Temporal Relation of ATP-Sensitive Potassium- Channel Activation and Contractility Before Cardioplegia

Transcription

1 Temporal Relation of ATP-Sensitive Potassium- Channel Activation and Contractility Before Cardioplegia Latha Hebbar, MD, Ward V. Houck, MD, James L. Zellner, MD, B. Hugh Dorman, MD, PhD, and Francis G. Spinale, MD, PhD Department of Anesthesia and Perioperative Medicine and Division of Cardiothoracic Surgery, Department of Surgery, Medical University of South Carolina, Charleston, South Carolina Background. Pharmacologic treatment using potassium-channel openers (PCOs) before cardioplegic arrest has been demonstrated to provide beneficial effects on left ventricular performance with subsequent reperfusion and rewarming. However, the PCO treatment interval necessary to provide protective effects during cardioplegic arrest remains to be defined. The present study was designed to determine the optimum period of PCO treatment that would impart beneficial effects on left ventricular myocyte contractility after simulated cardioplegic arrest. Methods. Left ventricular porcine myocytes were assigned randomly to three groups: (1) normothermic control 37 C for 2 hours; (2) cardioplegia K (24 meq/l) at 4 C for 2 hours followed by reperfusion and rewarming; and (3) PCO and cardioplegia 1 to 15 minutes of treatment with the PCO aprikalim (100 mol/l) at 37 C followed by hypothermic (4 C) cardioplegic arrest and subsequent rewarming. Myocyte contractility was measured after rewarming by videomicroscopy. A minimum of 50 myocytes were examined at each treatment and time point. Results. Myocyte velocity of shortening was reduced after cardioplegic arrest and rewarming compared with normothermic controls (63 3 m/s versus 32 2 m/s, respectively; p < 0.05). With 3 minutes of PCO treatment, myocyte velocity of shortening was improved after cardioplegic arrest to values similar to those of normothermic controls (56 3 m/s). Potassium channel opener treatment for less than 3 minutes did not impart a protective effect, and the protective effect was not improved further with more prolonged periods of PCO treatment. Conclusions. A brief interval of PCO treatment produced beneficial effects on left ventricular myocyte contractile function in a simulated model of cardioplegic arrest and rewarming. These results suggest that a brief period of PCO treatment may provide a strategy for myocardial protection during prolonged cardioplegic arrest in the setting of cardiac operation. (Ann Thorac Surg 1998;65: ) 1998 by The Society of Thoracic Surgeons Preconditioning is an adaptive response in which brief episodes of reversible ischemia render the myocardium more tolerant to subsequent prolonged intervals of ischemia. The activation of adenosine triphosphate sensitive potassium (K ATP ) channels within the myocardium has been proposed to be a fundamental mechanism involved in eliciting the protective effects of preconditioning [1, 2]. Furthermore, it has been demonstrated that activation of these K ATP channels can be achieved pharmacologically with the use of specific potassium-channel openers (PCOs) [3, 4]. Past reports showed that treatment of isolated hearts with PCOs before hyperkalemic cardioplegic arrest imparted a protective effect on functional recovery of the myocardium [5, 6]. However, the specific duration of PCO treatment necessary to impart this protective effect remains to be defined. This laboratory Accepted for publication Nov 25, Address reprint requests to Dr Spinale, Division of Cardiothoracic Surgery, Medical University of South Carolina, 171 Ashley Ave, Rm 418 CSB, Charleston, SC previously demonstrated that in an isolated myocyte system, hyperkalemic cardioplegic arrest with subsequent rewarming and reperfusion was associated with a reduction in contractile performance [7, 8]. Accordingly, the objectives of the present study were twofold: (1) to determine whether PCO treatment before hypothermic, hyperkalemic cardioplegic arrest would improve isolated myocyte contractile performance with subsequent reperfusion, and (2) to identify the optimal PCO treatment interval necessary to provide protective effects with subsequent cardioplegic arrest. Material and Methods Five age- and weight-matched pigs (Yorkshire, 25 to 28 kg) were included in the study. All animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC, 1996) by The Society of Thoracic Surgeons /98/$19.00 Published by Elsevier Science Inc PII S (98)

2 1078 HEBBAR ET AL Ann Thorac Surg POTASSIUM CHANNELS AND CARDIOPLEGIA 1998;65: Myocyte Isolation and Contractile Function Measurement Myocytes were isolated using previously described methods [7, 8]. Briefly, animals were anesthetized with 1.5% isoflurane in 50% oxygen and 50% nitrous oxide, a sternotomy was performed, and the heart was extirpated quickly and placed in cold oxygenated Krebs solution. A region of the left ventricular free wall (5 by 5 cm) incorporating the circumflex coronary artery was dissected free, cannulated, and perfused with an oxygenated collagenase solution (0.5 mg/ml, type II, 146 U/mg; Worthington Biochemical Corporation, Freehold, NJ). The isolated myocytes then were resuspended in standard cell culture medium (2 mmol/l of Ca 2, 2% bovine serum albumin, Medium M-199; Gibco Laboratories, Grand Island, NY) and placed on coverslips coated with a basement membrane substrate (Matrigel; Collaborative Research, Inc, Bedford, MA). The yield of viable myocytes was greater than 80% in all myocyte preparations and was not affected by cardioplegic arrest and rewarming. Viable myocytes were defined as those cells that maintained a rod shape, were Ca 2 tolerant, remained quiescent in culture, excluded trypan blue, and responded to electrical field stimulation. Myocyte contractile function was examined using computer-assisted videomicroscopic techniques described previously [7, 8]. Briefly, myocytes were imaged on an inverted microscope (Axiovert IM35; Zeiss Inc, Germany) in 2.5-mL thermostatically controlled chambers at 37 C. Myocytes were stimulated at 1 Hz and contractions were imaged with the use of a charge-coupled device (GPCD60; Panasonic, Secaucus, NJ). Myocyte motion signals were input through an edge detector system (Crescent Electronics, Sandy, UT), converted into a voltage signal, digitized, and input into a computer (80286, ZBV2526; Zenith Data Systems, St. Joseph, MI) for subsequent analysis. Stimulated myocytes were allowed a 5-minute stabilization period after electrical stimulation, and contraction data for each myocyte were recorded. Parameters computed from the digitized contraction profiles included percentage of shortening, velocity of shortening (in micrometers per second), velocity of relengthening (in micrometers per second), and time to 50% relaxation (in milliseconds). Myocyte percentage of shortening was determined as the percentage difference between the maximum and minimum cell length for each contraction. Myocyte velocity computations were obtained by differentiating the digitized contraction profiles. All parameters were calculated for each contraction and the results were averaged for a minimum of 20 contractions. Only those myocytes that maintained a rod-shaped morphology throughout the study protocols, remained quiescent in culture, and responded in a homogeneous fashion to electrical stimulation were included in the analysis. Experimental Protocol After a 1-hour stabilization period at 37 C, isolated myocytes were assigned randomly to one of four treatment groups: (1) normothermic control incubation of myocytes in 37 C Ringer s solution (Na, 130 mmol/l; Cl, 109 mmol/l; K, 4 mmol/l; Ca 2, 1.8 mmol/l) containing 30 meq/l of HCO 3 for 2 hours in a 95% oxygen environment (n 96); (2) cardioplegia incubation of myocytes in a Ringer s cardioplegia solution containing 24 meq/l of potassium and 30 meq/l of HCO 3 for 2 hours at 4 C (n 90); (3) PCO and cardioplegia treatment of myocytes at 37 C in standard cell culture media containing 100 mol/l of the PCO aprikalim for 1, 2, 3, 4, 5, 10, and 15 minutes (n 475, with a minimum of 50 myocytes included at each time point); or (4) PCO, glibenclamide, and cardioplegia concomitant treatment of myocytes at 37 C in standard cell culture media containing 100 mol/l of the PCO aprikalim and 1 mol/l of glibenclamide, a potassium channel antagonist, for 10 minutes (n 86). After the respective PCO treatment period, myocyte preparations were rinsed and placed in cell media at 37 C for 5 minutes and then subjected to simulated cardioplegic arrest in a Ringer s cardioplegia solution containing 24 meq/l of potassium and 30 meq/l of HCO 3 for 2 hours at 4 C. After each of these protocols, myocytes were resuspended in cell culture media and myocyte contractile function was determined after 10 minutes of rewarming. The concentration of aprikalim (100 mol/l) that was chosen for these experiments was based on the results of previous studies that documented sustained electromechanical arrest and improved cardioprotection after global ischemia in isolated heart preparations [9, 10]. After measurement of contractile function, myocytes were exposed to 25 nmol/l of isoproterenol and contractile function measurements were repeated. This concentration of isoproterenol (6.25 ng/ml) previously has been shown to produce a maximum response in isolated myocyte preparations [7]. Data Analysis Changes in indices of myocyte function between the control and cardioplegia groups were examined using multivariate analysis of variance. When the analysis of variance revealed statistically significant differences, pairwise tests of individual group means were compared using Bonferroni s probabilities. All statistical analysis was performed using standard statistical software programs (BMDP Statistical Software Inc; University of California Press, Los Angeles, CA). Results are presented as the mean plus or minus the standard error of the mean. Values of p of less than 0.05 were considered statistically significant. Results Simulated Cardioplegia Steady-state contractile function for myocytes treated with normothermia, cardioplegia, and PCO plus cardioplegia is summarized in Figure 1. There was a statistically significant reduction in myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest and rewarming compared with normothermic control values. For example, isolated myocyte percentage and velocity of shortening were reduced by more than 40% in the

3 Ann Thorac Surg HEBBAR ET AL 1998;65: POTASSIUM CHANNELS AND CARDIOPLEGIA 1079 Fig 1. Steady-state myocyte contractility (percentage and velocity of shortening; A) was reduced after cardioplegic arrest and rewarming (squares) compared with normothermic values (triangles). Similarly, myocyte relaxation (velocity of relengthening and time to 50% relaxation; B) was impaired after cardioplegic arrest and rewarming compared with normothermic values. With potassium-channel opener (PCO) treatment (circles) for 3 minutes, myocyte function was improved with subsequent cardioplegic arrest and was similar to normothermic values. Potassium-channel opener treatment for less than 3 minutes did not impart a protective effect, whereas treatment for more than 3 minutes did not increase the protective effect further. (*p 0.05 versus the normothermic control group; p 0.05 versus the cardioplegia group.) cardioplegia group compared with the normothermia group. Myocyte velocity of relengthening also was decreased by more than 55% after hyperkalemic cardioplegic arrest. The time to 50% relaxation, which is an index of active relaxation, was increased in cardioplegic myocytes compared with normothermic values. Potassium-Channel Opener Treatment Before Cardioplegia Potassium channel opener treatment for less than 3 minutes followed by cardioplegic arrest and rewarming did not impart a protective effect on myocyte contractile function and resulted in values similar to those of the cardioplegia group (Fig 1). With 3 minutes of PCO treatment followed by cardioplegic arrest and rewarming, however, myocyte percentage and velocity of shortening increased by more than 35% compared with cardioplegia group values. In addition, with a 3-minute treatment interval followed by cardioplegic arrest, velocity of relengthening increased and time to 50% relaxation decreased compared with the cardioplegia group. The beneficial effects of PCO treatment before cardioplegic arrest on myocyte contractile function were observed for all treatment intervals from 3 to 15 minutes (Fig 1). Treatment with a PCO for more than 3 minutes did not improve contractile function after cardioplegic arrest further compared with treatment for 3 minutes. -Adrenergic Response -Adrenergic responsiveness was assessed in myocytes treated with normothermia and in myocytes treated with cardioplegic arrest and rewarming (Fig 2). Isoproterenol (25 nmol/l) caused a statistically significant increase ( 50%) in myocyte percentage and velocity of shortening in all groups. However, all indices of myocyte contractile function in the cardioplegia group remained reduced relative to the normothermic control group. With less than 3 minutes of PCO treatment before cardioplegic arrest, the -adrenergic response was similar to cardioplegia group values. Three minutes of PCO treatment

4 1080 HEBBAR ET AL Ann Thorac Surg POTASSIUM CHANNELS AND CARDIOPLEGIA 1998;65: Fig 2. After -adrenergic stimulation with 25 nmol/l of isoproterenol, myocyte contractility (percentage and velocity of shortening; A) was reduced after cardioplegic arrest and rewarming (squares) compared with normothermic values (triangles). Similarly, myocyte relaxation (velocity of relengthening and time to 50% relaxation; B) was impaired after cardioplegic arrest and rewarming compared with normothermic values. With potassium-channel opener (PCO) pretreatment (circles) for 3 minutes, myocyte function was improved with subsequent cardioplegic arrest and was similar to normothermic values. Potassium-channel opener treatment for less than 3 minutes did not impart a protective effect, whereas treatment for more than 3 minutes did not increase the protective effect further. (*p 0.05 versus the normothermic control group; p 0.05 versus the cardioplegia group.) resulted in a significant increase in -adrenergic responsiveness after cardioplegic arrest and rewarming compared with the cardioplegia group. In addition, 3 minutes of PCO treatment resulted in improved velocity of relengthening and a reduced time to 50% relaxation compared with cardioplegia group values. The beneficial effects of PCO treatment before cardioplegic arrest on -adrenergic responsiveness were similar for all PCO treatment intervals of 3 minutes or more (Fig 2). Concomitant Potassium-Channel Opener and Glibenclamide Treatment To ensure that the effects of the PCO aprikalim on myocyte contractile function resulted specifically from K ATP channel activation, concomitant treatment with the K ATP channel antagonist glibenclamide was performed before simulated cardioplegic arrest. The results are summarized in Figure 3. Combined treatment with glibenclamide and aprikalim yielded steady-state myocyte velocity of shortening values similar to those of the cardioplegia group. Thus, glibenclamide abolished the beneficial effects of PCO treatment before hyperkalemic cardioplegic arrest, which indicates that the protective effects on myocyte contractile function achieved by aprikalim appear to be mediated by the K ATP channel. Comment Past studies have demonstrated that myocardial preconditioning before hyperkalemic cardioplegic arrest or prolonged ischemia results in improved myocardial protection [5, 6, 11 16]. It also has been demonstrated that a particular class of pharmacologic compounds, PCOs, could selectively open the K ATP channels and provide protective effects similar to myocardial preconditioning [1, 2, 13]. However, the duration of pharmacologic treatment with a PCO that is necessary to provide protective effects in the setting of hyperkalemic cardioplegic arrest remains to be defined. Therefore, the overall goal of this study was to determine the optimum duration of PCO treatment before cardioplegic arrest for protection of myocyte contractile function after reperfusion and re-

5 Ann Thorac Surg HEBBAR ET AL 1998;65: POTASSIUM CHANNELS AND CARDIOPLEGIA 1081 Fig 3. Treatment with the potassium-channel opener (PCO) aprikalim for 10 minutes resulted in a statistically significant increase in myocyte velocity of shortening compared with treatment with cardioplegia only. Simultaneous treatment with a potassiumchannel opener and the adenosine triphosphate sensitive potassium channel antagonist, glibenclamide (PCO/Glib/Cardioplegia), blocked the protective effects of potassium channel opener treatment. Further, these co-incubation studies resulted in values similar to those achieved with cardioplegia alone. (*p 0.05 versus the normothermic control group; p 0.05 versus the cardioplegia group; # p 0.05 versus the potassium-channel opener plus cardioplegia group. warming. The results of this study demonstrated that only 3 minutes of PCO treatment were required before hypothermic, hyperkalemic cardioplegic arrest to improve indices of myocyte contractility with subsequent reperfusion and rewarming. Past reports have demonstrated that pharmacologic treatment of the myocardium with a PCO before cardioplegic arrest or prolonged ischemia has cardioprotective effects [5, 6, 11 16]. Menasché and colleagues [6], in an isolated rabbit heart model, demonstrated that PCO treatment before hyperkalemic cardioplegic arrest imparted cardioprotective effects with subsequent rewarming. Using human atrial myocardial preparations, Speechly-Dick and associates [13] demonstrated that PCO treatment before prolonged ischemia provided protective effects with respect to contractile performance. The results of the present study demonstrate that at the level of the isolated left ventricular myocyte, PCO treatment before cardioplegic arrest provides beneficial effects on contractile performance with subsequent reperfusion and rewarming. More important, these protective effects of PCO treatment on myocyte contractile performance are time-dependent. Thus, the protective effects of PCO treatment before cardioplegic arrest do not appear to be dependent on nonmyocyte populations but can be induced directly at the level of the myocyte. Several past reports have demonstrated the cardioprotective effects of PCOs given before cardioplegic arrest [4, 5, 11, 12], incorporated in conventional cardioplegic solutions [17], or used as the sole agents for inducing and maintaining cardioplegic arrest [9, 10, 18 20]. It was shown that indices of myocyte contractility after reperfusion and rewarming improved when a PCO-augmented cardioplegic solution was used compared with a hyperkalemic cardioplegic solution [17]. In addition, several reports have provided evidence suggesting that the induction and maintenance of cardioplegic arrest with the use of PCOs provides cardioprotection compared with the use of hyperkalemic cardioplegic solutions [9, 10, 18, 19]. The present study builds on these past reports by demonstrating, in an isolated myocyte model of simulated cardioplegic arrest, that a brief period of PCO treatment before cardioplegic arrest provides protective effects on myocyte contractile processes with subsequent reperfusion and rewarming. The mechanism of action of PCOs is attributed to the activation of K ATP channels within the myocyte sarcolemma. The activation of K ATP channels is associated with hyperpolarization of the cell membrane, with subsequent reduction in the inward calcium current and resultant preservation of high-energy phosphate levels [9, 10, 20, 21]. This mechanism may not be the sole contributing factor to the protective effects observed in the present study because the PCO treatment was followed by a prolonged period of depolarizing cardioplegic arrest. However, the initial period of K ATP activation before cardioplegic arrest may have reduced the relative extent of potassium-induced myocyte depolarization, which in turn may have influenced intracellular calcium processes. The activation of K ATP channels also appears to be involved in the induction of myocardial preconditioning [1, 2]. An intracellular mechanism involved in the preconditioning response appears to be sarcolemmal translocation of protein kinase C [13, 22]. The activation and translocation of protein kinase C is associated with several intracellular events, including the phosphorylation of K ATP channels located in the myocyte sarcolemma [22 25]. In addition, recent findings have suggested that the activation of K ATP channels through PCOs may result in the activation and translocation of protein kinase C [26]. Therefore, in the present study, a potential contributing mechanism to the beneficial effect of PCO treatment before cardioplegic arrest is the activation of protein kinase C, which in turn induces intracellular protective events [22 25]. Although past reports have demonstrated the beneficial effects of PCO treatment before hyperkalemic cardioplegic arrest [5, 6, 11, 12], the treatment interval necessary to confer this cardioprotective effect remained unclear. For example, Menasché and co-workers [5, 6] treated isolated rat hearts with the PCO nicorandil for 5 minutes, followed by 5 minutes of reperfusion, before hyperkalemic cardioplegic arrest and demonstrated its protective effects. Sugimoto and colleagues [11], using guinea pig papillary muscle, demonstrated that 15 minutes of PCO treatment before cardioplegic arrest resulted in improved contractile performance with reperfusion. The present study, using an isolated myocyte model, demonstrated that a PCO treatment period of 3 minutes achieved protective effects with respect to contractile function that were not improved further by more prolonged treatment periods. This finding may have practi-

6 1082 HEBBAR ET AL Ann Thorac Surg POTASSIUM CHANNELS AND CARDIOPLEGIA 1998;65: cal applications in the setting of cardiac operation, in that a short treatment interval could be instituted before prolonged hyperkalemic cardioplegic arrest for improved cardioprotection. However, the current in vitro study did not examine whether PCO treatment before cardioplegic arrest could be elicited with different drug concentrations. Thus, the dose-dependence of PCO treatment with respect to cardioplegic arrest warrants further investigation. Although the isolated myocyte model used in the present study has some advantages over an in vivo preparation, it also has some important limitations. This system allows for maximum solute diffusion capacity between the cytosol and extracellular space and does not include the buffering and osmotic influences of the extracellular matrix that may be operative in vivo. This isolated myocyte system also differs from in vivo preparations in which capillary diffusion distances are affected by coronary artery disease and hypertrophy. In the present study, an extracellular potassium concentration of 24 meq/l was used to simulate hyperkalemic cardioplegic arrest. However, it is unlikely that the myocyte is exposed continuously to this high extracellular potassium concentration throughout the cardioplegic interval in vivo. In light of these limitations, direct extrapolation of the results of the present in vitro study to the in vivo use of PCOs before cardioplegic arrest should be done with caution. Nevertheless, the results of this study suggest that pharmacologic treatment of isolated myocytes before simulated cardioplegic arrest can provide protective effects on myocyte contractile processes after reperfusion and rewarming, and that these protective effects can be achieved through the activation of K ATP channels. This study also defined the temporal characteristics of the treatment window, showing that a PCO treatment interval of 3 minutes produced protective effects on myocyte contractility. Thus, this study provides direct evidence that brief pharmacologic activation of K ATP channels before hyperkalemic cardioplegic arrest may provide a method of cardioprotection. Supported by a South Carolina American Heart Association grant (B. Hugh Dorman), a National American Heart Association grant (B. Hugh Dorman and Francis G. Spinale), and National Institutes of Health grant HL (Francis G. Spinale). Francis G. Spinale, MD, is an established investigator of the American Heart Association. References 1. Parratt JR, Kane KA. K ATP channels in ischemic preconditioning. Cardiovasc Res 1994;28: Tan HL, Mazon P, Verberne HJ, et al. Ischemic preconditioning delays ischemia induced cellular electrical uncoupling in rabbit myocardium by activation of ATP sensitive potassium channels. 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