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Eur J Cardiothorac Surg 1998;13:467-474
© 1998 Elsevier Science NL

Influence of heat stress on myocardial metabolism and functional recovery after cardioplegic arrest: a 31P N.M.R study1

Jay Jayakumar, Ryszard T. Smolenski, Caroline C. Gray, Andrew T. Goodwin, Kameljit Kalsi, Mohamed Amrani, Magdi H. Yacoub

Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Medicine, Harefield Hospital, Harefield, Middlesex, UB9 6JH, UK

Received 1 October 1997; received in revised form 12 January 1998; accepted 19 January 1998.

Corresponding author. Tel.: +44 1895 828893; fax: +44 1895 828902.


   Abstract
Top
 Abstract
Introduction
Materials and methods
 Results
Discussion
Appendix A. Conference...
 References
 
Objective: Heat stress and induction of heat shock proteins confer protection against myocardial ischemia-reperfusion injury; however the precise mechanisms of this effect remain unknown. We investigated the influence of heat stress on metabolic and functional recovery after cardioplegic arrest, in a protocol mimicking clinical donor heart preservation. Methods: Langendorff perfused rat hearts in control group (C, n=6) and heat stressed (24 h prior to experiment) group (HS, n=6) were subjected to 4 h of ischemia at 4°C following cardioplegic arrest (St. Thomas’ No. 1). 31P nuclear magnetic resonance spectroscopy was used to follow changes in ATP, phosphocreatine and inorganic phosphate concentrations during the pre-ischemic, ischemic and reperfusion periods. Myocardial adenine nucleotide levels in hearts at the end of experiments and purine catabolite release in coronary effluent during reperfusion, were evaluated using high performance liquid chromatography. Mechanical function in the pre-ischemic and reperfusion periods was evaluated using an intraventricular balloon. Western immunoblotting was used to quantitate HSP70 expression. Results: Although baseline concentrations of ATP and phosphocreatine were similar in C and HS groups, the rate of high-energy phosphate depletion was attenuated during the early phase of ischemia in HS groups. On reperfusion, recovery of ATP was 10–20% greater in HS versus C groups; phosphocreatine levels also recovered better in the HS group, transiently reaching levels 40% higher in HS versus C groups. The concentrations of adenine nucleotides in hearts were significantly higher in the HS versus C groups. These changes were associated with an attenuation of total purine catabolite release in the coronary effluent in HS versus C groups. A significant improvement in relative recovery of developed pressure was shown in HS versus C groups in the post-ischemic periods. Conclusions: Heat stress causes beneficial changes in high-energy phosphate metabolism in the rat heart subjected to cardioplegic arrest and ischemia. Improved mechanical recovery in HS versus C groups was associated with a decreased rate of high-energy phosphate depletion and increased recovery of ATP and phosphocreatine levels during reperfusion. Changes in energy metabolism may play a role in the mechanism of cardioprotection by heat stress during prolonged hypothermic cardiac arrest.

Key Words: Heat shock proteins • Myocardial ischemia • Myocardial reperfusion injury • Cardioplegia • Energy metabolism • Nuclear magnetic resonance


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
Discussion
Appendix A. Conference...
 References
 
Heat stress enhances endogenous protective mechanisms against ischemia-reperfusion injury in the heart and the induction of heat shock proteins (HSPs) is believed to play a crucial role in this phenomenon [1] [2]. HSPs are a family of proteins which are constitutively expressed in cells and have an essential role in intracellular protein folding and translocation and are thus termed molecular chaperones [3]. HSP levels are increased under various conditions of stress, including heat stress, ischemia and toxins [4] [5] [6] and HSP70 is particularly important for cardioprotection.

We have previously shown that HSP70 is associated with improved recovery of both ventricular and coronary endothelial function following prolonged cardioplegic arrest and reperfusion [7] [8]. This recovery was greatest when the interval between heat stress and ischemia was 24 h, correlating with maximal levels of HSP70 induction [9].

The mechanisms by which heat stress confers cardioprotection after ischemia-reperfusion remains unclear [10]. In addition to increased expression of HSP70, proposed mechanisms include attenuation of free-radical damage due to induction of antioxidant enzymes [11] and enhancement of nitric oxide production [12]. Heat stress has also been shown to attenuate the calcium paradox following ischemia-reperfusion, indicating a possible role for HSP70 in intracellular calcium handling [13].

Some studies have indicated that changes in myocardial energy metabolism after heat stress is related to improved mechanical function following normothermic ischemia [14] [15]. However, detailed analysis of high-energy phosphate metabolism and its relationship to mechanical recovery has not been performed in hearts subjected to heat stress prior to cardioplegic arrest and prolonged hypothermic ischemia.

In this study we used nuclear magnetic resonance spectroscopy (N.M.R.) to investigate the sequential changes in ATP, phosphocreatine (PCr) and intracellular inorganic phosphate (Pi) levels in heat stressed (HS) and control (C) rat hearts in a model mimicking cardiac preservation for clinical transplantation together with evaluation of mechanical recovery.


   Materials and methods
Top
 Abstract
 Introduction
Materials and methods
Results
Discussion
Appendix A. Conference...
 References
 
Animals
Male Sprague-Dawley rats (280–320 g) were used in all studies. Animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research; the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the Institute of Laboratory Animal Resources (N.I.H. publication No. 86–23, revised 1985); and the ‘European Convention on Animal Care’ guide. The study was approved by the institutional ethics committee on animal research.

Experimental protocol
Control (n=6) and heat stressed (n=6) hearts were studied to determine metabolic and functional changes before, during and after 4 h cardioplegic arrest at 4°C, as shown in Fig. 1 . After an initial 40 min normoxic perfusion required for optimization of the N.M.R. and collection of initial spectra, pre-ischemic mechanical function was evaluated using a balloon catheter. Subsequently, hearts were arrested by infusion of 4°C cardioplegia at a constant pressure of 70 mmHg for 6 min. Hearts were immersed in cardioplegia and maintained at 4°C with the aid of the temperature control unit of the N.M.R. probe.



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  		Fig. 1. Experimental protocol.

 
After 4 h of cardioplegic arrest, hearts were reperfused with Krebs–Henseleit buffer at 37°C and coronary effluents were collected during the first 25 min of reperfusion. After 40 min of reperfusion, post-ischemic mechanical function was evaluated. At the end of the experiments, hearts were freeze-clamped in liquid nitrogen for high performance liquid chromatographic (H.P.L.C.) analysis.

Hearts were not paced during the entire protocol; pre- and post- ischemic heart rates were recorded after 60 min of perfusion and 60 min reperfusion, respectively.

Induction of heat stress
Rats were subjected to heat stress after induction of anesthesia with sodium pentobarbitone (intraperitoneal, 50 mg/kg) using a temperature-controlled heating pad (I.M.S., Cheshire, UK). Body temperature (monitored with a rectal probe) was maintained between 42 and 42.5°C for 25 min as described previously [7]. Control animals underwent anesthesia but not heat stress. On recovery, animals were re-hydrated with normal saline (intraperitoneal, 10 ml/kg) and used for experiments 24 h later.

Heart perfusion
Rats were anesthetized with diethyl ether and sodium heparin (1000 IU/kg) injected via the femoral vein. Hearts were rapidly excised, placed in ice-cold Krebs–Henseleit buffer, immediately attached to a Langendorff apparatus and perfused with filtered Krebs–Henseleit buffer (118 mM NaCl, 4.7 mM KC1, 1.2 mM MgSO4, 1.2 mM KH2PO4, 24 mM NaHCO3, 11 mM glucose, 1.2 mM CaCl2 pH 7.4) at a constant pressure of 100 cmH2O and continuously gassed with a 95%O2/5%CO2 mixture at 37°C, as described previously [16]. Perfusion was carried out inside the bore of the magnet to determine changes in high-energy phosphate levels, as described below.

Cardioplegic solution
St. Thomas’ Hospital cardioplegic solution No. 1, supplied as a concentrate (Martindale, Essex, UK), was diluted (1:50) in Ringer’s solution (Travenol Labs, Norfolk, UK) and filtered before use.

31P Nuclear Magnetic Resonance Spectroscopy
Changes in myocardial ATP, PCr and Pi were followed using 31P N.M.R. (Bruker AMX-400 wide bore vertical system, 31P frequency 161.9 MHz). Fully relaxed spectra were acquired at 20 min of normoxic perfusion (36 scans, 90° angle and 15 s interpulse delay). Subsequently, saturated spectra (24 or 280 scans, 60° angle, 2 s interpulse delay) were collected throughout the experiment. An initial ATP concentration of 23 µmol/g dry wt, as measured by HPLC in both normal and heat shocked hearts, was used for calibration of N.M.R. data. For calculation of high-energy phosphate levels in saturated spectra, saturation factors obtained from repeated fully relaxed and saturated spectra acquired during baseline conditions were used. Correction of the saturation factor obtained at 37°C versus 4°C was established in a solution of ATP, PCr and Pi with intracellular concentrations of inorganic ions.

Functional assessment
Assessment of mechanical function was performed using a balloon catheter inserted into the left ventricle to determine systolic and end-diastolic pressures, as previously described [17]. The balloon was inflated with water in 25 µl steps from 0 to 300 µl. Peak-systolic and end-diastolic pressures were recorded at each loading of the balloon and used to calculate developed pressure. Recovery of mechanical function was expressed as relative recovery of post-ischemic versus pre-ischemic developed pressure (RRDP). Pre- and post-ischemic coronary flows were obtained by timed collection of the coronary effluent into a measuring cylinder.

High performance liquid chromatography
At the end of perfusion hearts were freeze-clamped using aluminium clamps pre-cooled in liquid nitrogen and freeze-dried overnight. About 40 mg of left ventricle was extracted at 4°C with 0.6 M perchloric acid (25 µ1/mg dry tissue ratio), centrifuged (13 000xg for 3 min at 4°C) and the supernatant neutralized with 2M KOH. After removal of precipitated potassium perchlorate by centrifugation (13 000xg for 3 min at 4°C) extracts were immediately analysed using HPLC, with application of the reverse phase method as previously described [18], using a Hewlett-Packard series 1100 chromatograph (Hewlett-Packard, UK). The analytical column (150x4.6 mm ID) was packed with 3 µm ODS Hypersil (Shandon, UK). Chromatographic conditions were as follows: Buffer A consisted of 150 mM KH2PO4 containing 150 mM KC1 (pH 6.0); Buffer B consisted of a 15% (v/v) solution of acetonitrile in buffer A. The composition of the mobile phase changed from 0 to 100% of buffer B according to the gradient curve, as previously described [18]. Sample peaks were integrated and quantified using a HP-Chemstation chromatography data system (Hewlett-Packard, UK). Coronary effluent samples collected during reperfusion were also analysed using the above method; samples were injected directly onto the chromatograph without prior extraction.

Assessment of HSP70 concentration
HSP70 concentration was assessed by SDS-PAGE and Western immunoblotting, as previously described [9]. Whole heart homogenates were solubilised in 1% w/v SDS (sodium dodecyl sulphate), assayed for total protein using the Bradford assay, denatured by heating at 100°C in Laemmli buffer and separated on 10% SDS gels until the Bromophenol Blue tracking-dye reached the end of the gel. The gels were equilibrated for 30 min in transfer buffer before protein transfer at 500 mA for 1 h. Western blots were blocked for 1 h using 3% w/v skimmed milk powder (Marvel) in phosphate-buffered saline (PBS: 0.15 M NaCl, 0.05 M phosphate buffer, pH 7.2) containing 0.05% w/v Tween-20; this blocks non-specific binding sites. Blots were then probed with monoclonal mouse antibody (SPA-810) specific to inducible HSP70 (Bioquote, York, UK), diluted to a final concentration of 1/1000, for 1 h. Blots were washed three times and incubated with secondary horseradish-peroxidase-conjugated rabbit anti-mouse antibody for 1 h.

Blots were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham, UK). Hyperfilm MP (myoperoxidase) was exposed to blots treated with ECL for 30 s and developed in an automatic film processor; following ECL exposure, antibodies were removed from blots by incubation in a solution of 2% w/v SDS, 6.25% v/v 1 M Tris–HCL, pH 6.8 and 0.7% v/v 2-mercaptoethanol. Proteins were then visualized by staining with 0.01% Amido Black in a solution of methanol, water and acetic acid (45:45:10 v/v ratio). Amido Black-stained blots and ECL films were scanned using a Molecular Dynamics 300A laser densitometer and HSP70 levels determined as a proportion of total protein loaded using Quantity One software (P.D.I., New York, USA).

Statistics
Values are presented as means±standard error of the mean (S.E.M.). Statistical comparison was performed by unpaired Student’s t-test or using analysis of variance (ANOVA) for repeated measures followed by Bonferroni test to indicate individual significant differences. A value of P<0.05 was considered as a significant difference.


   Results
Top
 Abstract
 Introduction
Materials and methods
 Results
Discussion
Appendix A. Conference...
 References
 
ATP, PCr and Pi concentrations evaluated using N.M.R. are displayed in Fig. 2 . Although baseline levels of ATP and PCr were similar in HS and C groups, the rate of depletion of high-energy phosphates during the initial phase of ischemia was attenuated in the HS versus C groups; the rate of Pi accumulation was also lower in HS versus C groups. On reperfusion, the recovery of ATP was 10–20% greater in HS versus C groups. PCr levels also recovered on reperfusion significantly better in HS versus C groups; at 10–30 min reperfusion, PCr levels were 20–40% higher in HS versus C groups.




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  		Fig. 2. Concentration of (A) ATP, (B) phosphocreatine and (C) intracellular inorganic phosphate levels in control hearts (n=6) and heat-stressed hearts (n=6) subjected to cardioplegic arrest for 4 h at 4°C ischemia evaluated using 31P N.M.R. Values represent the mean (±S.E.M.). * P<0.05 versus control.

 
The concentrations of ATP, ADP, AMP and NAD in freeze-dried hearts are shown in Table 1. Again, significantly higher levels of ATP and total adenine nucleotides were found in HS versus C groups. Total purine catabolite (inosine, adenosine, hypoxanthine, xanthine and uric acid) release during the first 25 min of reperfusion is shown in Fig. 3 . There was 20% less total purine catabolite release from HS versus C groups.


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  		Table 1. Concentrations (µmol/g dry wt.) of adenine nucleotides and NAD in control and heat stressed hearts subjected to cardioplegic arrest and ischemia for 4 h at 4°C

 


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  		Fig. 3. Total purine release in coronary effluent collected over 25 min of reperfusion in control hearts (n=6) and heat-stressed hearts (n=6) subjected to cardioplegic arrest for 4 h at 4°C ischemia. Values represent the mean (±S.E.M.). * P<0.05 versus control.

 
The changes in pre- and post-ischemic peak systolic pressure (LVSP) and end diastolic pressures (LVEDP) are shown in Fig. 4 A and B; relative recovery of developed pressure (RRDP) for both groups is shown in Fig. 5 . There were no significant differences in LVSP and LVEDP before ischemia. Post-ischemic LVSP and LVEDP in HS were not different from post-ischemic C. Higher post-ischemic values of LVSP in HS versus pre-ischemic and higher values of LVEDP in C versus pre-ischemic were the only significant differences. However, the RRDP was 10–25% greater in HS versus C groups at all balloon loadings, indicating improved mechanical function following heat stress. The induction of HSP70 in HS versus C groups was confirmed by Western immunoblotting and laser densitometry; the relative intensity of HSP70 in HS versus C groups was 13.6±4.9 versus 3.9±0.5 (arbitary units). Pre- and post-ischemic coronary flow rates are reported in Table 2; the post-ischemic coronary flows were significantly different from pre-ischemic values but there was no difference between HS and C groups. Hearts were not paced during the protocol and pre- and post-ischemic heart rates are reported in Table 3; there were slight decreases in heart rates both in HS and C post-ischemia (approximately 6%).




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  		Fig. 4. Left ventricular peak systolic pressure, LVSP (A) and left ventricular end-diastolic pressure, LVEDP (B) in control hearts (n=6) and heat-stressed hearts (n=6) subjected to cardioplegic arrest for 4 h of ischemia at 4°C. Values represent the mean (±S.E.M.). * P<0.05 versus pre-ischemic heat stressed, ** P<0.05 versus pre-ischemic control and pre-ischemic heat stressed, *** P<0.05 versus pre-ischemic control, pre-ischemic heat stressed and post-ischemic control.

 


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  		Fig. 5. Relative recovery of developed pressure. Values represent the mean (±S.E.M.) for control hearts (n=6) and heat-stressed hearts (n=6). * P<0.05 versus control. ** P<0.01 versus control.

 

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  		Table 2. Pre- and post-ischemic coronary flow rates (ml/min) in control and heat stressed hearts subjected to cardioplegic arrest and ischemia for 4 h at 4°C

 

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  		Table 3. Pre- and post-ischemic heart rates (beats/min) in control and heat stressed hearts subjected to cardioplegic arrest and ischemia for 4 h at 4°C

 

   Discussion
Top
 Abstract
 Introduction
Materials and methods
 Results
Discussion
Appendix A. Conference...
 References
 
This study demonstrated significant changes in high-energy phosphate metabolism in heat stressed hearts which accompanies improved preservation of cardiac mechanical function following prolonged hypothermic cardioplegic arrest. A decreased rate of high-energy phosphate depletion during ischemia and an increased rate of recovery during reperfusion, were observed in hearts subjected to heat stress. These changes may play a role in the mechanism of cardioprotection by heat stress during preservation for cardiac transplantation.

A significant preservation of myocardial ATP and PCr in HS versus C groups, as assessed by N.M.R., was observed during the early phase of ischemia; after 3–4 h ischemia, PCr was slightly higher in HS versus C groups. A notable finding was that the PCr signal was still visible after 4 h ischemia, even in the control group, indicating excellent protection of high-energy phosphates by cardioplegia; however this protection was further enhanced in the heat stressed group.

Several mechanisms may be responsible for better preservation of high-energy phosphates in HS versus C groups. One possibility is improved regulation of mitochondrial H(+)-Transporting-ATP-Synthase (F1F0-ATPase). Under conditions of oxygen deficiency, delayed or inefficient inhibition of this enzyme by ATPase inhibitor (IF-1) may result in considerable ATP breakdown [19] [20]. It is possible that heat stress may interfere with ATPase activity under ischemia, via either HSP induction or another mechanism. A further possibility is optimized high-energy phosphate utilization for residual membrane transport activity, which is supported by recent studies indicating changes in calcium handling in heat stressed myocytes [10] [21].

On reperfusion, the enhanced overshoot of PCr in HS versus C groups was observed together with better recovery of ATP concentration. However, the initial rate of high energy phosphate restoration in the early period of reperfusion was similar. Results of adenine nucleotide levels in heart extracts are in agreement with the N.M.R. data: significantly higher levels of ATP and total adenine nucleotides were found in HS versus C groups.

Analysis of the coronary effluent confirms the decline in ATP catabolism following heat stress; 20% less total purine catabolites was released in HS versus C groups. Increased nucleotide catabolism is a consequence of an imbalance between energy production and energy utilization and is considered to be a sensitive marker of myocardial ischemia [22] [23] [24] [25] and previous studies have shown a close association between purine concentration in coronary effluent and preservation of ATP levels in the heart after ischemia [26]. As we were investigating the changes in energy metabolism after heat stress, we did not study markers of necrosis. However, previous studies have shown reduced creatine kinase release after heat stress [27].

Other studies concerning changes in high-energy phosphate metabolism after heat stress report conflicting results. Some studies show better preservation of ATP levels after heat stress [14] [28], while others show no changes in nucleotide levels at different stages of ischemia-reperfusion [29]. These differences may be due to varying experimental conditions, especially to the presence of pyruvate in the perfusion buffer [29]. Replacement or addition of pyruvate as metabolic substrate during perfusion markedly affects energy status in the heart [30]; furthermore, pyruvate metabolism is markedly affected after heat stress [15]. Differences in temperature during ischemia seems a less likely explanation for these differences, since we have observed similar changes in high-energy phosphate metabolism in a normothermic model of myocardial ischemia (unpublished observations).

A recent study concerning the effect of heat stress on cardiac function and metabolism after prolonged hypothermic storage [28] supports the results presented here with regard to better maintenance of high-energy phosphate levels during and after ischemia in hearts subjected to heat stress. However, our study provides detailed evaluation of the timing of the metabolic differences and evaluation of function in the same hearts, due to application of N.M.R. spectroscopy. We were able to additionally show that heat stress leads to a slower rate of decline of ATP levels during ischemia, with a significant PCr overshoot in the reperfusion period. Furthermore, our N.M.R. measurements provide more accurate estimation of in vivo concentrations, especially that of unstable PCr.

In the above report, baseline levels of ATP and especially of PCr were very low, which may be a result of different collection/storage/extraction procedures before HPLC analysis; high baseline levels of purine catabolites observed in analysed cardiac samples indicates a considerable breakdown of metabolites between collection and freezing of specimens. However, results of functional evaluation presented in this study are in agreement with our results, with regard to the degree of improvement of developed pressure in heat stressed hearts and with regard to the predominant effect of heat stress on systolic function as opposed to diastolic compliance.

An alternative metabolic mechanism by which heat stress may protect the myocardium is by attenuating intracellular acidosis. However, it was difficult to quantitate changes in intracellular pH in the present study because the intracellular inorganic phosphate NMR signals were very broad during ischemia; this complicates the accurate definition of the position of these peaks. Secondly, the relationship between pH and the chemical shift of inorganic phosphate at 4°C is not well established. Consequently, we are unable to present reliable measurements of intracellular pH in the present study. However, in another set of experiments which involved a protocol of 25 min of normothermic ischemia we have shown that pH is indeed higher during ischemia in hearts subjected to heat stress compared to controls (unpublished observations). Further limitations of our results in terms of clinical application are possible species differences in cardiac energy metabolism and the use of a crystalloid-perfused model.

In conclusion, this study demonstrates improved preservation of myocardial high-energy phosphates in heat stressed hearts subjected to cardioplegic arrest and prolonged hypothermic ischemia. The decreased rate of high-energy phosphate depletion and increased recovery of ATP and PCr levels during reperfusion in heat stressed hearts was associated with improved mechanical recovery. Changes in energy metabolism may thus play an important role in the mechanism of cardioprotection by heat stress during preservation for cardiac transplantation. Future investigations of these metabolic pathways may lead to techniques which can enhance current methods of prolonged donor heart storage.


   Acknowledgments
 
This study was supported by The British Heart Foundation (Grant No. FS 96/062).


   Footnotes
 
Presented at the 11th Annual Meeting of the European Association for Cardio-thoracic Surgery, Copenhagen, Denmark, September 28- October 1, 1997. Back


   Appendix A. Conference discussions
Top
 Abstract
 Introduction
Materials and methods
 Results
Discussion
Appendix A. Conference...
References
 
Dr D. Taggart (Oxford, UK) I have one comment and a question at the end. I did a small series of patients in Oxford looking for heat shock protein by Northern and Western-blotting techniques and I used for coronary bypass grafting an intermittent ischemic protocol, so 10 min to do a distal graft, reperfuse for 10 min, 10 min to do another graft. I compared five patients undergoing coronary artery bypass grafting, with one patient in whom I did an aortic valve replacement using cold crystalloid cardioplegia and we biopsied the left ventricle before and serially throughout the operation until about 40 min after the termination of bypass. In the patients who had the intermittent ischemic protocol we detected very significant increased levels of both heat shock protein itself and indeed marked upregulation of mRNA for that, but what was interesting was that in the one patient undergoing aortic valve replacement submitted to cold crystalloid cardioplegia at the temperature of 4°C we couldn’t find any evidence of upregulation. Could you comment on that.

Dr J. Jayakumar: That is quite an interesting study. Your protocol seems to be a preconditioning protocol; however, you are looking for heat shock proteins. Recent studies have shown that preconditioning can not only lead to the classical preconditioning effect but also lead to a heat shock protein induction effect as well and so what you might be observing is actually the initial stages of the heat stress response induced by your pre-conditioning protocol; the link between the classical and the delayed protection is now being elucidated.

Dr P. Taggart: If I may just pursue this a little bit, how quickly do you think you can induce heat shock protein in the human setting? Animal models would suggest you can detect upregulation of mRNA within about half an hour of ischemia. Do you think that is relevant to clinical practice?

Secondly, you mentioned that in your own study you used cold crystalloid cardioplegia, I think you said for 4 h and the point I was trying to make is do you think that degree of hypothermia may in fact switch off these potentially protective mechanisms.

Dr J. Jayakumar: In answer to the latter point, we don’t know the answer. We do know in animal models that HSP mRNA is increased very early after the ischemic period and this may occur in humans as well. The other point is that patients undergoing cardiac surgery and who have had previous ischemic events may also have increased baseline levels of HSP 70, so therefore we have to look at whether the HSP levels are induced prior to surgery or due to your intermittent ischemic protocol.


   References
Top
 Abstract
 Introduction
Materials and methods
 Results
Discussion
Appendix A. Conference...
 References
 

  1. Lindquist S The heat-shock response. Annu Rev Biochem 1986;55:1151-1191.[Medline]
  2. Lindquist S, Craig EA The heat-shock proteins. Annu Rev Genet 1988;22:631-677.[Medline]
  3. Ellis RJ, van der Vies SM Molecular chaperones. Annu Rev Biochem 1991;60:321-347.[Medline]
  4. Currie RW, Karmazyn M, Kloc M, Mailer K Heat-shock response is associated with enhanced post-ischemic ventricular recovery. Circ Res 1988;63:543-549.[Abstract/Free Full Text]
  5. Maulik N, Engelman RM, Wei Z, Liu X, Rousou JA, Flack JE, Deaton DW, Das DK Drug-induced heat-shock preconditioning improves post-ischemic ventricular recovery after cardiopulmonary bypass. Circulation 1995;92:II381-388.
  6. Brown JM, Grosso MA, Terada LS, Whitman GJ, Banerjee A, White CW, Harken AH, Repine JE Endotoxin pre-treatment increases endogenous myocardial catalase activity and decreases ischemia-reperfusion injury of isolated rat hearts. Proc Natl Acad Sci USA 1989;86:2516-2520.[Abstract/Free Full Text]
  7. Amrani M, Corbett J, Allen NJ, O’Shea J, Boateng SY, May AJ, Dunn MJ, Yacoub MH Induction of heat-shock proteins enhances myocardial and endothelial functional recovery after prolonged cardioplegic arrest. Ann Thorac Surg 1994;57:157-160.[Abstract]
  8. Amrani M, Allen NJ, O’Shea J, Corbett J, Dunn MJ, Tadjkarimi S, Theodoropoulos S, Pepper J, Yacoub MH Role of catalase and heat shock protein on recovery of cardiac endothelial and mechanical function after ischemia. Cardioscience 1993;4:193-198.[Medline]
  9. Amrani M, Corbett J, Boateng SY, Dunn MJ, Yacoub MH Kinetics of induction and protective effect of heat-shock proteins after cardioplegic arrest. Ann Thorac Surg 1996;61:1407-1411.[Abstract/Free Full Text]
  10. Hess ML, Kukreja RC Free radicals, calcium homeostasis, heat shock proteins and myocardial stunning. Ann Thorac Surg 1995;60:760-766.[Abstract/Free Full Text]
  11. Currie RW, Tanguay RM Analysis of RNA for transcripts for catalase and SP71 in rat hearts after in vivo hyperthermia. Biochem Cell Biol 1991;69:375-382.[Medline]
  12. Malyshev IY, Manukhina EB, Mikoyan VD, Kubrina LN, Vanin AF Nitric oxide is involved in heat-induced HSP70 accumulation. FEBS Lett 1995;370:159-162.[Medline]
  13. Marber MS, Walker JM, Latchman DS, Yellon DM Attenuation by heat stress of a submaximal calcium paradox in the rabbit heart. J Mol Cell Cardiol 1993;25:1119-1126.[Medline]
  14. Yellon DM, Pasini E, Cargnoni A, Marber MS, Latchman DS, Ferrari R The protective role of heat stress in the ischaemic and reperfused rabbit myocardium. J Mol Cell Cardiol 1992;24:895-907.[Medline]
  15. Marber MS, Walker JM, Latchman DS, Yellon DM Myocardial protection after whole body heat stress in the rabbit is dependent on metabolic substrate and is related to the amount of the inducible 70-kDa heat stress protein. J Clin Invest 1994;93:1087-1094.
  16. Smolenski RT, Yacoub MH, Seymour AM-L Reduced purine catabolite production in the post-ischemic rat heart: A 31P NMR assessment of cytosolic metabolites. Magma 1994;2:417-420.
  17. Weber KT, Janicki JS, Schroff S. Measurement of ventricular function in the experimental laboratory. In: Fozzard HA, Haber G, Jennings RB, Katz AM, Morgan ME, editors. The Heart and Cardiovascular System. Raven Press, New York, 1986:856–886.
  18. Smolenski RT, Lachno DR, Ledingham SJM, Yacoub MH Determination of sixteen nucleotides, nucleosides and bases using high-performance liquid chromatography and its application to the study of purine metabolism in hearts for transplantation. J Chromatogr 1990;527:414-420.[Medline]
  19. Rouslin W, Broge CW, Grupp IL ATP depletion and mitochondrial functional loss during ischemia in slow and fast heart-rate hearts. Am J Physiol 1990;259:1759-1766.
  20. Rouslin W, Broge CW Mechanisms of ATP conservation during ischemia in slow and fast heart-rate hearts. Am J Physiol 1993;264:C209-C216.[Abstract/Free Full Text]
  21. Cornelussen RN, Ver Donck L, Verellen G, Gorgers M, van der Vusse GJ, Reneman RS, Snoeckx LH Calcium homeostasis in cardiomyocytes isolated from heat-shocked rats. Am J Physiol 1996;271:H1938-H1946.[Abstract/Free Full Text]
  22. de Jong JW Diagnosis of ischemic heart disease with AMP-catabolites. In: De Jong JW, ed. Myocardial Energy Metabolism. Boston: Nijhof, 1988:237-244.
  23. Harmsen E, de Jong JW, Serrnys PW Hypoxanthine production by ischemic heart demonstrated by high pressure liquid chromatography of blood purine nucleosides and oxypurines. Clin Chim Acta 1981;115:73-84.[Medline]
  24. Smolenski RT, Yacoub MH Liquid chromatographic evaluation of purine production in the donor human heart during transplantation. Biomed Chromatogr 1993;7:189-195.[Medline]
  25. Smolenski RT, Schrader J, de Groot H, Deussen A Oxygen partial pressure and free intracellular adenosine of isolated cardiomyocytes. Am J Physiol 1991;260:C708-C714.[Abstract/Free Full Text]
  26. Smolenski RT, Lachno DR, Yacoub MH Adenine nucleotide catabolism in human myocardium during heart and heart-lung transplantation. Eur J Cardiothorac Surg 1992;6:25-30.[Abstract]
  27. Liu X, Engelman RM, Moraru II, Rousou JA, Flack III JE, Deaton DW, Maulik N, Das DK Heat shock. A new approach for myocardial preservation in cardiac surgery. Circulation 1992;86:358-363.
  28. Zhang J, Furukawa RD, Fremes SE The beneficial effects of heat-shock for prolonged hypothermic storage. J Surg Res 1996;63:314-319.[Medline]
  29. Currie RW, Karmazyn M Improved post-ischemic ventricular recovery in the absence of changes in energy metabolism in working rat hearts following heat-shock. J Mol Cell Cardiol 1990;22:631-636.[Medline]
  30. From AHL, Zimmer SD, Michurski SP, Mohanakrishnan P, Ulstad VK, Thoma WJ, Ugurbil K Regulation of the phosphorylation rate in the intact cell. Biochemistry 1990;29:3731-3743.[Medline]



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