was increased by rapid pacing [60] (Fig. 5).

Connecting the dots: how altered mitochondrial Ca

uptake leads to oxidative stress

Notably, the change in mitochondrial NADH redox potential

in the presence of high Nai+ persisted after the period of high

work ended, indicating a significant shift in the cellular redox

Figure 5. Effect of Nai+ on excitation–contraction bioenergetic coupling. (a) Infl

Elevated Nai+ can help to increase SR Ca2+ load, but will impair stimulation of th

Ca2+ efflux through mNCE. (b) Impaired energy supply and demand matching at 1


balance [60]. This could be the result of oxidative damage and

an inability of the mitochondria to prevent the accumulation

of toxic reactive oxygen species during rapid pacing. Net

oxidation of mitochondrial matrix NADH would be expected

to deplete the antioxidant defenses because NADPH regen-

eration in the matrix depends on NADH production and the

TCA cycle through several direct and indirect linked reactions

including the NADH:NADPH transhydrogenase, NADP+-

dependent isocitrate dehydrogenase and Malic enzyme

[61], through the following reactions (see Fig. 6)::

uen

e T

5

Transhydrogenase reaction:

NADH þ NADPþ þHþ

! NADþ þ NADPH (coupled to protonmotive force)

NADP+-dependent isocitrate dehydrogenase:

Isocitrate þ NADPþ þHþ ! a-ketoglutarate þ NADPH

Malic enzyme:

Malate þ NADPþ þHþ ! pyruvate þ NADPH

ce of cytosolic Na+ on Ca2+ distribution and mitochondrial energetics.

CA cycle by mitochondrial Ca2+ as a result of increased mitochondrial

mM versus 5 mM Nai+; adapted from Maack et al. [60] with permission.


Figure 6. Interactions between Ca2+ handling, metabolic and antioxidant systems. The antioxidant capacity of the mitochondrial matrix is linked to the

tricarboxylic acid (TCA) cycle through chains of redox reactions involving glutathione, NADPH and NADH via glutathione reductase (GR) and glutathione

peroxidase (GPX), which detoxifies hydrogen peroxide (H2O2) produced by the dismutation (through superoxide dismutase, SOD) of superoxide (O2��),

a byproduct of electron transport (electron transport chain; ETC). NADPH is maintained in the reduced form by at least three reactions, the

NADH:NADPH transhydrogenase, NADP+-dependent isocitrate dehydrogenase and malic enzyme. Inadequate Ca2+ loading (via the mitochondrial Ca2+

uniporter; MCU) of the mitochondrial matrix during increases in work may limit the activation of Ca2+-sensitive enzymes in the TCA cycle, which include

pyruvate dehydrogenase (PDH), a-ketoglutarate dehydrogenase (a-KGDH) and NAD+-dependent isocitrate dehydrogenase. Net oxidation of the NADH

and NADPH pools will result in oxidative damage (particularly in targets with reactive thiols) by limiting the ability to scavenge reactive oxygen species.

Enhancing mitochondrial Ca2+ influx or inhibition of Ca2+ efflux (mediated by the Na+/Ca2+ exchanger; mNCE) is postulated to restore energy supply and

demand matching that is impaired by high cytosolic Na+.

Maintaining the NADPH/NADP+ redox couple in the

reduced state is crucial for the detoxification of ROS produced

�

in the mitochondrial matrix because the negative redox

potential of NADPH (>�320 mV) is the main driving force

for the reduction of oxidized glutathione (GSSG), the most

abundant thiol buffer in the mitochondrion and is also

required to maintain the specific thiol buffer thioredoxin

(TRX) in the reduced form. GSH and reduced thioredoxin,

in turn, are utilized to remove hydrogen peroxide (H2O2),

which is generated as a product of superoxide (O2��) dismu-

tation. This chain of antioxidant reactions is required to

remove the O2�� that is produced as a byproduct of mito-

chondrial respiration through the following enzymatic reac-

tions (see Fig. 6):

Transhydrogenase reaction:

NADH þ NADPþ þHþ

! NADþ þ NADPH (coupled to protonmotive force)



� N

21

ADP+-dependent isocitrate dehydrogenase:

Isocitrate þ NADPþ þHþ ! a-ketoglutarate þ NADPH

� M
alic enzyme:
Malate þ NADPþ þHþ ! pyruvate þ NADPH

Maintaining the NADPH/NADP+ redox couple in the reduced

state is crucial for the detoxification of ROS produced in the

mitochondrial matrix because the negative redox potential of

NADPH (>�320 mV) is the main driving force for the reduc-

tion of oxidized glutathione (GSSG), the most abundant thiol

buffer in the mitochondrion and is also required to maintain

the specific thiol buffer thioredoxin (TRX) in the reduced form.

GSH and reduced thioredoxin, in turn, are utilized to remove

hydrogen peroxide (H2O2), which is generated as a product of

superoxide (O2��) dismutation. This chain of antioxidant

reactions is required to remove the O2�� that is produced as

a byproduct of mitochondrial respiration through the follow-

ing enzymatic reactions (see Fig. 6):

� G
lutathione reductase (GR):
NADPH þ GSSG þ Hþ ! NADPþ þ 2GSH

Thioredoxin reductase:
�
TRX�S2þNADPH þ Hþ ! NADPþ þTRX�ðSHÞ2Glutathione peroxidase (GPX):
�
H2O2þ2GSH ! H2O þ GSSG

Peroxiredoxin:
�
H2O2þTRX�ðSHÞ2 ! H2O þ TRX�S2

Superoxide dismutase (SOD):
�
O2�� þ2Hþ ! H2O2

The pathways described above illustrate how the balance of

Na+ and Ca2+ in the cytosolic and mitochondrial compart-

ments can have an impact on the redox state of the mito-

chondria and the ability to detoxify ROS. As we have

previously argued, a shift in the balance between the rate

of mitochondrial ROS production and the antioxidant capa-

city can lead to the abrupt deenergization of the mitochon-

drial network, which we referred to as mitochondrial

criticality [62]. This event can precipitate ventricular arrhyth-

mias because of the generation of heterogeneous regions of

inexcitability (metabolic sinks), as we have described pre-

viously [63].

It is also important to recognize that this feed forward

effect of Ca2+ on mitochondria is accompanied by feedback

of both the redox state and phosphorylation potential on the

ion channels and transporters involved in excitation–con-

traction coupling. Reactive thiols have been shown to mod-

ulate the activity of the SERCA [64], the ryanodine receptor

[65] and the L-type Ca2+ channel [66] and several other


sarcolemmal ion channels. In addition, the Ca2+ handling

proteins are subject to regulation by ATP, ADP and Mg2+ [67],

as is the classical sarcolemmal ATP-sensitive K+ (KATP) chan-

nel. Changes in the mitochondrial energy state are, therefore,

rapidly transmitted to changes in electrical excitability, Ca2+

handling and contractile function of the cardiomyocyte.

Taking all of these ideas into consideration, we have pro-

posed a novel mechanism that may contribute to functional

decompensation and/or sudden death in the context of

chronic metabolic stress, such as during postischemic remo-

deling, hypertrophy and heart failure [68]. Nai+ loading in the

diseased heart may partially compensate for impaired con-

tractility by enhancing SR Ca2+ load; however, the effect of

elevated Nai+ on mitochondrial Ca2+ loading induces a mis-

match between NADH supply and energy demand. The effect

on the mitochondrial redox state results in a state of chronic

oxidative stress because of the depletion of NADPH and GSH.

Over time, some myocytes reach a critical state and mito-

chondrial membrane potential collapses, triggering first, a

situation in which metabolic sinks may be created in the

heart, predisposing the heart to reentrant type arrhythmias

[63], and ultimately, resulting in necrotic or apoptotic cell

death. Attrition of myocytes in this manner will eventually

result in a decline and failure of contractile function.

It is important to note that the cascade of failures described

above will be greatly exacerbated by the impairment of Ca2+

handling already known to be present at the cellular level in

the failing heart [39]. Ca2+ transients with diminished ampli-

tudes and rates of rise are less likely to drive Ca2+ into the

mitochondria, especially in the presence of elevated Nai+.

This vicious cycle will be amplified by the negative effects of

the changes in redox state and energetics on SR Ca2+ hand-

ling. The integrative aspects of this hypothesis will require

further investigation to determine if cause and effect relation-

ships can be established and if key control points can be

identified. To strengthen interpretation of experimental

results and test these interactions, it will be important to

develop computational models that integrate the effects of

bioenergetics, electrophysiology, Ca2+ handling and contrac-

tion [69].

Summary and future directions

Recent studies demonstrating an increase in resting Nai+ in

humans and in animal models of cardiac hypertrophy and

failure have spurred renewed interest in understanding the

role of Nai+ in modulating the electrical and contractile

properties of the heart. The overall influence of Nai+ on

the action potential and the cytosolic Ca2+ transient is com-

plex and species-dependent, and depends on the balance of

several ion fluxes. The amplitude and direction of the sarco-

lemmal Na+/Ca2+ exchange current and Ca2+ flux will be

strongly influenced by the magnitude of triggered SR Ca2+

release and the voltage at the plateau of the action potential.


These factors, along with elevated Nai+, combine to enhance

reverse-mode Na+/Ca2+ exchange current during the action

potential plateau in myocytes from failing hearts. On the

contrary, depressed SERCA activity and enhanced NCX den-

sity conspire to deplete SR Ca2+ content during diastole in the

failing heart, which may be reversed by partial inhibition of

forward-mode Na+/Ca2+ exchange.

Positive inotropic interventions in heart failure will only be

effective if there is an adequate supply of energy to meet

increased levels of work. Elevated Nai+ might impair mito-

chondrial Ca2+ loading and limit the stimulation of oxidative

phosphorylation when there is an increase in the cytosolic

Ca2+ transient. Hence, the integrated effects of agents that

increase Nai+, such as digoxin, must be considered in light of

their impact on both sarcolemmal, SR and mitochondrial

Ca2+ movements. Moreover, a therapeutic approach aimed at

preventing or correcting the disruption of Nai+ balance in the

failing heart should be investigated in future studies.

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the role of na dysregulation in cardiac disease and how it impacts electrophysiology

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