Journal of Surgical Research 139, 176 –181 (2007) doi:10.1016/j.jss.2006.07.041 Heat Stress Attenuates ATP-Depletion and pH-Decrease During Cardioplegic Arrest Sebastian Vogt, M.D., Ph.D.,*,1 Dirk Troitzsch, M.D.,* Hashim Abdul-Khaliq, M.D.,† and Rainer Moosdorf, M.D., Ph.D.* *Biomedical Research Center, Cardiovascular Research Lab and Heart Surgery, University Hospital, Philipps-University Marburg, Marburg, Germany; and †German Heart Institute Berlin, Berlin, Germany Submitted for publication June 21, 2006 Background. The capacity of heat stress induction to improve myocardial tolerance against ischemia is well known. We investigated cardiac energy metabolism after hsp 72 ⴙ/73 ⴙ induction in isolated perfused neonatal rabbit hearts subjected to prolonged cold cardioplegic ischemia. Methods. Hearts from neonatal rabbits were excised, isolated perfused and arrested by 2-h cold cardioplegic ischemia. Rectal temperature of eight neonatal rabbits was raised to 42.0 to 42.5°C for heat shock protein expression in a whole body water bath for 15 min before the onset of arrest. Another set of eight rabbits without hyperthermia pretreatment served as control. Recovery of left ventricle function was assessed by aortic flow, cardiac output, and max dP/dt. Status of highenergy phosphates was measured by 31phosphorus nuclear magnetic resonance-spectroscopy. Results. Immunoblot analysis revealed clear hsp 72ⴙ/73ⴙ induction after a brief period of systemic hyperthermia. Heat stress pretreatment resulted in a better recovery of left ventricular function (aortic flow and cardiac output improvement P < 0.05, max dP/dt P < 0.01) than in controls at 60 min after reperfusion. During ischemia and reperfusion, myocardial energy metabolism was better preserved in hearts after hsp induction as a consequence of increased ␥-, ␣-, and ␤-ATP as well as phosphocreatine-values over controls. The ischemiainduced pH-decrease was attenuated. Conclusion. These data contribute to the evidence of heat stress mediated beneficial effects on functional myocardial recovery and improved cardiac energy metabolism after prolonged cold cardioplegic ischemia. More importantly, the attenuation of ischemic 1 To whom correspondence and reprint requests should be addressed at Klinik für Herzchirurgie, Universitätsklinikum, PhilippsUniversität Marburg, Baldinger Stra␤e 1, D-35043 Marburg, Germany. E-mail: vogts@med.uni-marburg.de. 0022-4804/07 $32.00 © 2007 Elsevier Inc. All rights reserved. pH reduction and better restoration suggest an involvement of mitochondrial membrane potential alterations. © 2007 Elsevier Inc. All rights reserved. Key Words: heat shock proteins; high energy phosphates. INTRODUCTION Induction of Heat Shock-Proteins (hsp) results in a cytoprotective effect. Different forms of acute and chronic stress induce production of abnormal and misfolded cellular proteins, which is neutralized by overexpression of these molecular chaperones, even in intact organs (e.g., in the heart). Our group demonstrated an improved functional recovery of the cardioplegic arrested ischemic hearts, when hsp 72 and 73 were induced after short term hyperthermia prior to ischemia [1]. These mechanisms, which confer protection against myocardial ischemia-reperfusion injury, have beneficial influence on high-energy-phosphate (HEP) metabolism [2]. Improved mechanical recovery after heat shock was associated with an decreased rate of HEP depletion and increased recovery of ATP and phosphocreatine levels during reperfusion. In an excellent paper Sammut et al. hypothesized an increased mitochondrial respiratory enzyme activity caused by hyperthermic stress. This study provides evidence for heat-stressmediated enhancement of mitochondrial energetic capacity [3]. Especially, the enzyme activities of respiratory chain complexes I, IV, and V were significantly increased. Only little is known about the simultaneous time course of the myocardial pH-value and HEP content monitored during heat shock protected ischemiareperfusion-injury. This circumstance is caused by technical problems. Until now, the only method to estimate HEP content and pH on parallel is the 31phos- 176 VOGT ET AL.: HEAT SHOCK PROTEINS AND HIGH ENERGY PHOSPHATES phorus nuclear magnetic resonance ( 31P-NMR) analysis. However, there are limitations in the assay. Jayakumar reports about difficulties to quantitate changes in intracellular inorganic 31P-NMR signals because of very broad peaks in the spectra [2]. Additionally, the relationship between pH and the chemical shift of inorganic phosphate at 4°C was not well established. In our experimental setting we reinvestigate the HEP-alterations depending on hsp 72/73 induction with special focus on the time course of pH-value during ischemia and reperfusion injury. MATERIALS AND METHODS Procedures for all animal were carried out in conformance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health. Hearts from neonatal New Zealand White rabbits (in each group n ⫽ 8, body weight 0.16 – 0.2 kg, age 8 –10 days) were perfused using a Langendorff perfusion apparatus. Heat shock treatment was identical to the setting described [1]. After being anesthetized a group of rabbits were gently heat stress treated with a temperature-controlled water bath and electric heating pad (heating rate of 0.5– 0.7°C/min, rectal temperature 42.0 – 42.5°C for 15 min, time of recovery 1 h). Hearts were rapidly excised and a cannula was placed into the aorta for perfusion in the non-recirculating Langendorff technique. The isolated working heart system with split circulation set up (Hugo Sachs Elektronik, March-Hugstetten, Germany) provides a physiological left ventricular pressure-volume work for neonatal rabbits and function mode was performed. Krebs-Henseleit buffer solution were used for heart perfusion. Cardioplegic arrest was induced by commercially available crystalloid cardioplegic solution (modified “Eppendorf cardioplegia”, Fresenius AG, Bad Hamburg, Germany). The experimental protocol was performed in two groups of eight neonatal rabbit hearts and identical with previous experiments [1]. Cold cardioplegic arrest (control group) was compared with cold cardioplegic arrest after whole-body hyperthermia and heat shock protein induction. Perfusion for 15 min in Langendorff technique and equilibration in “working heart mode” for 20 min were required for steady state conditions. Global hypothermic arrest was induced by 177 left atrial inflow/aortic outflow-clamping and retrograde single-dose infusion of cold crystalloid Eppendorf cardioplegic solution (4°C, volume 20 mL, aortic pressure between 30 and 50 mmHg, myocardial temperature 12–14°C, duration 2 h). Hearts were reperfused for 15 min in Langendorff mode and converted back to the working heart mode by restoring left atrial and aortic flows. Heat-shock protein analysis of constitutive hsp73 ⫹ and inducable hsp72 ⫹ was performed by myocardial biopsies from left heart tissue before the onset of cold cardioplegic arrest and after 60-min reperfusion. Evaluation with Western blot analysis and densitometry by a gel doc-reader (Software Quantity one; Bio-Rad Laboratories, Hercules, CA). The status of myocardial HEP were measured by time-resolved 31 P-NMR spectroscopy according [4]. Whole perfusion system were placed into a superconducting magnet (4.7 Tesla; Biospec, Bruker, Germany). A rectangular surface coil adopted to the phophorussignal (81 Mhz) was positioned upon the hearts. Signals for ATPpeaks (␣, ␤, and ␥), Phosphocreatine, and inorganic phosphorus were registered in equal time intervals. Intracellular pH was calculated using the calibrated chemical shift of the inorganic phosphate peak in the spectra. Data are plotted as mean ⫾ SEM. Statistical analysis within the groups were performed using paired Student’s t-test and between the two groups using the unpaired Student’s with a MannWhitney rank sum test as a non-parametric second step. Differences were considered statistically significant at P ⬍ 0.05 and marked in the figures with asterisks. RESULTS Immunoblot analysis of the biopsies from the left ventricles revealed high-level expression of heat shock proteins (hsp 72 ⫹/73 ⫹, Fig. 1–3). We found this expression in a time dependent manner. For the investigations in this report a heat stress of 15 min at 42.0 to 42.5°C was chosen and an optimal time of normothermic recovery of 60 min followed. After recovery the hearts were harvested and hemodynamic baseline data were evaluated. Values were equal in each group (Table 1). When cardioplegic arrest was induced, after 60 min reperfusion the aortic flow and cardiac output were FIG. 1. Densitometric analysis of hsp 72 ⫹/73 ⫹ expression detected by Western blot technique of control and the group after hyperthermic stress (n ⫽ 8 in each group). Data were evaluated before ischemia and after reperfusion. When animals were exposed to hyperthermic stress a clear expression of hsp 72 ⫹/73 ⫹ is seen. 178 JOURNAL OF SURGICAL RESEARCH: VOL. 139, NO. 2, MAY 15, 2007 TABLE 1 Hemodynamic Data of Both Groups After Myocardial Recovery at 60 min of Reperfusion LV max. dp/dt (mmHg/s) Baseline 60 min reperfusion Aortic flow (mL/min) Baseline 60 min reperfusion Cardiac output (mL/min) Baseline 60 min reperfusion Heart rate (beats/min) Baseline 60 min reperfusion FIG. 2. Examples for immuno-dot-analysis of 72 kDa-hspexpression after 15 min heat shock exposure (see “Materials and Methods”) at different time intervals for subsequent recovery: mon Ab: anti-hsp 70; clones brm-22 and h 5147, Sigma-Aldrich; detection by horseradish-peroxidase-staining and 4-chloro-1-naphtol (SigmaAldrich). With increased time of recovery, the hsp-expression is found to be increased. After optimization protocol of post heat shock hemodynamic data (data not shown) further investigations were carried out with 60-min recovery time. increased by 25.3% and 25.8%, respectively (P ⬍ 0.05). Contractility (dP/dt max) was reduced in the control group by 54.9% (P ⬍ 0.01). Heart rate between the groups and after reperfusion showed no differences. In 31 P-NMR-analysis, initial data of HEP and pH were equal. During cardioplegic arrest different reductions in HEP was found. At final ischemia, higher levels of phosphocreatine (Fig. 4) and ATP-derivates were detected after heat shock protein induction (P ⬍ 0.05, Fig. 4). After reperfusion, increased ATP peak areas and phosphocreatine values were measured, which returned to baseline. In this group, values were superior to the controls (P ⬍ 0.05). These data underline the FIG. 3. Example for comparative immuno-dot-analysis of hsp 72 ⫹-expression between heat shock compromised group and sham group. Two animals were sacrificed initially and investigated for hsp-expression before starting heat shock exposure (control *). Control group* HT-group* P-value 970 ⫾ 100 550 ⫾ 70 970 ⫾ 200 900 ⫾ 150 NS ⬍0.01 33.3 ⫾ 2.1 23.2 ⫾ 3.4 34.5 ⫾ 2.2 30.1 ⫾ 3.2 NS ⬍0.05 35.8 ⫾ 3.0 23.8 ⫾ 2.7 37.1 ⫾ 2.6 33.2 ⫾ 2.4 NS ⬍0.05 210 ⫾ 8 205 ⫾ 9 208 ⫾ 10 219 ⫾ 13 NS NS * Values as mean ⫾ SD; control group (n ⫽ 8, without heat shock pre-treatment) and HT-group (n ⫽ 8, with heat pre-treatment); LV max. dp/dt, maximum rate of left ventricular pressure rise; NS, no significance. heat shock response mediated beneficial effect on preservation of HEP during cold cardioplegic ischemia and restoration of myocardial energy metabolism during reperfusion. Parallel to these findings, the pH-decrease during ischemia is attenuated and pH-values returned to baseline data in reperfusion (P ⬍ 0.05, Fig. 5). DISCUSSION Molecular chaperones such as the heat shock family of stress proteins participate in different cellular processes, especially in cytoprotection. There is a stressinducible network, both at the transcriptional and posttranscriptional levels. At the present time we discuss their clinical significance in physiological and pathophysiological conditions such as ischemic preconditioning and aging [5]. Expression of hsps is an evolutionary relict and found in all species, but the hsp genes vary in the pattern of their expression. It correlates with the resistance to stress [6]. Up-regulation of protein expression establishes a defense system to maintain cellular protein homeostasis and tolerance to ischemia, hypoxia, and radical oxygen species responsible for cardiovascular disorders [7]. Currie and coworkers suggest a therapeutic value for the heat-shock response by its association with enhanced post-ischemic ventricular recovery and limitation of tissue necrosis during coronary occlusion and reperfusion [8, 9]. Although they found an absence of changes in energy metabolism [10] the increase of heat shock protein 70 mRNA in rat heart exposed to oxidant stress suggested an involvement of mitochondrial respiratory enzyme function [11]. Further investigations by Sammut and coworkers recover contribution of heat stress to the enhancement of cardiac mitochondrial complex activity VOGT ET AL.: HEAT SHOCK PROTEINS AND HIGH ENERGY PHOSPHATES 179 FIG. 4. Results of the 31P-NMR spectroscopy of both groups during perfusion, cold cardioplegic ischemia and reperfusion: on the base an example of ECG-triggered 31P-NMR-spectrum is documented. The relating time courses of each signal for HEP is plotted above. With ongoing ischemia a reduction of the myocardial HEP contents was registered, the restoration of these compounds begins during reperfusion. Differences between hsp 72 ⫹/73 ⫹ induction and control were found in the depth of HEP reduction in ischemia and restoration of them in reperfusion. These data suggest beneficial effects on myocardial energy metabolism in or after ischemia. [3, 12]. Our group contributes to the importance of cardiac heat shock protection by confirmation of clear correlation between heat shock protein induction and improved myocardial performance after cardioplegic arrest [1]. In this setting we addressed the enhanced protection of myocardial energy metabolism by heat stress pretreatment in newborn rabbit hearts. Concluding the results, we found higher myocardial HEP levels (␥-, ␣-, and ␤-ATP, phosphocreatine) at final ischemia when hsp 72 and hsp 73 were induced (Fig. 1). After reperfusion the ATP- and phosphocreatine-peak areas are increased in the hsp 72 ⫹/73 ⫹ group over control and reveal improved myocardial HEP-contents by heat shock (Fig. 4). More importantly, the fall of ischemic induced intracellular myocardial pH-values was attenuated (Fig. 5). These data correlate with previous experiments of Jayakumar and co-workers [2] and confirm the beneficial changes of heat stress in HEP metabolism. In their study it was difficult to define the intracellular inorganic 31P-NMR signals and additionally the chemical shift of inorganic phosphate at low temperatures was not established. We found an acute course of inorganic phosphate peak sufficient for intracellular pH calculation. Moreover, Kost worked out pH standardization protocols for 31P-NMR-spectroscopy at low temperatures [13]. Based on his measurements Bock et al. and Binzoni et al. defined a pH change of ⫺0.015 to ⫺0.016 pH units/°C [14, 15] that seems to be negligable for our investigation. Therefore, we point out an attanuation of pH-decrease in cardioplegic induced myocardial arrest. The importance of pH-decrease is very often discussed in the context of severity of ischemic tissue damage. Otherwise, the myocardial pH-value is relevant for the proton motive force, which itself is determined by the mitochondrial membrane potential. Three enzyme complexes of the mitochondrial respiratory chain (NADHubiquinone oxidoreductase ⫽ complex I, cytochrome c reductase ⫽ complex III, and cytochrome c oxidase ⫽ complex VI) are electron transport-driven proton pumps that transfer reducing equivalents from nutrition to dioxygen accompanied by transfer of protons across the inner mitochondrial membrane, and generate a proton motive force. The ATP-Synthase (complex V) in mito- 180 JOURNAL OF SURGICAL RESEARCH: VOL. 139, NO. 2, MAY 15, 2007 FIG. 5. Evaluation of myocardial pH-values during the experimental protocol. The data were calculated from the chemical shift between the signals of inorganic phosphate to phosphocreatine. After cold cardioplegic ischemia in both groups a pH reduction was found. In contrast to control, after hsp 72 ⫹/73 ⫹-induction the pH reduction was attenuated and in reperfusion the pH returned back to physiological values. chondria uses this driving element to produce ATP from ADP and inorganic phosphate. When an increased activity of cytochrome c oxidase is known in case of hypoxia [16] on the one hand and the mitochondrial membrane potential drops sharply in parallel with mechanical cardiac performance in ischemia [17], then our findings can only be interpreted as an involvement of hsp 72 ⫹/73 ⫹ in its function as chaperones for cytochrome c oxidase-translation. Cytochrome c oxidase is the terminal enzyme of the mitochondrial respiratory chain which transfers electrons from cytochrome c to molecular oxygen, coupled with the uptake of protons from the matrix forming water and translocating protons across the mitochondrial inner membrane. It is considered terminating enzyme complex for electron transfer and preservation of mitochondrial membrane potantial driving the activity of ATP-synthase and ATP-production. Corresponding to this point Piel et al. found increased ventricular cytochrome oxidase I mRNA and protein content in case of hypoxia. The Heme a,a3 content, as essential element of cytochrome c oxidase, and turnover number was also increased [16]. In our investigation the elevated myocardial level of HEP when hsp were induced, underline the thesis. Therefore, it seems to be evident that the beneficial action of hsp expression is caused by induction of protein translation and internalization [18] of the apoenzyme into the mitochondrium resulting in higher enzyme activities of cytochrome c oxidase. If other mitochondrial enzyme complexes are involved in this mechanism still remains open. It is suggested that this effect also concerns the level of gene expression and contains a pivotal importance in delayed ischemic preconditioning [19]. At least, we have to extent our view on other further hsp species: Selective temporal induction of different hsp (e.g., 70, 60, and 32) might cooperate and participate in the regulative defense program of mitochondrial enzyme complex induction [20, 21]. Moreover, hsp 90 is involved in up-regulation of NOsynthase-activity and NO production, which itself participate in post-ischemic myocardial oxygenation and oxygen consumption by modulation of mitochondrial electron transport [22]. 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