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DOES SODIUM SALICYLATE TREATMENT ENHANCE HSP 72 EXPRESSION AND MYOCARDIAL PROTECTION?
Joel William Atance
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Community Health University of Toronto ,
0 Copyright by Joel William Atance (1998)
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Does sodium salicylate treatment enhance HSP 72 expression arid myocardial protection?
Master of Science, 1998. Joel Wiiam Atance, Graduate Department of Community
Health, University of Toronto.
It was of interest to determine whether an in vivo administration of salicylate,
combined with a mild heat shock, could potentiate the heat shock response, and confer
signiticant myocardial protection. To test this hypothesis, 25 male Sprague-Dawley rats
were divided into five groups (n=5 per group): 1) unstressed (control); 2) sodium
salicylate treated., 3) mildly heat-shocked; 4) mildly heat-shocked plus sodium salicylate
treated; and 5) severely heat shocked. Hearts were analyzed for hernodynamic performance
on a Langendorff apparatus. Following an ischemic episode, hearts from animals that were
severely heat shocked recovered a greater percentage of left ventricular developed pressure,
and rate of contraction and relaxation, compared to unstressed (control) animals. These
animals also showed a significantly greater accumulation of left ventricular HSP 72.
Animals that were mildly heat shocked, with or without sodium salicylate treatment, were
not conferred myocardial protection, and did not show significant increases in left
ventricular HSP 72 content. With this experimental design, it can be concluded that
sodium salicylate treatment does not potentiate the myocardial heat shock response in viva
F i t and foremost, I would like to thank my supervisor, Dr. Marius Locke, who a l l along displayed an admirable level of patience, while starting a lab from scratch, and supervising two graduate students, and occasional summer students, all of whom had veIy Little experience in the field. Marius, I can't think of one time when you were too busy to offer help, and I know your plate has been full for the last three years. For that I am very grateful. I would also like to thank Dr. Michael Plyley, and Dr. Nancy McKee, the other members of my advisory committee. Almost four years ago, I was a little uncertain of my future direction, and contemplating a career change. It was suggested I speak to Dr. Plyley. He had encouraging words for me, which gave me the confidence to undertake a fairly drastic change in academic disciplines. Thereafter, he was always willing to share his expertise on all types of academic issues. Dr. McKee has been instrumental in providing much needed lab space, and expert hands-on advice. Her generosity is much appreciated. Special thanks to Dr. Carol Rodgers, whose many courses I followed were always excellent, well-researched, and well-taught.
Thank you to my fellow students. My time at the U of T really got better as I moved along, and this was due in no small part to the many friends I made in the program. Thanks Robert for making an effort to unite graduate students spread out in the big city. Thanks also for pushing me to do OEP '98. It really helped out down the road. Thanks Cora for being a willing participant in aIl the little functions. Good luck Adrian in wrapping things up, and with future plans. Thanks to the office staff, especially Ruby, Tim, and Wenda, for courteous help, work hours, and the odd supplies.
You may never read this, but a sincere thanks to the many AC people I first befriended when I knew not a soul in this town. You made a difference when the going was tough.
Finally, thank you to my family, who don't know much about HSPs, but were there long before this all began, and without whom I wouldn't be typing this up.
*.
A B ~ T ~ A C T -------------------------------------------------.-----.----------------------------------------------------- 11
-.- AcmowLEDGMENTs ------.---------------*- **-.** * .*- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ----- ----- IJl
mumE OF CONTENTS -------------------------------------- .*------------------* *.* *---.--- *.***.**** -.---- iv
I X T OF TABLES ---.-----------------------------.-------------------------------------------------------------- vi
LIsT OF FIGURES --.----.-* **.**-** -*--------*.-.-------------- *.**.*.* ---------.- **.** +...--.- * * - * **..- --*.*--* vii ..-
LIST OF APPENDICES..* -----*------------- .--------------- .----- .------ . . --*------------------- .----------- VlLl
LIST OF -BRE-TIoNS. -- -- -- - - *-. *. - - -*. - - - -. * * * *. . - *. . - - - - - - - - * - - ** - --. . * -- * - * * - * . * - +.. . -- - - ix
TABLE
1 . Pre-ischemic absolute values for hernodynamic variables ---- .--------..-.+.------amiamiamiamiami 30
LIST OF FIGURES
FIGURE
Pre-heat stress (baseline) rectal temperatures are similar between groups- - --- - - 24
Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, experienced heat shocks of similar duration ~ ~ ~ - - - ~ ~ ~ - ~ ~ a l a l a l a l a l a l a l a l a l a l a l a l a l a l 25
Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, showed similar average T, during entire heat stress --------------- 26
Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, showed similar average T, during 15 mio heat shock- -- -- - - --. 27
Graphical representation of left ventricular HSP 72 content following i=hemia-reperfusiontl - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - tl tl tl - - - - - - - - - - -. - - . -. . * * . + - * . - -. - - - - . . . * * * *a tl tl tl tl * tl tl . - - 45
vii
APPENDIX
ATF' AP ANOVA BSA sC T cm COX DNA DMF ddH20 g h i~ HSC HSE HSF HSP kD kg LVDP L mRNA
CLg
fi Cun mg min ml d . - l
rnm m g m m ~ g s-' mM nm NO NFDM NSAlD ID PGA, PGD, PGE, PGH +dP-dt -' -dP*dt " kdP*dt -' =r SDS
adenosine triphosphate alkaline p hosphatase analysis of variance bovine serum albumin degrees Celsius degrees Fahrenheit centimetre cyclooxygenase deoxyribonucleic acid N,N-dimethyl formamide double distilled de-ionized water gram hour(s) intraperitonealy heat shock cognate heat shock element heat shock factor heat shock protein kilodalton kilogram left ventricular developed pressure litre messenger ribonucleic acid micrograms microlitre micrometers milligram minu te(s) millilitre millilitres per minute millime= miltimetres of mercury millimetres of mercury per second millimolar nanometers nitric oxide non-fat dried milk powder non-steroidal anti-inflammatory drug one-dimensional prostaglandin A, prostaglandin 4 prostaglandin E, prostaglandin H synthase rate of contraction rate of relaxation rate of contraction and relaxation rectal temperature sodium dodecyl sulphate
SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis TBS tris-buffered saline Tr33S tris-buffered saline plus 0.05% Tween-20 U units v volt(s)
CHAPTER 1
1.1 Background
In response to elevated temperatures and other forms of protein damaging stresses,
both prokaryotic and eukaryotic cells temporarily suspend most gene transcription and
mRNA translation, while conspicuously increasing these processes in a select family of
genes (Lodish, 1995). These genes encode proteins which increase cell survival after
exposure to stress (for review see Morimoto et al., 1994). These proteins, originally
discovered in the salivary glands of fruit fly larvae subjected to temperature elevations,
were accordingly termed heat shock proteins (HSPs)(Ritossa, 1962; Tissieres et al., 1974).
Subsequent research has demonstrated the synthesis of HSPs to be a universal response to
a wide variety of protein damaging stresses, including hypoxia (Heacock and Sutherlmd,
1990), low pH (Whelan and Hightower, 1985), and in some cases, exercise (Locke et al.,
1990).
1.2 HSPs
The heat shock proteins are classified according to their molecular mass. Thus,
HSP 60, HSP 70, HSP 90, and HSP 110 are categorized as high molecular mass HSPs,
while those HSPs ranging in mass fiom 8 to 47 kD, are considered low molecular mass
HSPs (Mestril and Dillmann, 1995). HSP 70 is the most highly conserved HSP, both
within and between species (Hunt and Morimoto, 1985). Remarkably, 50% of the amino
acid sequence is conserved between E. coli and humans (Schlesinger, 1990). The term
'HSP 70' refers to one or more isofonns of the 70 kD family. Foremost among these are
the inducible isoform, HSP 72, and the cognate isoform, HSC 73, which can both be
elevated after exposure to various stresses (Locke, 1997). As well, both HSP 72, and
HSC 73, have been shown to be involved in various stages of protein synthesis
(Beckmann et al., 1990), transport (Chirico et al., 1988), and degradation (Chiang et al.,
1989). It is unclear why cells require both a cognate, and an inducible isoform of very
similar sequence, and function (Brown et al., 1993). However, HSC 73 possesses
intervening sequences not found in HSP 72 ( M a d et al,, 1994). It has been suggested
that the lack of intervening sequences in HSP 72 may facilitate the rapid transcription of
this protein during periods of stress (Hunt and Morimoto, 1985). In general, it is believed
that HSC 73 functions primarily during unstressed conditions, whereas HSP 72 is
synthesized inpponse to cellular demands during episodes of stress (Black and Subjeck,
1991).
1.3 Cellular function of HSP 70
In the cell, HSP 70 is thought to function as a protein chaperone. Using the
hydrolysis of ATP as an energy source, HSP 70 aids in the folding of newly synthesized
polypeptides (Ku et d., 1995), and the translocation of proteins across membranes
(Deshaies et al., 1988). HSP 70 has been shown to re-activate denatured proteins (often
the result of excessive heat or acidity) by restoring native conformation (for review see
Knowlton, 1995). This mechanism is thought to be critical for enhancing cell survival
during episodes of stress.
The heat shock response occurs rapidly and results in a robust induction of HSP
70. In fact, the heat shock gene is described as being in a constant state of readiness
(Mestril and Dilhqm, 1995), such that within 15 minutes of exposure to temperatures 3 to
5°C above normal, heat shock proteins axe preferentially synthesized (Lindquist and
Petersen, 1990). The exact means by which a cell 'senses' an increased temperature stress
still remains unknown. However, a model for the activation of the heat shock response has
been proposed (Abravaya et al., 1992).
1.4 HSP regulation
The promoter region of all heat shock genes contains a unique sequence termed the
heat shock element (HSE). In mammals, a constitutively expressed protein, d e d heat
shock factor (HSFl), binds to the HSE and mediates a stress-induced HSP induction. In
unstressed cells, the HSF is inactive, and is thought to be bound to HSP 70 (Abravaya et
al., 1992). Following stress, many proteins become denatured or unfold, and thus may
compete for HSP 70 binding. With a new pool of substrates available for binding, some
HSP 70 will release HSF. This allows the HSF to trirnerize to an active state, and
subsequently, to bind to the heat shock element, thus inducing HSP transcription.
Eventually, the amount of newly translated HSP 70 will outnumber the pool of denatured
proteins, and the excess HSP 70 is thought to rebind and deactivate HSF, preventing
further binding to the HSE,
1992).
1.5 Evidence for the
Thermotoleraflce is
temperatures. The synthesis
and effectively halting HSP 70 expression (Abravaya et al.,
role of HSP 70 in thermotolerance
described as the cell's ability to withstand
and degradation of HSPs precedes the acquisition
increased
and decay
of thermotolerance, suggesting a protective role for these proteins (Li and Mak, 1985).
After prior exposure to a brief, mild heat shock (-42OC), cells are able to withstand an
otherwise lethal heat shock ( - 4 5 O C ) (Li and Werb, 1982). This transient resistance to
elevated temperatures has been described as acquired thennotolerance (Henle and
Dethlefsen, 1978). Various experimental protocols have shown the importance of HSP 70
in conferring cellular thermotolerance. For example, when cultured fibroblasts were micro-
injected with antibodies to HSP 70, the cells were unable to survive even a brief incubation
at 45O C @abowol et al., 1988), suggesting that HSP 70 is indispensable for ceUula-r
survival during heat stress. In other studies, when cellular HSP 70 expression was
enhanced, a subsequent increase in thennotolerance was demonstrated. For example,
when an HSP 70 gene construct was placed under control of the p-actin promoter, and the
cells transfected (ensuring induction of HSPs at normal temperatures), the transfected cells
displayed a significantly greater degree of thermotolerance than non-transfected cells (Li et
al., 199 1; Angelidis et al., 199 1). In a similar experiment, myocytes were tramfected with
plasmids for various HSPs (under control of the p-actin promoter). Cells transfected with
an HSP 70 gene construct showed a significantly greater resistance to subsequent heat, or
ischemic stress (Heads et al., 1995). These results strongly suggest that HSP 70 can
confer protection to cells. Interestingly, whole body thermotolerance has also been
demonstrated in mice (L et al., 1983). A non-lethal preconditioning heat shock was
shown to protect C3H mice against thermal death during a subsequent, othenvise lethal,
hyperthermic episode, although the role of HSP 72 in the observed thermotolerance was
not investigated.
1.6 HSP 70 and myocardial protection
HSP 70 can be induced by several different types of stress (for review see
Morimoto et al., 1994). Accordingly, induction of HSPs by one stressor may confer
subsequent tolerance against another stressor. This phenomenon, known as cross-
tolerance, has- been demonstrated in several cases (for review see Yellon and Marber,
1994). Protection from ischemic stress through a prior heat stress is an example of cross-
tolerance that has been widely studied because of its potential for medical and general health
applications. In rat and rabbit models, whole body heat stress has been shown to result in
subsequent myocardial protection fiom ischemic insult (Currie et al., 1988; Karmazyn et
al., 1990; DonneJly et al., 1992; Hutter et al., 1994; Marber et al., 1994). Cunie et. al.
(1988) demonstrated that hearts from rats heated to 42°C for 15 min, 24 hours prior, had
better indices of hernodynamic function after an ischemic episode, than non-heat treated
controls. Creatine kinase release, a marker of cell injury, was significantly reduced in'
hearts from heat-shocked animals. Domeuy et al. (1992) observed a reduced infarct size in
hearts isolated from animals subjected to a 42OC heat shock for 20 min, 24 h prior. In
agreement with HSP 70 providing cell protection (Li and Werb, 1982; Li and Mak, 1985),
the cardiac tissue from heat treated animsls showed an elevated HSP 70 content.
In general, there is a correlation between HSP 70 content and myocardial
protection. Hearts from rats exposed to a progressively higher temperature heat shock
express greater amounts of HSP 72, and are subsequently better protected against
myocardial ischemia (Hutter et al., 1994). Thus, a heat shock of 42OC is more effective at
inducing myocardial HSP 72, and reducing myocardial infarct size, than a heat shock of
40°C. Similarly, Kannazyn et al. (1990) showed that myocardial protection was related to
myocardial HSP 72 content. Animals were heat-shocked for 15 min at 42*C, and
myocardial protection assessed 24, 48, 96, and 192 h post-heat shock. Myocardial
protection was greater in animals heat-shocked 24 or 48 h prior, than in animals heat-
shocked 96, or 192 h prior. The degree of myocardial protection corresponded to
myocardial HSP 70 content, which after 48 h, decreased progressively over time. .
Though hypethermia and exercise are effective inducers of HSP 72, these stresses
are also known to alter the expression of other HSPs and antioxidant proteins (Trost et al.,
1998). As such, the correlation between HSP 70 accumulation and subsequent myocardial
protection cannot be interpreted as absolute proof of the protein's role in heart promtion.
The use of transgenic mice has proved advantageous in isolating the direct effect of HSP 72
on myocardial protection. Studies using these animals have provided strong evidence
implicating HSP 72 in myocardial protection (Plumier et d., 1995; Marber et al., 1995;
Radford et al., 1996). Isolated hearts from transgenic mice over-expressing human HSP
70 (under control of a 8-actin promoter), showed an improved functional recovery and
reduced cell& injury following ischemia, when compared to hearts fiom transgene
negative littermates (Plumier et al., 1995). Marber et al. (1995), using transgenic mice
overexpressing rat HSP 70, observed a reduced infarct size in post-ischemic hearts of
transgene positive animals. Similar results confirming the role of HSP 70 in conferring
myocardial protection, by reducing infarct size, were also demonstrated by Hutter et al.
(1996). Subsequent to global ischemia, transgenic mice hearts also displayed an enhanced
recovery of high energy phosphate stores, and a greater correction of metabolic acidosis
relative to hearts of non-transgenic Litter mates (Radford et al., 1996). Recently, rat hearts
transfected with the human HSP 70 gene, using a virus delivery system, also showed
better functional recovery than control hearts, when perfused using the Langendorff
isolated heart technique (Suzuki et al., 1997). Taken together, these experiments provide
strong support for HSP 70 playing a critical role in conferring myocardial protection.
An ischemic challenge to the myocardium actually presents two distinct periods of
stress, ischemia, and reperfusion. Each stress is defmed by a characteristic set of events
which are either the result of unavailability of oxygen (as a result of ischemia), or re-
introduction of oxygen (re-perfusion). During ischemia, irreversible injury in highly
oxidative myocardial tissue is thought to be associated with ATP depletion, cessation of
glycolysis, and inability to clear waste products (Jennings and Reimer, 198 1; Jennings and
Reimer, 1983). Ischemia causes disruption of the electron transport chain, disappearance
of glycogen particles, and eventual morphological damage to the cell which is evident in
sarcolernma disruption, distortion of 2-lines, and mitochondria swelling (Jennings and
Reimer, 198 1; Jennings and Reimer, 1983; Hammond et al., 1985; Hearse et al., 1977).
Despite the obvious dangers posed by ischemia, reperfusion also presents a unique
challenge, which initially exacerbates existing cell damage. Research has focussed on the
'oxygen paradox' which occurs during reperfusion. In effecf the rapid reestablishment of
normal oxygen tension following an ischemic period results in more tissue injury than if
oxygen levels are gradually restored. The restoration of molecular oxygen, which is
ultimately necessary for cell recovery from ischemia, is accompaaied by a heavy burst of
free radical production. However, free radicals such as the superoxide anion (SO,'), and the
hydroxyl radical (-OH) further damage the sarcolemma and mitochondria during
reperfusion, through lipid peroxidation (Guameri et al., 1980; Opie, 1989). Disruption of
the sarcolemma is believed to facilitate massive calcium influxes, which decrease the
contractile response of the myocardium, and possibly induce arrhythmias (Gao et al.. 1995;
Opie, 1989; Jennings et al., 1983). Thus, ultrastructural injuries originally caused by
ischemia are extended by re-perfision (Hearse et al., 1977). As well, recovery of ATP
stores and the restoration of normal metabolism may be delayed even after several days
following reperfusion (Reimer et al., 198 1 ).
Despite a wealth of evidence demonstrating the role of HSP 70 in myocardial
protection, littIe is known about the mechanisms involved. The role of k radicals in
causing cellular damage during re-perfusion was confirmed in several experiments in which
the introduction of anti-oxidants, such as catalase, or superoxide dismutase, conferred
myocardial protection @as et al., 1986; Myers et al., 1985; Otani et al., 1986). Karmazyn
et al- (1990) demonstrated that myocardial protection conferred by accumulation of HSP 72
may be mediated by catalase. It was suggested that HSP 72 may modulate the activity of
catalase (Karmazyn et d., 1990). This theory is concordant with suggestions that HSPs
exert their protective effect by stabilizing, or solubilizing damaged proteins, and preventing 3
heat-induced insoluble aggregates within the nucleus, andor the cytoplasm @ o ~ e l l y et
al., 1992; Nguyen et al., 1989). Furthermore, HSP 72 may protect and restore protein
synthesis to normal levels during re-perfusion (Trost et al., 1998).
1.7 The salicylates
In 1838, the Italian chemist Piria split salicin, the extract of willow bark, into a
sugar and an aromatic component, and through various oxidative processes converted the
latter into salicylic acid (Vane and Bottin, 1992). Some s i x t y years later, this compound
was acetylated by Hoffmann, and acetylsalicylic acid, or aspirin, was created wane and
Botting, 1992). Although aspirin and salicylic acid have similar modes of action, these
chemical relatives also possess some distinct properties, suggesting aspirin is not simply a
pro-drug for salicylate.
The most documented action of the salicylates is the prevention of prostaglandin
synthesis (Vane, 1971). Prostaglandins are cyclic fatty acids derived from precursor
arachadonic acids. Found in nearly all mammalian tissue, prostaglandins are lrnown to have
important physiologic and phannacologic activities (Murray et al., 1988). Originally
discovered for their role as initiators of smooth muscle contraction, prostaglandins are also
involved in the inflammatory response, and platelet aggregation. Thus, humans have
traditionally used aspirin to relieve pain and inflammation, or to reduce the risk of clot
formation, and subsequent strokes or myocardial infarctions.
The salicylates prevent prostaglandin synthesis by inhibiting the cyclo-oxygenase
activity of prostaglandin H synthase (PGH), one of two catalytic activities performed by
this enzyme in the conversion of arachadonic acids to prostaglandins (Ohki et al., 1979).
Aspirin irreversibly inactivates cyclo-oxygenase activity by transfening its acetyl group to
the PGH enzyme (Roth and Majerus, 1975). There is evidence that aspirin may also
inhibit cyclo-oxygenase activity by a mechanism independent of acetylation. Salicylate is
thought to prevent de novo synthesis of prostaglandin H synthase, thus inhibiting the
cyclo-oxygenase activity of this enzyme (Wu et al., 199 1). More recently, different forms
of the cyclooxygenase enzyme (PGH) have been identified, including COX-1, the
constitutive isoform, and COX-2 the inducible isoform. It has been suggested that the
mode of action of the salicylates and other non-steroidal anti-Mammatory drugs
(NSAIDS) may depend on their interaction with the different COX isofom (Frolich,
1997).
Apart fiom variations in their mode of action, aspirin and salicylic acid also appear
to differ in rate of plasma clearance. In rats administered either aspirin or salicylic acid, the
peak plasma concentration of salicylic acid OCCLKS within 1 h (Higgs et d., 1987). In either
case, salicylic acid concentration declines by half in approximately 6 h, but aspirin is
undetectable after 1 h (Eggs et al., 1987).
1.8 Salicylates and the heat shock response
The salicylates, though well known for relief of pain and idammation, are believed
to have additional properties. Human erythroleukemic cells treated with aspirin during, or
immediately after, heat shock, synthesized greater amounts of HSP 70, and for a longer
duration than non-treated cells (Amid et al., 1995). Mesalamine, a compound related to
sodium salicylate, increased the the& induction of HSP 72 in rat intestinal epithelial cells
(Burress et al., 1997). Conversely, Lee et al. (1995) concluded that when HeLa cells were
treated with indomethacin, combined with mild heat shock, the transcription of heat shock
genes was induced to greater levels than mild heat shock done. Indomethacin was reported
to lower the temperahxe threshold for the heat shock response, such that Hela cells heat-
shocked at 40°C in combination with indomethacin treatment, were capable of surviving a
subsequent 44S°C heat shock. This acquired thennotolerance was as effective as that
observed in cells previously heat-shocked at 41°C, but without indomethacin treatment (Lee
et al., 1995). gdomethacin has also been shown to enhance the heat induced expression of 1
HSP 70 in rat ghoma cells (Ito et al., 1996). Interestingly, in both yeast and Drosophila,
sodium salicylate activation of the HSF has been shown to prevent subsequent heat-
induced transcription of the heat shock gene (Winegarden et al.. 1996; Giardina et al.,
1995).
The various salicylates have been shown to exert their regulatory effect at the level
of the HSF (Jurivich et al., 1992; Jurivich et al., 1995; Amici et al., 1995). In the absence
of hyperthermic treatment, Jurivich et al. (1992) found that sodium salicylate treatment to
HeLa cells activated the HSF to its DNA binding state. This resulted in an HSF:HSE
binding to levels similar to that observed after a 42°C heat shock (Jurivich et al., 1992;
Jurivich et al., 1995). However, the increased HSF activation did not lead to a subsequent
increase in HSP 70 gene transcription. A number of different reasons have been given for
this observation, including the possibility that multiple HSF isoforrns may respond
uniquely to a given stress (Cotto et al., 1996). Thus, heat may activate one specific HSF
isofom, while sodium salicylate would presumably activate another. Alternatively,
salicylate may induce tkeonine phosphorylation of HSFl, while heat induces serine
phosphorylation of HSF1. Only the latter condition transactivates heat shock gene
expression (Jurivich et al., 1995).
Salicylate mediated enhancement of the heat shock response is not fully understood,
and in vivo research in the area is limited to one study. In rats treated with aspirin, HSP
70 mRNA induction was not increased in liver, lung, and kidney (Fawcett et al., 1997).
However, when aspirin was administered to animals one hour prior to a 30 min heat shock
in an ambient temperature of 37OC, a significantly greater induction of HSP 70 mRNA was
observed compared to animals that were heat-shocked alone. Based on Western blot
analyses, the authors also reported an elevation in HSP 70 content in the liver of animals
heat-stressed in combination with aspirin treatment. It was also reported that aspirin
enhanced the elevation of core body temperature in animals exposed to heat stress; mildly
heat-shocked animals treated with aspirin, reached significantly higher core temperatures 3
than animals that were only mildly heat-shocked. Thus, Fawcett et al. (1997) concluded
that the potentiation of heat induced HSP 70 expression was likely the result of an aspirin-
mediated elevation in core temperature. Based on this fmding, it remains unclear whether
salicylate can potentiate the heat shock response in vivo, without affecting animal core
temperatures.
1.9 Thesis objectives
The degree of myocardial protection conferred by a prior heat shock is strongly
associated with HSP 72 content (Kannazyn et al., 1990; Hutter et al., 1994). A heat
shock of 42OC confers significant myocardial protection, but also presents a severe stress to
the animal. Various salicylates have been shown to enhance the heat-induced expression of
HSP 70 in v i m . Aspirin has also been shown to potentiate the HSP 72 induction by a
mild heat shock in vivo, in rat liver, lung, and kidney. At present, it remains unknown
whether salicylates can directly affect HSP 72 induction in the rat heart during mild heat
shock in vivo. Furthermore, it is also unknown if any salicylate enhanced induction of
HSP 72 at lower heat shock temperatures, can confer an HSP 72 mediated myocardial
protection. Thus, the specific aims of this thesis are as follows:
1) to examine the effect of a common non-steroidal anti-inflammatory drug (sodium
salicylate) on myocardial HSP 72 accumulation in vivo.
2) to determine whether in vivo treatment with sodium salicylate, in combination with a
mild heat shock of 4WC, increases myocardial HSP 72 content to levels similar to those
observed following a severe heat shock of 42°C.
3) to determine whether in vivo treatment with sodium salicylate, in combination with a
mild heat shock of 40°C, results in myocardial protection similar to that observed following
a severe heat shock of 42°C.
CHAPTER 2
2.1 Animals,
AND METHODS
sodium salicylate and heat treatments
Adult, male, Sprague-Dawley rats (300-350 g; Charles River) were used in these
experiments. Animals were maintained on a 12 h dark/light cycle, housed in pairs at 2 1" C ,
50% xdative humidity, and were provided food and water ad libitum. Animals were
divided into five groups (n=5 per group): 1) control (unstressed), 2) sodium salicylate only
(400 mgokg'l), 3) mild heat shock (4U°C for 15 min), 4) mild heat shock ( W C for 15 min)
combined with sodium salicylate (400 mg-kg-'), and 5) severe heat shock (42OC for 15
min) (figure 1).
Sodium salicylate (salicyic acid, sodium salt; Sigma Chemical Company,
Mississauga, Ontario, Canada) was dissolved in H,O, and administered intraperitonealy
(ip) in a 0.5 ml volume. Preliminary results indicated a sodium salicylate dosage of 400
mg-kg-', in combination with a mild heat shock, was the minimum amount necessary for
detection of an increased HSP 72 response (appendix VT). For animals treated with'
sodium salicylate and also subjected to heat shock, sodium salicylate was administered one
hour prior to heat shock. For the purposes of this experiment, 'heat shock' refers to the 15 7
min period where T, was maintained at 40°C or 42OC, while 'heat stress' refers to the entire
period required to raise T, , and retum to baseline temperature. AU animals subjected to
heat shock were anesthetized with sodium pentobarbital (30 mg-kg-' ip), and baseline rectal
temperature (T,) was recorded. Animals subjected to total body heat stress were
subsequently placed on a heating pad that consisted of a Fisher Standard Isoelectric
Focusing System and Control Unit (Fisher Scientific, Nepean. Ontario, Canada) lined with
bench coat. The surface of the heating pad was maintained at 50" C until T, was within
Figure 1: Schematic illustration of methods
male Sprague-Dawley rats divided into 5 groups, n=5 per group
1 sodium salicylate injection1 400 mg-kge1, ip) P
I hour
I ltransfer of animal to heating pad 'I for I . 15 min heat . shock of: I
24 hour recovery
I isolation and perfusion of heart in Langendorff model I
I heart biopsy I
separation of proteins by one-dimensional SDS-PAGE
Western blot analysis of HSP 72/HSC 73 content
OS°C of the desired value for the 15 min heat shock. The pad temperature was then
adjusted to maintain a constant core temperature for 15 rnin. At the conclusion of the 15
min period, pad temperature was lowered to 20° C, and animal T, was allowed to return to
baseline. In the case of the mildly heat-shocked animals, and those mildly heat-shocked in
combination with sodium salicylate treatment, T, was raised to approximately 40°C.
Animals subjected to a severe heat shock had T, raised to approximately 42OC. In all cases,
rectal temperatures were maintained for 15 min, and subsequently returned to previously
determined baseline values. Two minutes prior, and throughout the entire heat stress (total
time rectal'temperature was above resting value), rectal temperature was measured
continuously using a Thermistor TSD 102C Tube Robe Transmistor (manufacturer reports
accuracy of M.0002"C) connected to a Biopac data acquisition system. Prior to each
individual heat shock, the Biopac software was calibrated for temperature by assigning
specific voltage values to two fixed temperatures within the physiological range observed in
the rat, 96OF and 107OF. A standard mercury thermometer (gradings of 0.2OF), and the
Tube Probe Transmistor were both immersed in a beaker of water. At watex temperatures
of 96OF, and 1079 (determined by visual inspection of the thermometer), the Thermistor
registered a specific voltage. This established a scale for the Biopac software to accurately
translate ensuing voltage measures into rectal temperatures. Following sodium dcylate
and/or heat shckk treatment, all animals were returned to their cages and allowed to recover
for 24 h. Twenty four hours after heat shock and/or sodium salicylate treatment, rats were
anesthetized with sodium pentobarbital (65 mg-kg-' ip) and injected with 1000 U of heparin
(Hepalean; Organon Teknika, Toronto, Ontario, Canada) via the tail vein, 10 min prior to
removal of the heart. Animals were sacrifiiced in a random manner. This ensured that no
group received emphasis at any point during experimentation, and furthermore, no group
was over-represented in a specific batch of animals.
2.2 Isolated heart preparation
2.2.1 Langendorff technique
Various Langendorff isolated and perfused heart systems exist and have been
described elsewhere (Langendofl, 1895; Neely et al., 1967; Neely et al., 1973); a constant
pressure, non-recirculating model was used for the present experiments (figure 2). This is
a retrograde perfusion method, i.e., the perfusate flows down the aorta, and not through
the left ventricle and out the aorta as blood does in v iva The inner chamber of a 2 L
water-jacketed reservoir (Radnoti Glass Technology, Inc., Monrovia, California, USA),
equipped with an oxygenating bubbler, was connected by 114 x 3/32 tygon tubing (Norton,
Akron, Ohio) to a three-way luer valve attached to a cannula hanging above a height-
adjustable heart chamber (Radnoti Glass Technology, Inc., Monrovia, California, USA).
A separate tygon tubing system (3/16 x 1/16) linked the water jacketed portions of the
buffer reservoir and the heart chamber with a pump (VWR 1 1 10; Preston Ind., Inc., Niles,
Illinois, USA) fixed to a 3 L pail. The pump warmed the water in the pail to 37°C and
ensured flow through the chamber outer jackets, thus maintaining the buffer at 37°C.
2.2.2 Langendorff preparation
Prior to each experiment, a Krebs-Henseleit buffer solution (Sigma Chemical
Company, Mississauga, Ontario, Canada) containing 4.7 mM KCI, 1.2 mM KH,PO,, 1.2
m M Mg,SO,, 118 mM NaCl, and 11 mM glucose, was freshly prepared. In order to
maintain ionic; and pH balance for proper function of the isolated heart, 2.0 mM CaCI, and
25 rnM NaHCO, were added to the Krebs-Henseleit buffer. The modified buffer solution
was filtered through a 0.8 micron filter (Gelman Scientific, Ann Arbor, Michigan, USA).
The buffer was placed in a 2 L water-jacketed reservoir and oxygenated with a 95% 045%
C 4 mixture, and maintained at 37OC. The reservoir was fastened to a stand 45 cm above
50 mmHg perfusion pressure flo
pacing I electrodes
Krebs-Henseleit
Mac computer
Figure 2. Langendorff apparatus. Shown is a retrograde perfusion, non-recirculating model (see text for further details). Buffer flow is indicated by the arrow.
the heart, a sufkient elevation to generate 50 mmHg perfusion pressure through the
cannula, thus maintaining the isolated heart.
2.2.3 Heart preparation
Anesthetized animals were placed on a dissecting board and hearts were exposed by
median sternotomy followed by cutting and retraction of the rib cage. The heart was
rapidly excised with a single cut across the arch of the aorta and the vena cava, and
immediately immersed in an icecold saline bath (0.9% NaCl). The heart was carefully
lifted from the saline using a pair of curved forceps, and the aorta was fitted on a cannula
suspended from a stand. The heart was secured to the cannula by tying the aorta to the
cannula using surgical thread. Excess tissue (lungs, fat) was removed. The heart was
gently flushed with ice cold saline from a 10 ml syringe attached to the opposite end of the
cannula. The cannulated heart was disconnected fkom the syringe and transferred to the
Langendorff apparatus. Immediately, perfusion of the heart was initiated. A water-fded,
balloon-tipped catheter (size 4; Radnoti Glass Technology, Inc., Monrovia, California,
USA) was inserted through the left atrium, into the left ventricle, and inflated to a volume
of 50 pl. Once inserted, hernodynamic measurements were started using MP 100
software. The pressure developed by each left ventricular contraction was measured
through ball- compression, and the resulting signal translated to the Biopac system by a
pressure transducer (COBE Labs Inc., Lakewood, Colorado, USA). Prior to each isolated
heart preparation, the Biopac software was calibrated for pressure by assigning a specific
voltage to two hxed pressure values within the range of left ventricular developed pressure
values (LVDP) observed in the isolated rat heart; 60 mmHg, and 180 mmHg. The rubber
tubing at the cuff end of an aneroid sphygmomanometer (gradings of 2 mmHg; Mabis
Healthcare Inc., Lake Forest, Illinois, USA) was cut, and tightly fitted over the pressure
transducer. The bulb of the shygmommometer was compressed to attain the desired
pressures of 60 m d g , and 180 mmHg (determined by visual inspection of the
shygmomanometer needle). At each pressure value, the tranducer registered a specific
voltage. This established a scale for the Biopac software to accurately translate ensuing
voltage measures into pressure readings. Rates of contraction and relaxation (kdP-dt 'I)
were calculated as the first derivative of the curve depicting left ventricular developed
pressure (LVDP), over time. Coronary flow, or the flow of perfusing buffer prior to
entering the cannulated heart was measured using a GiImont Instruments shielded
flowmeter. Flow values were manually recorded. Hearts were electrically paced at 320
beatsaid using plunge electrodes originating from an output channel on the Biopac
sys tern.
2.3.4 Langendorff protocol
The following protocol was used for the heart experiments: after a 45 min
equilibration period, the hearts were subjected to 45 min of complete, warm (37*C), global
ischemia by halting coronary perfusion and electrical pacing. This was achieved by closing
the 3-way luer valve above the heart cannula, further pinching off the tygon tubing with a
clip, and finally disconnecting one of the electrodes from the heart. Following 45 min of
global ischemia at 37OC, flow and pacing were restored, and the heart reperfused for 30
min. Data were recorded continuously throughout the protocol using the Biopac system
and Acknowledge MP lOO software. Hernodynamic indices (LVDP, *dP-dt - I , coronary
flow) were evaluated 5 min prior to ischemia, and at 0, 5, 10, 15, 20, 25, and 30 min of
reperfhion. Following reperfusion, the hearts were tdmmed of excess tissue (atria, great
vessels), and fiozen at -80°C.
2.4 Protein determination
Frozen portions near the apex of the left ventricle (40-60 mg) were placed in 13 x
100 mm disposable test tubes (Fisherbrand; Fisher Scientific, Nepean, Ontario, Canada)
containing 15 volumes of 600 mM NaCI, and 15 mM Tris (pH 7.3, and homogenized at
19
4OC using an Ultra-Turrax T8 grinder @(A Labortechnik, S taufen, Germany). Protein
concentrations were determined by the method of Lowry et al. (195 I), using bovine senun
albumin (BSA) as a standard. Five pL, of sample homogenate were added to 495 pL of
ddH,O in 13 x 100 mm tubes set-up in triplicate. Five ml of Lowry reagent (2 mVlOO ml
of 2% W/V CuS04-H20, 2 mVlOO ml of 4% w/v sodium tartrate in 96 ml of 3% w/v
NqCO, made in 100 mM NaOH) were added, vortexed, and allowed to react for a
minimum of 15 min. After 0.5 ml of phenol reagent (Anachemia, Mississauga, Ontario,
Canada) diluted 1:2 with d-0 was added,
for a minimum of 30 min. Absorbance was
samples were vortexed
measured at 660 nm in
and allowed to react
a Turner model 340
Spectrophotometer. A standard curve consisting of 10, 20, 40, 60, 80, and 100 pg of
BSA was constructed, and sample protein concentrations determined using a linear
regression equation.
2.5 One-dimensional separation of proteins
One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) was conducted according to the method described by L a e d (1970), using
a Bio-Rad mini-protean I1 gel electrophoresis system (Bio-Rad Laboratories, Mississauga,
Ontario, Canada). SDS-PAGE consisted of a 5- 15% polyacrylamide gradient separating
gel, and a 3% stacking gel. The glass plates were cleaned with 70% ethanol, and
assembled with 1.5 mm Teflon spacers in gel moulds, according to the manufacturer's
instructions. The separating gel was poured from equal amounts of 5% and 15%
acrylamide mixtures. A dualchambered mixer was placed on a stir plate, and tubing run
from the proximal chamber to the gel mould. The stopcock separating the two chambers
was initially closed, and the 5% mixture was added to the distal chamber. The stopcock
was opened very briefly to evacuate air between the two chambers. A small stir-bar was
added to the proximal chamber, followed by the 15% mixture, which immediately began
running into the gel mould. When the bottom of the mould was covered by the 15%
acrylamide, the stopcock was opened fully, and the acrylamide solutions mixed and drained
into the mould. When the pouring was complete, the separating gel was overlayed with
40-saturated butanol and allowed to polymerize for 30-45 min. The butanol was then
carefully rinsed off, a 10 well 1.5 mm Teflon comb was placed between the glass plates,
and a 3% stacking gel (pH 6.8) overlayed and allowed to polymerize for 30-45 min.
Protein samples were placed in 600 pl microtubes containing an equal volume of sample
buffer (0.5 M Tris (pH 6.8). 10% glycerol, 10% 2-f!-mercaptoethanol, 4% SDS, 0.05%
bromophenol blue), vortexed, and loaded into wells. SDS running buffer (192 mM
glycine, 25 mM Tris-Cl (pH 8.3), 0.1% SDS) was carefully added, and samples were
initially electrophoresed for 30 min at 70V, approximately until the loading dye front had
reached the separating gel, at which point the voltage was increased to 110V until the dye
front had reached the bottom. Human purified HSP 70, or bovine HSC 73 (product
#SPP-750, Stress-Gen, Victoria, British Columbia, Canada), were coelectrophoresed.
2.6 Protein transfer and immunoblotting
2.6.1 HSP 72
Following eelectrophoretic separation, proteins were transferred to nitrocellulose
membranes (0:22 jm thick, Bio-Rad Laboratories), as described by Towbin et al. (l979),
using the Bio-Rad mini-protean II gel transfer system. The gels were equilibrated in
transfer buffer (192 mM glycine, 25 mM Tris-C1 (pH 8.3), 0.1 % SDS and 20 % methanol)
for 10 min, removed and placed into a sandwich consisting of a Brillo pad, 3 pieces of
filter paper (Fisher Scientific, Nepean, Ontario, Canada), the nitrocellulose membrane, the
gel, three more pieces of filter paper, and a second Brillo pad. AU components of the
sandwich were immersed in transfer buffer prior to, and during, assembly. Two gel
sandwiches and an ice pack were placed in the gel transfer system. The proteins were
transferred to the nitrocellulose membrane at a constant 40 V for 4 h, with an ice pack
change at 2 h. Following protein transfer, the nitrocellulose membrane was blocked with
5% non-fat dried milk powder (NFDM) in Tris-buffered saline (TBS; 500 mM NaCl,
20mM Tris-C1 (pH 7.5)) for one hour, after which blots were washed twice, for 5 min
each time, in TTBS (TBS with 0.05 % Tween-20). The gels were stained to verify that
complete protein transfer had occurred. The blots were incubated overnight with a
polyclonal antibody 799 (1:2000 dilution in TTBS with 2% NFDM; a generous g& from
R.M. Tanguay, Lava1 University, Ste.-Foy, Quebec) specific for HSP 72. Following two
5 min washes in TTBS, the blots were immersed for 1 h in a solution of goat-anti-rabbit
IgG conjugated to alkaline phosphatase secondary antibody (BioRad, 1: 1000 dilution in
TI'BS with 2% NFDM). The blots were washed twice in ' I T B S , and once in TBS for 5
min each time, and immersed in a bicarbonate buffer (100mM Na&O,, 1 mM MgCl, (pH
9.8)) containing 3% w/v pnitro-blue-tetrazolium chloride ptoluidine salt in 70% N,N-
dimethyl-fonnamide (Dm and 1.5% wlv 5-bromo4chloro-3-indolyl phosphate in 100%
DMF. After development, blots were washed in d W 0 and allowed to dry. Immunoblots
were scanned using an AGFA Arcus 2 scanner, and HSP bands on the image were
quantified using Kodak 1D 1.0 image analysis software. Standard curves were constructed
to assure Linearity. The lane assignment for all blots run, whether showing HSP 72, or
HSC 73, was identical. On all blots, a control sample was run on the left most lane.
Proceeding rightward, in successive adjacent lanes, samples were run from the sodium 3
salicylate group, the mild heat shock group, the mild heat shock plus sodium salicylate
group, and the severe heat shock group. On all blots, a common sample was run, in order
to allow for comparison of quantified bands between blots. Bands representing left
ventricular HSP 72 content for all animals were run on a total of 4 blots, each of which was
subsequently quantified. Each experiment was repeated twice.
2.6.2 HSC 73
The protocol for analysis of HSC 73 (the HSP 70 cognate isoform) foilowing
protein transfer was identical to the one described above for HSP 72, with the following
exception. Analysis of HSC 73 was performed by incubating the blots for 3 hours with an
AP-conjugated monoclonal antibody specific for HSC 73 (1:5000 dilution in ' I T B S with
2% blotto; catalog #SPA-8 ISAP, Stress-Gen, Victoria, British Columbia, Canada). Thus,
following incubation with this antibody, the blots were simply washed twice in 'ITBS, and
once in TBS for 5 min each time, and developed in the bicarbonate buffer, as previously
described for HSP 72.
2.7 Statistical analysis
InStat 2.01 was used to analyze all data. For all heat shock and hernodynamic
variables (see appendices p. 63 for a list of alI variables analyzed), a one-way ANOVA
was performed, followed by a Tukey's post-hoc test to determine where s i m c a n t
differences (pc0.05) existed between the twatment groups. Statistical analysis was
performed using data points obtained fiom all 25 animals, i.e., five treatment groups where
n=5 per group.(refer to appendices, p. 63, for all individual measurements).
Chapter 3
3.1 Heat Shock
Resting rectal temperatures CT, ) during unstressed conditions for animals in the
three heat shock treatment groups [mild heat shock (40°C), mild heat shock plus sodium
salicylate (40°C and 400 mg-kg-'), and severe heat shock (42OC)], prior to the 15 min heat
shock, were 37.0+0.3OC, 36.8M.1°C, and 36.9rt0.4OC, respectively (figure 3). Tr was
not significantly different between groups.
The total heat stress duration (total time rectal temperature was above resting value)
was not significantly different between mildly heat-shocked animals, and those subjected to
a mild heat shock in combination with sodium salicylate treatment (44.4fi.3 vs 45.5s. 1
min, figure 4). Conversely, animals subjected to a severe heat shock required an average
of 65.3f 1.8 min to raise Tr to 42°C for 15 min, and return T, to baseline. This was (by
design) a sigaiFicady longer heat stress period than that experienced by the two milder heat
shock groups @cO.OOl).
The average Tr of mildly heat-shocked animals, and mildly heat-shocked animals 7
treated with sodium salicylate, was not significantly different during the entire heat stress
(39.1f0.17 vs 39.W.O7OC, figure 5), and during the 15 rnin heat shock period
(40.lf0.02 vs 40. 1+0.02"C, figure 6). As expected, average Tr during the entire heat
stress (figure 5), and during the 15 min heat shock (figure 6), was significantly higher for
animals subjected to severe heat shock than for animals subjected to mild heat shock, or
mild heat shock plus sodium salicylate treatment (p4.001).
mild mild severe heat heat heat
shock shock shock PIUS
sodium salicy late
Figure 3. Re-heat stress (baseline) rectal temperatures are similar between groups. No significant differences in baseline T, were detected among the three groups. Data are expressed as meansfSE, n=5 per group.
Figure 4. Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, experienced heat shocks of similar duration. Shown is the time required for animals from each group to reach desired heat shock T,, then return to pre-heat stress baseline Tr. Mildly heat shocked animals, and midly heat shocked animals treated with sodium salicylate, required a similar amount of time to reach 40°C and return to baseline. Predictably, severely heat shocked animals required significantly more time to reach 42"C, and return to baseline. *p<0.001 compared to mild heat shock and mild heat shock plus sodium salicylate groups. Data are expressed as meansSE, n=5 per g r o w
mild mild severe heat heat heat
shodc shock shock P I ~
sodium saiicy Iate
Figure 5.' Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, showed similar average T, during entire heat stress. The severely heat shocked animals had a significantly higher average T, than animals from the other two groups for the total heat stress period, which includes the time to ascend and descend from the 15 min heat shock plateau. *p<0.001 compared with mild heat shock and miId heat shock plus sodium salicylate groups. Data are expressed as meansSE, n=5 per group.
mild mild severe heat heat heat shock shock shock
elus sodium salicy late
Figure 6. Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, showed similar average T, during 15 min heat shock. Tr during the 15 minute heat shock was controlled using the isoelectric pad (see text). Thus, animals that were severely heat shocked maintained T, very close to 42OC, while Tr of animals that were mildly heat shocked, and those that were mildly heat shocked plus sodium salicylate treated, slightly surpassed 40°C, as desired. *p<0.001 compared with mild heat shock and mild heat shock plus sodium salicylate groups. Data are expressed as meanskSE, n=5 per group.
Animais subjected to a mild heat shock, and animals subjected to a mild heat shock
plus sodium salicylate treatment, reached a similar peak T, during the heat stress
(40.=.04 vs. 40.3M.02, respectively; figure 7). The peak T, of animals subjected to a
severe heat shock was significantly greater than the peak T of animals in all other groups
(pc0.00 1 ) . Thus, mildly heat-shocked animals, and mildly heat-s hocked plus sodium
salicylate treated animals, were subjected to very similar heat stresses, while severely heat-
shocked animals were subjected to a greater heat stress.
3.2 Coronary flow
The pre-ischemic absolute value for coronary flow (of perfusion buffer) in hearts of
unstressed animals was 6.2M.6 ml-mid (table 1). Prior to ischemia, hearts from
salicylate treated animals had a coronary flow of 5 .M. 2 ml- mid'. Re-ischemic flow in
hearts of mildly heat-shocked animals was 6. 1f 1.1 rnl-mid, while in hearts from animals
that were mildly heat-shocked plus sodium salicylate treated, flow was 5% 1.1 ml-min-' .
Finally, in hearts from animals subjected to severe heat shock, coronary flow was
5.8H.5 ml-mh-'. No simcant differences were detected between groups.
To allow for comparisons between the five groups, the post-ischemic recovery of
coronary flow during the 30 min reperfusion period was expressed as a percentage of
absolute pre-ischemic values (figure 8). Following this data normalization, hearts from
unstressed (control) animals recovered 78.3k5.65 of pre-ischemic coronary flow at 5 min
of reperfusion. The recovery of coronary flow in hearts from unstressed animals did not
change ~ i ~ c a n t l y during the remainder of reperfision.
Although there were no statistically sigruficant differences between groups, hearts
from the control, sodium salicylate, and mild heat shock plus sodium salicylate treated
animals regained less of their pre-ischemic flow than hearts from animals subjected to
severe heat shock, or hearts from animals subjected to mild heat shock. This trend was
mild mild severe heat heat heat
shock shock shock plus
sodium saiicylate
Figure 7. Mildly heat shocked, mildly heat shocked plus sodium salicylate treated animals, showed similar peak T, during heat stress. Shown is the peak Tr reached by each group, at some point during the 15 min heat shock. Animals that were severely heat shocked reached a significantly higher peak T, than mildly heat shocked, or mildly heat shocked plus sodium salicylate treated counterparts (*p<0.001). Data are expressed as meansfSE, n=5 per group.
unstressed (controi)
sodium salicylate
mild heat shock
d d heat
I shock plus sodium salicylate
severe heat 17
pre-ischemic rate of contraction I relaxation (+dP*dt 'I. (-dP-dt *I; ~ ~ H ~ - s - & s E ) ~ ~ H ~ - s - ~ * s E )
pre-ischemic left venbricular developed pressure amp; r n & S E )
Table 1. he-ischemic absolute values for hernodynamic variables are shown. Values were collected 5 min prior to ischemia Data are expressed as meanskSE.
10 io 3b L
reperfusion time (min)
control
q* sodium salicylate
,+ mild heat shock
mild heat shock ,+ plussodium
salicy late
-- severe heat shock
Figure 8. Post-ischemic recovery of coronary flow is unchanged by heat, or heat and sodium salicylate treatment. No significant differences were detected between groups. Data are expressed as a percentage of absolute pre-ischemic d u e s (meanskSE, n=5 per group).
most apparent after 10 min, and continued throughout the remainder of reperfusion. After
only 5 min of reperfusion, hearts fYom animals subjected to severe heat shock recovered
87.5&4.9% of pre-ischemic flow, and hearts from animals subjected to mild heat shock
recovered 83.2+4.7% of pre-ischemic flow values. After 5 min of reperfusion, hearts
from control, sodium salicylate, and mild heat shock plus sodium salicylate treated animals
recovered 78.3k5.68, 80.5&4.2%, and 74.7+4.5% of pre-ischemic flow, respectively.
Thus, at 5 min of reperfusion, no trend in recovery was immediately discernable, and there
was no indication of possible differences in degree of recovery. .However, after 10 min of
reperfusion, a trend was observed in the difference in flow recovery between groups. At
10 min, hearts from animals subjected to severe heat shock, and hearts from animals
subjected to mild heat shock, recovered over 80% of pre-ischemic coronary flow
(86.(W4.9% and 8 2.4k6.5, respectively). Conversely, hearts fkom unstressed (control)
animals, sodium salicylate treated animals, and mildly heat-shocked plus sodium salicylate
treated animals recovered less than 80% of pre-ischemic coronary flow (74.2H .3,
75.2B.3 and 74.4&4.7%, respectively). In all groups, there was fluctuation in the
percentage of absolute pre-ischemic coronary flow recovered during the reperfusion period.
Nevertheless, hearts from animals subjected to severe heat shock, and hearts from animals
subjected to mild heat shock, showed a trend towards a greater recovery of coronary flow
than hearts fkom all other groups.
In summary, the combinations of heat stress and/or sodium salicylate treatment
used here do not appear to confer any simcant advantage in a heart's ability to recover
coronary flow following ischemia.
3.3 Rate of contraction and relaxation
3.3.1 Rate of contraction
The pre-ischemic absolute value for rate of contraction, as indicated by +dP-dt -', in
hearts of unstressed animals was 1848k50.8 &gd (table 1). Prior to ischemia, hearts
fiom salicylate treated animals had a rate of contraction of 17793A40.2 mrnHg=s-'. h e -
ischemic +dP-dt in hearts of mildly heat-shocked animals was 1767S3.0 mm~g-s- ' ,
while in hearts fiom animals that were mildly heat-shocked plus sodium salicylate treated,
+dP-dt was 1498k109.3 mmHgd. Finally, in hearts from animals that were subjected
to severe heat shock, the pre-ischemic rate of contraction was 1637H40.9 rn~nHg-s-I. No
sigmficant differences were detected between groups.
When the post-ischemic recovery of +dP-dt -' was expressed as a percentage of
their respective absolute pre-ischemic values, hearts fiom control animals recovered
30.3&4.2% of their pre-ischemic +dl?& -' a . r 5 minutes of reperfusion (figure 9).
Thereafter, the recovery of +dP-dt -' in heats from control animals increased in a linear
manner. At 30 min of reperfusion, the recovery of rate of contraction in control hearts
reached 48.3&9.0% of pre-ischemic values.
As previously mentioned, hearts from unstressed (control) animals recovered
30.3&4.2% of pre-ischemic values at 5 min of reperfusion, while hearts from sodium
salicylate treated animals recovered 23.4&4.4%. Hearts from mildly heat-shocked animals
recovered 3 1 .=. 1 %, while those from mildly heat-shocked plus sodium salicylate treated
animals recovered 35.5s. 1%. Hearts from animals subjected to severe heat shock
recovered 24.5k4.295 of pre-ischemic +dP-dt -' values. At 5 min of reperfusion, there
were no significant differences in the recovery of +dP& " between groups
After 10 min, and throughout the remainder of reperfusion, differences were
observed in the recovery of +dP& between groups. Hearts from severely heat-shocked
animals, and those mildly heat-shocked plus sodium salicylate treated, recovered +dl?-dt '' to a greater extent than all other groups. This appeared to be the result of a more rapid rate
of +dP-dt " recovery. By 10 min, hearts fiom animals subjected to a severe heat shock
reached 54.7+10.3% of pre-ischemic +dP-dr -', more than twice the value observed at 5
min of repefision (24.5k4.2). In addition, hearts fiom mildly heat-shocked plus sodium
salicylate treated animals increased recovery of +dP-dt -' from 35.5&5.1% at 5 min, to
10 20 30
reperfusion time (min)
control
sodium salicylate
mild heat shock
miId heat shock plus sodium salicytate
severe heat shock
Figure 9. A 42°C heat shock, and a 40°C heat shock lus sodium salicylate enhanced ! post-ischemic recovery of rate of contraction (+dP-dr - ). Compared to hearts from unstressed (control) animals, hearts from severely heat shocked animals recovered a significantly greater percentage of re-ischemic +dP-dt -l (*p<0.05 after 15 rnin reperfusion). At 30 min of r e p e d i o n , +dP& was significantly greater in hearts from mildly heat shocked animals treated with sodium salicylate, than hearts from unstressed (control) animals (+pc0.05). Data are expressed as means+SE, n=5 per group.
50.5+9.3% at 10 min. Nonetheless, at LO min of reperfusion, the only si@cant
difference in recovery of +dP.dt " observed between groups was between hearts of animals
subjected to severe heat shock, which had recovered 54.7f 10.3% of +dP.dt 'I, and those
of sodium salicylate treated animals, which had recovered only 2 1.4--8% @<0.05).
Compared to all other groups at 10 min of reperfusion, the recovery of +dP-dt -' in hearts
fiom severely heat-shocked animals was not siplicantly different. Hearts from animals
that were mildly heat-shocked, mildly heat-shocked plus sodium salicylate treated, and
severely heat-shocked, showed a trend of an increasing recovery of +dP-dt throughout
reperfusion. In contrast, hearts fiom both unstressed, and salicylate treated animala
actually showed a decrease in recovery of idP-dt -' between minutes 5 and 10.
Subsequently, both groups exhibited continuing increases in recovery of +dP-dt " . Thus,
at 10 min of reperfusion, the significant difference observed in restoration of rate of
contraction between hearts fiom severely heat-shocked animals, and hearts from sodium
salicylate treated animals, was the result of a large increase in +dP& -' recovery in the
former group, and a small decrease in +dP-dt -' recovery in the latter group, between 5 and
10 minutes of reperfusion.
At 15 min of reperfusion, the recovery of rate of contraction in hearts from animals
subjected to severe heat shock reached 72Sf 10.9% of pre-ischemic values, and was
significantly greater than that observed in hearts of sodium salicylate treated animals
(23.6k3.3%, p<O.01), and also that observed in hearts from unstressed (control) animals
(39.1&6.5%, p<0.05). In addition, the recovery of +dP-dr for hearts from mildly heat-
shocked plus sodium salicylate treated animals was significantly greater than observed for
hearts from sodium salicylate treated animals (58.5f9.4% vs. 23.6*3.3%, pc0.05).
Hearts fiom animals in all groups increased their recovery of +dP/dt throughout the
remainder of reperfusion, and the significant differences were consistent for the rest of the
30 min period. In addition, +dP-dt -' recovery was sigruficantly greater in hearts from
severely heat-shocked animals compared to hearts from mildly heat-shocked animals, at 20
rnin of reperfbsion and thereafter (p4.05). Furthermore, after 30 min of reperhion,
hearts £iom mildly heat-shocked plus sodium salicylate treated animals, recovered a
significantly greater percentage of their pre-ischemic absolute +dP-dt - I , than hearts fkom
unstressed animals (75.8M.5% vs. 48.3B.0%, p<0.05). This difference was not
observed when comparing +dP& recovery of hearts fiom mildly heat-shocked animals,
to +dP& " recovery of hearts fiom unstressed animals.
A heat shock of 42OC for 15 min is capable of conferring myocardial protection, in
terms of recovery of rate of contraction, following an ischemic insult (figure 9). A milder
heat shock of 40°C for 15 min, in combination with sodium salicylate treatment,
significantly kproves +dP& -' recovery when compared to unstressed controls, but this is
only evident after 30 min of reperfusion. Conversely, a heat shock of 40°C for 15 min, in
the absence of sodium salicylate treatment, does not significantly improve +dP-dt -' recovery, compared to unstressed controls.
3.3.2 Rate of relaxation
The pre-ischemic absolute value for rate of relaxation, as indicated by -dP-dt -I, in
hearts of unstressed animals was 1162+68.8 mmHg-s" (table 1). Prior to ischemia, hearts
from salicylate treated animals had a rate of relaxation of 983k107.4 mm~g-s-' . Pre-
ischemic - d ~ & -' in hearts of mildly heat-shocked animals was lOO3H 1.4 mmHg-s",
while in hearts fkom animals that were mildly heat-shocked plus sodium salicylate treated,
-dP-dt -' was 91 1B7.6 mmHg-s-l. Finally, in hearts from animals that were subjected to
severe heat shock, the pre-ischemic absolute rate of relaxation was 1034k86.7 mmHgd.
No significant differences were detected between groups.
When the post-ischemic recovery of -dP& -' was expressed as a percentage of their
respective absolute pre-ischemic values, hearts from unstressed (control) animals recovered
35.6&5.0% of their pre-ischemic -dl?-dt '' after 5 min of reperfusion (figure 10).
Thereafter, hearts fkom unstressed animals recovered progressively more of their pre-
+ control
.I_Q1_ mild heat shock
mild heat shock plussodium salicylate * severe heat shock
reperfusion time (min)
Figure 10. A 42°C heat shock, and a 40°C heat shock plus sodium salicylate enhanced post-ischemic recovery of rate of relaxation (-dPdt -1). Compared to hearts fiom unstressed (controls) animals, hearts fiom severely heat shocked animals recovered a significantly greater percentage of pre-ischemic -dP& - I (*p<0.05 after 20 min reperfusion). At 30 rnin of reperfusion, -dP.dt -'is significantly greater in hearts from mildly heat shocked animals treated with sodium salicylate, than in hearts from unstressed (control) animals (+p<0.05). Data are expressed as meansfSE, n=5 per group.
ischemic -dP-dt -I. Following the 30 min reperfusion period, these hearts had recovered
53.4B.396 of their pre-ischemic -dP-dt -'. Considering all groups as a whole, the
recovery of rate of contraction differed somewhat fiom the recovery of rate of relaxation.
For example, at 5 min of reperfusion, no statistically significant differences existed
between groups in terms of both -dP& " and +dP-dr -'. After 10 min of reperfusion, there
was still no significant difference between groups in terms of recovery of rate of relaxation.
In contrast, at 10 min, the hearts from animals subjected to severe heat shock recovered
sipdicantly more of their pre-ischemic rate of contraction than hearts from sodium
salicylate treated animals. At 15 min of reperfusion, hearts fiom both severely heat-
shocked animals, and mildly heat-shocked plus sodium salicylate treated animals had
recovered significantly more of their pre-ischemic -dP.dt -' than hearts from sodium
salicylate treated animals (77.M 12.5% and 68 Ski 0.1 %, respectively vs. 3 1 .w .4%;
p<O.01 and p<0.05, respectively). This difference appears to be attributabIe to a slower
rate of recovery of -dP-dt -' in the hearts of sodium salicylate treated animals compared to
all other groups. Similarly, hearts from unstressed animals also showed a slower rate of
recovery during the middle portion of reperfusion. Thus, at 20 min, -dP.dt " recovery of
hearts fiom animals subjected to severe heat shock was significantly greater than that of
hearts from unstressed (control) animals. In the last 10 min of reperfusion, hearts fiom all
groups progressively increased their recovery of -dP-dt ". This was particularly evident in
hearts from sodium salicylate treated animals. As a result, at 25 and 30 min of reperfision,
there was no longer a significant difference in rate of relaxation recovery between hearts
from animals subjected to mild heat shock plus sodium salicylate treatment, and those fiom
animals only treated with sodium salicylate. Furthermore, at 30 min of reperfusion, there
was no sigruticant difference in -dP-dt -' recovery between hearts from severely heat-
shocked animals, and hearts from sodium salicylate treated animals. At the end of the 30
min reperfusion period, hearts fiom animals subjected to a severe heat shock, and those
fiom animals subjected to a mild heat shock plus sodium salicylate treatment, had recovered
a siacantly greater percentage of their pre-ischemic -dP-dt " than hearts fiom unstressed
animals (87.8k7.61 and 86.2+7.6%, respectively, vs. 53.4&9.3%, p<0.05).
A heat shock of 42°C for 15 min is capable of conferring myocardial protection, in
terms of recovery of rate of relaxation, following an ischemic insult (figure 10). A milder
heat shock of 40°C for 15 min, in combination with sodium salicylate treatment, also
significantly improves -dP-dt -' recovery, but this was only observed at the conclusion of
the 30 min reperfusion period. Conversely, a heat shock of W C for 15 min, without
sodium salicyate treatment, does not significantly improve -dP-dr " recovery, compared to
unstressed controls.
3.4 Left ventricular developed pressure
The pre-ischemic absolute value for left ventricular developed pressure (LVDP) in
hearts fkom unstressed animals was 85.7kl.7 mmHg (table 1). Prior to ischemia, hearts
from salicylate treated animals had an LVDP of 78.1S.2 mrnHg. Pre-ischemic LVDP in
hearts of mildly heat-shocked animals was 77.632.2 mmHg, while in hearts from animals
that were mildly heat-shocked plus sodium salicylate treated, LVDP was 67 .=.O rnmHg.
Finally, in hearts from animals that were subjected to severe heat shock, the pre-ischemic
absolute left ventricular developed pressure was 7426 .4 mrn Hg. No significant
differences were detected between groups.
When the post-ischemic recovery of LVDP was expressed as a percentage of their
respective absolute pre-ischemic values, hearts fkom unstressed (control) animals recovered
33.W4.7% of their pre-ischemic LVDP after 5 minutes of reperfusion (figure 1 1). After
10 min, recovery of left ventricular developed pressure had increased to 42.%7.2%. In
the middle portion of the reperfusion period, from 10 min to 20 min, the restoration of
LVDP plateaued (43.5+6.9% LVDP recovery at 20 min). Thereafter, these hearts showed
an increased recovery of pre-ischemic LVDP values. However, after 30 rnin of
control
* sodium salicylate - mild heat shock
mild heat shock --m- plus sodium
salicylate * severe heat shock
reperfusion time (min)
Figure 11. A 42OC heat shock enhanced post-ischemic recovery of left ventricular developed pressure (LVDP). In comparison to controls, recovery of LVDP is significantly greater in hearts from severely heat shocked animals after 15 min of reperfusion (*p<0.01). Hearts from animals in the mild heat shock plus sodium salicylate group did not exhibit significantly greater restoration of LVDP relative to hearts from unstressed (control) animals. Data are expressed as a percentage of absolute pre-ischemic d u e s (meanskSE, n=5 per group).
reperfusion, hearts fkom unstressed animals had regained only about half of their pre-
ischemic LVDP (5O.P+lO. 1 %). As previously mentioned, after 5 min of reperhsion,
hearts from unstressed animals recovered 33.01t4.795 of pre-ischemic LVDP, while hearts
from sodium salicylate treated animals, d d l y heat-shocked animals, mildly heat-shocked
plus sodium salicylate treated animals, and severely heat-shocked animals recovered
26.824.0%, 36.4rU.2, 39.1fi.4, and 25.W4.0 of their pre-ischemic LVDP, respectively.
At 5 min of reperfusion there were no significant differences in LVDP recovery between
groups. However, at 10 min of reperfusion, hearts fkom severely heat-shocked animals
recovered a significantly greater amount of pre-ischemic LVDP than those fiom sodium
salicylate treated animals (59.1H.6 vs. 23 .= .6%, p<0.05). The differences between
these two groups continued throughout the 30 minute reperfusion period. At 10 min of
reperfusion, the hearts of mildly heat-shocked plus sodium salicylate treated animals
recovered a similar percentage of LVDP when compared to hearts of severely heat-shocked
counterparts (53.2393% vs. 59.1B.6). However, recovery of LMlP in the hearts from
mildly heat-shocked plus sodium salicylate treated animals was not significantly different
from any other group at 10 min. Nevertheless, at this point and for the remainder of the
reperfusion period, a trend was observed where hearts fiom animals subjected to a mild
heat shock plus sodium salicylate treatment, recovered LVDP to a greater extent than hearts
fiom mildly heat-shocked animals, but to a lesser extent than hearts from severely heat-
shocked animals. After 15 min of reperfusion, the hearts fiom animals subjected to a
severe heat shock recovered LVDP to a significantly greater extent than hearts from
unstressed animals, this continued for the remainder of reperfusion, such that at 30 min,
values were 94.5k4.695 vs. 50.9&10.1% (pc0.05). In addition, the percentage of LVDP
recovered at 15 min of reperfusion was significantly greater in hearts from mildly heat-
shocked plus sodium salicylate treated animals than in hearts from animals treated only with
sodium salicylate (64.5k10.4 vs. 26.3&3.6%, p ~0.05). This observation was also made
at 20 minutes, but not thereafter. This difference existed when the rate of LVDP recovery
was increasing in the hearts of mildly heat-shocked plus sodium salicylate treated animals,
but decreasing in the hearts of sodium salicylate treated animals. LVDP recovery in the
hearts of sodiuk salicylate treated animals was low immediately upon reperfusion, and by
25 rnin reached values similar to those observed in unstressed animals.
In general, the recovery of LVDP during reperfusion was greatest in the hearts fkom
animals subjected to severe heat stress, followed by hearts from mildly heat-stressed plus
sodium salicylate treated animals, then in hearts from mildly heat-stressed animals only.
However, when compared to hearts fiom unstressed animals, only hearts fkom animals
subjected to severe heat shock differed significantly in terms of LVDP recovery. These
results demonstrate a heat shock of 42°C for 15 min is capable of conferring subsequent
myocardial protection. Other treatment forms u r n in this experiment did not confer
myocardial in terms of LVDP recovery.
3.5 HSP 70 content
3.51 ESP 72
To evaluate differences in HSP 72 content, portions (40-60 mg) of the left
ventricle, near the apex, were homogenized, total protein separated by SDS polyacryIamide
gel electrophoresis, and transferred to nitrocellulose membrane, as described in materials
and methods. A representative Western blot (figure 12A) shows HSP 72 content was
detectable in f l hearts examined. When purified human HSP 70 (catalog #SPP-755;
Stress-Gen, Victoria, British Columbia, Canada) was co-electrophoresed ( h e 6 ), and
used as a standard, it co-migrated to approximately the same position as proteins from
myocardial tissue, thus confirming the specificity of the HSP 72 antibody. HSP 72 content
was barely detectable in the heats of unstressed (control) animals (lane I). Conversely,
HSP 72 content was noticeably elevated in hearts fiom animals heat-shocked to 42O C
(lane 4 ). HSP 72 content in the hearts from animals in all other groups was similar to that
observed for controls, however, some trends were detected. Visual inspection suggested
HSP 72+
Figure 12. A) HSP 72 content is increased in left ventricle following heat shock to 42OC. A representative Western blot illustrates left ventricular HSP 72 content following heat and/or sodium salicylate treatment (100 pg protein loaded per lane). Portions of the left ventricle were homogenized, total protein separated by SDS polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane, as described in materials and methods. Only the heart of the animal heat shocked to 42°C has a discernably higher HSP 72 content (lane 4 ). Lane I : control; lane 2 : sodium salicylate only (400 mgkgL); lane 3 : mild heat shock (40°C); lane 4: severe heat shock (42°C); lane 5: mild heat shock (40°C) combined with sodium salicylate (400 mg-kg-I); lane 6 : human HSP 70 standard. B) HSC 73 content in left ventricle is unchanged following heat shock and/or sodium salicylate treatment. A representative Western blot illustrates left ventricular HSC 73 content following heat andlor sodium salicylate treatment (100 pg protein loaded per lane). Portions of the left ventricle were homogenized, total protein separated by SDS polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane, as described in materials and methods. Lane I: control; lane 2 : sodium s alicylate only (400 mg-kg-'); lane 3 : mild heat shock (40°C); lane 4: severe heat shocp (42°C); lane 5: mild heat shock (40°C) combined with sodium salicylate (400 mg-kg ).
myocardial HSP 72 content of sodium salicylate treated animals was less than that of
unstressed animals (compare [mte 2 to lrme I). Conversely, myocardial HSP 72 content
from mildly heat-stressed plus sodium salicylate treated animals was greater than
myocardial HSP 72 content in unstressed animals (compare Inne 5 to h e I).
To fuaher assess left ventricular HSP 72 content, quantification of bands
representing HSP 72 fiom Western blots was performed using 1-dimensional Kodak image
analysis software. In all cases, HSP 72 content was expressed as a percentage of the mean
value determined fiom the hearts of the unstressed (control) aniqals. Hearts from animals
subjected to severe heat shock showed a significant increase in HSP 72 content compared
to hearts f?om unstressed animals (Figure 13, p<0.001). No significant differexes were
observed in HSP 72 content between hearts from animals subjected to mild heat shock and
those from animals given the combined treatment of mild heat shock plus sodium salicylate.
Although left ventricular HSP 72 content was greater in the hearts from mildly heat-
shocked plus sodium salicylate treated animals, relative to hearts from unstressed animals,
no significant difference was detected. Hearts from sodium salicylate treated animals
showed a decreased HSP 72 content in comparison to unstressed animal hearts, but again,
this difference was not statistically significant These results demonstrate that a heat shock
of 42°C for 15 min is capable of sigmficantly increasing myocardial HSP 72 content.
However, a mild heat shock of 40°C for 15 min, alone or combined with sodium salicylate,
does not increase left ventricular HSP 72 content compared to unstressed animals.
3.5.2 HSC 73 content
To evaluate differences in HSC 73 content, portions (40-60 mg) of the left
ventricle, near the apex were homogenized, total protein separated by SDS polyacrylarnide
gel electrophoresis, and transferred to nitroceUulose membrane, as described in materials
and methods. A representative Western blot (figure 12B) shows HSC 73 content was
detectable in all hearts examined. While HSP 72 content was noticeably elevated in hearts
control sodium mild mild severe salicy late heat heat heat
shock shock shock PIUS
sodium salicy late
Figure 13. Graphical representation of left ventricular HSP 72 content following ischemia-reperfusion. HSP 72 content of the lefi ventricle was quantified using 1-dimensional image analysis software. Data are expressed as a percent of control. Left ventricular HSP 72 content in animals heat shocked to 42OC is significantly elevated compared to all other groups (*p<0.001). Data are expressed as means+SE, n=5 per group.
fkom severely heat-stressed animals, visual inspection suggested that the various treatments
did not affect left ventricular KSC 73 content. Thus, hearts fkom animals in ail groups
showed similar HSC 73 content.
To fuaher assess left ventnicdar HSC 73 content, quantification of bands
representing HSC 73 from Western blots was performed using 1-dimensional Kodak
image analysis software (figure 14). In all cases, HSC 73 content was expressed as a
percentage of .values determined for hearts from unstressed (control) animals. Various (r
combinations of heat andlor salicylate treatment did not affect the accumulation HSC 73 in
the left ventricle. HSC 73 is constitutively expressed, and generally detected following
stressed, or unstressed conditions. Thus, it was not surprising to observe no sigmficant
differences in myocardial HSC 73 content between groups.
r
- control sodium mild mild
salicylate heat heat shock shock
plus sodium salicy I ate
Severe heat
shock
Figure 14. Graphical represenration of left ventricular HSC 73 content following ischemia-repefision. HSC 73 content of the left ventricle was quantified using I-dimensional image analysis software. Data are expressed as a percent of control. Left ventricular HSC 73 content is srnilar in animals from all groups. Data are expressed as meanskSE, n=5 per group.
Chapter 4
The heat shock response is universal, and has been demonstrated in many
organisms (for review, see Morirnoto et al., 1994). Cells exposed to temperature
elevations, either in vitro or in vivo, respond by rapidly synthesizing HSP 72 and other
HSPs (Ritossa, 1962; Tissieres et d., 1974; Currie et al., 1982; Li et al., 1982; Barb et
aI. 1988; Blake et al., 1990). Heat-induced accumulation of HSP 72 has been shown to
confer subsequent myocardial protection (Currie et al., 1988; Kamxizyn et al., 1990;
Hutter et ai. 1994; Lofke et al., 1995). Recent evidence has suggested that various
salicylates potentiate the heat shock response in vitro (Amici et al., 1995; Ito et al., 1996;
Burress et al., 1997). However, only one study has examined this concept in vivo.
Fawcett et al. (1997) have reported that aspirin potentiated the heat shock response in rat
liver, lung, and kidney, but in that study, the myocardium was not examined.
Furthermore, Fawcett et al. (1997) did not determine whether potentiation of the heat shock
response provided any protective effect. Thus, it was of interest to propose two novel
questions. Firstly, does salicylate have an effect on the heat-induced HSP 72 accumulation
in the myocardium? And secondly, does any such accumulation of HSP 72 provide
myocardial protection?
In the present study, neither sodium salicylate treatmat alone, nor sodium
sulicylate treatment in combination with a mild heat shock of 409C for 1.5 min, were found
to increase lefr ventricular HSP 72 content signt~cuntly in rhe animals observed (figures
12A and 13). This result differs from that reported by Fawcett et al. (1997), who
concluded that aspirin potentiated the heat shock response in vivo. Reasons for this
discrepancy are not clear, but certainly, some rnethodologicaI differences between the two
studies need to be addressed.
First, in the present study, as in the research undertaken by Fawcett et al. (1 997),
the animals were treated with a 'salicylate' 1 h prior to the heat stress. However, in that
study, Fawcett et al. (1997) used aspirin (100 mg-kg1), whereas in the present study,
sodium salicylate was used (400 mg-kg-'). Thus, there were differences in both dosage
and in the type of salicylate used between the two studies. L
While .it is not clear whether the difference in dosage had an effect on the
potentiation of the heat shock response, it has been noted that sodium salicylate is
commonly administered in greater doses than aspirin (Vane and Botting, 1992). There are
differences in the chemical properties of aspirin and sodium salicylate which affect the
mode of action of the two drugs. In mammals, aspirin prevents prostaglandin production
by inhibiting cyclo-oxygenase through acetylation (Roth and Majerus, 1975). In contrast,
there is evidence that sodium salicylate stops prostaglandin production primarily through
the prevention of de novo synthesis of prostaglandin H synthase (Wu, 1991). It is unclear
whether the putative differences in the mode of action of these two drugs could affect
potentiation of the heat shock response. Furthermore, while sodium salicylate can be
dissolved and administered in an &O medium, aspirin is not easily dissolved in H,O, and
presumably for this reason, Fawcett et al. (1997) administered the aspirin via an ethanol
vehicle. However, it should be noted that ethanol itself has been shown to be an inducer of
HSPs (Li, 1983). Furthermore, it has been demonstrated that two or more stressors acting
in concert can have a synergistic effect on the expression of certain HSPs (Rodenhiser et
al., 1986; Hahn et al., 199 1). Thus, the administration of ethanol in combination with a
heat shock may produce a confounding induction of HSP 72.
Secondly, in comparing the present study to that of Fawcett et al. (1997), the
method of heat stress exposure should be closely evaluated. Fawcett et al. (1997) exposed
the animals to a constant, ambient temperature of 37OC for 30 min. ; this method allowed T,
to rise in an uncontrolled manner. In the present study, an adjustable thermal plate was
used to control the change in T, more precisely. In this way, a rise in T, above the desired
value was quickly corrected, thus ensuring that any observed potentiation of the heat shock
response by salicylate would not simply be the result of a higher T, Animals that were
mildly heat-stressed, and animals that were mildly heat-stressed in combination with
sodium salicylate treatment, required similar time periods to elevate rectal temperatures CT,)
to 400C for 15 min, and for T, to return to baseline Levels (figure 4). In addition, the
animals from both of these groups had identical average T, during the 15 min heat shock
(figure 6), and similar average T, during the entire heat stress (figure 5). This finding
stands in contrast to that reported by Fawcett et al. (1997), who observed a sigdicant
difference in core temperature between the mildly heat-shocked rats, and those mildly heat-
shocked in combination with aspirin treatment. In the Fawcett study, at the conchsion of
the 30 min heat shock period, the animals treated with aspirin reached an average core
temperature of 40.3"C, whereas the animals that experienced heat-shock without the
corresponding aspirin treatment reached an average core temperature of only 39.4OC. This
result would suggest that the aspirin potentiated a rise in core body temperature during heat
shock. In fact, Fawcett et al. (1997) concluded that aspirin enhanced the accumulation of
HSP 70 by mediating a rise in core body temperature. However, the accumulation of HSP
70 through a rise in core body temperature makes it difficult to isolate any independent
effect that aspirin may have had on HSP 70 induction. Indeed, a rise in core body
temperature alone is likely sufficient to enhance the heat shock response. Hutter et al.
(1994) demonstrated a correlation between core temperature during heat shock and
subsequent HSP accumulation. Following heat shocks of 40, 41, and 42"C, there was an
incremental elevation in myocardial HSP 72 (Hutter et al., 1994). Such a relationship
would imply that a significant increase in core body temperature, similar to the one
observed in the study by Fawcett et al. (1997), could in itself, result in a greater
accumulation of HSP 72.
Thirdly, as previously mentioned, Fawcett et al. (1997) observed a signifcant
elevation in core body temperature in the miIdly heat-shocked animals previously
administered aspirin, relative to counterparts that were only mildly heat-shocked. The
authors did not pursue this observation, giving no indication of how long core body
temperature remained elevated following the 30 min heat shock. It is possible that since
aspirin enhanced the rise in core temperature, the drug may have also prolonged the return
of core temperature to unstressed values. Thus, the animals would have been exposed to
an overall greater heat stress, which could possibly cause a greater induction of HSP 72.
Although a brief, but intense heat shock of 42°C for 15 min is known to elicit a robust
induction of HSP 72, heat shocks of lesser temperatures, but of longer durations have also
been shown to effectively induce HSP 72 (Blake et d., 1990; Kregel et al., 1995). Blake
et al. (1990) reported appreciable levels of HSP 70 mRNA in tissues of heat-shocked rats
whose core body temperature was less than 40°C. In the Blake study, animals were
exposed to a 90 min heat stress in which core body temperature remained less than 40°C.
Kregel et al. (1995) reported a four-fold elevation in both hepatic and myocardial HSP 72
content foilowing a heat shock of 4I0C for 30 min. Importantly, Kregel et al. (1995)
employed a heating system requiring approximately 45-55 min to raise the colonic
temperature of the rat to 41°C, and maintained this temperature for 30 rnin. Thus, in these
studies (Blake et al., 1990; Kregel et al., 1995), the animals were exposed to heat stresses
in which the core temperature of the animal remained elevated above the resting value for at
least 90 min. It is not clear how long animal core temperature remained elevated in the
study by Fawcett et al. (1997). However, it is likely that animals in the present study were
exposed to an overall shorter heat stress than those in the study by Fawcett et d. (1997). A
distinguishingefeature in the present study was the use of a heating pad which dowed a
rapid cooling of the animal following the 15 min heat shock, hence Limiting the duration of
the heat stress. Thus, the core temperature of the mildly heat-shocked animals, with or
without salicylate treatment, was elevated above resting values for only 45 min.
52
In the present study, the animalR treated with a mild heat shock plus sodium
salicylate showed a trend towards a higher peak T, than mildly heat-shocked only
counterparts, (40.3'C vs 40.Z°C; Figure 6). The observation of a higher peak T, in the
former group, albeit not statistically significant, may be noteworthy. Fawcen et al. (1997)
observed that aspirin potentiated the rise in core body temperature of animals at the end of a
30 min exposure to an elevated temperature. As mentioned previously, animals in the
study by Fawcett et al. (1997), subjected to a combination of heat stress and aspirin
treatment, exhibited an elevation in T, of approximately 1°C, compared to animals that were
only heat-strqsed (40.3OC vs 39.4"C, respectively). It should be reiterated that in that
study, animal T, was allowed to rise freely in response to an elevated temperature. In the
present study, the trend of a higher peak T, in animals that were mildly heat-shocked in
combination with sodium salicylate treatment occurred despite efforts to adjust plate
temperature, and keep T, of both groups as close to 4OT as possible. In effect, it seemed
more difficult to minimi;re the overshoot of T, in the animals that were subjected to mild
heat shock plus sodium salicylate katment after T, reached 40°C. In the present study, it is
unlikely that a dBerence of 0. 1°C in peak T, between animals in the two mild heat shock
groups carried any physiological significance, especially in terms of HSP 72 content.
Moreover, it .is debatable whether this trend in higher peak T, would be observed
repeatedly. Nevertheless, the present study may offer some support to the observation that
salicylate may enhance the rise in core body temperature during a mild heat shock, a s
reported by Fawcett et al. (1997).
It is not clear how the salicylates could mediate a heat-induced rise in core body
temperature. Though well known for their antipyretic properties, the salicylates are thought
to have little effect in the afebrile state (Vane et al., 1992). In response to pyrogens derived
from invading organisms, animais produce interleukin- 1, which stimulates arachidonic acid
metabolism, and eventual prostaglandin production. Through complex thermoregulatory
mechanisms, the prostaglandins increase body temperature. Accordingly, the antipyretic
effect of salicylate is achieved by inhibiting prostaglandin synthesis. Studies in which
animals were heat-stressed have shown that the prostaglandins are not involved in normal
body temperature regulation (Vane et ai., 1992). Thus, salicylate enhancement of heat-
induced elevations in body temperahue are probably not mediated by the drug's effect on
prostaglandins. Moreover, if the prostaglandins were involved in the temperature
elevations caused by the imposed heat stress, it is reasonable to assume that salicylate
would act to oppose the rise in core body temperature. It is possible that the salicylates
exert a direct effect on the animal's temperat regulation centers, such as the
hypothalamus, the medulla oblongata, or the spinal cord. If salicylate were to affect the
ability of any of these centers to control heat loss, then animal core temperature may rise
faster than normal. Thus, an animal treated with salicylate in combination with a mild heat
shock, may experience a greater rise in core body temperahlre than a mildly heat-shocked
only counterpart.
The adxninistration of salicylate to exercising animals may be helpful in gaining
insight into the drug's effect on core body temperature. In an exercise modeI, animal core
temperature would presumably rise freely, as in the study by Fawcett et al. (1997). It
would be of interest to determine whether those animals treated with salicylate prior to
exercise would attain a higher core temperature than untreated animals. In addition, would
higher core temperatures lead to an increased accumulation of HSP 72?
Fawcett et al. (1997) provided evidence that aspirin potentiated the heat-induced
accumulation of HSP 72 mRNA in rat liver, lung, and kidney. Nevertheless, the authors
only documented an increase of the protein in the liver. This was perhaps surprising, given
that the hepatic tissue in question was harvested only 30 min after heat shock. Currie et al.
(1982) demonstrated that HSP 72 synthesis is not detectable immediately following heat
shock, nor within 30 min post-exposure, in several tissues, including liver. In any case,
observations regarding HSP 72 accumulation in Liver should not be extended to the
myocardium, since there is evidence that the heat shock response is tissue specific. Blake
et al. (1990) found discordance of in vivo expression of HSP 72 among different tissues.
The authors noted that HSP 70 mRNA accumulated in a time dependent manner in liver. In
this tissue, HSP 70 mRNA accumulation was several fold higher at 6 h post-heat shock
than at one hour post-heat shock. Hanagan et al. (1995) demonstrated that the liver was
sensitive to the rate of heating. The authors showed that a high rate of heating during heat
shock could induce greater hepatic HSP 72 induction than a lower rate of heating,
irrespective of-total heat load. More importantly, Hanagan et al. (1995) suggested that the
liver may be a tissue susceptible to early thermal damage. Thus, it is possible that a whole
body heat stress of a given intensity would elicit HSP induction more vigorously in the
liver than in the heart. Taken together, these results indicate that interpretation of the heat
shock response should be k t e d to the specific tissue under investigation. Accordingly,
this reasoning could partially explain why Fawcett et al. (1997) observed salicylate
potentiation of HSP 72 induction in the liver, while in the present study, salicylate did not
potentiate HSP 72 induction in the myocardium.
In the present study, animal core temperature was precisely controlled during a heat
shock of 40°C; ensuring that sodium salicylate had no effect on core temperature. Thus, it
was demonstrated that in the absence of a temperature elevation, salicylate did not potentiate
HSP 72 induction during a mild heat shock Accordingly, it is suggested that aspirin
potentiation of the heat shock response observed by Fawcett et al. (1997) was solely the
result of the drug's enhancement of the rise in core temperature during heat shock It is not
known why the actions of salicylate observed in vitro were not observed in vivo. The
evidence supporting an in vitro potentiation of the heat shock response by the salicylates
must be carefully interpreted, as the type of cell line studied, and salicylate type and dose
used, as well as timing of administration, all appear to be of importance. Aspirin,
mesalamine, ahd indomethacin, administered either during or after a heat shock, were al l
found to potentiate the heat shock response in various human cell lines (Amici et al., 1995;
Lee et al., 1995; Buress et al., 1997). Taken together, these results provide encouraging
evidence that various non-steroidal anti-inflammstory drugs (NSAIDs) can potentiate the
heat shock response in vitro. However, it is possible that in vivo, in the presence of
complex physiological systems, the effects of salicylate are quite different, and cannot be
compared to those observed in isolated cell systems.
While the studies discussed above have reported that the various salicylates enhance
the heat shock response, others have suggested that these drugs may only partially activate
the heat shock response (Jurivich et al., 1992; Jurivich et al., 1995). Jurivich et al. (1992)
found that sodium salicylate activated the heat shock fztor (HSF) to its DNA binding state
in HeLa cells. However, the increased activation of HSF did not lead to a subsequent
elevation in tripscription of the HSP 70 gene. In the present study, HSF activation was
not examined. However, Fawcett et al. (1997), in their work on the aspirin-mediated in
vivo potentiation of the heat shock response, did not observe any effect of the drug on
HSF:HSE binding. There is some evidence that salicylate activation of HSF inhibits
subsequent heat-induced HSP 70 gene transcription in both Drosophih and yeast
Winegarden et al., 1996; Giardina et al., 1995). However, both the study by Fawcett et
al. (1997), and the present study, did not fmd evidence indicating salicylate inhibits heat-
induced transcription of the HSP 70 gene.
In the present study, and in the study conducted by Fawcett et al., salicylate was
administered htraperitonedy. It is unknown whether orai administration could have
potentiated a heat shock response. I . man, plasma concentrations of aspirin rise rapidly,
and peak 20 min after oral ingestion (Rowland et al., 1972). Plasma salicylate levels reach
a peak approximately 1 h following ingestion (Rowland et al., 1972). Similarly, and as
mentioned previously, peak plasma salicylate levels were observed in the rat approximately
1 h post-oral ingestion (Higgs et al., 1987). It seems reasonable to assume that
intrapentoned administration of salicylate would accelerate the appearance of the drug in
the plasma. Consequently, it appears unlikely that oral administration of salicylate, one
hour prior to heat shock, would be favourable in potentiating the heat shock response,
when compared to intraperitoneal administration.
A possibility not explored in the present study, nor in the study by Fawcett et aI.
(1997), is that saiicylate requires a longer time period in vivo to affect the HSP 70 gene. A
rapid appearance of the drug in plasma might not necessarily lead to an immediate effect on
the HSP 70 gene. For example, it may be insightful to observe HSP 72 accumulation in
the myocardium following whole body heat shocks at 6, 12, and 24 h post-salicylate
administration. It is also unknown how chronic use of aspirin may affect the heat shock
response. There is evidence showing that distribution of salicylate in vivo is tissue-
dependent, with high concentrations occuring in the liver, and the kidney ( B m e , 1974;
B m e et d., 1976). Thus, salicylate may not accumulate preferentially in the myocardium.
In the present study, animals treated with sodium salicylate only showed a trend
towards a decreased left ventricular HSP 72 content compared to controls (figure 13). It is
unclear whether a similar trend existed in the tissues examined by Fawcett et al. (1997).
Visual inspection of their blots suggested reduced levels of HSP 70 and HSP 70 rnRNA in
the liver of rats breated with aspirin only, but a lack of quantification makes reasonable
observations difficult. It is tempting to speculate about possible explanations for the
decreased left ventricular HSP 72 content observed in the animals that were treated with
sodium salicylate. Sodium salicylate may have an indirect effect on the synthesis of HSP
72. The anti-proliferative prostaglandins PGA, and P G 4 have been implicated in the
induction of HSP 72 (Amici et al., 1992; Holbrook et al., 1992). It is possible that the
inhibition of these prostaglandins by salicylate could result in a decreased synthesis of the
protein. Recently, it was reported that following heat shock, a sharp increase in nitric
oxide (NO) precedes, and is necessary for HSP 70 accumdation in rat heart (Malyshev et
al., 1995). Interestingly, additional research has demonstrated that both aspirin and
sodium saticylate inhibit the production of NO in murine macrophage cell Lines (Kepka-
Lenhart et d., 1996). Furthermore, Farivar et al. (1996) reported that salicylate is a
transcriptional inhibitor of nitric oxide synthase in cardiac fibroblasts. This finding was
confirmed in murine macrophage cells (Amin et al., 1995). Thus, it is possible that even in
the absence of heat shock, attenuation of NO synthesis by salicylate may adversely affect
HSP 72 expression.
In mammals, an episode of whole body heat stress, and attendant accumulation of
HSP 70, has been shown to confer subsequent myocardial protection. This has been
demonstrated successfixlly by several researchers using various models (Cunie et al., 1988;
Karmazyn et al., 1990; Donnelly et al., 1992; Hutter et aI., 1994; Locke et al., 1995). In
the present study, it was of interest to determine a) whether in vivo administration of
sodium salicyIate could potentiate the heat shock response, and b) if so, would the
response be sufficient to confer myocardial protection following a mild heat shock of 40°C.
This question had not previously been examined.
Hearts from mildly heat-shocked plus sodium salicylate treated animals showed a
significantly improved recovery of the rates of contraction and relaxation compared to
control hearts;but only after 30 min of reperfusion (figure 8, A and B). However, hearts
fkom mildly heat-shocked plus sodium salicylate treated animals did not show a
significantly greater post-ischemic recovery of LVDP at any time during reperfusion
compared to their control counterparts (figure 9). Animals exposed to a 15 min heat shock
at 42"C, 24 h prior were conferred myocardial protection. The hearts from these animals
recovered a significantly greater percentage of their pre-ischemic left ventricular developed
pressure (LVDP), and the rate of contraction and relaxation (kdP-dt-'), than hearts from the
unstressed (control) animals. These results were in agreement with others reported
previously (Currie et al., 1988; Karmazyn et al., 1990; Locke et al., 1995; Lncke et al.,
1996). The hearts isolated from rats exposed to 15 min of 42OC hyperthermia, 24 h prior,
and subjected to complete global ischemia, showed an improved recovery of contractile
function, and reduced indices of reperfkion injury, as evidenced by a decreased creatine
kinase efflux (Currie et al., 1988). The hearts fiom rats exposed to whole body
hyperthermia of 42OC for 20 min, 24 h prior, showed a signif~cant reduction in infarct size,
and a greater degree of myocardial salvage (Domelly et al., 1992). Similarly, the stress of
3 consecutive days of treadmiU running has been shown to confer myocardial protection
equal to that provided by a single heat shock of 4Z0C for 15 min W k e et al., 1995).
Studies showing a significant myocardial recovery from an ischemic episode have
reported a significant elevation of myocardial HSP 70 follohhg a brief, but severe,
hyperthennic stress (Currie et al., 1988; Karmazyn et al., 1990; Domelly et al., 1992;
Hutter et al., 1994). Using two-dimensional gels, Currie et al. detected a 7 1 kD protein in
hearts from rak that were subjected to a heat shock of 42OC for 15 min, 24 h prior, and
subsequently conferred myocardial protection. The protein was undetectable in hearts from
unstressed animals (Currie et al., 1988). Karmazyn et al. (1990) subjected rats to a 42°C
heat shock for 15 min, and examined myocardial HSP 70 content 24, 48, 96, and 192 h
post-heat shock On a two-dimensional gel, a protein of 7 1 k D was easily detectable at the
four time intervals, but its intensity diminished in a time dependent manner, following a
peak at 24 h post-heat shock. Again, this protein was undetectable in hearts fkom
unstressed controls. Hutter et al. (1994) demonstrated a direct correlation between HSP 70
accumulation and myocardial protection by observing infarct size in hearts fkom animals
exposed to a heat shock of either 40-41, or 42OC. The hearts fiom animals heat-shocked to
41 or 42OC had a sigmflcantly higher HSP 72 content than hearts from the unstressed
controls, and conversely, showed a marked reduction in infarct size. In the present study,
the animals heat-shocked for 15 min at 42"C, and allowed to recover for 24 h, showed a
significant elevation in left ventricular HSP 72 content, compared to the unstressed
controls, and compared to animals that were mildly heat-shocked, with or without salicylate
treatment (figures 12A and 13). The left ventricular HSP 72 content in hearts fiom
severely heat-stressed animals (42OC for 15 min) was elevated approximately five-fold
relative to hearts fiom the unstressed animals (figures 12A and 13), and accordingly, the
hearts from the severely heat-stressed animals were conferred protection, in terms of LVDP
and 2dPd.t -'. These findings are in agreement with results of other studies in which HSP
72 content was quantified following heat shock. In rats heat-shocked for 20 min at 42"C,
24 h prior, an approximate four-fold increase in myocardial HSP 72, above unstressed
levels, was observed (Huner et al., 1994).
Previous studies have shown low levels of myocardial HSP 72 are insufficient to
confer myocardial protection (Domelly et d., 1992; Hutter et al., 1994; Locke et al.,
1995). The hearts of animals heat-shocked to 40°C for 15 or 20 min, 24 h prior, did not
show a significant elevation of HSP 72, and accordingly were not protected (Hutter et al.,
1994; Locke et al., 1995). Similarly, rats exposed to a 20 min ischemic pre-treatment
showed a modest elevation in HSP 72 content 24 h later, and were not conferred
myocardial protection (Do~e l ly et id., 1992). In the present study, a mild heat shock of
40°C for 15 min, with or without sodium salicylate treatment, did not sigmficantly increase
left ventricular HSP 72 content above the levels observed in the unstressed controls
(figures 12A and 13). Not surprisingly, these hearts were not conferred any significant
myocardial protection in terms of LVDP. Hearts from animals that were mildly heat-
shocked plus sodium salicylate treated were conferred myocardial protection in terms of
+dP-dt 'l, but only at 30 min of reperfusion.
In comparison to controls, the hearts fkoq the animals treated with sodium
salicylate only initially showed a decreased recovery of LVDP and +dP-dt -' during reperfusion than was observed subsequently (figures 7 & 8). It is tempting to speculate on
possible explanations for this trend, which although noticeable, was not statistically
signifcant. The impaired recovery of LVDP and &dP& -' observed in the hearts from the
sodium salicylate treated animals, at the onset of reperfusion, may be explained by a lower
HSP 72 content relative to the unstressed animals. However, such reasoning does not
account for the eventual improvement in myocardial function observed in these hearts
(figures 7 & 8).
organism 24 h
Some effects of aspirin are known to be
beyond administration. For instance,
long lasting,
it has been
and could affect the
shown that aspirin
irreversibly inactivates some enzymes, such as cyclo-oxygenase in platelets (Pedersen and
FitzGerald, 1984), and glutamate decarboxylase (Gould et al., 1965). In the former
scenario, platelet function is altered for severd days (Vane and Botting, 1992).
Furthermore, salicylate in rat plasma is thought to have a half-life of approximately 6h
(Higgs et al., 1987). Clearly, certain effects of salicylate could persist for 24 h or longer, L
or perhaps until washout by reperfusion in the isolated heart. In a model of local ischemia
using isolated-rabbit hearts, aspirin was shown to be proarrhythmic (a proarrhythmia is a
drug-aggravated, or drug-induced, cardiac arrhythmia) (Dhein et al., 1997). Thus, hearts
from animals given sodium salicylate may be susceptible to electrophysiological side
effects. Aspirin is an inhibitor of certain vasodilators, such as the prostaglandins PGE, and
PGD, (Feinrnan et al, ; Vane and Botting, 1992), and an antagonist of the powerfd
vasodilator, bradykinin (Vane and Botting, 1992). Initial constriction of critical heart
vessels during reperfusion may explain a slowed recovery of contractile function.
Winegarden et al. (1996) proposed that lowered cellular ATP levels were the result of
sodium salicylate intedering with oxidative respiration. A reduced energy state in the cell
could explain the impaired contractility at the start of reperfusion in the h e m from the
animals treated with sodium salicylate. Finally, the role of the salicylates as modifiers of
transcriptional activity in certain genes has been documented and discussed above. Recent
research has indicated that the constitutively expressed HSP 25 is located adjacent to the
myofiblils in the heart, and thus, is thought to play a part in contractile function (Hoch et
al., 1996). In an isolated rat heart model, the expression of heme oxygenase, an
antioxidant protein, was found to be stimulated by reperfision, a period of consequential
fke radical production (M-aulik et al., 1996). A salicylate-mediated impairment in the
synthesis of either of these two proteins could impair myocardial function. In summary, it
is possible that some of the aforementioned effects could be deleterious to the contractile
function of the heart, which may initially be compromised during reperfusion, until the
aspirin is washed out. The dysfunction observed during early reperfusion in the hearts of
the animals treated with sodium salicylate, and hypothesized to be a result of the drug, was
not observed in the hearts of the animals treated with sodium salicylate in combination with
mild heat shock. It is possible that a heat shock, given concurrently with salicylate
treatment, may have a stabilizing effect on myocardial function.
Conclusion C
Animals subjected to a heat shock of 42°C- as brief as 15 min, are conferred
myocardial protection, as evidenced by a significant post-ischemic recovery of both left
ventricular developed pressure, and rates of contraction and relaxation. This protective
effect appears'to be mediated, at least in part, by an increased expression of the heat
inducible isofom of the HSP 70 family, HSP 72. In the present study, administration of
sodium salicylate, either alone, or in combination with a mild heat shock of 40°C for 15
min, did not potentiate the induction of HSP 72 in vivo. The HSP 72 content in the h e m
from mildly heat-shocked animals, whether treated or untreated with sodium salicylate, was
similar to that observed in the hearts from unstressed controls. Furthermore, the animals
that were mildly heat-shocked plus sodium salicylate treated were not coderred myocardial
protection. This result was not surprising given the strong association between HSP 72
accumulation and myocardial protection. It is not clear whether past results obtained in
vitro, and showing a potentiation of the heat shock response by various salicylates, can be
reproduced in v i v a Prior to the present study, only one other group had attempted to
demonstrate that a salicylate could potentiate the heat shock response in vivo (Fawcett et
al., 1997). In the presence of aspirin, the heat-induced accumulation of HSP 72 was
enhanced; however, it was concluded that this was the result of a rise in core body
temperature, somehow caused by aspirin (Fawcett et al., 1997). Similarly, the present
study was unable to show that sodium salicylate potentiated the heat shock response in vivo
by some direct action on the HSP 72 gene. The actions of salicylate in the complex
environment of a multiceuular organism are not fully understood. More research will be
needed to fully elucidate the action of this drug in viva
I APPENDIX I I Selected temperature, and heat stress duration values for all animals subjected to heat shock
animal unstressed duration ave temp (heat ave temp (heat temp(OC) (-1 stress) ( T I shock) ("C)
. mhs 1 36.0 53.89 38.6 40.1 mhs2 36.8 43.47 39.0 40.1
I
mhs 5 37 -9 37.76 39.5 1 40.1 mhss 1 37.0 45.26 39.0 1 40.2 mhss 2 37.0 40.76 39.1 40.1 mhss 3 36.6 47.44 39.0 40.2 mhss 4 36.3 52.20 38.8 40.1 mhss 5 37.1 41.57 39.2 40.1 sb 1 37.0 59.6 1 40.2 42.1 shs 2 3 5 -4 68.64 39.8 42.1 shs 3 37.4 69.36 40.4 42.0 shs 4 37.0 64.37 40.2 42.1 shs 5 37.5 64.58 40.4 42.0
mhs: mildly heat shocked animal rnhss: mildly heat shocked and sodium salicyIate treated animal shs: severely heat shocked animal
APPENDIX I1
Absolute LVDP values (mmHG), for al l animals, at end of equilibration period (40 min), and thoughout reperhion
ss: sodium salicylate treated animal mhs: mildly heat shocked animal mhss: mildly heat shocked and sodium salicylate treated animal shs: severely heat shocked animal
20 min
33.1 35.0 23.8 32.9 63.4 18.0 31-5 21.8'- 28.0 29.3 34.2 32.3 51.0 51.2 58.3 48.1 49.9 68.2 35.8- 28.9 56.5 82.2 82.9 38.4 48.4
animal
control 1
10 min 32.5
25 min
32.5 43.3 19.4 46.6 64.0 27.4 36.7 45.9 33.0 32.0 43.6 51.7 57.8 41.2 62.4 50.1 49.1 74.8 41.4 38.2 62.8 82.2 82.9 44.7 56.6
control 2 control 3 control 4 control 5 ss 1 ss 2 ss 3 ss 4 ss 5 mhs 1 mhs 2 mhs 3 mhs4 mhs 5 mhss 1 mhss 2 mhss 3 mhss 4 mhss 5 shs 1 shs 2 shs 3 shs 4 shs 5
40 min equilibration
87 -7
15 min
32.2
30 rnin
31.1 47.1 17.1 59-5 63.3 30-1 40.4 45.0 37.7 34.6 57.5 61.5 59.0 40.9 62.8 49.3 50.6 79.7 45.6 43.8 62.8 82.2 82.9 52.8 67.2
5min reperfusion
14.7 84.2 84.5 81.1 91.1
34.4 25.1 31.5 62.2
34.1 23.9 31.5 36.9
12.2 26.4 15.5 22.6 25.8 29.3 28.8 37.9 39.8 53.9' 47.2 54.1 65.8 30.8 21.7 47.9 82.2 82.0 33.3 40.1
38.2 22.3 29.8 62.7 10.8 24.7 12.6 21.2 24.2 29.3 15.5 36.3 38.3 43.3 40.1 52.0 42.4 26.0 18.2 31.7 73.0 56.5 31.6 27.1
69.2' 67 -6 67.5 95.1 91.3 72.4 77.7 75.4 76.9 85.8 57 .O 67.2 8 1 .O' 77.5 56.7 62.8 82.2 82.9 55.1 87.9
13.8 28.5 14.3 25.7 21.5 26.3 22.7 35.5 29.5 26.4 33.7 22.3. 33.3' 23.7 17.8 8.5
18.9 20.5 21.4 21.9
APPENDIX III
Absolute K O W values (d-mid'), for all animals, at end of equilibration period (40 min), and thoughout reperfusion
animal 1 40 min 1 5 min I 1 0 I15 120 125 I30 equilibration reperfusion m h min 1 min min rnin
control 1 6.0 5 .4 5.6 5.6 1 5.8 5.6 5.4 I I I I 1 I I
control 2 I 5.6 1 3.11 3.11 3.11 3.11 2.91 2.9 I I 1 I I I I
control 3 I 4.8 1 2.01 1-61 1.81 2.01 1.81 1.3 I 1
control 4 6.6 3.3 2.3 3.1 3.3 3.5 3.5 control 5 8.1 6.6 6.0 6.8 6.4 6.0 6.0 ss 1 4.6 3.6 3.3 3.8 3.8 3.8 3.6 ss 2 5.2 3.1 3.1 3.6 3.6 4.0 3.8 ss 3 4.4 3.6 2.4 2.3 3.3 2.5 2.3
I
ss 5 5.6 2.5 2.3 2.3 2.4 2.5 2.5 mhs1 4.4 2.9 3.3 3.8 4.0 4.0 3.8 mhs 2 5.6 5.2 5.6 5.4 5.2 4.8 4.8 mhs 3 4.8 4.0 3.8 4.2 4.2 4.0 3.6 mhs4 5.6 3.3 3 . 1 3.6 4.0 4.0 4.2 mhs5 10.4 5.8 4.6 4.8 5.2 5.2 5.2 mhss 1 I 4.6 2.9 2.9 3.3 3.3 3.3 3.1 mhss 2 1 9.9 5.2 5.6 6.8 7.0 6.8 6.8 . I
mhss 3 5.2 4.0' 4.0 5.0 5.2 5.2 4.8 mhss 4 6.4 3.8 3.6 3.6 3.6 3.6 3.5 mhss 5 3.5 1.3 1.2 1.4 1.5 1.5 1.4 sfis 1 7.6 6.8 6.4 6.8 6.8 6.4 6.4 shs 2 4.8 4.8 1 4.8 4.8 4.8 4.8 4.8
k
shs 3 5.6 4.8 4.6 5.0 5.2- 5.2 5.2 shs 4 5 -6 3.3 3.3 3.6 3.6 3.6 3.6
I I I 1 - -
I I
shs 5 1 5.6 1 3.31 3.11 3.31 3.31 3.51 3.5
b.
ss: sodium salicylate treated animal mhs: mildly heat shocked animal mhss: mildly heat shocked and sodium salicylate treated animal shs: severely heat shocked animal
I APPENDIX IV
Absolute +dP-dr -' values (mmHG-s-I), for all animals, at end of equilibration period (40 min), and thoughout reperfusion
ss: sodium salicylate treated animal mhs: mildly heat shocked animal mhss: mildly heat shocked and sodium salicylate treated animal shs: severely heat shocked animal
animal
control 1 control 2 control 3 control 4 control 5 ss 1 ss 2 ss 3 ss 4 ss 5 mhs I
40 min. equilibration
1700.2 1842.1 1857.6 1823.5 2018.3 16 16.5 1500.7 1543.4
5 min reperhion
270.8 721.8 503.9 572.7 760.6 255.6 603.3 263.5
285.5 684.4 804-1 875-8 842.1
1040.0 920.3 558.8 361.1
480.8 676.9 601.2 536.0 676.7 482.3 656.3 506.0 308.4 166.1 601.0 399.4 398.8 423.5
mhs2 mhs 3 mhs4 mhs5 mhss 1 mhss 2 mhss 3 mhss 4 mhss 5 shs 1 shs 2 shs 3 shs 4 s h 5
10 min
579.3 714.7 503.9 610.4 317.9 240.6 527.9 233.4
2165.2 2069.8 1549.7
- 1803.0 1789.9 1750.9 1940.3 1263.1 1386.5 18 10.4 1706.7 1324.0 1449.5 1795.5 1861.2 1 173.9 1905.7
401-2 483-4 586.8
548.4 714.5 826.6
1072.1 962.4
1047.4 1259.8 611.7 466.4
514.8 415.4 51 1.6
15 min
571.7 654.1 518.9 617.9
1302.9 278.2 520.4 278.6
634.2 1563.0 1168.0 647.1 468.9
25 min 20 min
594.3 646.6 556.5 617.9 1333
383-5 610.9 399.0
454.2 ' 559.0
1322.2 1256.C 796.5
1283.4 1045.1 949.4
1719.9 966.6
1008.1
586.0 1000.3 901.8
1155.1 1037.6 934.4
1463.4 725.0 586.8
1019.2 1795.5 1725.6 692.3 771.3
30 min
560.2 589.2
1449.5 1795.5 1861.2 1106.2
651.0 679.8
1051.8 1158.2 841.6
1291.0 1030.1 91 1.8
1606.8 883.6 850.1
1230.6 1795.5 1861.2 767.5
601.8
726.8 702.5
1419.3 1795.5 1861.2 903.0
594.3 87-9.7 473.8 926.8
1355.6 586.5 731.5 948.7
960.4
579.2 962-4 398.f
1205.f 1363.1 669.2 852.2 963.7
1173.6 677.1
1141.9
872.6
1436.8
APPENDIX V
Absolute -@I& '' values (mmHGd), for a l l animals, at end of equilibration period (40 min), and thoughout reperfusion I
animal l40rnin 15min I 1Ominl 15 min i2Omin 125 min equilibration reperhion
control 1 1324.0 233.2 473.9 526-6 541.6 534.1
ss: sodium salicylate treated animal mhs: mildly heat shocked animal mhss: mildly heat shocked and sodium salicylate treated animal shs: severely heat shocked animal
APPENDIX VI
Determination of sodium salicylate dosage
Myocardial HSP 72 content 24 hours after treatment with I S min heat shock (hs) plus sodium salicylate. Total protein content from left ventricle was separated by SDS-PAGE, transferred to nitrocellulose, and reacted with HSP 72-specific antibody. Shown is a western blot. Lane 1, control (unstressed); lane 2, mild hs (40°C); lane 3, severe hs (42°C); lanes 4 & 5, mild hs (40°C) plus 200 mg+kgl sodium salicylate; lanes 6 & 7, mild hs (40°C) plus 300 mg.kg1sodiurn salicylate; lanes 8 & 9, mild h: (40°C) plus 400 mg-kg1 sodium salicylate; lane 10, mild hs (40°C) plus 100 mg-kg sodium salicylate. When compared to lane 3 (severe heat shock), only lanes 8 & 9 show a similar elevation in HSP 72 content. Therefore, on the basis of this preliminary data, a sodium salicylate dosage of 400 mg-kglwas chosen for hrther study.
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