Drug-Induced Heat-Shock Preconditioning Improves Postischemic Ventricular Recovery After Cardiopulmonary Bypass
Background Heat-stress preconditioning of mammalian heart has been found to confer protection against ischemic reperfusion injury. Heat shock is generally provided by warming the animal by mechanical means, which is often impractical in a clinical setting. Amphetamine, a sympathomimetic drug, can elevate the body temperature as a result of enhanced endogenous lipolysis. In this study, we examined the effects of heat shock induced by amphetamine on postischemic myocardial recovery in a setting of coronary revascularization for acute myocardial infarction.
Methods and Results Adult Yorkshire swine were injected with amphetamine (3 mg/kg IM) (n=12), and body temperature was continuously monitored. For control studies, the pigs were injected with saline (n=12). Five swine in each group were killed after 3 hours to obtain biopsies of vital organs to measure heat-shock protein (HSP) mRNAs. After 40 hours, the remaining 7 pigs in each group were placed on cardiopulmonary bypass, and the isolated, in situ heart preparations were subjected to 1 hour of occlusion of the left anterior descending coronary artery followed by 1 hour of global hypothermic cardioplegic arrest and 1 hour of reperfusion. Postischemic myocardial performance was monitored by measuring left ventricular (LV) pressure, its dP/dt, myocardial segment shortening, and coronary blood flow. Cellular injury was examined by measurement of creatine kinase release. The antioxidant enzymes superoxide dismutase and catalase were also assayed. Amphetamine treatment was associated with the induction of mRNAs for HSP 27, HSP 70, and HSP 89 in all the vital organs, including heart, lung, liver, kidney, and brain. Amphetamine also enhanced superoxide dismutase and catalase activities in the heart. Significantly greater recovery of LV contractile functions was noticed, as demonstrated by improved recovery of LV developed pressure (61% versus 52%), LV dP/dtmax (52% versus 44%), and segment shortening (46.2% versus 10%) and reduced creatine kinase release in the amphetamine group.
Conclusions The results demonstrate that amphetamine can induce whole-body heat shock that can precondition the heart, enhancing cellular tolerance to ischemia-reperfusion injury. Amphetamine is a sympathomimetic drug that may be used for preconditioning.
Recent years have witnessed the development of a novel concept for myocardial preservation, based on the fact that enhancement of the endogenous cellular defense system provides each cell with new protein synthesis and thereby the means to protect itself when it is more susceptible to injury. By use of this concept, a variety of preconditioning protocols have been developed. Preconditioning of the heart by repeated stunning can delay the onset of further irreversible injury1 or even reduce the subsequent postischemic ventricular dysfunction2 3 and incidence of arrhythmias.4 Such myocardial preservation by repeated short-term reversible ischemia led to the development of the concept of stress adaptation. Consequently, a number of investigators developed new ideas of preconditioning, which include adenosine,5 potassium channel opening,6 hypoxia,7 heat shock,8 and oxidative stress.9
Currie and colleagues10 were probably the first group to demonstrate enhanced postischemic ventricular recovery after subjecting the heart to heat shock. The results of many recent studies11 12 13 now support the earlier observation by Currie and further imply that the heart can be preconditioned by heat shock to prevent ischemia-reperfusion injury.14
The conventional method of inducing whole-body heat shock is by warming the animal by immersing it in warm water15 or by wrapping it with an electric blanket.16 However, it is extremely difficult to raise the temperature of an internal organ such as the heart to 42°C to 43°C without causing severe burns. This is not only because the heart is protected by skin, muscle, and other tissues but also because of the ability of a live animal to maintain its own body temperature against hypothermia or hyperthermia.17 Moreover, it is practically impossible to raise the body temperature of large animals, such as pigs or dogs, by this method. In this study, we attempted to resolve this problem by inducing heat shock with a sympathomimetic drug, amphetamine, that has been known to increase the body temperature by enhancing endogenous lipolysis.18 Amphetamine was found to induce the expression of the mRNAs for HSP 27, HSP 70, and HSP 89 within 3 hours in all the vital organs of the pig, demonstrating its ability to induce whole-body heat shock.
Isolated In Situ Swine Heart Preparation
Yorkshire pigs of either sex weighing 20 to 25 kg were used in our study. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 86-23, revised 1985). Pigs were divided into two groups. The experimental group, consisting of 12 pigs, received amphetamine sulfate (3 mg/kg IM), and the rectal temperature was monitored. The control animals (n=12) received saline injection only. Five animals from each group were killed after 3 hours for collection of tissue biopsies for subsequent analysis of HSP gene expression. Forty hours after the injection, the remaining animals were tranquilized with ketamine (Ketaject, 50 mg/kg) and anesthetized with an intravenous injection of sodium pentobarbital (Nembutal, 25 mg/mL). Each animal was supported by controlled respiration with room air by a Harvard respirator, and the chest was opened through a median sternotomy as described elsewhere.19 After heparinization with sodium heparin (500 U/kg), cardiopulmonary bypass with a pediatric bubble oxygenator (Bentley BOS-5, Baxter Healthcare Corp) primed with 1 L of plasmalyte solution was begun. Arterial inflow was via the ascending aorta, and venous drainage was via cannulas placed in the right atrium. The heart was isolated from the systemic circulation and maintained in a perfused, oxygenated state by cross-clamping of the cannulated ascending aorta as described previously.19 The systemic perfusion was then discontinued. After the systemic circulation was drained into the oxygenator, both the superior and inferior venae cavae and the azygos vein were ligated. The coronary effluents were collected through a cannula inserted into the right ventricular outflow tract. The heart was perfused at a constant pressure of 75 mm Hg with normothermic (37°C) blood.
LV Global and Regional Functions
Isovolumetric LV pressure measurements were performed with a Millar Mikro-Tip catheter transducer (Millar Instruments, Inc), which was placed within a 10-mL compliant balloon and inserted into the left ventricle through an apical stab wound.19 Balloon volume was maintained at 10 mL for each global function measurement. The LV dP/dtmax and LVDP were also measured. LV segment function in the LAD-distributed region was measured with two piezoelectric crystals (Triton Technology) implanted in a circumferential plane. The transducers were placed near the endocardium through small epicardial punctures, and the two crystals were separated by ≈1 cm. For the measurements of segment shortening, the balloon was deflated, and measurements were made in an empty beating heart. Tracings of segment length were recorded on a Honeywell AR-6 recorder (PPG Biochemical Systems, Honeywell, Inc) interfaced with an oscilloscope (Tektronics, Inc) coupled to a sonomicrometer (Triton Technology). End-diastolic length (EDL) and end-systolic length (ESL) were identified on the recordings. The percent segment shortening was calculated as [(EDL−ESL)/EDL]×100.
Baseline measurements were taken after 15 minutes of stabilization at normothermic perfusion, consisting of LVDP, LV dP/dtmax, myocardial segment shortening, CBF, and CK release. The LAD was then occluded distal to the first diagonal branch for 60 minutes. After 60 minutes of LAD occlusion, the aortic perfusion was stopped, and 50 mL of cold (4°C) potassium crystalloid cardioplegic solution was infused into the aortic root to arrest the heart. Cardioplegia reinfusion was made every 15 minutes during 60 minutes of cardioplegic arrest. The LAD snare was removed just before the second injection of cardioplegia. The myocardial temperature was monitored by a thermistor inserted into the ventricular septum and was maintained with cold saline between 6°C and 10°C during cardioplegia. Normothermic reperfusion was performed for 60 minutes after cardioplegic arrest. In each study, the heart was paced both before and after ischemia at 120 beats per minute by ventricular pacing. Hemodynamic and metabolic measurements were taken after 15 minutes and 60 minutes of reperfusion (Fig 1⇓). At the end of each experiment, LV biopsies were taken for biochemical determination.
CBF and CK Release
CBF was measured by timed collection of coronary effluent.19 The CK activity of coronary effluent was determined by the enzymatic assay method by use of a CK assay kit (Sigma Diagnostics). CK release was determined by measurement of CK accumulation over time.20
Induction of HSP mRNA Expression
Total RNA was extracted from the tissues by the acid guanidinium thiocyanate–phenol–chloroform method, and the concentration of RNA was estimated by measurement of the optical density (OD) of the preparation at 260 nm (1 OD=40 μg RNA/mL).21 The ratio between the readings at 260 and 280 nm (OD260/OD280) provided the estimate of the purity of the isolated RNA. The isolated RNAs had a ratio between 1.94 and 2.05 (pure preparation should have a ratio of 2.0). This preparation was further verified by running RNA on 1% agarose gel stained with ethidium bromide. RNA (10 μg) from each sample was subjected to 1% agarose/2.2 mol/L formaldehyde gel electrophoresis and then transferred to nylon membrane (Gene Screen Plus, NEN Research Products) by capillary blot as described previously.20 The membranes were baked under vacuum at 80°C for 2 hours and prehybridized in a solution that contained 50% formamide, 6×SSPE, 5×Denhardt’s reagents, 0.9% SDS, and 100 μg/mL denatured salmon testes DNA at 42°C for 1 hour. They were then hybridized in a solution containing 5% formamide, 6×SSPE, 1% SDS, and 100 μg/mL of denatured salmon testes DNA with HSP cDNA probes (StressGen) at 42°C for 20 hours. HSP 27 probe was a 400-bp Pst I fragment of the human cDNA, HSP 70 (inducible) probe was a 4-kb EcoRI fragment of the human cDNA, and HSP 89-α probe was a 1.3-kb Pst I fragment of the human cDNA. β-Actin probe (Oncor), a 770-bp fragment of chicken cDNA, was used as a housekeeping gene. All the cDNA probes were labeled with [α-32P]dCTP by use of a random-primed DNA labeling kit (Boehringer Mannheim Biochemica). The specific activity was ≈0.7×109 dpm/μg.
After the membranes were washed with 2×SSC at room temperature for 5 minutes (twice), with 2×SSC and 1% SDS at 60°C for 30 minutes (twice), and with 0.1×SSC at room temperature for 30 minutes (once), autoradiograms were obtained after exposure to Kodak X-Omat RP films at −70°C (NEN Research Products). Each hybridization was repeated at least three times with different membranes. After each hybridization, the residual cDNA was removed and rehybridized with β-actin cDNA probe, the results of which served as control. The autoradiogram was quantitatively evaluated by computerized gel scanning as described previously. The results of densitometric scanning were normalized relative to the signal obtained for β-actin cDNA.
The tissue homogenate was prepared by homogenization of weighed amounts of tissue in a measured volume of ice-cold Tris-sucrose buffer containing 0.25 mol/L sucrose, 10 mmol/L Tris-HCl, 1 mmol/L EDTA, and 0.5 mmol/L DTT, pH 7.5. After centrifugation at 1000g for 15 minutes, the supernatant was centrifuged at 10 000g for 20 minutes to obtain the mitochondrial pellet that was used as a source of Mn-SOD9 The supernatant was again subjected to centrifugation at 105 000g for 1 hour. The supernatant was used to assay Cu/Zn-SOD. Catalase was estimated as described previously.9 SOD activity was assayed by its inhibitory action on the superoxide-dependent reduction of ferricytochrome by xanthine–xanthine oxidase.13 The final concentrations in the assay medium (total volume, 1 mL) were cytochrome C 100 μmol/L, hypoxanthine 100 μmol/L, Tris/HCl 10 mmol/L, and enzyme protein 50 to 80 μg. The reaction was initiated by addition of 8 mU xanthine oxidase. Catalase was estimated by measurement of the decreases in the absorbance of H2O2 at 240 nm.21 Final concentrations in the assay medium (total volume, 1 mL) were phosphate buffer 35 mmol/L, pH 7.2, Triton X-100 0.02%, and enzyme protein 30 to 50 μg. The reaction was started by the addition of 30 μL of 1% H2O2 and monitored by the decrease in absorbance of H2O2 at 240 nm.
All results were expressed as mean±SEM. Student’s t test was performed for comparison of differences between the two groups, and a two-way ANOVA for repeated measures followed by Bonferroni’s correction was performed to compare the differences within the group by use of computerized statistical analysis software (primer, ©1988, McGraw-Hill Inc). A difference was considered significant only at values of P<.05.
Increase in Body Temperature by Amphetamine
Initially, we used several doses of amphetamine to examine the induction of heat shock. Doses of 1 and 2 mg/kg amphetamine could not elevate the body temperature and did not induce heat-shock mRNA. Doses >5 mg/kg amphetamine made the pigs extremely hyperactive and restless for several hours. A dose of 10 mg/kg was lethal to the pig. The optimal dose to induce the heat shock was found to be 3 mg/kg.
The rectal temperature of pigs before amphetamine injection ranged from 37°C to 39°C. One hour after the amphetamine injection, the rectal temperature increased markedly, to a range of 41.5°C to 42.5°C. This hyperthermic state lasted up to 1 hour, gradually decreased to 40°C after 2 hours, and finally returned to the basal temperature in another 3 to 4 hours (Fig 2⇓). The animals were slightly hyperactive after the amphetamine injection for a period of 3 to 6 hours, and then they behaved normally.
Improved Postischemic LV Function
The baseline values for global and regional functions were comparable between amphetamine and control groups (Table⇓). LAD occlusion caused dyskinesis in the LAD region, and the dyskinesis persisted throughout the duration of 60 minutes of occlusion in all hearts. Then the hearts were rapidly arrested with cold cardioplegia, and the heart temperature was maintained between 6°C and 10°C in both groups. During reperfusion, the heat-shock group showed better recovery of LVDP and LV dP/dtmax, as shown in Figs 3⇓ and 4⇓. The recovery of LVDP was significant after 60 minutes of reperfusion, 72% versus 52% (amphetamine versus control, P<.05), whereas LV dP/dtmax became significant after 15 minutes of reperfusion (58% for amphetamine and 40% for control, P<.05). This difference was maintained during the rest of reperfusion such that at the end of 60 minutes of reperfusion, the recovery of LV dP/dtmax for the amphetamine group was 60% versus 44% for the control group (P<.05) (Fig 4⇓) when determined at the end of reperfusion.
The baseline percent segment shortening was considered 100% (actual value varied between 1 and 2 mm), which is shown in Fig 5⇓. Percent segment shortening in both groups showed steady recovery during reperfusion: 46.2% recovery was observed in the amphetamine group compared with 10% in the control group (P<.05) at 60 minutes of reperfusion.
CBF and CK Release
The change of CBF was not significantly different between the two groups (Fig 6⇓); responsive hyperemia was observed during reperfusion in both groups. CK, which reflects cellular injury and membrane permeability, was significantly increased during reperfusion in both groups, but the CK release was significantly higher at 15 and 60 minutes of reperfusion in the control group compared with the heat-shock group (P<.05) (Fig 7⇓).
Induction of the Expression of HSP mRNAs by Amphetamine
To examine whether amphetamine could induce heat-shock genes, biopsies from heart, lung, liver, kidney, and brain were examined for the expression of the mRNAs for HSPs. Northern blots were hybridized with different probes for HSP 27, HSP 70, and HSP 89. β-Actin served as control. Amphetamine induced the expression of the mRNA for all the HSPs tested. The observed HSP 27, HSP 70, and HSP 89 mRNA had signals at 0.9, 2.4, and 3.0 kb, respectively, compared with the RNA marker (28S and 18S) (results not shown). After autoradiography, the hybridization signals were quantified by scanning densitometry. Each hybridization signal was normalized relative to the signals obtained for β-actin signal. These results are depicted in Figs 8 through 10⇓⇓⇓.
Stimulation of SOD and Catalase Enzyme Activities
Forty hours of amphetamine treatment significantly enhanced SOD and catalase enzyme activities in the heart (Fig 11⇓). Thus, 40 hours of pretreatment with amphetamine increased the myocardial catalase activity by 63.7% (control, 13.25±1.9 versus amphetamine, 21.28±1.6; P<.005). The activity of Cu/Zn-SOD was increased by 43.1% (control, 5.41±0.75 versus amphetamine, 7.73±0.63; P<.04), and Mn-SOD was increased by 50% (control, 19.59±1.01 versus amphetamine, 40.0±0.9 nmol · min−1 · mg protein−1; P<.05) by amphetamine treatment compared with control.
Myocardial preservation by preconditioning1 is a new concept; it is believed to be mediated by the endogenous protective mechanism.22 Ischemia is by far the most widely studied preconditioning stimulus and has led to the conclusion that such preconditioning can render the heart more tolerant to ischemia-reperfusion injury.1 2 3 4 5 6 Hypoxic preconditioning, similar to ischemic preconditioning, can also reduce myocardial ischemic reperfusion injury.7 Among other methods, adenosine preconditioning has met with success in animal models.5 Preconditioning is also achievable by oxidative stress.9 23 Heat-shock preconditioning has previously been found to be effective in the animal model but has been limited to only small animals, such as the rat.10 11 The surface of the body must be warmed up several degrees higher and for a much more prolonged period (which often can cause severe burns) to induce heat shock to an internal organ such as the heart.
In the present study, we used the sympathomimetic drug amphetamine to elevate the body temperature. It is well known that amphetamine stimulates cellular metabolism by accelerating endogenous lipolysis.18 In agreement, we demonstrated marked elevation of body temperature within hours, which is instrumental for the induction of heat-shock response. Amphetamine is one of the most potent sympathomimetic amines with respect to stimulation of the central nervous system. Prevention and reversal of fatigue by amphetamine has been studied extensively in experimental laboratories, military field studies, and athletics.24 The optimal therapeutic dose (3 mg/kg) was established after injection of different groups of pigs with several doses of amphetamine ranging from 0.5 to 10 mg/kg. Amphetamine at 10 mg/kg caused ventricular fibrillation in the pigs, whereas 0.5 to 1 mg/kg was quite ineffective in elevating the body temperature. The therapeutic dose used in this experiment caused a minimal amount of restlessness but was enough to induce the heat-shock response. Amphetamine caused the induction of the expression of HSP genes in all the vital organs, including heart, lung, liver, brain, kidney, and intestine, indicating that this drug can cause a whole-body heat-shock response. Thus, induction of heat shock by amphetamine seems to be superior to other commonly used methods, such as direct heating of the body or immersing the animals in a hot-water bath.
In addition to enhancing endogenous lipolysis, resulting in the elevation of body temperature, amphetamine also stimulates the α-receptors and provokes the release of catecholamines. This may induce a preconditioning phenomenon resulting in myocardial preservation. However, it is well established that such a preconditioning effect can last for only a few hours. We specifically selected a time of 40 hours after amphetamine treatment to study myocardial preservation to eliminate any sympathomimetic or preconditioning effects of the drug. A growing body of evidence now suggests that myocardial protection is observed after 24 to 48 hours (called a second window of protection or adaptation) by a mechanism likely to be mediated by the expression of heat-shock, antioxidant, and/or other stress proteins.
The induction of the expression of HSPs has been observed in a variety of cells and tissues in response to many diseases, including oxidant stress, ischemia-reperfusion, inflammation, and fever.25 26 It is generally believed that the expression of HSPs represents the acute response of the cell to the pathophysiological condition in an attempt to adapt itself to that particular disease state. Thus, heat shock can induce the expression of specific stress-related genes, including heat-shock genes, that are often translated into heat-shock proteins and other stress proteins that are protective against the subsequent injury to the cells and tissues.25 26 27 The major HSPs have been classified into four families: the HSP 90 family (83 to 90 kD), the HSP 70 family (66 to 78 kD), the HSP 60 family, and the HSP 27 family (15 to 30 kD).28 These stress proteins are functionally different and therefore are likely to differ in their ability to enhance cellular tolerance to subsequent stress.
The induction of heat shock has been demonstrated to be protective against ischemic injury in several models. For example, Currie and associates10 reported that heat shock induced by physical heating improves the recovery of LV contractile force after 30 minutes of ischemia in rat hearts. The expression of heat-shock protein and induction of catalase activity were also demonstrated in their experiment. Heat-shock pretreatment also improved the function in hypothermically preserved kidney transplants.29 Rapid expression of heat-shock proteins has been shown in liver and heart subjected to ischemic insult.30 31 Knowlton and coworkers,15 from the results of their study of the enhanced postischemic ventricular recovery after the heart was exposed to brief periods of ischemia, also suggested that induction of HSP 70 might play a role in myocardial stunning and preconditioning. A recent study by Donnelly and associates11 indicated a cardioprotective effect of whole-body hyperthermia against ischemic injury. In agreement with these results, our laboratory also demonstrated that perfusing an isolated heart with warm blood could protect it against ischemia-reperfusion injury.8 Contrary to that, in a study by Wall et al,32 the beneficial effects of heat shock were not found after severe myocardial ischemia, although the HSP synthesis was enhanced. Yellon and coworkers33 also demonstrated the induction of HSP 72 after whole-body heat shock, but without any cardioprotection. To the contrary, irrespective of the species differences, almost all the investigators found cardioprotective effects of heat shock when isolated heart was used as the experimental model.8 10 11 As mentioned earlier, it is extremely difficult to raise the temperature of an internal organ such as the heart. Warming the large animals does not usually raise the myocardial temperature high enough to induce heat shock, whereas in the isolated heart model this can be easily achieved by perfusing the heart at 42°C to 43°C.
The results of the present study demonstrate that pretreating the hearts with amphetamine elevated the body temperature to 42°C to 43°C; induced whole-body heat shock, as evidenced by the induction of the expression of HSP genes; improved postischemic LV global as well as regional contractile functions; and reduced myocardial cellular injury after LAD artery occlusion and hypothermic cardioplegic arrest. Sixty minutes of regional ischemia followed by 60 minutes of hypothermic cardioplegic arrest was designed to mimic the clinical event of acute coronary occlusion (thrombosis) leading to ischemia followed by revascularization.
The exact mechanisms of the protective effects of HSPs against cellular injury remain unknown. The results of our study also demonstrated activation of two antioxidant enzymes, SOD and catalase. A recent study from our laboratory demonstrated activation of SOD when an isolated in situ pig heart was subjected to heat shock by 42°C blood cardioplegia.8 In another recent study, mRNAs for catalase and Mn-SOD simultaneously with the mRNAs of HSPs were induced after four brief episodes of ischemia.21 In contrast, Karmazyn et al34 found no detectable change in mRNA for catalase after heat shock or during recovery after heat shock, but they demonstrated an increase in catalase enzyme activity at 24 and 48 hours after the induction of heat-shock response. The authors concluded that catalase activity is either translationally or posttranslationally regulated. Several recent studies also suggest that heat shock enhances the cellular antioxidative defense system.35 In another related study, heat shock increased the tolerance of polymorphonuclear neutrophils to oxidative injury mediated by H2O2.36 Substantial evidence supports the formation of oxygen free radicals and development of oxidative stress in ischemic-reperfused myocardium.37 38 Ischemia and reperfusion also cause the inactivation of natural antioxidant enzymes such as SOD and catalase. Thus, the stimulation of SOD and catalase could reflect a mechanism of myocardial adaptation to oxidative stress.39 Although it is tempting to speculate that heat shock–mediated stimulation of antioxidant enzymes is instrumental for the enhanced postischemic functional recovery of the heart, further study is required to confirm such a possibility.
In summary, we have demonstrated that a sympathomimetic drug such as amphetamine can induce the heat-shock response, inducing the expression of several HSP genes. This study has some relevance to the concept of warm heart surgery, in which heart is directly subjected to heat shock by warm blood cardioplegia. However, the myocardial preservation by warm blood cardioplegia resembles the preconditioning phenomenon, whereas induction of the antioxidant defense system through heat shock by amphetamine reflects the adaptive response. Amphetamine, either directly or indirectly, through the stimulation of the antioxidant defense system, can adapt the heart to ischemic stress and improve postischemic ventricular recovery in a setting of coronary revascularization for acute myocardial infarction.
Selected Abbreviations and Acronyms
|CBF||=||coronary blood flow|
|I-60||=||60 minutes after LAD occlusion|
|LAD||=||left anterior descending coronary artery|
|LV dP/dtmax||=||maximum of the first derivative of LV pressure|
|LVDP||=||LV developed pressure|
|R-15||=||15 minutes after reperfusion|
|R-60||=||60 minutes after reperfusion|
This study was supported by NIH grants HL-34360 and HL-22559. Dr N. Maulik was supported by a grant from the American Heart Association.
- Copyright © 1995 by American Heart Association
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