Heat-stressed Hearts Research Articles

Do mitochondria limit hot fish hearts? Understanding the role of mitochondrial function with heat stress in Notolabrus celidotus.

Hearts are the first organs to fail in animals exposed to heat stress. Predictions of climate change mediated increases in ocean temperatures suggest that the ectothermic heart may place tight constraints on the diversity and distribution of marine species with cardiovascular systems. For many such species, their upper temperature limits (Tmax) and respective heart failure (HF) temperature (THF) are only a few degrees from current environmental temperatures. While the ectothermic cardiovascular system acts as an “ecological thermometer,” the exact mechanism that mediates HF remains unresolved. We propose that heat-stressed cardiac mitochondria drive HF. Using a common New Zealand fish, Notolabrus celidotus, we determined the THF (27.5°C). Haemoglobin oxygen saturation appeared to be unaltered in the blood surrounding and within heat stressed hearts. Using high resolution respirometry coupled to fluorimeters, we explored temperature-mediated changes in respiration, ROS and ATP production, and overlaid these changes with THF. Even at saturating oxygen levels several mitochondrial components were compromised before THF. Importantly, the capacity to efficiently produce ATP in the heart is limited at 25°C, and this is prior to the acute THF for N. celidotus. Membrane leakiness increased significantly at 25°C, as did cytochrome c release and permeability to NADH. Maximal flux rates and the capacity for the electron transport system to uncouple were also altered at 25°C. These data indicate that mitochondrial membrane integrity is lost, depressing ATP synthesis capacity and promoting cytochrome c release, prior to THF. Mitochondria can mediate HF in heat stressed hearts in fish and play a significant role in thermal stress tolerance, and perhaps limit species distributions by contributing to HF.

Heat Stress Contributes to the Enhancement of Cardiac Mitochondrial Complex Activity

Hyperthermic stress is known to protect against myocardial dysfunction after ischemia-reperfusion injury. It is unclear however, what energetic mechanisms are affected by the molecular adaptation to heat stress. We hypothesized that mild hyperthermic stress can increase mitochondrial respiratory enzyme activity, affording protection to mitochondrial energetics during prolonged cardiac preservation for transplantation. Rat hearts were excised after heat-stress or sham treatment and subjected to cold cardioplegic arrest and ischemia followed by reperfusion in an ex vivo perfusion system. Cardiac function, mitochondrial respiratory, and complex activities were assessed before and after ischemia. Heat shock protein (Hsp 32, 60, and 72) expression was increased in heat-stressed hearts. This was associated with increased mitochondrial complex activities in heat-stress versus sham-treated groups for complex I-V. During reperfusion, higher complex activities and respiratory control ratios were observed in heat-stressed versus sham-treated groups. Recovery of ventricular function was improved in heat-stressed hearts. Furthermore, mitochondria in reperfused heat-stressed myocardium exhibited intact membranes with packed, parallel, lamellar cristae, whereas in sham-treated myocardium, mitochondria were severely disrupted. This study provides the first evidence of heat-stress-mediated enhancement of mitochondrial energetic capacity. This is associated with increased tolerance to ischemia-reperfusion injury. Protection by heat stress against myocardial dysfunction may be partially due to enhancement of mitochondrial energetics.

Age dependence of heat stress mediated cardioprotection

Background. To study the influence of age on heat stress cardioprotection, functional recovery, nucleotide concentrations, and heat stress protein 70 (Hsp70) levels were compared in the heat stressed (HS) and control (C) hearts at different ages, in a protocol mimicking donor heart preservation for transplantation. Methods. Control and heat stressed (24 hours before experiment) rat hearts were divided into three age groups: (I) 1 month, (Y) 4 months, and (M) 16 months (n = 6). Left ventricle balloon catheter was used to determine systolic and end-diastolic pressure/volume relations before and after 4 hours of cardioplegic arrest at 4°C. Another identical set of isolated hearts underwent 5 minutes of normoxic perfusion to obtain preischemic Hsp70 content and metabolite concentrations. Results. The postischemic recovery was highest in group HS-Y as compared to C-Y, HS-I, C-I, HS-M, and C-M. There were no differences in preischemic adenine nucleotides or creatine metabolite concentrations between the three age groups. In contrast, the nicotinamide adenine dinucleotide (oxidized form) (NAD +) and nicotinamide adenine dinucleotide phosphate (oxidized form) (NADP +) concentrations were significantly raised in group HS-Y. Hsp70 content was increased in all HS groups with no difference between the age groups. Conclusions. Improved postischemic functional recovery after cardioplegic arrest was observed in the young adult HS hearts. This was associated with highest NAD + and NADP + concentrations and did not correlate with increased Hsp70 content.

Enhanced protection of heat shock in myocardial infarction: inhibition of detrimental effect of systemic hyperthermia.

We have shown that isolated blood-perfused heat-stressed hearts are protected only when the blood donor animal has not been exposed to hyperthermia. Systematic hyperthermia results in larger infarction of both isolated control and heat-stressed hearts. In this study we investigated whether indomethacin inhibits in vivo the detrimental effect of hyperthermia. Male rabbits were divided into four groups, that is A(30), B(30), C(30), and D(30), representing hearts that ultimately received 30 minutes of ischemia. In a second series, rabbits were divided into groups A(45), B(45), (C45), and D(45) representing hearts that ultimately received 45 minutes of ischemia, and in a third series were divided into groups A(HSP), B(HSP), C(HSP), and D(HSP) representing animals that were heat shocked and their hearts were used to measure heat shock proteins. All the A groups (heat shocked) were subjected to 42 degrees C hyperthermia, all the B groups to the same procedure but with the addition of indomethacin (heat shocked + indomethacin), all the C groups served as controls, and all the D groups were treated with indomethacin only (control + indomethacin). Twenty-four hours later, all (30) and (45) groups were subjected to ischemia, whereas hearts from all (HSP) groups were harvested for heat shock protein measurements. When the animals were exposed to 30-minute ischemia, a significant difference in the infarcted to risk zone ratio (%I/R) was observed: A(30): 33.0 +/- 5.2, B(30): 16.1 +/- 4.4 [conferring a 51.2% reduction in infarct size, P < 0.05], C(30): 48.9 +/- 4.0, and D(30): 47.8 +/- 3.8 [P < 0.001 vs. B (30) and P < 0.05 vs. A(30)]. However, the %I/R did not differ among any of the (45) groups. Heat shock proteins themselves were seen to increase in A(HSP) and B(HSP) groups. Indomethacin enhances the beneficial effect of heat shock after 30-minute ischemia in vivo, reducing the infarct size by 51.2% in comparison with heat shock.

Relative induction of heat shock protein in coronary endothelial cells and cardiomyocytes: Implications for myocardial protection

Objectives: Induction of the 70 kd heat shock protein in the heart is known to exert a protective effect against postischemic mechanical and endothelial dysfunction. However, the exact site of induction and the mechanisms involved remain unknown. The aim of this study was to investigate the relative capacity of endothelial and myocardial cells to express the 70 kd heat shock protein in response to heat stress, as well as their significance. Methods: (1) Postischemic recovery of cardiac mechanical and endothelial function was studied in isolated rat hearts with and without endothelial denudation with saponin. (2) Semiquantitative determination of induction of 70 kd heat shock protein by Western immunoblotting was performed in the whole cardiac homogenate, in isolated cardiac myocytes, and in coronary endothelial cells. (3) Immunocytochemistry was used to visualize the distribution of induction of 70 kd heat shock protein in both cell types. Results: Postischemic recovery (percent preischemic value ± standard error of the mean) of cardiac output in hearts from heat-stressed animals was significantly improved (66.7 ± 6.9 vs 44.5 ± 4.5 in the control group, p < 0.01). In heat-stressed hearts treated with saponin no improvement in the recovery of cardiac output was noted (44.7 ± 6.9 in heat-stressed hearts vs 38.0 ± 4.0 in heat-stressed, saponin-treated hearts, p = not significant). Endothelial function (as assessed by the vasodilatory response to the endothelium-dependent vasodilator 5-hydroxytryptamine) improved from 31.0 ± 5.2 in the control group to 65.8 ± 7.1 in heat-stressed hearts ( p < 0.02 vs control) and dropped to –1.9 ± 3.8 in heat-stressed hearts treated with saponin. Immunocytochemistry showed that only sections of hearts from heat-treated rats showed a strong specific reaction with heat shock protein antibody. The positive staining was seen in endothelial cells. Induction of 70 kd heat shock protein content in the whole cardiac homogenate from heat-stressed rats as measured by Western immunoblotting was 5.2 ± 1.9 (vs 0.0 in non-heat–stressed rats, p < 0.0001) and dropped to 0.0 in heat-stressed hearts treated with saponin. The tentative amount of 70 kd heat shock protein was 18.1 ± 7.8 in isolated endothelial cells from heat-stressed hearts and 2.3 ± 2.3 in isolated cardiac myocytes ( p < 0.01 vs endothelial cells). Conclusions: Coronary endothelial cells are the main site of induction of 70 kd heat shock protein in the heart and appear to contribute to the protective effects of heat stress on the recovery of mechanical and endothelial function. (J Thorac Cardiovasc Surg 1998;115:200–9)

A novel mitochondrial protease converts xanthine dehydrogenase to xanthine oxidase: [formula omitted], Risto Lapatto and Kari O. Raivio. Children's Hospital, University of Helsinki

Prior heat stress leads to an enhancement of postischemic mechanical function and improvement in biochemical indices of injury in the rat heart, associated with an elevation in endogenous catalase activity. We have examined the effect of heat stress on free radical release during reperfusion in the isolated rat heart using electron spin resonance (ESR). Twenty four hours after heat stress or sham treatment, hearts were perfused in the Langendorff mode and subjected to 10 min of no-flow global ischemia followed by 10 min of reperfusion. Coronary effluent was collected at specific time points in PBN for ESR measurement. A PBN adduct was identified with characteristics consistent with an alkoxyl radical: PBN-LO. In sham hearts there was a rapid rise in adduct release to a maximuum (228 ± 15% of stabilization values, p < .05) occurring 1 min into reperfusion. In heat stress hearts there was no significant rise in adduct release during the reperfusion period. Pretreatment of hearts with 3-amino triazole, an inhibitor of catalase, failed to clarify whether the protection seen in heat stress hearts was a result of the elevation in catalase activity. These results suggest that heat stress protects the myocardium against the oxidative stress of ischemia-reperfusion.

Heat stress induces rapid recovery of mechanical function of ischemic fatty acid perfused hearts by stimulating glucose oxidation during reperfusion

Whole-body heat stress (HS) in rats leads to the accumulation of myocardial heat shock proteins and subsequent protection against ischemic injury in glucose-perfused hearts. We determined whether HS treatment would confer protection against ischemia in hearts perfused with high levels of fatty acids. In addition, since fatty acids can potentiate ischemic injury by inhibiting glucose metabolism, the effects of HS on glucose utilization were also determined. Anesthetized rats were subjected to whole-body hyperthermia by raising body temperature to 41-42 degrees C 15 min. Twenty four hours later, their hearts were perfused with buffer containing either 11 mM glucose alone or 11 mM glucose and 1.2 mM palmitate, and then subjected to ischemic conditions followed by reperfusion. In hearts perfused with glucose only, HS improved aortic flow (expressed as percent change from preischemic aortic flow) late into the reperfusion period. Rates of overall glucose utilization under these conditions were similar between control and HS hearts. When hearts were perfused with 1.2 mM palmitate, the benefits of HS on aortic flow occurred at the onset of the reperfusion period. This beneficial effect was associated with a significant increase in glucose oxidation. Our results show that HS induces a faster rate of recovery in fatty acid perfused hearts but does not offer more protection against ischemic damage when compared with hearts perfused with glucose as a sole substrate.

In vivo gene transfection with heat shock protein 70 enhances myocardial tolerance to ischemia-reperfusion injury in rat.

Heat shock protein 70 (HSP70) has been reported to be involved in the myocardial self-preservation system. To obtain the evidence that HSP70 plays a direct role in the protection from myocardial ischemia-reperfusion injury, rat hearts were transfected with human HSP70 gene by intracoronary infusion of hemagglutinating virus of Japan (HVJ)-liposome containing human HSP70 gene. The control hearts were infused with HVJ-liposome without the HSP70 gene. The hearts from whole-body heat-stressed or nontreated rats were also examined. Western blot and immunohistochemical analysis showed that apparent overexpression of HSP70 occurred in the gene transfected hearts and that gene transfection might be more effective for HSP70 induction than heat stress. In Langendorff perfusion, better functional recovery as well as less creatine phosphokinase leakage after ischemia were obtained in the gene transfected hearts with HSP70 than in the control or nontreated hearts. Furthermore, the gene transfected hearts showed better functional recovery than the heat-stressed hearts. These results indicated that overexpressed HSP70 plays a protective role in myocardial injury, suggesting the possibility that gene transfection with HSP70 may become a novel method for myocardial protection through enforcing the self-preservation systems.

Heat stress protects the perfused rabbit heart from complement-mediated injury.

We determined if heat stress induction of heat shock protein (HSP) 70 modulates complement activation in an experimental model of xenograft rejection. Male New Zealand White rabbits were heat stressed (core body temperature to 42 degrees C for 15 min; n = 9). Control rabbits (n = 13) were not exposed to heat stress. Hearts were removed 18 h later and perfused by the Langendorff method. After equilibration, human plasma (source of human complement) was added to the perfusion medium. Hemodynamic variables recorded during perfusion with human plasma were improved in hearts from heat-stressed animals compared with control hearts. Assembly of the soluble membrane attack complex was reduced in the interstitial fluid effluent from the heat-stressed hearts. Electron microscopic evidence of ultrastructural changes was attenuated in the hearts from heat-stressed rabbits. Myocardial tissue from heat-stressed animals exhibited an increase in inducible HSP 70 that was virtually absent in the hearts of control rabbits. Previous whole body hyperthermia protects the rabbit heart against the detrimental effects of heterologous plasma, suggesting that heat-stress induction of HSP 70 limits the extent of complement activation by a discordant vascularized tissue (xenograft). Induction of heat stress proteins by the donor organ might be an important mechanism affecting the outcome of xenograft transplants.

Diminished heat shock response in the aged myocardium.

Induction of heat shock proteins (Hsps), Hsp72 in particular, has been associated with myocardial protection. Since a decreased Hsp response has been reported to occur with aging, it was of interest to determine if hearts from aged animals also demonstrate an altered heat shock response and subsequent myocardial protection. Adult (6 months old) and aged (22 months old) Fischer 344 rats were heat stressed by raising their rectal temperatures to 41 degrees C for 10 min. At selected times following heat stress (0-24 h) hearts were examined for heat shock transcription factor trimerization and DNA-binding activity (Hsf1 activation), Hsp72 mRNA accumulation, Hsp72 and Hsf1 protein content, as well as, protection from ischemia using the Langendorff isolated heart model. Following heat stress, hearts from aged animals demonstrated a 47% reduction in Hsf1 activation, a reduction in Hsp72 mRNA and a 35% reduction in Hsp72 protein content, compared to hearts from adults. Interestingly, myocardial Hsf1 protein content was similar between aged and adult animals. Hearts from heat stressed adult animals (24-h prior) demonstrated an enhanced postischemic recovery as indicated by a greater recovery of left ventricular pressure and rate of contraction (P < 0.05), while hearts from heat stressed aged animals failed to demonstrate an enhanced postischemic recovery. These results suggest that hearts from aged animals exhibit an impaired ability to produce the protective Hsps and thus, may explain, at least in part, the increased susceptibility of aged hearts to stress.

The Protective Effect of Heat Stress Against Reperfusion Arrhythmias in the Rat

Heat stress provides protection against mechanical dysfunction and myocardial necrosis after prolonged ischemia. We have investigated whether this protection extends to reperfusion arrhythmias occurring after a short (non-lethal) ischemic insult. Anaesthetized open chest rats were subjected to a 5-min occlusion of the left coronary artery. The incidence and duration of reperfusion arrhythmias and the duration of sinus rhythm were assessed in the first 5 min of reperfusion. Prior heat stress led to a reduction in the incidence (100-63%, P ⩽ 0.05) and duration (66.2 ± 15.8 to 9.4 ± 2.9s, P ⩽ 0.05) of ventricular tachycardia and a non-significant reduction in the incidence (76-50%) and duration (74.3 ± 23.4 to 42.9 ± 24.4s, P = 0.09) of ventricular fibrillation. This resulted in a significant increase in the duration of sinus rhythm (142.1 ± 27.6 to 216.7 ± 24.8s, P ⩽ 0.05) and reduction in arrhythmia score ( P ⩽ 0.05) in heat stressed rats compared with controls. This protection against reperfusion arrhythmias was associated with a two-fold increase in endogenous catalase activity and expression of the inducible heat stress protein HSP 70. Inhibition of catalase with pre-administered 3-amino triazole resulted in a paradoxical protection in both sham and heat stress hearts. We conclude that heat stress leads to protection against reperfusion arrhythmias; however, we have been unable to resolve whether the changes in catalase activity of HSP expression are the mediators of the demonstrated cardioprotection.

Heat stress attenuates free radical release in the isolated perfused rat heart

Prior heat stress leads to an enhancement of postischemic mechanical function and improvement in biochemical indices of injury in the rat heart, associated with an elevation in endogenous catalase activity. We have examined the effect of heat stress on free radical release during reperfusion in the isolated rat heart using electron spin resonance (ESR). Twenty four hours after heat stress or sham treatment, hearts were perfused in the Langendorff mode and subjected to 10 min of no-flow global ischemia followed by 10 min of reperfusion. Coronary effluent was collected at specific time points in PBN for ESR measurement. A PBN adduct was identified with characteristics consistent with an alkoxyl radical: PBN-LO. In sham hearts there was a rapid rise in adduct release to a maximuum (228 ± 15% of stabilization values, p < .05) occurring 1 min into reperfusion. In heat stress hearts there was no significant rise in adduct release during the reperfusion period. Pretreatment of hearts with 3-amino triazole, an inhibitor of catalase, failed to clarify whether the protection seen in heat stress hearts was a result of the elevation in catalase activity. These results suggest that heat stress protects the myocardium against the oxidative stress of ischemia-reperfusion.

Attenuation by Heat Stress of a Submaximal Calcium Paradox in the Rabbit Heart

Heat stress limits the injury associated with myocardial ischaemia and reperfusion, an effect previously attributed to enhanced endogenous anti-oxidant activity. We examined the influence of heat stress on the calcium paradox, and injury in which oxidant stress in not thought to play a major role. Twenty-four hours following sham or true heat stress, rabbits were re-anaesthetized and hearts either removed for stress protein analysis (n = 8), or Langendorff-perfused (n = 20) and subjected to a calcium paradox. Ten minutes following calcium repletion (Ca2+ = 1.3 mM), left ventricular developed pressure was better preserved in heat stress vs control hearts (38.3 ± 5.0 vs 18.8 ± 4.1 mmHg, respectively, P = 0.003) whilst contracture, measured by left ventricular enddiastolic pressure, was diminished (21.6 ± 4.7 vs 39.9 ± 5.2 mmHg, respectively, P = 0.02). Creatine phosphokinase release at 1 min was less in heat stress vs control hearts (10.6 ± 8.6 vs 86.4 ± 33.7 U/min/g, respectively, P = 0.01). The myocardial content of the 72 kDa stress protein was elevated eight-fold in heat stress vs control hearts (2.8 ± 0.02 vs 0.4 ± 0.1 U, respectively, P = 0.01), This study suggest that some portion of the stress protein response represents a form of cardiac adaptation capable of limiting myocyte injury independent of anti-oxidant mechanisms.

Heat stress limits infarct size in the isolated perfused rabbit heart.

Heat stress, with the expression of heat stress proteins, has been shown to protect the rabbit heart in vitro against global ischaemia/reperfusion injury, though no benefit is apparent in an in vivo rabbit model of infarct size. The aim of this study was therefore to investigate this discrepancy and to discover whether heating itself has any effect which could negate the protection derived from myocardial stress protein synthesis. (1) To ascertain whether heat stress could limit infarct size in the absence of blood, isolated buffer perfused hearts, with or without prior heat stress, were subjected to 45 min of regional ischaemia and 120 min reperfusion, and the resulting infarct size was expressed as a percentage of the risk area (I/R%). (2) The observations were repeated in an isolated blood perfused heart model in which a support rabbit (heat stressed or control) was used to perfuse the isolated heart. In the buffer perfused heart, prior heat stress reduced I/R from 70.8(SEM 4.4)%, n = 10, in controls to 51.5(5.7)%, n = 12 (p < 0.05). In hearts perfused by support rabbits, prior heat stress reduced I/R [from 34.7(3.7)%, n = 16, to 23.5(3.3)%, n = 15 (p < 0.05)] only when the perfusing rabbit was a control (not heat stressed). If the perfusing rabbit had been heated, I/R was greater in both heat stressed and control hearts [51.9(7.0)% and 44.9(3.3)%, p < 0.05 v control support rabbit]. Heat stress limits infarct size in this rabbit model. However it appears to have additional adverse effects, probably on the blood, which may override any benefit associated with myocardial stress protein synthesis.

The protective role of heat stress in the ischaemic and reperfused rabbit myocardium

Cells subjected to increases in temperature induce the expression of several proteins known as heat shock or stress proteins. This process enhances the cell's ability to overcome the effects of further stress. In this respect, the effects of heat stress have been reported to protect the hearts of rats following ischaemia and reperfusion. We have confirmed and extended this observation, not only using different indices of myocardial injury but also in another species, namely the rabbit. Animals were anaesthetized and the body temperature raised to 42°C for a 15-min period. Controls were treated in the same way but without heating. Twenty-four hours later the rabbits were re-anaesthetized and the hearts removed for either heat stress protein analysis or perfusion with Krebs buffer using an isolated perfused heart apparatus. Hearts were subjected to 60 min of low flow (1 ml/min) ischaemia followed by 30 min of reperfusion. All hearts subjected to heat stress showed an enhanced recovery of function upon reperfusion as measured by improvements in developed pressure (27.3 ± 3.6 vs 16.3 ± 3.0 mmHg) and diastolic pressure (37.3 ± 7.4 vs 54.7 ± 3.1 mmHg). In addition, creatine kinase release, associated with reperfusion, was significantly reduced in the heat-stressed hearts (532 ± 102 vs 1138 ± 73 mU/min/g wet wt). Myocardial accumulation and release of oxidized glutathione, an index of oxidative stress, was significantly reduced in the heat-stressed group (0.003 ± 0.003 vs 0.376 ± 0.113 nmol/min/g wet wt). The improved metabolic status of the reperfused heat-stressed hearts was further demonstrated by a significant conservation in the levels of ATP (6.1 ± 0.9 vs 2.8 ± 0.8 μmol/g dry wt) and CP (36.9 ± 6.4 vs 16.4 ± 5.1 μmol/g dry wt). Finally, isolated mitochondrial function in terms of respiratory control index (RCI) was maintained in the heat-stressed hearts (9.2 ± 0.9 vs 5.7 ± 0.2) and overloading with calcium was reduced. These data extend the hypothesis that heat stress protects the heart following ischaemia and reperfusion in this in vitro model, in a way as yet undetermined.