Diazoxide Provides Protection to Human Myocardium In Vitro That Is Concentration Dependent

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1 Diazoxide Provides Protection to Human Myocardium In Vitro That Is Concentration Dependent Marek A. Deja, MD, Krzysztof S. Golba, MD, PhD, Maciej Kolowca, MD, Kazimierz Widenka, MD, Jolanta Biernat, MD, and Stanislaw Wos, MD, PhD Second Department of Cardiac Surgery and Department of Cardiology, Medical University of Silesia, Katowice, Poland Background. Diazoxide has been shown to confer significant myocardial protection in many experiments. This study was designed to assess its influence on the structural injury and functional recovery of human myocardium subjected to hypoxia/reoxygenation in vitro. Methods. The isolated electrically driven human right atrial trabeculae, obtained during cardiac surgery, were studied. The tissue bath was oxygenated with 95% oxygen and 5% carbon dioxide, hypoxia being obtained by replacing oxygen with argon. The influence of diazoxide on atrial contractility was studied first. Next, the two trabeculae from one atrial appendage were studied simultaneously, adding diazoxide to the tissue bath 10 minutes before hypoxia in one, with another serving as a control. We tested mol/l and 10-4 mol/l diazoxide in three sets of experiments testing 30, 60, and 90 minutes of hypoxia. We continued reoxygenation for 120 minutes (in 60-minute and 90-minute hypoxia experiments) and subsequently tested reaction to 10-4 mol/l norepinephrine. Apart from continuous recording of the contraction force, we measured the troponin I release into the tissue bath after ischemia and reoxygenation. Results. Diazoxide exerted a negative inotropic effect in human atrial muscle (pd ). Both concentrations of diazoxide studied resulted in better functional recovery of atrial trabeculae subjected to 30 minutes of hypoxia. With longer hypoxia, only the higher diazoxide concentration provided significant protection as assessed by contractility. After 120 minutes of reoxygenation, only diazoxide-treated muscle was viable enough to respond to norepinephrine. Only 10-4 mol/l diazoxide resulted in lower troponin I release during hypoxia and reoxygenation. Conclusions. This study shows that diazoxide provides significant concentration-dependent protection against hypoxia/reoxygenation injury to human myocardium in vitro. (Ann Thorac Surg 2004;77:226 32) 2004 by The Society of Thoracic Surgeons The ATP-sensitive potassium (K ATP ) channels were first linked to preconditioning by Gross and associates [1]. The protection achieved was initially ascribed to the action of sarcolemmal K ATP channels, and the role of shortening of the action potential was postulated [2]. It was subsequently convincingly shown in different models [3, 4, 5] that it was the mitochondrial, not the sarcolemmal, K ATP channel that afforded protection against myocardial injury. Mitochondrial K ATP channels became considered an end-effector of preconditioning by the way of either making the mitochondria more resistant to Ca 2 entry [6] or by improving cell energy management or energy transfer to the cytoplasm [7], or by some other unknown mechanism. More recently, it was proposed that mitochondrial K ATP channel acts as a trigger or mediator rather than an effector of preconditioning [8, 9]. Its activation leads to free radical formation, and only Presented at the Thirty-ninth Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 31 Feb 2, Address reprint requests to Dr Deja, Second Department of Cardiac Surgery, Medical University of Silesia, Ul. Ziolowa 47, Katowice, Poland; narizol@slam.katowice.pl. subsequently protein kinase C or tyrosine kinase is activated. This view is, however, still debatable [10]. Whether the mitochondrial K ATP channel is the effector or mediator of preconditioning, it plays an important role in cardioprotection [11]. Importantly, it may be an attractive target for eliciting the protection against ischemia in human myocardium. The basic, selective mitochondrial K ATP channel activator, used in most laboratory studies examining the role of mitochondrial K ATP channels in preconditioning [3 13] (diazoxide), had been used for years in humans for the treatment of hypertensive crisis and hyperinsulinemia. It has been shown to confer significant myocardial protection in many experiments [3 13], and it has been proposed as a possible adjunct to myocardial protection during cardiac surgical procedures [15 19]. Nevertheless, diazoxide use for myocardial protection during cardiac surgery in real patients has not been reported yet. Before it can be used clinically, its influence on the contractility of human myocardium has to be established. It is important to find what level of protection can be afforded by diazoxide in human myocardium and what concentration is necessary to provide significant myocardial protection 2004 by The Society of Thoracic Surgeons /04/$30.00 Published by Elsevier Inc doi: /s (03)

2 Ann Thorac Surg DEJA ET AL 2004;77: DIAZOXIDE PROTECTS HUMAN MYOCARDIUM in human tissue. These are the questions addressed by the present study. Material and Methods The experiments were performed on the isolated electrically driven human right atrial trabeculae acquired from the right atrial appendages discarded during cardiac surgery. The Local Bioethics Committee approval for the use of human tissue was obtained. The tissue was transferred in ice-cold Krebs-Henseleit solution to the laboratory. The single trabecula less than 1 mm in diameter was mounted in the organ chamber (Schuler Organbath, Hugo Sachs Elektronik, March- Hugstetten, Germany [HSE]) containing Krebs-Henseleit solution of the following composition [mol/l]: NaCl, 118.0; KCl, 4.70; CaCl 2, 2.52; MgSO 4, 1.64; NaHCO 3, 24.88; KH 2 PO 4, 1.18; glucose, 5.55; sodium pyruvate, 2.0 (ph 7.4). It was oxygenated with carbogen (95% oxygen, 5% carbon dioxide) and maintained at 37 C. The trabecula was driven with 1 Hz, 50 ms square stimuli using platinum field electrodes and the potential of 150% of the threshold for given preparation. The stimulator type 215 (HSE) was used. The contraction force was measured with F30 isometric force transducer type 372 (HSE). The signal was enhanced with bridge amplifier type 336 (HSE) and recorded with Graphtec Linearcorder WR 3320 (HSE). The trabecula was gradually stretched to seek for the optimal preload according to the Frank-Starling relationship. Once the maximal contraction force was achieved, the preparation was relaxed to 90% of optimal tension and left for 30 minutes of stabilization. During this period, it was thoroughly washed. In the first set of experiments (n 20), the influence of diazoxide (Sigma Chemical Co, St. Louis, MO) on myocardial contractility was assessed. Diazoxide was dissolved in dimethyl sulfoxide (DMSO) (Sigma Chemical Co). The concentration of DMSO in tissue bath was always kept below 0.4%. We checked that this concentration of DMSO did not influence the myocardial contractility. Diazoxide was applied in increasing concentrations, starting with 10-8 mol/l and increasing in negative logarithm half-molar cumulative steps up to 10-3 mol/l to establish the concentration-effect relationship. The contractility after diazoxide was expressed as a percentage of the initial contraction force, and presented at each concentration level as a mean SEM. The concentrationeffect relationship was obtained from a regression analysis to the general logistic equation of Michaelis and Menten (1913): Cn E C n K n, c where E is effect, C is concentration, K C is drug-receptor complex dissociation constant equal to the concentration causing half-maximal effect (EC 50 ), and n is the Hill coefficient. From the above, the mean pd 2 -log(ec 50 ) and Hill coefficient ( SEM) were estimated. 227 During subsequent experiments, the hypoxia was simulated by substituting oxygen in carbogen with argon (95% argon, 5% carbon dioxide). This resulted in the drop of tissue bath oxygen partial pressure from mm Hg to mm Hg (p 0.001) and caused significant and rapid impairment of muscle inotropism. On reoxygenation, the carbogen (95% oxygen, 5% carbon dioxide) was added again and the tissue was washed several times. The two trabeculae from one atrial appendage were always studied simultaneously, adding diazoxide to the tissue bath 10 minutes before simulated hypoxia in one, with another serving as a control. We tested mol/l (31.6 mol/l) and 10-4 mol/l (100 mol/l) diazoxide. Six separate sets of experiments (10 patients each) were performed testing two different diazoxide concentrations added 10 minutes before 30, 60, and 90 minutes of hypoxia respectively. In the experiments with a longer (60 or 90 minutes) hypoxia period, we kept the reoxygenation period for 120 minutes and subsequently added 10-4 mol/l norepinephrine [(-)-arterenol bitartrate (Sigma Chemical Co)] to test for stunning. We recorded the force of contraction continuously. We compared the recovery of contraction force after 40 minutes of reoxygenation. This was the time after which contractility achieved plateau and before it started declining again. In experiments with 60 and 90 minutes of hypoxia, we additionally analyzed the contraction force after 120 minutes of reoxygenation and after adding norepinephrine. For each preparation, the contraction force recorded was compared with its initial contraction force (after stabilization period) and expressed in percent, the initial contraction force being 100%. To allow for troponin I accumulation, the tissue bath was not replaced throughout the hypoxia period, and apart from the initial washout, it was not replenished throughout the reoxygenation as well. The sample of tissue bath was obtained for troponin I concentration measurement after the hypoxia period and after 120 minutes of reoxygenation (not in the 30-minute hypoxia experiments). The troponin I concentration was measured using the immunoenzymatic method (Diagnostic Resort Group International Co, Mountainside, NJ) and expressed in nanograms per milligram wet tissue weight. Statistical Analysis We first used two-way repeated measures (RM) analysis of variance (ANOVA) with the time of hypoxia as one factor and the concentration of diazoxide (10-4 vs vs 0 mol/l [control]) as another. Because two-way RM ANOVA showed influence of the hypoxia time on contractility, we analyzed separately the influence of diazoxide concentration on the outcomes (both contractility and troponin I release) in experiments with different hypoxia times using one-way RM ANOVA. We used the Holm- Sidak method for the post hoc multiple comparison procedure. We considered p less than 0.05 significant. SigmaStat for Windows Version 3.0 (SPSS Inc, Cary, NC) was used for statistical analysis. CARDIOVASCULAR

3 228 DEJA ET AL Ann Thorac Surg DIAZOXIDE PROTECTS HUMAN MYOCARDIUM 2004;77: Fig 1. The influence of diazoxide on contractility of human right atrial trabecula. Points depict mean SEM. E EC 50 SEM (n 20). Results We found diazoxide to exert a negative inotropic effect in human atrial myocardium (Fig 1). This was observed with relatively high diazoxide concentrations. Diazoxide pd 2 equaled This relates to an EC 50 of 110 mol/l. Diazoxide given 10 minutes before hypoxia caused some depression of contractility. It dropped to 79% 2.2% (p 0.001) with mol/l and to 54% 3.6% (p 0.001) with 10-4 mol/l diazoxide. The period of hypoxia resulted in a significant depression of contractile force to average of 8.8% 0.5%. The contraction force recovered after 40 minutes of reoxygenation depended not only on the length of hypoxia (p 0.001) but also on the concentration of diazoxide (p 0.001), as shown by two-way RM ANOVA. We analyzed separately the recovery of contractility in experiments with different hypoxia times using one-way RM ANOVA with post hoc testing (Fig 2). Both concentrations of diazoxide allowed significantly better recovery of contractility after 30 minutes of hypoxia and 40 minutes of reoxygenation (65% 7% vs 47% 5%; p 0.002; and 62% 4% vs 45% 6%; p 0.013). With 60 minutes of hypoxia, the recovery was significantly improved by 10-4 mol/l diazoxide. In experiments with 90 minutes of hypoxia, the contractility of atrial muscle treated with 10-4 mol/l diazoxide was not only higher than the control (56% 9% vs 24% 3%, p 0.001) but also on average by 30% higher (p 0.001) than in the muscle treated with a lower ( mol/l) diazoxide concentration. Prolonged reoxygenation led to gradual decline in contraction force. Still, the contractility maintained depended on both the hypoxia time (p 0.019) and on the diazoxide concentration (p 0.001). With 60 minutes of hypoxia, the contractility at the end of the 120-minute reoxygenation period was significantly higher in 10-4 mol/l diazoxide treated myocardium than in control (26% 5% vs 9% 1%, p 0.02). The difference failed to reach the significance (p 0.07) with the lower diazoxide Fig 2. The recovery of contractility (as the percentage of the initial contraction force) after a period of hypoxia and 40 minutes of reoxygenation. Bars depict mean contractility SEM. The p values given in boxes are the result of one-way RM ANOVA for experiments with given hypoxia time. The p values above the bars come from post hoc testing using the Holm-Sidak method. n 10 for each bar. concentration (Fig 3). In experiments with 90 minutes of hypoxia, we showed again a higher contractility of trabeculae treated with 10-4 mol/l diazoxide not only in comparison with the control (20% 6% vs 6% 2%, p 0.018) but also in comparison with the lower diazoxide concentration (11% 2%, p 0.014). At the end of the 120-minute reoxygenation period, the muscle treated with diazoxide responded to norepinphrine with an increased contractility, whereas the muscle not protected failed to respond. Appreciably, the increase in contractility of muscle treated with mol/l diazoxide failed to attain statistically significant difference over control in the 90-minute hypoxia experiment (Fig 4). We observed a tendency towards lower troponin I release during hypoxia in the diazoxide-treated myocardium. However, we could prove a statistically significant difference only in experiment with 90 minutes of hypoxia, and only when using the higher (10-4 mol/l) diazoxide concentration ( vs ng/mg, p 0.003) (Fig 5). The same was true regarding troponin I release throughout the 120-minute reoxygenation period ( vs ng/mg, p 0.003) (Fig 6). Comment The first important finding of our study is the negative inotropic action of diazoxide in human atrial muscle. To our knowledge, this has not been described in the experiments of other authors. We have therefore extended our

4 Ann Thorac Surg DEJA ET AL 2004;77: DIAZOXIDE PROTECTS HUMAN MYOCARDIUM 229 CARDIOVASCULAR Fig 3. The contractility (as the percentage of the initial contraction force) after period of hypoxia and 120 minutes of reoxygenation. Bars depict mean contractility SEM. The p values given in boxes are the result of one-way RM ANOVA for experiments with given hypoxia time. The p values above bars come from post hoc testing using the Holm-Sidak method. n 10 for each bar. Fig 4. The contractility (as the percentage of the initial contraction force) in response to 10-4 mol/l of norepinephrine (NE) after a period of hypoxia and 120 minutes of reoxygenation. Bars depict mean contractility SEM. The p values given in boxes are the result of one-way RM ANOVA for experiments with given hypoxia time. The p values above bars come from post hoc testing using the Holm- Sidak method. n 10 for each bar. experiment to 20 preparations, and the results were farily consistent throughout all atrial trabeculae studied. We have checked that DMSO used as a solvent for diazoxide, when added alone to tissue bath, caused no contractility change. Characteristically, the concentration necessary for negative inotropic action was rather high. Diazoxide pd 2 equaled 3.96, which relates to EC 50 of 110 mol/l. This is more than 100 times more than the K 1/2 value for mitochondrial K ATP channel (0.8 mol/l), but still less than K 1/2 value for sarcolemmal K ATP channel (840 mol/ L), as shown by Garlid and associates in reconstituted bovine heart mitochondrial and sarcolemmal K ATP channels [3]. We cannot explain the negative inotropic effect of diazoxide in our preparations, and we continue to study it in our laboratory. Having established the diazoxide influence on human atrium contractility, we chose to study the myocardioprotective effects of its two different concentrations. We elected to study the influence of mol/l (approximately 30 mol/l) and 10-4 mol/l (100 mol/l) of diazoxide for several reasons. First, these very concentrations were used in various laboratory and animal studies before. Second, the authors generally used either the concentration of 30 mol/l [14, 15, 19, 20] or 100 mol/l [9, 12 13], or concentrations in between the two [8, 16 18, 25], and there is little comparison between the cardioprotective action of them. In the work of Garlid and associates [3], both doses possessed similar cardioprotective effect in isolated rat hearts, and the authors elected to use 30 mol/l diazoxide in their further experiments. On the other hand, Liu and associates [4] studied the cardioprotection and opening of mitochondrial K ATP channels mediated by diazoxide in rabbit ventricular myocytes. They showed that at concentrations up to 100 mol/l, diazoxide dose-dependently opened the mitochondrial without activating the sarcolemmal K ATP channel. K 1/2 value was 27 mol/l, suggesting marked cardioprotective benefit of increasing the diazoxide concentration above this value. Last but not the least, 100 mol/l diazoxide caused 50% contractility drop in our experiments (EC 50 ), whereas 30 mol/l caused only little (approximately 20%) decrease in contractility. Therefore, we considered it important, especially in view of potential clinical use, to find whether increasing diazoxide concentration would confer significant cardioprotective benefit. Our experimental model of myocardial hypoxia and reoxygenation is based on always studying a pair of atrial trabeculae from the same patient. This is necessary, as we observed a different level of myocardial contractility suppression with hypoxia and a different level of recovery with reoxygenation in different patients. It was not surprising, as the patients differed from the point of view of not only exact cardiac pathology but also from the point of view of their medication, demographics, and so on. We have reported previously, for instance, that the atrial trabeculae of patients with different disease states differ considerably in their inotropic responses [24]. To study cardioprotection with diazoxide, we elected not to use tissue from diabetic patients, but we decided that further exclusion criteria would not be practically feasi-

5 230 DEJA ET AL Ann Thorac Surg DIAZOXIDE PROTECTS HUMAN MYOCARDIUM 2004;77: Fig 5. The troponin I release to the tissue bath over the hypoxia period in nanograms per milligram wet mass of the atrial trabecula. Bars depict mean SEM. The p values given in boxes are the result of one-way RM ANOVA for experiments with given hypoxia time. The p values above bars come from post hoc testing using the Holm-Sidak method. n 10 for each bar. Fig 6. The troponin I release to the tissue bath over the 120-minute reoxygenation period in nanograms per milligram wet mass of the atrial trabecula. Bars depict mean SEM. The p values given in boxes are the result of one-way RM ANOVA for experiments with given hypoxia time. The p values above bars come from post hoc testing using the Holm-Sidak method. n 10 for each bar. ble. After all, one could never achieve the situation close enough to the clean laboratory animal experiment. We confirmed, nevertheless, in earlier sham experiments that the results analyzed pairwise were consistently showing functional (contractility) and structural (troponin I release) deterioration in preparations subjected to ischemia compared with preparations oxygenated throughout the experiment. To be able to assess the troponin I release to the tissue bath, time was needed to allow it to leak from the tissue to the Krebs-Henselait solution and to accumulate. Therefore, we did not analyze troponin I release in the 30-minute hypoxia experiments. For the same reason, we extended the reoxygenation period to 120 minutes and we did not replace the tissue bath throughout this time. This is in contrast to atrial trabecula being continually superfused during the reperfusion period in the very similar experimental model by Yellon and associates [20]. Yellon s group concentrated, however, on functional recovery only, and no attempt to collect the indicator enzymes was made. Our study showed significant myocardial protection against hypoxia and reperfusion conferred by diazoxide. The recovery of contractility after 40 minutes of reoxygenation did not depend on diazoxide concentration if the trabeculae were subjected to the short period of hypoxia. However, a clear advantage of using higher diazoxide concentration was visible when assessing the functional recovery after longer, particularly 90-minute, hypoxia. The longer reoxygenation period resulted in a gradual loss of contractile function, probably related to reoxygenation injury. However, 10-4 mol/l diazoxide protected, to some extent, against this injury. The functional recovery of human atrial trabeculae from hypoxia/reoxygenation insult in our study should be compared with the results of the already cited Yellon s group study, performed in a very similar model [20]. They studied the classic ischemic preconditioning and the role of protein kinase C and K ATP channels. The functional recovery of the human atrial trabeculae from 90 minutes of simulated ischemia in their study was of the same level as the recovery we observed with 10-4 mol/l of diazoxide. However, they did not observe the decline of myocardial function over the 120-minute reperfusion period. As mentioned earlier, their preparation was continually superfused during the reperfusion, whereas we did not replace the bath solution to allow for troponin I accumulation. This might have enhanced the reoxygenation injury in our experiments. Second, the tissue bath solution used in Yellon s laboratory contained a high concentration (10 mmol/l) of pyruvate. Pyruvate has been shown to possess cardioprotective properties [21 22], particularly against the reperfusion injury. And, indeed, when we used a high pyruvate level Krebs-Henseleit solution in our laboratory, it enhanced recovery (data not published). Therefore, we resigned from using it so as not to confound the results. To test whether the deteriorating contractility during the reoxygenation period depended on the loss of cellular integrity or on the increasing stunning, we added

6 Ann Thorac Surg DEJA ET AL 2004;77: DIAZOXIDE PROTECTS HUMAN MYOCARDIUM norepinephrine to the tissue bath. The reversibility of contractility loss in human atrial trabeculae under positive inotropic challenge fulfils the definition of stunned myocardium [23]. We found that all diazoxide-protected trabeculae responded with an increase of contraction force, whereas no response in control muscle was observed. This finding would suggest that the function decline with reoxygenation depended on structural damage in control group, and on growing stunning in diazoxide-treated group. In other words, the diazoxide did not prevent stunning development, but it preserved structural integrity necessary for inotropic response to norepinephrine. We draw no conclusion from the magnitude of the response to norepinephrine, as we have shown previously in our laboratory that this may vary from patient to patient [24]. However, the rise in contractility of muscle treated with mol/l diazoxide and subjected to 90 minutes of hypoxia failed to reach statistical significance compared with the control. This confirms once more the benefit of using the higher diazoxide concentration. Although we observed the tendency to lower troponin I release after diazoxide treatment in both hypoxia and reoxygenation in all experiments, the 90-minute ischemia period was necessary to make differences statistically significant. Perhaps 60-minute ischemia was too short to allow for significant troponin I accumulation in tissue bath. Still, in 90-minute hypoxia experiments, the differences were significant only with higher diazoxide concentration. This parallels the results of functional recovery in 90-minute hypoxia experiments. Based on the above, we conclude that diazoxide protected human atrial myocardium from hypoxia/ reoxygenation injury in a concentration-dependent fashion. It both improved functional recovery and maintained cellular integrity. This is opposite to earlier reports showing that mitochondrial K ATP channel opening reduces infarct size but has little, if any, effect on functional recovery [16 17, 26], unless we speculate that diazoxide might have other points of action in human myocardium, different from mitochondrial K ATP channel [26]. The last issue to address is the difference between our results and the results obtained in study of Pomerantz and associates [14]. They found that subjecting the myocardium to diazoxide for a short period before ischemia provided significant protection, whereas leaving the diazoxide in the tissue bath throughout the ischemic period resulted in loss of protective effect. However, the authors studied 30 mol/l diazoxide only. Perhaps with 10-4 mol/l diazoxide, the authors would observe the protective effect similar to our results. We chose to leave the diazoxide in the tissue bath for the period of ischemia to mimic the situation in which the diazoxide is one of the components of cardioplegic solution. In this case, it would be infused at the beginning of (or hopefully with blood cardioplegia before) the ischemic period. It would, with a bit of luck, stay in coronaries in between the infusions, and would be replenished with maintenance cardioplegia doses. There are data from other authors suggesting that diazoxide given in cardioplegia, and even reaching the myocardium only at the beginning of reperfusion, may be beneficial for myocardial preservation [16 19]. We suggest that to achieve maximal cardioprotection, the concentration of diazoxide reaching the myocardium should be rather high (ie, at the level of 10-4 mol/l [100 mol/l]. It may cause a significant negative inotropic effect, but at least in vitro, this effect easily disappears with washout. 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7 232 DEJA ET AL Ann Thorac Surg DIAZOXIDE PROTECTS HUMAN MYOCARDIUM 2004;77: condition human myocardium. J Thorac Cardiovasc Surg 2000;120: Toyoda Y, Levitsky S, McCully JD. Opening of mitochondrial ATP-sensitive potassium channels enhances cardioplegic protection. Ann Thorac Surg 2001;71: Wakiyama H, Cowan DB, Toyoda Y, Federman M, Levitsky S, McCully JD. Selective opening of mitochondrial ATPsensitive potassium channels during surgically induced myocardial ischemia decreases necrosis and apoptosis. Eur J Cardiothorac Surg 2002;21: McCully JD, Wakiyama H, Cowan DB, Federman M, Parker RA, Levitsky S. Diazoxide amelioration of myocardial injury and mitochondrial damage during cardiac surgery. Ann Thorac Surg 2002;74: Kavelaitis E, Oubenaissa A, Mouas C, Peynet J, Menasche P. Ischemic preconditioning with opening of mitochondrial adenosine triphosphate-sensitive potassium channels or Na /H exchange inhibition: which is the best protective strategy for heart transplants? 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J Physiol Pharmacol 1995;46: Toyoda Y, Friehs I, Parker RA, Levitsky S, McCully JD. Differential role of sarcolemmal and mitochondrial K ATP channels in adenosine enhanced ischemic preconditioning. Am J Physiol Heart Circ Physiol 2000;279:H Hanley PJ, Mickel M, Loffler M, Brandt U, Daut J. K ATP channel-independent targets of diazoxide and 5-hydroxydecaonate in the heart. J Physiol 2002;542: DISCUSSION DR MARSHALL L. JACOBS (Philadelphia, PA): It is a lovely experiment and a very, very fine presentation. I am afraid my naivete will be revealed by the question, but I am struck most by the response to norepinephrine stimulation and the apparent implications of that. Could you elaborate further on that? I mean, if one had looked not at postreoxygenation function but just that poststimulation function, you would have an earthshatteringly dramatic result. What are the implications of this response to norepinephrine stimulation? DR DEJA: We actually hoped to show that diazoxide prevented stunning to some extent. There were physiological studies done previously using this kind of tool (inotropic stimulation for finding or proving the stunning). To our surprise, we found a significant response to norepinephrine in diazoxide-treated muscle, which means that the muscle was simply stunned when losing the contractile force over 120 minutes of reoxygenation, and no response at all or no significant response in the muscle not treated with diazoxide, which, in our interpretation, together with the troponin level, means that probably the structural damage to the muscle was big enough not to allow for any significant inotropic response. Yes, we think it is a very interesting finding. Unfortunately, we have not found a group where there was a smaller response to norepinephrine in myocardium not treated with diazoxide and a bigger response in treated, which probably would look even more elegant. DR TOMASZ TIMEK (Redlands, CA): Thank you for the presentation. It was beautiful. The question I had was, could you comment on the mechanism of the negative effect of diazoxide before hypoxia, and is that effect also dose-dependent? DR DEJA: Yes, it is dose-dependent. As you have probably noticed, there is around 10% to 15% drop of contractile force with the lower concentration ( mol/l) and almost 30% to 40% contractile force drop with the higher concentration (10 4 mol/l) of diazoxide. Now, it is very difficult to comment on it, and what we are planning to do is to try and exclude the possibility that diazoxide was actually acting via the sarcolemmal channel at this high concentration. To do this, we need a sarcolemmal ATP-dependent potassium channel blocker. In the experiments I know, there is no proof that diazoxide in such a concentration can work on sarcolemmal channels. At least Gross and Garlid s group studies show that with this concentration and with the relative affinity to sarcolemmal channels being 2,000 times less than to the mitochondrial, one should not expect it. So, the only thing I can think of is the sarcolemmal channel being involved, and we obviously need to test for it.