Catalase Inhibition With 3-Amino-1,2,4-Triazole Does Not Abolish Infarct Size Reduction in Heat-Shocked Rats
Background Recent studies have shown that improved myocardial salvage after heat-shock pretreatment correlates with the amount of induced cardiac heat-shock protein (HSP)72. However, heat shock also induces myocardial catalase activity, potentially reducing free radical–mediated ischemic injury. The aim of the present study was to determine whether catalase inhibition with 3-amino-1,2,4-triazole (3-AT) abolishes the reduction of infarct size conferred by heat-shock treatment in rats.
Methods and Results Myocardial catalase activity was measured in both heat-shocked and control rats 60 minutes after either 3-AT (1000 mg/kg IV) or saline infusion. In separate experiments, heat-shocked and control rats were treated with 3-AT or saline 60 minutes before being subjected to 35 minutes of left coronary artery occlusion and 120 minutes of reperfusion. Infarct size was determined by dual perfusion with triphenyltetrazolium chloride and phthalocyanine blue dye. Heat-shock treatment significantly increased myocardial catalase compared with control animals (180.5±4.8, n=6, versus 86.2±14.7, n=5, units/g wet wt; P<.05). Treatment with 3-AT significantly reduced myocardial catalase activity in both heat-shocked and control animals (29.6±5.7, n=5, and 36.4±15.3, n=6, respectively). Heat-shock treatment significantly reduced infarct size in rats that were both treated and untreated with 3-AT compared with respective control groups (22.5±3.7%, n=26, 28.2±4.0%, n=22, 52.0±3.0%, n=23, and 48.6±3.2%, n=26, respectively; P<.0001 for both heat-shocked groups versus both control groups; infarct mass/risk area mass×100).
Conclusions Catalase inhibition with 3-AT does not abolish the reduction of infarct size in heat-shocked rats.
When exposed to heat shock or hyperthermia, cells in most organisms increase production of a family of proteins called HSPs, or stress proteins.1 Previous studies have demonstrated that induction of HSPs protects from subsequent exposure to an adverse environmental stress.1 2 3 4 5 6 For example, cells initially exposed to sublethal hyperthermia induce HSPs and are then able to tolerate a subsequent, more severe episode of hyperthermia that otherwise would have been lethal.2 3 4 5 6 This phenomenon is called acquired thermal tolerance. In addition, HSP induction by one environmental stress can confer cross-tolerance to a different environmental stress. For example, HSPs induced from exposure to a toxin may protect against a subsequent heat-shock challenge.7
Cardiac cells produce HSPs in response to hyperthermia and various other adverse environmental stresses, including ischemia.8 9 10 11 12 13 14 Furthermore, the heart appears to exhibit cross-tolerance to ischemia after heat-shock treatment and subsequent HSP induction.11 12 13 14 Previous work by Donnelly and coworkers in our laboratory13 demonstrated that rats heat shocked to 42°C and allowed to recover for 24 hours showed marked induction of cardiac HSP72. In addition, we showed that heat-shocked rats had smaller infarcts than control animals when subjected to a prolonged episode of ischemia and reperfusion. Subsequently, Hutter and coworkers14 demonstrated a progressive amount of cardiac HSP72 induction in rats exposed to progressive degrees of hyperthermia. When these rats were subsequently exposed to ischemia and reperfusion, there was a direct correlation between the amount of cardiac HSP72 induced and the reduction in infarct size.
On the other hand, heat shock also increases a number of antioxidants within the heart,16 which could potentially offer protection from oxidative stress such as ischemia and reperfusion. Recently, Currie et al11 and Karmazyn et al15 demonstrated that heat shock induces myocardial catalase activity as well as HSPs. Furthermore, in both studies, isolated perfused hearts obtained from rats that had been heat shocked showed improved functional recovery after global ischemia and reperfusion. In the latter study,15 this enhanced ventricular recovery was abolished when catalase activity was inhibited with 3-AT, an irreversible catalase inhibitor.17 18 19 Thus, it is unclear whether protection against ischemic injury by heat shock is due to induction of HSPs or increased antioxidant activity in the heart. The purpose of the current study was to assess whether catalase inhibition with 3-AT abolishes the reduction of infarct size by heat-shock treatment in rats.
All experiments performed in this study were approved by the Committee on Animal Research at UCSF and were performed in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23, revised 1985).
Animal Model of Heat Shock and Acute Myocardial Infarction
The procedure of heat-shock treatment and a rat model of acute ischemia and reperfusion were used as previously described.13 14 Female Sprague-Dawley rats (weight, 225 to 250 g) were anesthetized with sodium pentobarbital (50 mg/kg body wt IP). An intravenous catheter was introduced into the tail vein of each animal for saline administration. Each rat was heat shocked to 42°C for 20 minutes using a heating lamp and a warming blanket. The core body temperature was monitored using a rectal thermometer. After heat shock, the rats were allowed to cool and recover at room temperature for 24 hours. Control rats were anesthetized at the same time as heat-shocked rats and were allowed to recover at room temperature.
Assessment of Myocardial Catalase Activity
After 24 hours of recovery, both heat-shocked and control rats were reanesthetized (pentobarbital, 50 mg/kg body wt IP) and randomized to receive either 3-AT (1000 mg/kg IV) or normal saline intravenously via the tail vein. The animals were then intubated via a tracheotomy and ventilated with a Harvard rodent respirator. Sixty minutes after 3-AT or saline infusion, a portion of the LV was sampled via a midline sternotomy for myocardial catalase determination in selected animals in each group. Myocardial catalase activity was measured as described previously by Cohen et al.20 Briefly, 100 mg of heart tissue was dounce homogenized on ice in 1 mL of isotonic sodium phosphate buffer, pH 7.4, with 1% ethanol. After centrifugation and the addition of Triton X-100, the supernate was diluted 10-fold with phosphate buffer and 1% ethanol. In one aliquot (test), the reaction was begun with the addition of 6 mmol/L H2O2 in potassium phosphate buffer, pH 7.0, and allowed to proceed for exactly 3 minutes before being terminated by the addition of 6 N H2SO4. In a second aliquot (blank), H2SO4 was added before H2O2. In both aliquots, the remaining H2O2 was quenched by the addition of 0.01 mol/L KMnO4. All reactions were performed in an ice bath. In addition, a standard was prepared containing 0.01 mol/L KMnO4, phosphate buffer, and 6 N H2SO4. Absorption was recorded at 480 nm. Catalase activity was defined as log[(Absstd−Absblank)/(Absstd−Abstest)]×K and expressed as units per gram of wet weight.
Protein Isolation and Western Blot Analysis
In selected rats in each group, a small sample of the LV was harvested for assessment of the presence of HSP72. After each sample was minced with a razor blade and alternately homogenized (Wheaton dounce homogenizer, 15 mL) in 1 mL of lysis buffer (5% sodium dodecyl sulfate, 1% 2-mercaptoethanol) and boiled until tissue particles were no longer present, the samples were quickly frozen in liquid nitrogen. Later the samples were thawed and centrifuged, and precipitate was discarded. The total protein concentration in each sample was determined by a modified Lowry procedure.21 Equal amounts of protein (45 μg) were loaded into lanes of 12.5% polyacrylamide gels. In addition, protein samples from REF maintained at 37°C and heat shocked to 43°C were used as negative and positive controls. After electrophoresis was performed, the proteins were transferred to nitrocellulose paper, and equal protein loads were confirmed by Ponceau staining. The Ponceau stain was then washed off, and the proteins were probed with a primary antibody that is specific for the inducible HSP72 (C92 antibody). The nitrocellulose was then blotted with a rabbit anti-mouse second antibody and developed for visual inspection.
Animal Model of Acute Myocardial Ischemia and Reperfusion
Separate groups of heat-shocked and control rats were randomized to receive either 3-AT (1000 mg/kg IV) or normal saline intravenously as described above and subjected to a protocol of myocardial ischemia and reperfusion.13 14 The animals were intubated via a tracheotomy and ventilated with a Harvard rodent respirator. With a midline sternotomy, a reversible snare occluder was placed around the proximal LCA and tested with a brief (2- to 3-second) occlusion to ensure correct placement of the occluder. After a subsequent 20-minute stabilization period, the LCA was occluded for 35 minutes and then released for 120 minutes. During the period of surgical instrumentation, core body temperature was maintained with a heating pad.
The ischemic risk area and infarct size in each animal were measured as described previously.13 14 22 After 120 minutes of reperfusion, the LCA was reoccluded, and 1 mL of phthalocyanine blue dye was infused into the tail vein, allowing dye to stain the nonischemic portion of the heart. The heart was then excised, rinsed of excess dye, trimmed of right ventricular and atrial tissue, sliced transversely into 2-mm-thick sections, and immersed in a 1% solution of TTC until the viable myocardium stained brick red. The LV sections were fixed in 10% formalin for 24 hours, photographed (Olympus OM-2 camera with a 90-mm macrolens and 2× teleconverter), and weighed. In each photograph, the ischemic risk area (unstained by phthalocyanine blue dye) and the infarcted area (unstained by TTC) were outlined and measured by planimetry. The area from each region was averaged from photographs from each side of each section and multiplied by the weight of tissue in that section. Infarct size was expressed both as a percentage of total LV mass as well as a percentage of the ischemic risk area.
All values are expressed as mean±SEM. Comparisons between groups were assessed by one-way ANOVA with post hoc analysis using the Student-Newman-Keuls test. Statistical significance was defined as P<.05.
Myocardial Catalase Activity
As displayed in Table 1⇓, myocardial catalase activity was significantly increased in rats that were heat shocked and then allowed to recover for 24 hours compared with non–heat-shocked control animals (180.5±4.8 [n=6] versus 86.2±14.7 [n=5], respectively; P<.05). After infusion of 3-AT, myocardial catalase levels were reduced to equivalent levels in both heat-shocked and control rats (29.6±5.7 [n=5] and 36.4±15.3 [n=6], respectively; P<.05 versus rats not treated with 3-AT, P=NS between groups treated with 3-AT).
Stress Protein Levels
Western blot analysis was performed using the C92 antibody, which recognizes only the inducible HSP72. As demonstrated in Fig 1⇓, LV samples from heat-shocked rats demonstrated marked HSP72 induction that was not affected by 3-AT administration. In contrast, LV samples from non–heat-shocked control rats that were either untreated or treated with 3-AT did not demonstrate significant induction of HSP72 (Fig 1⇓). These findings were noted in all hearts studied (n=4 for each group, data not shown). REF cells maintained at 37°C did not show any HSP72 (negative control). In contrast, REF cells that were heat shocked to 43°C and allowed to recover for 24 hours demonstrated the presence of HSP72 (positive control).
Myocardial infarct size was significantly reduced in heat-shocked rats, both treated and untreated with 3-AT, as demonstrated in Table 2⇓ and Fig 1⇑. Furthermore, catalase inhibition with 3-AT had no significant effect on infarct size in either heat-shocked or control rats (Table 2⇓, Fig 1⇑). There were no differences in the size of the ischemic risk area among the four groups (Table 2⇓).
Our results indicate that catalase inhibition with 3-AT does not abolish infarct size reduction in heat-shocked rats. Heat-shocked rats that were treated with 3-AT had a similar reduction in infarct size compared with heat-shocked rats that were untreated with 3-AT before prolonged coronary artery occlusion and reperfusion. These data indicate that infarct size reduction noted in heat-shocked rats is not dependent on induced myocardial catalase activity.
A number of investigators have demonstrated a potential role for heat-shock treatment and the induction of HSPs to protect the heart from subsequent ischemic injury. Currie and coworkers11 12 and Karmazyn and coworkers15 demonstrated that isolated, perfused hearts obtained from heat-shocked rats had better functional recovery after global ischemia and reperfusion than did hearts obtained from control rats. Donnelly et al13 showed that rats heat shocked to 42°C expressed a high level of myocardial HSP72 and had smaller infarcts after prolonged coronary artery occlusion and reperfusion than did control animals. This observation by Donnelly et al was confirmed by Currie et al23 and Walker et al24 in the rabbit model of ischemia and reperfusion. Furthermore, Hutter et al14 demonstrated a correlation between the amount of HSP72 induction and the degree of infarct size reduction. Rats subjected to progressively increased amounts of whole body hyperthermia demonstrated stepwise increases in the amount of cardiac HSP72 induction. After prolonged coronary artery occlusion and reperfusion, there was a progressive reduction in infarct size that correlated with the amount of HSP72 induced. Similarly, the degree of functional recovery of an isolated rabbit papillary muscle subjected to hypoxia and reoxygenation has been correlated with the amount of HSP70 induced by heat shock.25 Finally, a recent study by Marber et al26 demonstrated reduced contractile dysfunction and reduced infarct size in isolated, perfused transgenic mouse hearts that overexpress inducible HSP70 after being subjected to global ischemia and reperfusion. Although these studies indicated a possible relation between the induction of myocardial HSPs and protection from ischemic injury, the mechanism of protection is unclear.
On the other hand, there are considerable data to suggest that heat-shock treatment induces antioxidant activity within the heart11 12 15 16 and may attenuate the release of free radicals.27 However, the importance of this increased antioxidant activity in protecting the heart from ischemic injury is unclear. Currie et al11 and Karmazyn et al15 suggested that catalase induction may mediate the cardiac protection in animals that have undergone prior heat-shock treatment. Currie et al11 demonstrated that rat hearts obtained from heatshocked animals had enhanced postischemic ventricular recovery. These rat hearts had both increased levels of HSPs and increased myocardial catalase activity.11 The apparent importance of myocardial catalase activity in promoting postischemic ventricular recovery was demonstrated by Karmazyn et al,15 who showed that catalase inhibition with 3-AT abolished this effect in isolated, perfused rat hearts obtained from heat-shocked rats. These investigators concluded that the improved postischemic recovery in heat-shocked rats was in large part due to induction of catalase activity and its ability to neutralize H2O2, thereby decreasing membrane peroxidation. More recently, Plumier et al28 demonstrated that transgenic mouse hearts that overexpress human HSP70 exhibited improved recovery of contractile force after ischemia and reperfusion compared with nontransgenic mouse hearts. Because the two groups had similar myocardial catalase activity, it appears that myocardial HSP70 rather than catalase activity may have played a more important role in promoting functional recovery after ischemia and reperfusion.
Although catalase inhibition abolished the enhanced postischemic functional recovery in isolated hearts obtained from heat-shocked rats,15 the current study indicates that it does not affect infarct size in heat-shocked hearts in the in vivo rat model of ischemia and reperfusion. Treatment with 3-AT and inhibition of catalase activity failed to increase infarct size in either heat-shocked or non–heat-shocked control rats. This apparent lack of influence of myocardial catalase activity on infarct size in our study is consistent with the observations of others.26 29 In dogs subjected to 90 minutes of ischemia followed by 6 hours of reperfusion, pretreatment with catalase failed to reduce infarct size.29 Furthermore, in isolated transgenic mouse hearts that overexpress HSP70, infarct size was significantly reduced after ischemia and reperfusion, yet myocardial catalase activity was unaltered.26 Thus, although catalase activity may play a role in enhancing functional recovery of postischemic stunned myocardium, it does not appear to be an important determinant of infarct size in the rat and canine models of myocardial ischemia and reperfusion.
Although the mechanism of protection from ischemic injury after heat-shock treatment is unclear, there is a growing body of evidence to suggest that it may be related to the induction of HSPs and their ability to bind to damaged proteins within the cell. Recently, Mestril and coworkers30 demonstrated that transfected cell lines that overproduce inducible HSP70 were protected from subsequent exposure to hypoxia and hypoglycemia, a protocol designed to simulate ischemia. Beckmann and coworkers31 showed that HSP72/73 recognized and bound to mature polypeptides that were rendered nonnative due to thermal denaturation. Potentially, HSP72/73 may stabilize denatured or partially denatured proteins during conditions of ischemic stress, facilitating either their repair or removal and enhancing cell viability.32 However, it should be emphasized that heat-shock treatment results in the synthesis of a variety of proteins of varying molecular weights that may play a protective role in subsequent exposure to adverse environmental stress such as ischemia. Furthermore, it should be noted that the heat-shock treatment can alter intracellular calcium levels,33 34 ATP concentration,34 and pH,33 35 all of which could affect cell viability. Further studies are needed to further define the protective nature of the heat-shock response.
The present study indicates that catalase inhibition with 3-AT does not abolish the reduction of infarct size in heat-shocked rats. Therefore, infarct size reduction after heat-shock treatment does not appear to be dependent on the induction of myocardial catalase activity. Although data exist to suggest a role for induction of HSP72 in reducing ischemic injury in heat-shocked rats, further studies are needed to define the mechanism(s) of protection and the potential role of other inducible HSPs.
Selected Abbreviations and Acronyms
|LCA||=||left coronary artery|
|LV||=||left ventricle; left ventricular|
|REF||=||rat embryo fibroblasts|
This study was supported in part by Grants-in-Aid from the American Heart Association (92007600) and the California Affiliate of the American Heart Association (95-225). We thank Aileen Holmes for helping to prepare the manuscript.
Reprint requests to Christopher L. Wolfe, MD, University of California, San Francisco, Moffitt Hospital, M-1186, San Francisco, CA 94143-0124.
- Received April 26, 1995.
- Revision received June 5, 1995.
- Accepted July 24, 1995.
- Copyright © 1995 by American Heart Association
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