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. 2000 Oct;5(4):359–368. doi: 10.1379/1466-1268(2000)005<0359:tmhsrf>2.0.co;2

The myocardial heat shock response following sodium salicylate treatment

Marius Locke 1,2, Joel Atance 1
PMCID: PMC312865  PMID: 11048658

Abstract

In cultured cells, salicylate has been shown to potentiate the induction of Hsp72 so that a mild heat stress (40°C) in the presence of salicylate induces an Hsp72 response that is similar to a severe heat stress (42°C). To determine whether salicylate can potentiate the myocardial Hsp70 response in vivo and confer protection from an ischemic stress, male Sprague-Dawley rats (250–300 g) were placed into 5 groups: (1) control, (2) salicylate only (400 mg/kg), (3) mild heat stress (40°C for 15 minutes), (4) mild heat stress plus salicylate, and (5) severe heat stress (42°C for 15 minutes). Twenty-four hours following salicylate treatment and/or heat stress, animals were anesthetized, their hearts rapidly isolated, and hemodynamic function evaluated using the Langendorff technique. Hsp72 content was subsequently assessed by Western blotting. Although salicylate in combination with a mild heat stress induced heat shock factor activation, only the hearts from severely heat-stressed animals (42°C) demonstrated a significantly elevated myocardial Hsp72 content and a significantly enhanced postischemic recovery of left ventricular developed pressure and rates of contraction and relaxation. These results support the role for Hsp72 as a protective protein and suggest that neither salicylate treatment alone nor salicylate in combination with a mild heat stress potentiates the myocardial Hsp72 response.

INTRODUCTION

Following exposure to elevated temperatures and other protein-damaging stressors, cells from all organisms respond by rapidly synthesizing a group of highly conserved proteins known as heat shock proteins (Hsps; Currie and White 1983; Welch 1987; Lindquist and Craig 1988). Members of the 70-kDa Hsp family have been shown to confer protection to cells against various stresses (Li and Werb 1982; Currie et al 1988). In addition, induction of Hsps by one stressor, such as heat, has been shown to confer subsequent tolerance against another stressor, such as ischemia (Li 1983; Currie et al 1988). This phenomenon, known as cross-tolerance, may have important health implications, since it suggests a method by which the myocardium's own endogenous protective mechanisms may be harnessed to diminish the stresses encountered during ischemia and reperfusion.

In the mammalian heart, elevated levels of both the inducible Hsp70 isoform (Hsp72) and, to a lesser extent, the constitutively expressed Hsp70 isoform (Hsc73) have been shown to reduce ischemia and reperfusion injury (Currie et al 1988; Karmazyn et al 1990; Chong et al 1998). An elevated myocardial Hsp70 content by heat or transgenic means has also been shown to confer protection from an ischemic insult (Donnelly et al 1992; Hutter et al 1994; Marber et al 1995; Plumier et al 1995; Radford et al 1996). For example, isolated hearts from transgenic mice overexpressing either human (Plumier et al 1995) or rat Hsp70 (Marber et al 1995) demonstrate an improved functional recovery and reduced cellular injury following ischemia when compared with hearts from transgene negative litter mates. Taken together, these studies strongly suggest Hsp70 members play an important role in protecting the myocardium during episodes of stress.

Despite a large amount of evidence demonstrating an important role for Hsp70 in myocardial protection, few studies have investigated nonstressful methods of elevating the protective Hsps. At present, most methods capable of increasing Hsp content in cultured cells involve extreme physiological and pharmacological conditions that are not well tolerated by whole animals. It is well established from in vitro studies using cultured cells that simultaneously administering 2 distinct mild stressors can elevate Hsp content to levels that are comparable to a single severe stress (Li 1983; Rodenhiser et al 1986). For example, treating cultured cells with clinically relevant doses of salicylate during or immediately after heat shock results in a greater synthesis of Hsp70 than nontreated cells (Amici et al 1995). This potentiation of the heat shock response has recently been examined in vivo. Fawcett et al (1997) treated rats with aspirin plus a mild heat shock and demonstrated an increased Hsp70 expression in liver, lung, and kidney. Since, to our knowledge, the effect of salicylate on the myocardial heat shock response has not been examined, the present study was designed to determine whether an in vivo treatment with salicylate in combination with a mild heat shock could enhance heat shock factor (HSF) activation and Hsp accumulation in the rat myocardium. In addition, if salicylate could potentiate the myocardial heat shock response, would the level of Hsp content following salicylate and/or mild heat stress be sufficient to confer myocardial protection?

MATERIALS AND METHODS

Animals, sodium salicylate, and heat treatments

Adult, male Sprague-Dawley rats (300–350 g; Charles River) were used in these experiments. All experiments and procedures were approved by the Animal Care Committee of the University of Toronto. Animals were maintained on a 12-hour dark-light cycle, housed in pairs at 21°C (50% relative humidity), and provided food and water ad libitum. Animals were divided into 5 groups (n = 5 per group): (1) control (unstressed), (2) sodium salicylate only (400 mg/kg), (3) mild heat shock (40°C for 15 minutes), (4) mild heat shock (40°C for 15 minutes) combined with sodium salicylate (400 mg/kg), and (5) severe heat shock (42°C for 15 minutes).

Sodium salicylate (salicylic acid, sodium salt; Sigma Chemical Company, Mississauga, Ontario, Canada) was dissolved in water and administered intraperitoneally in a 0.5-mL volume. For animals treated with sodium salicylate and also subjected to heat shock, sodium salicylate was administered 1 hour before heat shock. For the purposes of this experiment, heat shock refers to the 15-minute period where rectal temperature (Tr) was maintained at 40°C or 42°C, whereas heat stress refers to the entire period required to raise Tr and return to baseline temperature. Before and during the entire heat stress, Tr was measured using a Thermistor TSD 102C Probe Transmistor connected to a Biopac data acquisition system. All animals subjected to heat shock were anesthetized with sodium pentobarbital (65 mg/kg intraperitoneally) and placed on a heating pad until Tr was within 0.5°C of the desired value for the 15-minute heat shock. The pad temperature was then adjusted to maintain a constant core temperature for 15 minutes. At the conclusion of the 15-minute period, animal Tr 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, Tr was raised to 40°C. Animals subjected to a severe heat shock had Tr raised to 42°C. In all cases, Trs were maintained for 15 minutes and subsequently returned to previously determined baseline values. Following sodium salicylate and/or heat shock treatment, all animals were returned to their cages and allowed to recover for 24 hours, except for animals used to evaluate HSF activation by gel shift analyses that were sacrificed immediately after heat shock or 1 hour following sodium salicylate treatment. Twenty-four hours after heat shock and/or sodium salicylate treatment, rats were anesthetized with sodium pentobarbital (65 mg/kg intraperitoneally) and injected with 1000 U of heparin (Hepalean; Organon Teknika, Toronto, Canada) via the tail vein 10 minutes before removal of the heart. Hearts were removed and immediately placed in ice cold saline in preparation for the Langendorff apparatus.

Preparation of protein extracts

Protein extracts were prepared according to the method of Mosser et al (1988). Briefly, portions of the left ventricle were thawed and homogenized in 15 volumes of extraction buffer (25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA [pH 8.0], 20 mM N-2-hydroxyethylpiperazine-N′-2-ethane-sulfonic acid [pH 7.9], 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsufonylfluoride) at 4°C at 5000 rpm using a Polytron. Tissue lysates were centrifuged at 14 000 rpm at 4°C (16 000 × g) for 20 minutes in an Eppendorf centrifuge. The supernatant was removed and stored at −70°C.

Mobility shift analyses

Protein extracts (100 μg) from control, salicylate-treated, and/or heat-shocked rat hearts were incubated with a 32P-labeled, self-complementary, ideal heat shock element (HSE) oligonucleotide (5′-CTA GAA GCT TCT AGA AGC TTC TAG-3′) in binding buffer (10% glycerol, 50 mM NaCl, 1.0 mM ethylenediamine-tetraacetic acid [EDTA] [pH 8.0], 20 mM Tris [pH 8.0], 1.0 mM DTT, 0.3 mg/mL of bovine serum albumin) with 0.1 ng (50 000 cpm) of 32P-labeled oligonucleotide and 2.5 μg of poly (dI dC) (Pharmacia Fine Chemicals, Piscataway, NJ, USA) for 30 minutes at room temperature. Samples were electrophoresed on 4% acrylamide gel at 200 volts for 2–3 hours. Gels were dried and exposed to Kodak Biomax magnetic resonance film. The HSF1 protein was identified by the addition of anti-HSF1 antibody (PA3–017, Affinity Bioreagents Inc, Golden, CO, USA) to heart extracts from severely heat-shocked animals and the correct HSF1-HSE interaction confirmed by incubating extracts with a 200-fold molar excess of unlabeled HSE as previously described (Locke et al 1995).

Isolated heart preparation

Following removal of the heart, the aorta was cannulated and the coronary arteries perfused under constant pressure conditions (50 mm Hg) with a modified Krebs-Henseleit buffer solution (Sigma) containing 4.7 mM potassium chloride, 2.0 mM calcium chloride, 1.2 mM monobasic potassium phosphate, 1.2 mM magnesium sulfate (Mg2SO4), 25 mM sodium bicarbonate, 118 mM sodium chloride, and 11 mM glucose. The buffer was bubbled with a 95% oxygen and 5% carbon dioxide mixture and maintained at 37°C. A water-filled, balloon-tipped catheter was inserted into the left ventricle, inflated to a volume of 50 μL, and used to assess left ventricular developed pressure (LVDP) and rates of contraction (+dP/dt) and relaxation (−dP/dt). Coronary flow (CF) or the flow of perfusing buffer before entering the cannulated heart was measured using a Gilmont Instruments flowmeter. Hearts were electrically paced at 320/min using plunge electrodes originating from an output channel on the Biopac system.

The following protocol was used for the heart experiments. After a 45-minute equilibration period, the hearts were subjected to 45 minutes of complete, warm (37°C), global ischemia by halting coronary perfusion and electrical pacing. Following 45 minutes of global ischemia at 37°C, flow and pacing were restored, and the heart underwent reperfusion for 30 minutes. Hearts that fibrillated excessively (>2 minutes) during reperfusion were excluded from the study. Data were recorded continuously throughout the protocol using the Biopac system and Acknowledge MP100 software. Hemodynamic indices (LVDP, ±dP/dt, CF) were evaluated 5 minutes before ischemia and at 0, 5, 10, 15, 20, 25, and 30 minutes of reperfusion. Following reperfusion, hearts were trimmed of excess tissue and frozen at −80°C for subsequent Hsp analysis.

Polyacrylamide gel electrophoresis and immunoblotting

Frozen portions (40–60 mg) of the left ventricle were homogenized at 4°C in 15 volumes of 600 mM sodium chloride and 15 mM Tris (pH 7.5), and protein concentrations were determined by the method of Lowry et al (1951). One-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was conducted according to the method described by Laemmli (1970), using a Bio-Rad mini-protean II 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. Following electrophoretic separation, proteins were transferred to nitrocellulose membranes (0.22 mm thick, Bio-Rad Laboratories), as described by Towbin et al (1979), using the Bio-Rad mini-protean II gel transfer system. Proteins were transferred to the nitrocellulose membrane at a constant 40 V for 4 hours. Following protein transfer, nitrocellulose membranes were blocked with 5% nonfat dried milk powder (NFDM) in Tris-buffered saline (TBS; 500 mM sodium chloride, 20 mM Tris-hydrochloride [pH 7.5]) for 1 hour, after which blots were washed twice, for 5 minutes each time, in TBS with 0.05 % Tween 20 (TTBS). Blots analyzed for Hsp72 were incubated with a polyclonal antibody (1:2500 dilution in TTBS with 2% NFDM; SPA-812, Stress-Gen, Victoria, Canada) specific for Hsp72. Following two 5-minute washes in TTBS, blots were immersed 1 hour in a solution of goat-anti-rabbit immunoglobulin G conjugated to alkaline phosphatase secondary antibody (Bio-Rad, 1:1000 dilution in TTBS with 2% NFDM).

Analysis of Hsc73 was performed by incubating blots for 3 hours with an alkaline phosphatase–conjugated monoclonal antibody specific for Hsc73 (1:5000 dilution in TTBS with 2% blotto; SPA-815AP, Stress-Gen). Following incubation with antibodies (Hsp72) or primary antibody alone (Hsc73), blots were washed twice in TTBS and once in TBS for 5 minutes each time and immersed in bicarbonate buffer (100 mM sodium carbonate, 1 mM magnesium chloride (MgCl2) [pH 9.8]) containing 3% (wt/vol) p-nitro-blue-tetrazolium chloride p-toluidine salt in 70% N,N-dimethyl-formamide (DMF) and 1.5% (wt/vol) 5-bromo-4-chloro-3-indolyl phosphate in 100% DMF. After development, blots were washed in double distilled water (ddH2O) and allowed to dry. Immunoblots and autoradiograms were scanned using an AGFA Arcus II scanner, and Hsp or HSF bands on the image were quantified using Kodak 1D 1.0 image analysis software. Standard curves were constructed to assure linearity.

Statistical analysis

InStat 2.01 was used to analyze all data. For each variable, analysis of variance was performed followed by Tukey's post hoc test to determine where significant differences (P < 0.05) existed among the treatment groups.

RESULTS

Pre–heat shock temperatures

Thermal responses recorded before, during, and after heat stress are listed in Table 1. Resting Trs during unstressed conditions for animals in the 3 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 [42°C]) before the 15-minute heat shock were 37.0°C ± 0.32°C, 36.8°C ± 0.14°C, and 36.9°C ± 0.37°C, respectively. No significant difference in resting Tr was detected among groups (Table 1).

Table 1.

Summary of heat stress and shock temperature and durationsa

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Total heat stress duration

Total heat stress duration, defined as the total time Tr was above resting value, was similar between mildly heat-shocked animals and those subjected to a mild heat shock in combination with sodium salicylate treatment (44.4 ± 3.34 minutes vs 45.5 ± 2.08 minutes). In contrast, animals subjected to a severe heat shock required an average of 65.3 ± 1.75 minutes to raise Tr to 42°C for 15 minutes and return Tr to baseline (Table 1). This was (by design) a significantly longer heat stress period than that experienced by the 2 milder heat-shocked groups (P < 0.001).

Average temperature

The average Tr of mildly heat-shocked animals and mildly heat-shocked animals treated with sodium salicylate was also similar during the 15-minute heat shock period (40.1°C ± 0.02°C vs 40.1°C ± 0.02°C) and during the entire heat stress (39.1°C ± 0.17°C vs 39.0°C ± 0.07°C; Table 1). As expected, average Tr during the 15-minute heat shock and during the entire heat stress was significantly higher for animals subjected to severe heat shock (42.1°C ± 0.02°C and 40.2°C ± 0.11°C) than for animals subjected to mild heat shock or mild heat shock plus sodium salicylate treatment (P < 0.001).

Peak temperature

Peak Tr during the 15-minute heat shock was similar between mildly heat-shocked animals and mildly heat-shocked animals treated with sodium salicylate (40.2°C ± 0.04°C vs 40.3°C ± 0.02°C). Again, as expected, peak Tr was significantly higher for animals subjected to severe heat shock (42.1°C ± 0.00°C) than for animals subjected to mild heat shock or mild heat shock plus sodium salicylate treatment (P < 0.001). These results demonstrate that mildly heat-shocked animals and mildly heat-shocked plus sodium salicylate treated animals were subjected to very similar heat stresses, whereas severely heat-shocked animals were subjected to a greater heat stress.

HSF activation following salicylate and heat shock

To determine if salicylate alone or in combination with mild heat shock caused trimerization and DNA binding of the heat shock transcription factor (hereafter termed HSF activation), rats were subjected to either salicylate treatment alone (100, 200, and 400 mg/kg intraperitoneally) or salicylate in combination with a mild heat stress (40°C for 15 minutes). Hearts were extracted 1 hour after salicylate treatment and/or directly after heat shock. No evidence of HSF activation was observed in heart extracts from controls or in heart extracts from animals treated with salicylate (400 mg/kg; Fig 1, lanes 2 and 3). However, when salicylate (200 or 400 mg/kg) was administered in combination with a mild heat shock (40°C), HSF activation was observed (Fig 1, lanes 4 and 5). Heart extracts from animals treated with a mild heat shock (40°C for 15 minutes) and a severe heat shock (42°C for 15 minutes, lane 7) also demonstrated HSF activation. However, compared with heart extracts from rats treated with both salicylate and a mild heat shock, the magnitude of HSF activation was greater in hearts from severely heat-shocked animals and lower in hearts from mildly heat-shocked animals. In comparison to the HSF activation observed in hearts from severely (42°C) heat-shocked animals, the level of HSF activation in hearts from mildly (40°C) heat-shocked animals and mildly heat-shocked animals in combination with either 200 or 400 mg/kg of salicylate was 31%, 60%, and 86%, respectively. The addition of 200 mM excess of unlabeled HSE abolished all HSF activation (Fig 1, lane 8), and the addition of HSF1 antibody to heart extracts from severely heat-shocked animals before electrophoresis resulted in a supershifted HSF1 complex, confirming HSF1 activation. These results demonstrate that 400 mg/kg of salicylate in combination with a mild heat shock potentiates HSF activation in the rat myocardium.

Fig 1.

Fig 1.

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HSF activation following salicylate and heat shock. Protein extracts were incubated with a 32P-labeled heat shock element and analyzed by MS-PAGE as described in the “Materials and Methods” section. Shown here is the top portion of the autoradiogram. Lane 1, free probe; lane 2, unstressed (control) rat heart; lane 3, rat heart 1 hour after 400 mg/kg of salicylate treatment; lane 4, rat heart after 200 mg/kg of salicylate treatment and a 15-minute, 40°C heat shock; lane 5, rat heart after 400 mg/kg of salicylate treatment and a 15-minute, 40°C heat shock; lane 6, rat heart after a 15-minute, 40°C heat shock; lane 7, rat heart after a 15-minute, 42°C heat shock; lane 8, rat heart after a 15-minute, 42°C heat shock with a 200 mM excess of unlabeled HSE added; lane 9, rat heart after a 15-minute, 42°C heat shock with anti-HSF1 antibody added to the extract. HSF, heat shock transcription factor complex; SS, supershifted heat shock transcription factor complex.

Hemodynamic function following ischemia

To determine if salicylate and/or heat shock is associated with an increased protection from ischemia, hearts from control, heat-shocked, and salicylate-treated animals were evaluated for hemodynamic function using the Langendorff isolated heart model. The preischemic absolute values for CF, +dP/dt, and LVDP for control, heat-shocked, and/or salicylate treated animals are listed in Table 2. No significant differences in absolute values for CF, ±dP/dt, or LVDP were detected among groups.

Table 2.

Preischemic absolute values for hemodynamic variablesa

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Coronary flow

No statistically significant differences in normalized CF values (percentage of preischemia) were detected between hearts from sodium salicylate–treated, mildly heat-shocked, mildly heat-shocked plus sodium salicylate–treated, and severely heat-shocked animals (Fig 2). Hearts from unstressed (control) animals recovered 78.3% ± 5.6% of preischemic coronary flow at 5 minutes of reperfusion and did not change significantly during the remainder of the reperfusion period. After 5 minutes of reperfusion, coronary flow recovery plateaued for all groups, and throughout the remainder of reperfusion, no differences in the recovery of coronary flow were detected among groups.

Fig 2.

Fig 2.

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Postischemic recovery of coronary flow is unchanged by heat or heat and sodium salicylate treatment. Data are expressed as a percentage of absolute preischemic values (means ± SE, n = 5 per group). No significant differences were detected among groups. Solid circles, control; open circles, mild heat shock; solid squares, mild heat shock plus salicylate; open square, severe heat shock; open triangles, salicylate treatment

Rate of contraction and relaxation

When the postischemic recovery of +dP/dt was expressed as a percentage of their respective absolute preischemic values, hearts from control animals recovered 30.3% ± 4.2% of their preischemic +dP/dt after 5 minutes of reperfusion (Fig 3A). At 5 minutes of reperfusion, there were no significant differences in the recovery of +dP/dt among groups. Thereafter, the recovery of +dP/dt in hearts from control animals increased in a linear manner so that at 30 minutes of reperfusion the recovery of +dP/dt reached 48.3% ± 9.0% of preischemic values. Hearts from sodium salicylate–treated animals exhibited a slightly lower but similar pattern of recovery during reperfusion. Hearts from mildly heat-shocked and mildly heat-shocked plus sodium salicylate–treated animals demonstrated a slightly higher, but not significantly different, rate of recovery during reperfusion. At 30 minutes of reperfusion, hearts from mildly heat-shocked plus sodium salicylate–treated animals demonstrated a significant difference compared with hearts from controls (75.8% ± 6.5% vs 48.3% ± 9.0%, P < 0.05). The recovery of +dP/dt in hearts from severely heat-shocked animals was similar to controls at 5 and 10 minutes of reperfusion, but at 15 minutes, +dP/dt was significantly greater than that observed in hearts from unstressed (control) animals (72.5% ± 10.9% vs 39.1% ± 6.5%, P < 0.05). This pattern continued throughout the remainder of reperfusion.

Fig 3.

Fig 3.

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A 42°C heat shock enhanced ±dP/dt following ischemic stress. Data are expressed as mean ± SE, n = 5 per group. Statistical significance from control (P < 0.05) is indicated by an asterisk. Solid circles, control; open circles, mild heat shock; solid squares, mild heat shock plus salicylate; open square, severe heat shock; open triangles, salicylate treatment

Similar results were obtained for −dP/dt (Fig 3B). The differences in −dP/dt observed among groups were consistent with the differences in +dP/dt among groups. However, the −dP/dt recovery for hearts from severely heat-shocked animals plateaued sooner than +dP/dt recovery.

Left ventricular developed pressure

During reperfusion, hearts from severely heat-shocked animals also recovered LVDP to a greater extent than hearts from controls. When expressed as a percentage of their respective preischemic values, hearts from control animals recovered 33.0% ± 4.7% of their preischemic LVDP after 5 minutes of reperfusion (Fig 4) and slowly increased so that at 30 minutes of reperfusion hearts from controls had regained only about half of their preischemic LVDP (50.9% ± 10.1%). In comparison, at 5 minutes hearts from animals that were treated with sodium salicylate alone, mild heat shock alone, mild heat shocked plus sodium salicylate, or severe heat shocked recovered 23.4% ± 4.4%, 39.1% ± 5.4%, and 25.0% ± 4.0% of LVDP, respectively. At 5 and 10 minutes of reperfusion, there were no significant differences in LVDP recovery among groups. However, at 15 minutes of reperfusion, hearts from severely heat-shocked animals demonstrated a significantly greater recovery of LVDP than hearts from unstressed animals (P < 0.05). This pattern continued for the remainder of reperfusion so that at 30 minutes hearts from severely heat-shocked animals recovered 94.5% ± 4.6% of LVDP, whereas hearts from controls recovered only 50.9% ± 10.1% of LVDP (P < 0.05). These results suggest that severe heat shock enhances postischemic myocardial recovery, but a mild heat shock and/or salicylate treatment does not enhance postischemic myocardial recovery.

Fig 4.

Fig 4.

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A 42°C heat shock enhanced postischemic recovery of LVDP. Data are expressed as a percentage of absolute preischemic values (mean ± SE, n = 5 per group). Statistical significance from control (P < 0.01) is indicated by an asterisk. Solid circles, control; open circles, mild heat shock; solid squares, mild heat shock plus salicylate; open square, severe heat shock; open triangles, salicylate treatment

Hsp70 content

To evaluate differences in Hsp72 and Hsc73 content, portions of the left ventricle were homogenized, and total protein was separated by SDS-PAGE and transferred to nitrocellulose membrane as described in the “Materials and Methods“ section. A representative Western blot (Fig 5A) shows Hsp72 was detectable in the hearts from all animals examined, including unstressed (control) animals (lane 1). As expected, Hsp72 content was noticeably elevated in hearts from severely (42°C) heat-shocked animals (lane 4). However, Hsp72 content in the hearts from animals in all other groups was similar to controls.

Fig 5.

Fig 5.

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(A) Hsp72 content is increased in left ventricle following heat shock to 42°C. A representative Western blot illustrating left ventricular Hsp72 content following heat and/or sodium salicylate treatment (100 μg of protein loaded per lane). Lane 1, control; lane 2, sodium salicylate only (400 mg/kg); 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). (B) Hsc73 content in left ventricle is unchanged following heat shock and/or sodium salicylate treatment. A representative Western blot illustrating left ventricular Hsc73 content following heat and/or sodium salicylate treatment (100 μg protein loaded per lane). Lane 1, control; lane 2, sodium salicylate only (400 mg/kg); 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). Portions of the left ventricle were homogenized, and total protein was separated by SDS-PAGE and transferred to nitrocellulose membrane as described in the “Materials and Methods” section

To further assess left ventricular Hsp72 content, quantification of bands representing Hsp72 from several Western blots was performed. When expressed as a percentage of the control value from unstressed animals, Hsp72 content in hearts from animals subjected to severe heat shock was significantly elevated (Fig 6A; P < 0.001). However, no significant differences were detected in Hsp72 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. These results demonstrate that a heat shock of 42°C for 15 minutes is capable of significantly elevating myocardial Hsp72 content. However, a mild heat shock of 40°C for 15 minutes, either alone or in combination with sodium salicylate, does not significantly increase left ventricular Hsp72 content compared with unstressed (control) animals.

Fig 6.

Fig 6.

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Graphic representation of left ventricular Hsp72 content obtained from densitometric scanning of Western blots reacted with antibody for Hsp72 (A) and Hsc73 (B). Data are expressed as mean ± SE of control (n = 5 per group). Statistical significance (P < 0.001) is indicated by an asterisk

A representative Western blot (Fig 5B) shows Hsc73 content was detectable in all hearts examined. However, none of the treatments, including severe heat shock, resulted in an accumulation of Hsc73. When Hsc73 bands on blots were quantified and expressed as a percentage of values determined for hearts from unstressed controls, no treatment resulted in an accumulation of Hsc73 in the left ventricle (Fig 6B).

DISCUSSION

Cells exposed to temperature elevations (heat shock), either in vitro or in vivo, respond by rapidly synthesizing Hsp72 and other Hsps (Tissieres et al 1974; Li and Werb 1982; Currie and White 1983; Blake et al 1990). An elevated myocardial Hsp72 content has been shown to confer myocardial protection (Currie et al 1988; Karmazyn et al 1990; Hutter et al 1994; Locke and Tanguay 1996b). Recent evidence has suggested that sodium salicylate and other nonsteroidal anti-inflammatory agents (NSAIDS) may potentiate the heat shock response both in vitro (Jurivich et al 1992; Amici et al 1995; Ito et al 1996; Burress et al 1997) and in vivo (Fawcett et al 1997). Thus, it was of interest to determine whether salicylate used in conjunction with a mild heat shock might increase myocardial Hsp72 content and confer myocardial protection. The results from the present study suggest that although salicylate in combination with a mild heat stress is capable of potentiating HSF activation, neither sodium salicylate treatment alone nor sodium salicylate treatment in combination with a mild heat shock (40°C for 15 minutes) significantly increases myocardial Hsp72 content. In addition, neither salicylate treatment alone nor salicylate in combination with a mild heat shock provides myocardial protection in the form of an enhanced postischemic LVDP recovery.

A number of studies (Jurivich et al 1992; Amici et al 1995) have shown that when cultured cells are treated with a mild heat shock in combination with salicylate, the heat shock response is potentiated so that the temperature threshold for Hsp induction is lowered. Lee et al (1995) treated HeLa cells with indomethacin combined with a mild heat shock and observed that transcription of heat shock genes was induced to greater levels than with mild heat shock alone. In addition, mesalamine, a compound related to sodium salicylate, has been shown to increase and accelerate the thermal induction of Hsp72 in rat intestinal epithelial cells (Burress et al 1997). Although salicylate treatment appears to potentiate the heat shock response in cultured cells, at present only one study (Fawcett et al 1997) has examined whether salicylate can potentiate the heat shock response in vivo. Fawcett et al (1997) reported that aspirin potentiated the heat shock response in rat liver, lung, and kidney. However, a significant difference in core temperature between the mildly heat-shocked rats and those mildly heat shocked in combination with aspirin treatment was observed. The authors concluded that aspirin increased Hsp70 content by mediating a rise in core body temperature. In the present study, the temperature stress provided was precisely controlled so that animals that were mildly heat stressed and animals that were mildly heat stressed in combination with sodium salicylate treatment exhibited similar average Trs during the 15-minute heat shock and during the entire heat stress. In addition, similar lengths of time were required for Tr to reach 40°C and to return to baseline levels. This ensured that any observed potentiation of the heat shock response by salicylate was not simply the result of a higher Tr. Thus, differences in heating protocols may explain why an increased Hsp content was observed by Fawcett et al (1997) but not in the present study. Alternatively, it may also be possible that Hsp72 might accumulate differently in the liver, kidney, and lung than in the myocardium. The heat shock response is known to be tissue specific and the liver a more heat sensitive organ than the heart (Blake et al 1990; Flanagan et al 1995; Locke and Tanguay 1996a).

Various salicylates and NSAIDS administered either during or after a heat shock have been shown to potentiate the heat shock response in various cell lines (Amici et al 1995; Lee et al 1995; Burress et al 1997). Taken together, these results provide evidence that the heat shock response can be potentiated in vitro. It might follow that a potentiated Hsp response might be expected in vivo. Although the exact reason(s) for a lack of a potentiated Hsp response in the present study remains unclear, a number of factors may explain this discrepancy. First, although both the mild heat stress of 40°C and the combination of salicylate and the mild heat stress may have been a sufficient stress to cause HSF activation, they may not have induced transcription and translation of the Hsps. An uncoupling of HSF activation and subsequent Hsp transcription has been reported when cultured cells are treated with a mild heat shock in combination with salicylate (Jurivich et al 1992). Second, the effects of salicylate on the more complex physiological systems present in a whole organism may be different than those observed in cell cultures.

In the myocardium, a severe heat stress (42°C), a mild heat stress (40°C), and a mild heat stress (40°C) in combination with salicylate all resulted in HSF activation, yet an accumulation of the Hsp72 protein was only observed following a severe heat stress (42°C). Why some stressors cause HSF activation and Hsp accumulation whereas others only cause HSF activation remains unknown. However, it may be that when exposed to reduced levels of stress (lower temperatures with or without salicylate) the extent of protein denaturation is insufficient to maintain the heat shock response and hence Hsp accumulation. Thus, although a stress may be sufficient to activate the HSF, once the myocardium realizes it can accommodate the stress, Hsp accumulation is arrested. Given the vital function of the myocardium to an organism, it might be an adaptive strategy to initially respond to all stressors by activating the HSF and later alter the level of Hsp protein as required.

In mammals, an episode of whole body heat stress, and the concomitant accumulation of Hsp70, has been shown to confer subsequent myocardial protection (Currie et al 1988; Karmazyn et al 1990; Donnelly et al 1992; Hutter et al 1994). Studies that show a significant myocardial recovery from an ischemic episode have reported a significant elevation of myocardial Hsp70 following a brief but severe hyperthermic stress (Currie et al 1988; Karmazyn et al 1990; Donnelly et al 1992; Hutter et al 1994). In the present study, animals exposed to a 15-minute heat shock at 42°C 24 hours earlier demonstrated an increased Hsp72 content and were also conferred myocardial protection as indicated by a greater percentage of their recovery of LVDP and ±dP/dt. These results are in agreement with others (Currie et al 1988; Karmazyn et al 1990; Locke and Tanguay 1996b) and support the concept that Hsp72 plays an important role in protecting the heart. In addition, previous studies have also shown that low levels of myocardial Hsp72 are insufficient to confer myocardial protection (Donnelly et al 1992; Hutter et al 1994; Locke and Tanguay 1996b). In the present study, a mild heat shock of 40°C for 15 minutes, with or without sodium salicylate treatment, did not significantly increase left ventricular Hsp72 content above the levels observed in the unstressed controls. Not surprisingly, these hearts were not conferred any significant myocardial protection, since both the hearts from mildly heat-shocked animals and the hearts from animals that received mild heat shocked plus sodium salicylate treatment did not show a significantly greater postischemic LVDP recovery at any time during reperfusion than control counterparts. However, hearts from mildly heat-shocked plus sodium salicylate–treated animals did show a significantly enhanced recovery of ±dP/dt compared with control hearts at 30 minutes of reperfusion. The exact reason for this remains unclear, but since Hsp72 content remained unchanged, it does not appear to be Hsp72 mediated. This protection may be related to other properties of sodium salicylate.

In conclusion, the results from the present study support the notion that Hsp72 plays an important role in protecting the myocardium during episodes of stress. Hearts from animals that were subjected to a severe heat shock (42°C for 15 minutes) demonstrated both an elevated Hsp72 content and an enhanced postischemic recovery. In contrast, the in vivo administration of sodium salicylate alone or in combination with a mild heat stress did potentiate HSF activation in the myocardium but did not potentiate the Hsp72 or Hsc73 protein response and, therefore, was not sufficient to confer myocardial protection. Thus, an in vivo treatment with sodium salicylate coupled with a mild heat shock does not potentiate the myocardial heat shock response and provide myocardial protection in whole animals.

Acknowledgments

This work was supported by Ontario Heart and Stroke Foundation Operating Grants NA-3438 and B4098 and a University of Toronto Connaught Grant. The authors wish to acknowledge Scott Driscoll for his help with the gel shift experiments.

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