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(Circulation. 1996;94:1408-1411.)
© 1996 American Heart Association, Inc.

Articles

Overexpression of Heat Shock Protein 72 in Transgenic Mice Decreases Infarct Size In Vivo

Jonathan J. Hutter, BS; Ruben Mestril, PhD; Eunice K.W. Tam; Richard E. Sievers, BS; Wolfgang H. Dillmann, MD; Christopher L. Wolfe, MD

the Cardiovascular Division, Department of Medicine and the Cardiovascular Research Institute, University of California, San Francisco (J.J.H., E.K.W.T., R.E.S., C.L.W.), and the Endocrinology Division, Department of Medicine, University of California, San Diego (R.M., W.H.D.).

Correspondence to Christopher L. Wolfe, MD, 505 Parnassus Ave, San Francisco, CA 94143-0124. E-mail WOLFE@CARDIO.UCSF.EDU.

	Abstract

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Background Previous studies have demonstrated that induction of heat shock protein (HSP) 72 by whole-body hyperthermia reduces infarct size in an in vivo model of ischemia and reperfusion. Furthermore, hearts obtained from transgenic mice that overexpress HSP72 demonstrate improved functional recovery and decreased infarct size in vitro after global ischemia and reperfusion.

Methods and Results To test the hypothesis that overexpression of HSP72 in transgenic mice reduces infarct size in vivo, transgenic mice that were heterozygous for a rat HSP70i gene ([+]HSP72) and transgene-negative littermate controls ([-]HSP72) were subjected to 30 minutes of left coronary artery occlusion followed by 120 minutes of reperfusion. Core body temperature was monitored with a rectal thermometer and maintained between 36.5°C and 37.0°C with a heating pad. Infarct size, determined by dual staining with triphenyltetrazolium chloride and phthalocyanine blue dye, was smaller in [+]HSP72 mice compared with [-]HSP72 mice (12.7±2.8% [n=7] versus 33.4±4.5% [n=6], infarct size/risk area, respectively; P<.05; mean±SEM).

Conclusions Overexpression of HSP72 reduces infarct size in this in vivo transgenic mouse model of myocardial ischemia and reperfusion.

Key Words: myocardial infarction • ischemia • reperfusion • proteins

	Introduction

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Recent advances in molecular genetics have allowed further elucidation of the protective role of HSP72 against myocardial infarction induced by ischemia. This class of proteins has been the focus of several investigations in the past decade because of the ability of these proteins to decrease cell vulnerability to hyperthermia and other environmental insults and to enhance cellular function.^{1 2 3 4} More recent studies^{5 6 7 8 9 10} have demonstrated the protection that these proteins confer in the heart after global ischemia and reperfusion. Using an in vivo model in rats that had been exposed to whole-body hyperthermia before ischemia and reperfusion, we and others have demonstrated reduced infarct size in association with the induction of HSP72.^{11 12 13 14} Furthermore, Hutter et al¹³ demonstrated a direct correlation between the amount of induced HSP72 and improved myocardial salvage assessed as infarct-size reduction. A similar correlation between the concentration of induced HSP72 and resistance to substrate deprivation was demonstrated in the isolated papillary muscle model.¹⁵

The specific role of HSP72 in myocardial protection has been difficult to ascertain thus far because the model of hyperthermic induction includes increased expression of a variety of stress proteins along with cellular changes in ATP,¹⁶ pH,^{17 18} and calcium.^{16 17} In addition, a study by Walker et al¹⁰ suggests the possibility of alterations in the blood of heat-shocked animals that may diminish the protective effect of HSP72 induction. These changes potentially alter the functional state of a cell, which makes a definitive causal relationship between HSP72 and myocardial protection difficult to prove.

The recent generation of myogenic cell lines and transgenic mice that overexpress HSP72 has been a major development in overcoming these limitations.^{19 20 21} Mestril et al¹⁹ showed that transfected embryonic rat heart-derived cells that overexpress HSP72 had improved survival after being subjected to hypoxia and hypoglycemia, an ischemic-like stress. Using transgenic mice that overexpress HSP72, Marber et al²⁰ demonstrated an increase of resistance to ischemic injury in Langendorff-perfused isolated heart preparations subjected to global ischemia and reperfusion. A similar study by Plumier et al²¹ showed improved postischemic myocardial recovery, again using an in vitro mouse heart preparation. As of yet, an in vivo model of ischemia and reperfusion in transgenic mice has not been accomplished. The present study used an in vivo model of coronary artery occlusion and reperfusion in transgenic mice that overexpress HSP72 to establish a direct role for the overexpression of HSP72 and protection from ischemic injury.

	Methods

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Animal Model of Acute Myocardial Ischemia and Reperfusion
These experiments were conducted within the guidelines established by the Committee on Animal Research at the University of California, San Francisco. Transgenic mice that were heterozygous for a rat HSP70i gene ([+]HSP72) and transgene-negative littermate controls ([-]HSP72) were provided by Dr Wolfgang Dillmann at the University of California at San Diego. As previously described, the transgenic mice were generated by use of a chimeric transgene that consisted of the rat inducible HSP72 gene inserted into the pCAGGS vector, which is under the control of the human cytomegalovirus enhancer and chicken ß-actin promoter.²⁰ This results in overexpression of HSP72 in cardiac muscle, skeletal muscle, and brain (R. Mestril, PhD, and W.H. Dillman, MD, unpublished observation, 1995).

Both [+]HSP72 (n=7) and [-]HSP72 (n=8) mice were anesthetized (pentobarbital 50 mg/kg IP), tracheostomized, and ventilated on a Harvard rodent respirator before midline sternotomy. After the heart was exposed and the pericardium removed, a reversible snare occluder, consisting of 9.0 Dermalon (Davis and Geck) and a short length of polyethylene tubing, was placed around the proximal LCA with the aid of a dissecting microscope. Optimal suture placement enters just inferior to the midline of the left atrial appendage and exits 2 mm superiorly and 1 mm laterally into the right ventricular outflow tract. Once in place, a brief occlusion was performed to visually confirm induced ischemia and reversibility. The mice then underwent 30 minutes of LCA occlusion followed by 120 minutes of reperfusion. During this time, temperature was monitored by use of a rectal thermometer and maintained between 36.5°C and 37.0°C with use of a heating pad and heating lamps.

Infarct Sizing
Infarct sizing was performed as described in previous studies.^{11 13 14 22} After 120 minutes of reperfusion, the LCA was reoccluded and 0.2 mL phthalocyanine blue dye was injected into the LV cavity with a 22-gauge needle positioned in the apex of the heart. The needle was kept in place while the dye perfused all nonischemic tissue. The heart then was excised immediately and rinsed in water to remove excess dye, the atria and right ventricular free wall were removed, and the remaining LV was sectioned transversely from apex to base into five 2-mm-thick slices. These samples were incubated in a TTC solution for 12 minutes to stain the viable myocardium brick red and then fixed in a 10% formalin solution for 24 hours. Each slice was then photographed (Olympus OM-2 camera with a 90-mm macro lens and a 2x teleconverter) and weighed (Mettler LAE 200 balance, Mettler Instrument Corp). We prepared photographs of both sides of each slice (five per heart) by outlining the total slice area, the risk area (unstained by the phthalocyanine blue), and the infarcted regions (unstained by the TTC). These areas were quantified by use of planimetry, averaged from both sides of each slice. During planimetry, the operator was blinded as to the genotype of the animal. The fractions of both risk area to total slice size and infarct size to total slice size were calculated and multiplied by the weight of that slice to determine risk area and infarct weight per slice. Infarct size was expressed as a percentage of LV mass and as a percentage of the ischemic risk area.

Protein Isolation and Western Blot Analysis
In separate experiments, mouse hearts from [+]HSP72 mice (n=3) and [-]HSP72 mice (n=3) were perfused on a Langendorff-perfusion apparatus with perfusion buffer (M199 [GIBCO, BRL] 9.6 g/L, taurine 0.625 g/L, carnitine 0.3 g/L, creatine 0.4 g/L, BSA 1.0 g/L, 1% penicillin, streptomycin, fungizone, and HEPES 2.0 g/L) containing collagenase (1 mg/mL) for 45 minutes. The hearts were then cut into small pieces and placed into a beaker that contained perfusion buffer and BSA. We further disrupted the tissue by pipetting with a wide-bore pipette tip, and the supernatant was stored at 37°C. Supernatants that contained disrupted cardiac cells were centrifuged at 300 rpm for 2 minutes. The supernatants were saved as the nonmyocytic fractions, as confirmed by light microscopy. The pellet was resuspended in additional buffer, washed, and recentrifuged until >90% of the cells in the pellet were cardiomyocytes, as determined by light microscopy. Protein concentrations of both the nonmyocytic (supernatant) and myocytic (pellet) fractions were determined by a modified Bradford method²³ (Bio-Rad protein assay kit, Bio-Rad Laboratories, Inc).

Western blot analysis was performed as previously described.²¹ Protein samples (40 µg) were loaded onto 8% polyacrylamide gels. After electrophoresis was performed, the proteins were transferred to nitrocellulose paper and probed with an alkaline phosphatase-conjugated mouse monoclonal primary antibody that is specific for inducible HSP72 (SPA-810 AP, Stress Gen, Biotechnologies Corp). The nitrocellulose was then developed with diaminobenzidine tetrahydrochloride (alkaline phosphatase substrate kit I, Vector Laboratories) for visual inspection. Alternatively, on separate gels, the proteins were probed with a rabbit polyclonal primary antibody that recognizes both constitutive HSP73 and inducible HSP72.⁷ The nitrocellulose was then developed with use of the biotin-streptavidin-peroxidase kit (Vectastain, ABC kit, Vector Laboratories) and diaminobenzidine tetrahydrochloride (DAB kit, Vector Laboratories).

Statistics
The presented values are expressed as mean±SEM. Comparisons between the two groups were assessed for significance by an unpaired Student's t test. Statistical significance was defined as a value of P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Western blot analyses of protein extracts from the myocyte-enriched fraction and the nonmyocytic fraction of hearts obtained from representative [+]HSP72 and [-]HSP72 mice are demonstrated in Fig 1. Both the myocyte-enriched and nonmyocytic fractions obtained from the hearts of [+]HSP72 mice contained inducible HSP72 (Fig 1A and 1B). However, there was substantially more HSP72 in the myocyte-enriched fraction compared with the nonmyocytic fraction (Fig 1A and 1B). Although constitutive HSP73 was found in both myocyte-enriched and nonmyocytic fractions of [-]HSP72 mice (Fig 1B), no inducible HSP72 was detected in either the myocyte-enriched or nonmyocytic fractions obtained from [-]HSP72 mice (Fig 1A and 1B). Myocardial protein extracts from all [+]HSP72 (n=3) and [-]HSP72 mice (n=3) studied demonstrated similar patterns as above (data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 1. Western blots of protein extracts from [+]HSP72 and [-]HSP72 mice. Mouse hearts were perfused on a Langendorff-perfusion apparatus with buffer that contained collagenase and centrifuged, and the supernatant was saved as the nonmyocytic (non-myoc. or non-myo) fraction. The pellet was resuspended, washed, and recentrifuged until >90% of the cells in the pellet were cardiomyocytes, which made up the myocyte-enriched (myocyte) fraction. Equal amounts of protein (40 µg) were loaded onto 8% polyacrylamide gels for PAGE and transferred to nitrocellulose. A, The proteins were probed with an alkaline phosphatase-conjugated mouse monoclonal primary antibody specific for inducible HSP72 (HSP70i). The nitrocellulose was then developed with diaminobenzidine tetrahydrochloride. B, The proteins were probed with a rabbit polyclonal primary antibody that recognizes both constitutive HSP73 (HSP70c) and inducible HSP72. The nitrocellulose was then developed by use of the biotin-streptavidin-peroxidase kit and diaminobenzidine tetrahydrochloride. Note that although both myocyte-enriched and nonmyocytic fractions of transgene-positive mice contain HSP72, there is substantially more in the myocyte-enriched fraction. No HSP72 was detected in either myocyte-enriched or nonmyocytic fractions of transgene-negative mice.

After mice were subjected to 30 minutes of LCA occlusion and 120 minutes of reperfusion, there were two deaths in the [-]HSP72 group and none in the [+]HSP72 group. Myocardial infarct size was significantly smaller in [+]HSP72 mice compared with [-]HSP72 mice, both when expressed as a percentage of LV mass and as a percentage of risk area (Fig 2). There was no significant difference in ischemic risk area between the two groups (Fig 2).

View larger version (16K): [in this window] [in a new window]   Figure 2. Infarct size in [+]HSP72 and [-]HSP72 mice after 30 minutes of LCA occlusion and 120 minutes of reperfusion. [+]HSP72 mice demonstrated significantly smaller infarcts compared with [-]HSP72 mice despite similar ischemic risk areas between the two groups.

	Discussion

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The induction of a class of proteins termed HSPs in cells exposed to environmental stimuli has been well established. These proteins have been shown to protect against subsequent insults, including a cross-tolerance to stimuli of a different type than the original; ie, a cell exposed to hyperthermia will acquire resistance to damage from a later ischemic episode. Previous studies have revealed an association between the induction of HSP72 and myocardial salvage after ischemia and reperfusion. In the present study, the protective role of HSP72 was investigated by use of an in vivo model of coronary artery occlusion and reperfusion in transgenic mice that overexpress HSP72. We found a significant decrease in infarct size in these transgenic mice compared with transgene-negative controls.

A correlation between HSPs induced by a preliminary hyperthermic episode and subsequent thermotolerance has been demonstrated in earlier investigations. Subjeck et al³ monitored HSP induction after 45°C pretreatment via [³⁵S]-methionine incorporation and demonstrated a relationship between protein synthesis and cell survival after a second hyperthermic episode. Landry et al⁴ further tested the hypothesis that HSPs cause thermotolerance. In that study, the time course of HSP synthesis and degradation in Morris hepatoma 7777 cells after 45°C heat shock was shown to parallel the onset and decay of thermotolerance.

These initial studies that compared induction kinetics implied a distinct relationship between HSP induction and thermotolerance in cell cultures. That similar heat-shock treatment could confer myocardial protection to ischemic injury became the focus of more recent studies that used whole-heart preparations. Currie and coworkers⁸ used heart contractility and creatine kinase release as measures of postischemic ventricular recovery in an isolated, perfused heart preparation. Rats previously exposed to 15 minutes of 42°C hyperthermia demonstrated improved recovery of contractile force and reduced creatine kinase release after ischemic perfusion. Furthermore, Currie et al⁹ went on to show that this improved recovery in isolated hearts from heat-shocked rats was unrelated to high-energy phosphate levels during the ischemic period in heat-shocked versus control hearts.

Other studies that used infarct sizing to infer the protective role of inducible HSPs after ischemia and reperfusion demonstrated improved myocardial salvage after hyperthermic pretreatment. Using an in vivo model of ischemia and reperfusion, Donnelly et al¹¹ found decreased infarct size in rats exposed to prior heat shock. In addition, heat-shocked rats demonstrated greater myocardial salvage than those rats pretreated with a 20-minute ischemic episode. A qualitative assessment of the degree of HSP72 induction revealed higher levels of HSP72 induction in response to heat shock compared with ischemic pretreatment, which indicates a potential association between the degree of HSP72 induction and protection from ischemic injury. Currie et al¹² used a similar model in rabbits to demonstrate reduced infarct size in response to HSP71 induction with heat shock and to show that this cardioprotection is transient. Both infarct-size reduction and HSP71 levels abated after 40 hours. Finally, a direct correlation between HSP72 induction and infarct-size reduction was established in a study by Hutter et al¹³ that used an in vivo model of global ischemia and reperfusion in rats with and without prior heat shock. Using a quantitative assay of HSP72 induction, they demonstrated progressive degrees of HSP72 induction with heat-shock pretreatment to progressively higher temperatures. Furthermore, there was a linear correlation between the degree of HSP72 induction and the amount of infarct-size reduction in groups of animals heat shocked to progressively higher temperatures.

Recently, Marber et al²⁰ used a strain of transgenic mice that overexpress HSP70i to assess the role of HSP72 in protecting the heart from ischemic injury. Isolated, perfused heart preparations from these mice revealed a marked recovery in contractile function as well as significant reductions in infarct size and creatine kinase release after ischemia and reperfusion. Similar results were found by Plumier and coworkers.²¹ However, both of these studies used nonworking, buffer-perfused hearts. Thus, the applicability of these findings to the in vivo working, blood-perfused heart, in which ischemic injury might be exacerbated by neutrophil-mediated free radical injury during reperfusion, is uncertain. The present study used the in vivo model of ischemia and reperfusion in transgenic mice that overexpress HSP72. Our results demonstrate that overexpression of HSP72 in transgenic mice leads to reduced infarct size. This study indicates a cause-and-effect relationship between the overexpression of HSP72 and infarct-size reduction because it avoids the cellular and physiological changes that have been shown to accompany hyperthermic pretreatment.

The mechanisms by which HSP72 confers myocardial protection are speculative. The pathological changes that occur in cells during ischemia include alterations in ionic balance, pH, and ATP levels. Subsequent reperfusion results in calcium influx and free radical stress. This milieu likely denatures existing proteins and halts further translation. Beckman et al²⁴ have shown that HSP72 and HSP73 transiently bind cells that have been rendered nonnative because of thermal denaturation. After ischemic stress, it is hypothesized that HSP72 functions as a molecular chaperone, stabilizing denatured and partially denatured proteins until they are repaired or excluded from the cell, thereby decreasing irreversible ischemic injury.¹¹

Conclusions
Transgenic mice that overexpress HSP72 had reduced infarct size after ischemia and reperfusion compared with transgene-negative controls. These results indicate a causal relationship between the overexpression of HSP72 and protection from ischemic injury in this in vivo model of coronary artery occlusion and reperfusion.

	Selected Abbreviations and Acronyms

 

HSP = heat shock protein [-]HSP72 = transgene-negative mice [+]HSP72 = transgene-positive mice LCA = left coronary artery LV = left ventricle, left ventricular TTC = triphenyltetrazolium chloride

	Acknowledgments

 
This study was supported in part by the California Affiliate of the American Heart Association (grant No. 95-225 to Dr Wolfe), an American Heart Association Grant-in-Aid (No. 94/1564) to Dr Mestril, and a Career Development Award from the NIH (No. HL-03150/01) to Dr Mestril.

	Footnotes

 
Presented in part at the 45th Annual Scientific Session of the American College of College of Cardiology, Orlando, Fla, March 1996.

Received January 29, 1996; revision received March 18, 1996; accepted April 1, 1996.

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