Heat-Shock Response Is Associated With Enhanced Postischemic Ventricular Recovery

Transcription

1 543 Heat-Shock Response Is Associated With Enhanced Postischemic Ventricular Recovery R. William Currie, Morris Karmazyn, Malgorzata Kloc, and Kathleen Mailer In cells, hyperthermia induces synthesis of heat-shock proteins and the acquisition of thermotolerance. Thermotolerant cells are resistant to subsequent oxidative stress. In this study, heat-shocked hearts were examined for evidence of protection during ischemia and reperfusion. Rats were exposed to 15 minutes of 42 C hyperthermia. Twenty-four hours later their hearts were isolated and perfused and the contractility examined during and after ischemic perfusion. No protection was observed during ischemic perfusion. However, upon reperfusion heatshocked hearts had recovery of contractility within 5 minutes of reperfusion, while control hearts showed no contractility at this time. Throughout 30 minutes of reperfusion heat-shocked hearts had significantly unproved recovery of contractile force, rate of contraction and rate of relaxation. Creatine kinase release, associated with reperfusion injury, was significantly reduced from a high of ±78.9 mu/min/g heart wt for controls to ±82.9 mu/min/g heart wt for heat-shocked hearts at 5 minutes of reperfusion. Following 30 minutes of reperfusion, ultrastructural examination revealed less damage of mitochondrial membranes in the heat-shocked hearts. Further biochemical investigations revealed that the antioxidative enzyme, catalase, was significantly increased to 137 ± 12.7 U/mg protein in the heat-shocked hearts while the control value was 64.8 ±8.3 U/mg protein. Hyperthermic treatment, which induces the heat-shock response, may be therapeutic for salvaging ischemic myocardium during reperfusion, through a mechanism involving increased levels of myocardial catalase. (Circulation Research 1988;63: ) Many forms of metabolic stress, including hyperthermia, induce the synthesis of heat-shock proteins. 1-4 Stressed cells, which synthesize and accumulate heat-shock proteins, acquire a transient resistance to subsequent episodes of oxidative stress This resistance to oxidative stress (often referred to as thermotolerance) is linked with an increased ability to remove free radicals. 6-7 The rapid generation of oxygen free radicals is postulated to be the major cause of organ damage during reperfusion. 8 Reperfusion injury occurs following periods of ischemia when there is reintroduction of molecular oxygen to tissues. Since tolerance to oxidative stress is associated with the heat-shock response, we reasoned that a preinduc- From the Departments of Anatomy (R.W.C.), Pharmacology (M. Karmazyn) and Biochemistry (M. Kloc), Dalhousie University, Halifax, Nova Scotia, Canada and Department of Chemistry (K.M.), St. Mary's University, Halifax, Nova Scotia, Canada. This study was supported by three grants from the New Brunswick Heart Foundation (awarded to R.W.C., M. Karmazyn, and K.M.) and a grant from Natural Sciences and Engineering Research Council of Canada (awarded to K.M.). Address for correspondence: R. William Currie, PhD, Department of Anatomy, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada. Received October 21, 1987; accepted March 23, tion of the heat-shock response could result in a protective influence against the oxidative stress of myocardial ischemia and reperfusion. To test this hypothesis we challenged isolated rat hearts (control and heat-shocked) with 30 minutes of low-flow ischemia followed by reperfusion. Contractility of hearts and creatine kinase (CK) efflux were measured to assess injury and recovery. Injury was also estimated by ultrastructural examination. Because reperfusion injury may be mediated by free radical generation, 8 hearts were examined for antioxidative enzymes, which control the normal generation of free radicals. Materials and Methods Male Sprague-Dawley rats ( g; Canadian Hybrid Farms, Centreville, Nova Scotia) were anesthetized with sodium pentobarbital (50 mg/kg body wt i.p.) and placed on a temperature-controlled heating pad until body temperature, monitored with a rectal thermometer, reached 42 C.^This hyperthermic temperature was maintained for 15 minutes after which the animals were allowed to recover for 24 hours. Control animals were subjected to a similar protocol except that the heating pad was not turned on; rectal temperature for these animals was 37 C.

2 544 Circulation Research Vol 63, No 3, September 1988 After 24 hours of recovery, the animals were killed by decapitation. Hearts were excised and either perfused by the Langendorff method or homogenized and assayed for superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH.Px) activity. Perfusion Protocol Excised hearts were perfused retrogradely using the Langendorff method. Hearts were perfused with Krebs-Henseleit buffer consisting of (ram) NaCl 120, NaHCO 3 20, KC1 4.63, KH 2 PO , MgCl 2 1.2, CaCl , and glucose 8. The ph of the buffer was 7.4, and the perfusion system was maintained at 37 C using a water-jacketed chamber and coil. The hearts were electrically paced at 5 Hz with a Grass S44 stimulator (Grass Instruments, Quincy, Massachusetts) at twice threshold voltage. Buffer flow was controlled with a peristaltic pump. Hearts were equilibrated for 30 minutes at 10 ml/min coronary flow. Hearts were subjected to 30 minutes of ischemic perfusion by reducing the flow rate to 1 ml/min. At the end of the 30-minute ischemic period, normal flow (10 ml/min) was reinstituted for a further 30 minutes. Mechanical activity was measured throughout each perfusion experiment according to previously described methods. 10 Apicobasal displacement and resting tension were obtained by attaching a Grass FT.03 strain gauge transducer to the heart apex. The transducer was positioned to yield an initial resting tension of 2 g. Recordings of mechanical activity were obtained on a Grass Model 7 polygraph. Creatine Kinase CK release from hearts was measured in the effluent buffer. Samples (1 ml) of effluent buffer were collected during the last minute of both preischemic perfusion and ischemic perfusion and at 1, 2, 5, 10, 20, and 30 minutes of reperfusion. CK content was determined by the spectrophotometric method of Rosalki 11 using CK kits from Sigma Chemical, St. Louis, Missouri. Two-Dimensional Gel Electrophoresis Immediately after perfusion, approximately 250 mg of ventricular muscle from each heart (except those fixed for electron microscopy, see below) was placed in 1 ml of lysis buffer for extraction of proteins. Proteins in 130 fi\ of lysis buffer were loaded on 2 x 100 mm columns for isoelectric focusing according to the method of O'Farrell 12 and as previously described. 13 The isoelectric gels were equilibrated in sodium dodecyl sulfate sample buffer for 1 hour and proteins were further separated in the second dimension by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. 14 Gels were stained for proteins with Coomassie brilliant blue R. Electron Microscopy Immediately following perfusion, two hearts from each group were perfusion-fixed (10 ml/min) with 2% glutaraldehyde, 0.5% paraformaldehyde, 1.5 mm CaCl 2, in 0.07 M sodium cacodylate buffer for 20 minutes. Portions of ventricular muscle were further fixed for 2 hours in the same solution. Tissue samples were post-fixed with osmium tetroxide, stained en block with uranyl acetate and embedded in Spurr resin. Thin sections were stained with lead citrate and examined on a Zeiss 10A electron microscope (Carl Zeiss, Toronto, Canada) at an accelerating voltage of 60 kv. Antioxidative Enzymes SOD, catalase, and GSH.Px levels were determined in freshly excised hearts. In addition, catalase levels were also determined in reperfused hearts. Hearts were homogenized in 10 mm KH 2 PO 4 (ph 7.4), 30 mm KC1, and 1% ethanol. SOD and GSH.Px were assayed in the 12,000g supernatant. SOD was assayed in 34 mm KH 2 PO 4 (ph 7.8), which contained 23 fxm cytochrome c and 56 JAM xanthine. The reaction was begun by the addition of sufficient xanthine oxidase to cause cytochrome c reduction Abs changes/min according to the method of McCord and Fridovich. 15 GSH.Px was assayed in 140 mm Tris-HCl (ph 7.0, 37 C), which contained 0.25 mm nicotinamide adenine dinucleotide phosphate reductase and glutathione reductase 50 fig. Before addition to the assay mixture, the test solution was incubated at 37 C for 5-10 minutes with reduced glutathione. The final concentration of reduced glutathione in the assay was 1 mm. The assay was begun by the addition of t-butyl hydroperoxide (final concentration 1 mm) following the method of Paglia and Valentine. 16 Catalase was assayed in the 2,000g supernatant according to the method of Del Rio et al 17 in 8.75 mm KH 2 PO 4 (ph 7.4, 37 C) containing diethylenetriaminepentaacetic acid. Samples were diluted with a solution containing 1% ethanol and 0.65% Triton X-100 in 50 mm KH 2 PO 4 (ph 7.4). The assay was begun by the addition of H 2 O 2 to a final concentration of 4.6 mm and diluted sample. The rate of oxygen production was measured using a YSI Model 53 oxygen monitor (Yellow Springs, Ohio). Protein was measured by the method of Lowry et al. 18 Statistical Analysis All values expressed are mean±sem. Differences were determined by comparison of the calculated test statistic with the two-tailed significance limits of Student's distribution. Results Contractility Contractile data for isolated hearts from both groups of animals is illustrated in Figure 1. None of the parameters differed before starting ischemia although force for heat-shocked hearts was slightly higher (6.4±0.9 g vs. 5.3 ±0.5 g) at the end of the 30-minute preischemic period. Ischemia produced a

3 Currie et al Enhanced Postlschemic Ventricular Recovery g 2^? 40 20H ' CONTROL O -O WAT SHOCKED 100 O 80 4) CP ' FIGURE 1. Graphs showing mechanical responses of isolated hearts from control and heatshocked rats. Each point represents the mean±sem of six hearts from each treatment group. Ischemia (I) was initiated at 0 minutes and maintained for 30 minutes as depicted by vertical dotted lines. *p<0.05; **p<0.01; ***p<0.001 from control values using Student's t test for paired data. RT, resting tension ' TIME (MIN) TIME (MIN) total reduction in contractility by 20 minutes with a corresponding progressive elevation in resting tension. The responses to ischemia alone were not affected by prior hyperthermic treatment. However, substantial differences in contractile recovery were evident following restitution of normal coronary flow. After 30 minutes of reperfusion (t = 60 minutes), hearts of animals subjected to hyperthermia had significantly increased recovery of force (2.4 ± 0.4 vs. 1.1 ± 0.3 g), rate of contraction (+ df/dt; 78.0±4.5 vs. 44.7± 10.8 g/sec) and rate of relaxation (-df/dt; 73.3±6.3 vs. 36.0±9.1 g/sec) when compared with control hearts. Relative to preischemic values the recovery of force was 38% vs. 21%, + df/dt was 85% vs. 49%, and - df/dt was 77% vs. 38% for heat-shocked vs. control hearts after 30 minutes of reperfusion. In addition, in hearts of treated animals, the first regular contractions occurred within 5 minutes of reflow, a time when no contractility was ever observed in control hearts. Resting tension changes associated with reperfusion were unaffected by prior hyperthermia. Two-Dimensional Gel Electrophoresis The major inducible heat-shock or stress protein of molecular mass 71 kda (SP71) was detectable by two-dimensional gel electrophoresis and Coomassie brilliant blue R staining in all hearts from hyperthermically treated rats. SP71 was not detectable in any of the control hearts. Representative examples of these differences are shown in Figure 2. Creatine Kinase CK release during preischemic perfusion was undetectable in hearts from both treatment groups. Ischemia resulted in measurable CK in the coronary effluent although the values were consistently low (<20 mu/min/g heart wt). Furthermore, coronary effluent CK content during ischemia was identical (<5% difference) for both groups (n = 3, data not shown). Reperfusion potently stimulated CK release from control hearts which peaked at 5 minutes of reflow at ±78.9 mu/min/g heart wt (Figure 3). However, hyperthermic treatment reduced the release of the enzyme at 5 minutes of reflow to ±82.9 mu/min/g heart wt. The diminution of CK release from the heat-shocked hearts was significant (p<0.05) at all sampling times during reflow, with the exception of the initial 1-minute reperfusion value. Electron Microscopy Two hearts from each treatment group were perfusion-fixed immediately after the 30-minute reflow and examined by transmission electron microscopy. Both control hearts demonstrated substantial mitochondrial swelling and in many mitochondria severe cristae disruption (Figures 4A and 4B). Although some degree of swelling was evident, mitochondria of heat-shocked hearts had a more normal morphology (Figure 4C). Heat-shocked hearts clearly had less ultrastructure disruption than control hearts. Antioxidative Enzymes Table 1 summarizes the SOD, catalase and GSH.Px activity in freshly excised control and heat-shocked hearts. After hyperthermic treatment, catalase activity was increased significantly (p<0.05) to ±12.7 U/mg protein which was more than twice the control value of 64.8 ±8.3 U/mg protein.

4 S46 Circulation Research Vol 63, No 3, September 1988 Mrx10"3 A I -43 B t.u FIGURE 2. Protein profiles of isolated hearts from control and heat-shocked rats. Arrow indicates the position of SP71, which was undetectable in control hearts (Panel A) and present in heat-shocked hearts (Panel B). Related and constitutively synthesized heatshock protein of M, 73 kda is indicated by arrowhead. Asterisks indicate HSP78, which has been identified as a glucose-regulated protein.13 a, act in After hyperthermic treatment, GSH.Px and SOD activity was almost identical with control values. Catalase activity was also determined in hearts after ischemia and reperfusion. After reperfusion, catalase activity appeared elevated at 108.2± 10.9 U/mg protein (n = 6) in heat-shocked hearts while the control value after reperfusion was 82.5 ±4.7 U/mg protein (n = 6). This apparent difference in catalase activity after reperfusion was not statistically significant. Discussion Here we have demonstrated that a brief period of whole-body hyperthermia enhances the recovery of ischemic hearts following reperfusion. Ischemia produced a complete cessation of contractility within 20 minutes with a corresponding progressive elevation of resting tension. These responses to ischemia alone were not affected by prior hyperthermic treatment. However, improved contractile recovery for the heat-shocked hearts was evident within 5 minutes of reperfusion. Reperfusion-related injury is also associated with the release of CK, and the efflux of this enzyme has been used as an index of the degree of tissue injury Within 2 minutes of reperfusion, heat-shocked hearts had signifi- cantly diminished creatine kinase efflux compared with control hearts. Consistent findings associated with ventricular dysfunction following reperfusion of ischemic myocardium are ultrastructural damage of mitochondria.2021 In the present experiments, heat-shocked hearts had reduced ultrastructural injury of mitochondria. It has been suggested that reperfusion injury may be caused by free radical generation and lipid peroxidation of membranes.822 This reperfusion injury may be mediated by depressed catalase activity in reperfused hearts.23 To further explore the nature of the protection seen in the hearts of hyperthermia-treated rats, we assayed the activity of the enzymes that are known to afford protection during oxidative stress This study revealed that the level of the antioxidative enzyme catalase was significantly increased in heatshocked hearts. These observations all indicate a protective influence of the hyperthermic treatment on reperfusion-associated myocardial dysfunction. These findings have implications for understanding the role of the heat-shock response under pathological conditions. Indeed, ischemia induces increased synthesis of mrna encoding for myocardial (dog) SP71.26 Reperfused rat hearts synthesize increasing amounts of SP71 in response to increasing ischemic

5 Currie et al Enhanced Postischemic Ventricular Recovery CONTROL o - - o HEAT-SHOCKED 400 to _) C 200- <-> REPERFUSION DURATION (MIN) FIGURE 3. Graph showing creatine kinase (CK) release from isolated hearts during reperfusion. Each point represents the mean±sem. Description of statistical significance as for Figure I. time.4 In the present study, by preinducing the heat-shock response, indicated by the synthesis and accumulation of SP71, cardiac recovery following ischemia was improved. A possible mechanism for this enhancement of recovery may be related to the induction of the heat-shock response: synthesis and accumulation of SP71 and/or increased concentration of antioxidative enzyme(s). SP71 corresponds to the major inducible heat-shock protein (hsp70-72) seen in mammalian and other cells In most unstressed rat tissues, including heart, the presence (Coomassie stainable) and synthesis of SP71 is undetectable. Two other heat-shock proteins observed in rat hearts are hsp73 and hsp78, each of which is constitutively synthesized and present in hearts.13 In live rats, after a 15-minute episode of hyperthermia (42 C) SP71 is a major product of protein synthesis. However, its synthesis is transient, persisting for less than 24 hours, at which time (24 hours posthyperthermia) there are large accumulations of SP71 in all rat tissues.9 Cells exposed to nonlethal heat treatment developed a transient protection against a subsequent lethal heat treatment. An important function of one or more of the heat-shock proteins might be to confer this measure of protection on cells during a subsequent metabolic trauma. In fact, the development of thermotolerance correlates with the increased synthesis of the heat-shock proteins More specifically, a good correlation exists between the level of the 70 TABLE 1. Antloxidative Enzyme Content (U/mg protein) in Hearts SOD Catalase GSH.Px Control Heat-shocked 17.0±2.2(8) (8) (8) 17.7 ±1.9 (8) 137.0±12.7(8)» 33.2 ±2.4 (8) Values are mean ± SEM (n). *p<0.05fromcontrol value using Student's t test. One unit activity for SOD, catalase, and GSH.Px was previously defined.43 V '. FIGURE 4. Electron photomicrographs showing mitochondrial ultrastructure following reperfusion of isolated rat hearts. Panel A: An example of severely swollen and disrupted mitochondria from a reperfused control heart. This severity of damage was seen in both control hearts but in neither of the two heat-shocked hearts subjected to ultrastructural analysis. Panel B: Mitochondria from a control heart. Both intramitochondrial and intracellular swelling are evident. Panel C: Mitochondria from a heat-shocked heart. Intramitochondrial and intracellular swelling are evident although less severe than that seen in control hearts. Magnification for all panels: x25,000.

6 548 Circulation Research Vol 63, No 3, September 1988 kda heat-shock proteins (SP71) and the sensitivity of cells to thermal stress. Thermotolerance decayed as the concentration of the 70 kda heat-shock proteins (SP71) decreased. 353 «In addition, numerous studies have demonstrated not only an association between the synthesis of heat-shock proteins and the acquisition of thermotolerance, * but also an association between thermotolerance and an increased ability to metabolize free radicals 6-7 by the increased activities of antioxidative enzymes Catalase and SOD added to the perfusion buffer enhance myocardial recovery during reperfusion by limiting lipid peroxidation and improving myocardial energy metabolism Addition of catalase to the perfusion buffer also significantly reduces CK efflux during reperfusion, by limiting membrane damage caused by free radicals. 39 Interestingly, consequences of ischemia and reperfusion are depletion of endogenous catalase, 23 SOD, and GSH.Px 40 activities and, hence, suppressed functional recovery. Ischemia causes a reduction of the protective mechanisms against oxygen toxicity. 41 In the present experiments, whether endogenous catalase was depleted during ischemia and reperfusion remains unresolved. Comparison of freshly excised hearts with reperfused hearts suggests a depletion of catalase after reperfusion for the heat-shocked hearts. On the other hand, the control hearts revealed an apparent increase of catalase after reperfusion. However, even after reperfusion catalase activity was apparently elevated in the heat-shocked hearts compared with the control hearts. There is controversy over the effectiveness of administered antioxidative enzymes. It may be that administered antioxidants, restricted to the extracellular space, are less effective in controlling toxic oxygen radicals than endogenous antioxidative enzymes. In the present experiments, hyperthermic treatment significantly increased the endogenous levels of catalase, and it is attractive to suggest that in the heart, increased catalase affords a salvaging influence following postischemic reperfusion. Here we propose that heat-shock induces tolerance, which affords protection during oxidative stress. Hyperthermia, which induced the major heat-shock protein, also increased the activity of catalase, which is protective during reperfusion. Whether the improved ventricular recovery is due to increased catalase activity or the accumulation of SP71 is not yet resolved and other possibilities remain. One possibility is that SP71, other heat-shock proteins or the hyperthermia itself, is modulating the activity of catalase. Very little is known about rat heart catalase; and so a further possibility exists that SP71 itself is a catalase subunit. We are presently attempting to purify rat heart catalase to check this possibility. The level of catalase in heart tissue is very much lower than the level of GSH.Px when both are normalized to nanomoles H 2 O 2 consumed per minute per milligram protein. Therefore, the relevance of increased catalase activity to improved postischemic ventricular recovery can be raised. There are three reasons why we believe that catalase is an important feature of hyperthermiainduced protection of the myocardium. First, Thayer, 42 using the catalase inhibitor aminotriazole, showed that catalase is a major route for H 2 O 2 detoxification in rat hearts. Second, catalase does not require a cofactor for enzymatic activity whereas GSH.Px requires reduced glutathione (GSH). Should GSH be depleted, GSH.Px cannot function as an antioxidant. Third, catalase is found in the cytosol and also in subcellular particles such as peroxisomes, but GSH.Px is found only in the cytosol. It is reasonable to conclude that compartmental localization of these enzymes may be important in their protective influence. Indeed, in heatshocked hearts, mitochondria appeared to have less ultrastructural damage after ischemia. The mechanistic basis responsible for cardiac dysfunction due to ischemia and reperfusion is extremely complex. One feature that may separate ischemic from reperfusion injury is that the latter, by virtue of rapid reintroduction of molecular oxygen, is associated with the formation of oxyradicals Free radicals have been implicated as major contributing factors to various types of tissue trauma. 44 Increased levels of antioxidative enzyme(s) would decrease the levels of toxic oxygen species. The elevated level of catalase which we observed following hyperthermia provides a very attractive explanation for the general development of tolerance to various types of oxidative stress. Induction of the heat-shock response may be therapeutic for salvaging ischemic myocardium during reperfusion though a mechanism involving increased levels of endogenous catalase. Acknowledgments The authors thank Sandra Dibb, Sheryl Crosby, George Lafontaine, and Brenda Ross for excellent technical assistance. We also thank Dr. James R. Neely for helpful discussion. References 1. Lindquist S: The heat-shock response. Ann Rev Biochem 1986;55: Pelham HRB: Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell 1986; 46: Currie RW, White FP: Trauma-induced protein in rat tissues: A physiological role for a "heat shock" protein. Science 1981 ;214: Currie RW: Effects of ischemia and perfusion temperature on the synthesis of stress-induced (heat shock) proteins in isolated and perfused rat hearts. 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