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J Thorac Cardiovasc Surg 1997;114:651-659
© 1997 Mosby, Inc.
CARDIOPULMONARY BYPASS, |
Supported by National Institutes of Health grant HL-45024, a Basic Research Grant from Pfizer Inc., a Grant-in-Aid from the South Carolina Heart Association, and a Grant-in-Aid from the American Heart Association. M. H. C. performed this work as a Medical Student Research Fellow of the American Heart Association. F. G. S. is an Established Investigator of the American Heart Association.
Received for publication August 6, 1996 revisions requested Dec. 5, 1996; revisions received April 21, 1997 accepted for publication April 22, 1997. Address for reprints: Francis G. Spinale, MD, PhD, Cardiothoracic Surgery, Room 418 CSB, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425.
Abstract
Objective: A potential intracellular mechanism for the protective effects of myocardial preconditioning is the activation of protein kinase C. The present study tested the hypothesis that a brief period of protein kinase C activation before cardioplegic arrest would provide protective effects on myocyte contractility with subsequent reperfusion and rewarming. Methods: Left ventricular porcine myocytes were assigned to the following treatments: (1) Protein kinase C/cardioplegia: Protein kinase C activation in myocytes (n = 39) for 3 minutes with a phorbol ester (10-9 mol/L of phorbol 12-myristate 13-acetate) in oxygenated, normothermic (37° C) cell media. Protein kinase C activation was followed by 2 hours of cardioplegic arrest (K+, 24 mEq/L; HCO3 –, 30 mEq/L; 4° C) and a 5-minute reperfusion period (37° C media). (2) Cardioplegia: Myocytes (n = 31), 2 hours of cardioplegic arrest, and a 5-minute reperfusion and rewarming period. Myocyte contractility was measured by means of high-speed videomicroscopy. For comparison purposes, contractile function was examined in myocytes (n = 70) under normothermic control conditions. Results: Myocyte shortening velocity was reduced after cardioplegic arrest when compared with normothermic values (22.3 ± 1.6 vs 48.8 ± 2.0 µm/sec, p < 0.0001). Protein kinase C activation before cardioplegic arrest normalized myocyte shortening velocity (48.8 ± 2.5 µm/sec). Co-incubation with phorbol 12-myristate 13-acetate and chelerythrine (10-6 mol/L), an inhibitor of protein kinase C, before cardioplegic arrest abolished the protective effects of phorbol 12-myristate 13-acetate pretreatment. Conclusion: These results suggest that an endogenous means of providing improved myocardial protection during prolonged cardioplegic arrest can be achieved through a brief period of protein kinase C activation.
Delivery of a hypothermic, hyperkalemic cardioplegic solution is a common means to obtain myocardial quiescence during cardiac surgical procedures. However, transient left ventricular (LV) dysfunction can occur with subsequent reperfusion and rewarming. Myocardial preconditioning describes a phenomenon in which myocardium made transiently ischemic becomes more tolerant to a subsequent and prolonged period of ischemia.
1-3 Experimental studies have demonstrated that ischemic preconditioning before the induction of cardioplegic arrest can improve LV pump function with reperfusion and rewarming. 4-6 Identification of the intracellular mechanism(s) that invoke the preconditioning phenomenon may provide important information for developing novel strategies for myocardial protection during prolonged periods of cardioplegic arrest. Recent studies have demonstrated that activation of the serine-threonine protein kinase C (PKC) may be an intracellular triggering mechanism for the preconditioning phenomenon. 7-13 However, whether and to what extent direct activation of PKC at the level of the myocyte provides protective effects in the setting of cardioplegic arrest and rewarming remains unknown. Accordingly, the present study was designed to test the central hypothesis that a short interval of PKC activation before a prolonged period of hypothermic, hyperkalemic cardioplegic arrest would improve myocyte contractile function on reperfusion and rewarming.
Pharmacologic activation of PKC has been demonstrated to occur in the presence of phorbol esters. 7,10-13 This laboratory has described previously that cardioplegic arrest with subsequent reperfusion and rewarming can be successfully performed in isolated LV myocytes. 14-16 This isolated myocyte system provides an uncomplicated means to examine cellular and molecular events that occur with respect to myocyte contractile processes during cardioplegic arrest and rewarming. Past reports have demonstrated that preconditioning can be successfully induced in isolated myocyte systems. 12,13,16,17 A brief exposure of isolated myocytes to phorbol esters has been shown to increase PKC activity. 13,18,19 Accordingly, the present study used an isolated myocyte system of cardioplegic arrest to determine whether a short pretreatment interval with a phorbol ester and subsequent PKC activation would provide protective effects on myocyte contractile processes.
Methods
Rationale.
The goal of the present study was to examine whether a brief interval of PKC activation before a prolonged period of simulated cardioplegic arrest would protect myocyte contractile function with rewarming. The first series of experiments was designed to develop a protocol for PKC activation in this myocyte system by examining the time and dose-dependent effects of phorbol ester administration on myocyte contractile function under steady-state normothermic conditions. The next series of experiments examined whether a brief PKC activation period before cardioplegic arrest would provide protective effects on myocyte contractile function with subsequent rewarming. The final series of experiments was performed to determine more carefully that the effects of phorbol ester treatment on myocyte function were due to specific PKC activation.
Myocyte isolation and contractile function.
Adult Yorkshire pigs (n = 5) were the source of LV myocytes. 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, D.C., 1996). The animals were anesthetized with isoflurane (2%/1.5 L/min) and their lungs ventilated through a nonrecirculating anesthesia circuit. A sternotomy was then performed, and the heart was quickly extirpated and placed in cold oxygenated Krebs solution. The region of the LV free wall perfused by the left circumflex coronary artery (5 x 5 cm) was dissected free, cannulated, and prepared for myocyte isolation as described previously. 14-16 In brief, oxygenated modified Krebs solution containing aerobic substrates and collagenase (0.5 mg/ml, type II; 146 U/mg, Worthington Biochemical Corp., Freehold, N.J.) was perfused and recirculated through the cannulated circumflex artery for 20 minutes. The tissue was then minced into 2 mm sections and added to an oxygenated solution containing bovine serum albumin (2%, Sigma Chemical Co., St. Louis, Mo.), deoxyribonuclease (DNase, 51 Kunitz units/ml, type IV, Sigma), CaCl2 (400 x 10-6 mol/L, Sigma), and collagenase (0.5 mg/ml, Worthington) and gently agitated. The isolated myocytes were then suspended in fresh culture medium (media 199, 2 mmol/L Ca2+, Gibco Laboratories, Grand Island, N.Y.). A 2 ml aliquot of the isolated myocyte suspension (5 x 104 cells/ml) was then plated onto coverslips previously coated with a laminin/fibronectin matrix (Matrigel, Collaborative Research, Inc., Bedford, Mass.) and incubated at 37° C for 1 hour in the presence of 95% oxygen and 5% carbon dioxide.
Myocyte contractile function was examined with the use of computer-assisted videomicroscopy techniques described previously. 14-16 In brief, myocytes were imaged on an inverted microscope (model PIM, World Precision Instruments, Sarasota, Fla.) in a 2.5 ml tissue chamber with a thermoregulator to maintain media temperature at 37° C. Myocytes were stimulated at 1 Hz and contractions were imaged with the use of a charge-coupled device (GPCD60, Panasonic, Secaucus, N.J.). Myocyte motion signals were input through an edge-detector system (Crescent Electronics, Sandy, Utah), converted into a voltage signal, digitized, and input into a computer (80286, ZBV2526, Zenith Data Systems, St. Joseph, Mo.) for subsequent analysis. Stimulated myocytes were allowed a 5-minute stabilization period, after which contraction data for each myocyte were recorded for a minimum of 20 consecutive contractions. Parameters computed from the digitized contraction profiles included percent shortening (percent), peak velocity of shortening (micrometers per second), peak velocity of relengthening (micrometers per second), total contraction duration (milliseconds), time to peak contraction (milliseconds), and time to 50% relaxation (milliseconds).
PKC activation and myocyte contractile function.
To develop a protocol for PKC activation in isolated myocytes, we used the initial series of experiments to examine the effects of a phorbol ester, phorbol 12-myristate 13-acetate (PMA, Sigma), on myocyte contractile function under normothermic conditions. The phorbol ester PMA was selected for these studies because it has been demonstrated previously to activate PKC in isolated myocyte preparations 11 and readily enters the intracellular compartment. Stock PMA was solubilized with 100% ethanol (Pharmco Products Inc., Brookfield, Conn.) and then diluted with a Krebs buffer solution. Myocyte contractile function and viability were examined before and after incubation (3 to 30 minutes) with PMA (10-6 to 10-9 mol/L). In the present study, a significant reduction in myocyte contractile function occurred at 10-9 mol/L of PMA with no change in myocyte viability (Fig. 1). However, myocyte viability, as defined in the previous section, was substantially reduced with PMA at concentrations greater than 10-8 mol/L and for incubation intervals greater than 10 minutes. Accordingly, incubation of myocytes with PMA at a concentration of 10-9 mol/L for 3 minutes was selected as the pretreatment protocol for the simulated cardioplegia experiments.
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14-16 Specifically, myocytes were exposed to a crystalloid cardioplegic solution (lactated Ringer's solution: K+, 24 mEq/L; HCO3 –, 30 mEq/L; oxygen tension > 300 mm Hg) for 2 hours at 4° C. After cardioplegic arrest, the cardioplegic solution was rapidly changed with normothermic cell culture medium. After 5 minutes of normothermic reperfusion, steady-state myocyte contractile function was examined. The experimental protocol for this series of studies is shown in Fig. 2.
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20 In a past report, chelerythrine at a concentration of 10-6 mol/L abolished the protective effects of preconditioning in human atrial tissue preparations. 7 Accordingly, myocytes were incubated in normothermic, oxygenated media containing both chelerythrine (10–6 mol/L) and PMA (10–9 mol/L) together for 3 minutes, then subjected to identical treatment as described in the previous paragraph. In an additional set of experiments, myocytes were exposed to chelerythrine (10–6 mol/L) for 3 minutes and then subjected to cardioplegic arrest. The experimental protocol for these studies is outlined in Fig. 2.
PKC translocation in isolated myocytes.
A generalized intracellular event that occurs after activation of PKC is translocation from intracellular cytosolic compartments to the cell membrane. 21-23 Furthermore, it has been demonstrated previously that PMA causes PKC translocation in isolated myocyte preparations. 23 Accordingly, localization of PKC in isolated myocytes was performed by means of fluorescent techniques. 24 Isolated myocytes from three different preparations were assigned to one of the following treatment groups: (1) incubation in oxygenated, normothermic media (37° C) for 3 minutes; (2) incubation in oxygenated, normothermic media containing PMA (10–9 mol/L) for 3 minutes; and (3) incubation in oxygenated, normothermic media containing both chelerythrine (10-6 mol/L) and PMA (10-9 mol/L) together for 3 minutes. After these treatments, myocytes were fixed in 3.7% molecular grade formaldehyde for 10 minutes and then transferred to a stabilization buffer containing 1,4 Peperazinedithanesulfonic acid (PIPES) (65 mmol/L), HEPES (25 mmol/L), ethyleneglycol tetraacetic acid (EGTA) (10 mmol/L), and MgCl2 (3 mmol/L). The myocytes were then incubated in the presence of a fluorescently conjugated PKC probe (BIODIPY FL bis-indolylmaleimide; 100 µmol/L, B7485 Molecular Probes, Eugene, Ore.) 24 for 10 minutes. Fluorescently conjugated bis-indolylmaleimide has been demonstrated previously to be membrane-permeant, bind to the catalytic domain of PKC, and demonstrate translocation of PKC from the cytoplasm to membranes. 24 In preliminary studies, the concentration and incubation period for this PKC probe provided specific intracellular staining in isolated LV myocyte preparations. After the incubation step, the coverslips containing the myocytes were placed on glass slides containing an antifade glycerol solution (4%, 1,8 diazabicyclo[2.2.2]octane, Sigma). The myocyte preparations were then examined by means of confocal scanning microscopy (MRC-1000, Bio-Rad Laboratories, Hercules, Calif.) with excitation/emission wavelength settings of 509/520, respectively. Myocytes were imaged at an original magnification of 600x and images digitally captured at 1.0 µm increments through the myocyte. A minimum of 25 myocytes were examined from each preparation.
Data analysis.
Indices of myocyte contractile function for the different treatment groups were compared by means of analysis of variance. If analysis of variance detected significant differences with respect to treatment groups, mean separation was performed with the use of Bonferroni bounds. All statistical procedures were performed with the use of the BMDP statistical software package (BMDP Statistical Software, Inc., Los Angeles, Calif.). Results are presented as mean ± standard error of the mean. Values of p < 0.05 were considered statistically significant.
Results
Steady-state myocyte contractile function under normothermic conditions, after simulated cardioplegic arrest with rewarming, and with PMA pretreatment followed by cardioplegic arrest and rewarming are presented in Table I. Indices of contractile function obtained under steady-state normothermic conditions are consistent with previous values obtained with the use of this myocyte preparation. 14-16 After cardioplegic arrest and rewarming, steady-state myocyte percent and velocity of shortening were reduced by more than 40% from normothermic control values. The reduction in myocyte contractility after simulated cardioplegic arrest and rewarming is consistent with previous reports from this laboratory. 14-16 Pretreatment with PMA before cardioplegic arrest and rewarming resulted in improved indices of myocyte contractile function with subsequent reperfusion and rewarming. Specifically, myocyte percent shortening, shortening velocity, and velocity of relengthening were significantly increased from cardioplegia-alone values. More important, these indices of contractile function with PMA pretreatment were similar to normothermic control values (p > 0.60). However, myocyte total duration of contraction, time to peak contraction, and time to 50% relaxation were prolonged with PMA pretreatment and cardioplegic arrest when compared with normothermic values. These results suggest that pretreatment with PMA before simulated cardioplegic arrest protected indices of myocyte contractile function with subsequent reperfusion and rewarming, but defects in the temporal aspects of myocyte contractile process persisted. Co-incubation with chelerythrine and PMA before cardioplegic arrest and rewarming abolished the protective effects of PMA pretreatment on steady-state myocyte shortening velocity (Fig. 3). In myocytes pretreated with chelerythrine before cardioplegic arrest (n = 21), myocyte percent and velocity of shortening were similar to cardioplegia-alone values (2.5% ± 0.2%, 26 ± 3 µm/sec, respectively). Therefore the protective effects on myocyte contractile function with PMA pretreatment before cardioplegic arrest and rewarming were likely mediated by specific activation of PKC.
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21-24 In myocytes treated with both PMA and chelerythrine, the fluorescent staining for PKC was similar to that in normothermic control myocytes (Fig. 4). Specifically, strong cytosolic staining could be detected in perinuclear and myofilament compartments with minimal diffuse staining along the sarcolemma. These results provide further evidence to suggest that PMA treatment caused PKC activation in this isolated myocyte preparation.
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Cessation of myocardial contractile activity is commonly required for the conduct of many cardiac surgical procedures and is routinely achieved through the administration of a hypothermic, hyperkalemic cardioplegic solution. However, transient LV dysfunction can occur after cardioplegic arrest, which may persist in the early postoperative period. Transient, brief periods of myocardial ischemia have been demonstrated to confer a protective effect to the myocardium with subsequent prolonged periods of ischemia; this protective effect has been defined as the preconditioning phenomenon. 1-3 A proposed intracellular mechanism for the protective effects of preconditioning is activation of PKC. 7-10 Past studies have demonstrated that a brief interval of PKC activation before a prolonged period of ischemia provided protective effects similar to those achieved by ischemic preconditioning. 7-10 The present study was designed to determine whether a brief interval of PKC activation before prolonged cardioplegic arrest would provide protective effects on myocyte contractile function with subsequent reperfusion and rewarming. Using an isolated myocyte model of simulated cardioplegic arrest, we made two important observations. First, a brief period of PKC activation achieved through exposure of myocytes to the phorbol ester PMA before cardioplegic arrest provided protective effects on myocyte contractile function with rewarming. Second, the protective effects of PMA pretreatment before cardioplegic arrest and rewarming were abolished by concomitant treatment with a specific inhibitor of PKC. Therefore the present study provides direct evidence that a brief period of PKC activation within the LV myocyte before cardioplegic arrest confers protective effects on myocyte contractile processes with subsequent reperfusion and rewarming.
In a past report, Illes and associates 4 demonstrated that preconditioning in the rabbit heart improved LV pump function after hypothermic cardioplegic arrest and rewarming. A number of recent studies have provided evidence that an intracellular event necessary for inducing the preconditioning phenomenon is through activation of PKC. 7-10,12,13 Past studies have demonstrated that preconditioning can be successfully elicited in isolated myocyte systems. 12,13,16-19 In a recent report from this laboratory, it was demonstrated that hypoxic preconditioning of isolated myocytes before simulated cardioplegic arrest improved contractile function with subsequent reperfusion and rewarming. 16 However, it remained unclear whether PKC activation before cardioplegic arrest would provide similar protective effects with respect to myocyte contractile function. In the present study, treatment with the phorbol ester PMA before prolonged cardioplegic arrest, significantly improved indices of myocyte contractile function with subsequent reperfusion and rewarming. Treatment of this isolated myocyte preparation with PMA caused a detectable intracellular shift in PKC from the cytoplasm to the sarcolemmal surface, consistent with PKC activation. 21-24 Furthermore, co-treatment with a PKC inhibitor and PMA eliminated the protective effects induced by PMA pretreatment. Taken together, the results from the present study build on past reports and provide direct evidence that induction of PKC activation in isolated myocytes immediately before cardioplegic arrest will result in improved contractile function with subsequent reperfusion and rewarming.
Consistent with past reports, 25-28 simulated cardioplegic arrest in an isolated LV myocyte system caused a significant reduction in the indices of myocyte contractile performance with subsequent reperfusion and rewarming. In the present study, a brief (3-minute) interval of PKC activation before the induction of cardioplegic arrest normalized myocyte percent and velocity of shortening with subsequent reperfusion and rewarming. This observation suggests that activation of PKC within the myocyte provided protective effects on myocyte homeostatic processes during the period of cardioplegic arrest. Activation of PKC results in a number of intracellular events within the myocyte, which include influencing ion exchange systems and pumps, activity of mitogen-activated protein kinases, phosphorylation states of contractile proteins, and cytoskeletal protein conformation. 21,22,25,26 The activation of PKC has also been shown to influence gene expression and myocardial growth properties. 21,22Results from the present study, as well as past reports, 7-10 demonstrated significant protective effects after only a brief period of PKC activation. Therefore the acute effects of PKC activation that were observed in the present study were probably not due to changes in transcriptional or translational processes, but more likely due to alterations in intracellular ionic homeostasis and phosphorylation states. PKC activational status has been demonstrated to influence the activity of the Na+/H+ exchanger, which will cause intracellular alkalinization. 21,22 PKC activation has also been demonstrated to influence intracellular Ca+2 levels by modulating the function of the l-type Ca+2 channel on the myocyte sarcolemma, 26 as well as the sarcoplasmic reticulum Ca+2-adenotriphosphatase. 21 Thus, in the present study, it is likely that PKC activation in isolated LV myocytes changed intracellular pH and Ca+2 levels, which provided a protective effect when subjected to prolonged hypothermic, hyperkalemic cardioplegic arrest. However, future studies that directly measure intracellular pH and Ca+2 with PKC activation during cardioplegic arrest will be necessary to directly examine this possibility.
Pretreatment of isolated myocytes with the phorbol ester PMA, and therefore activation of PKC, before cardioplegic arrest improved myocyte shortening and velocity of shortening with subsequent reperfusion and rewarming. However, abnormalities in temporal indices of the contraction process, such as time to peak contraction, duration of contraction, and time to 50% relaxation, were prolonged with PMA pretreatment when compared with normothermic values. Myocyte percent shortening reflects the maximal extent of myofilament interaction and velocity of shortening reflects the rate of cross-bridge cycling. PMA pretreatment increased the time interval to peak myocyte contraction, but myocyte percent shortening was similar to normothermic values. These findings suggest that PKC activation before cardioplegic arrest likely influenced mechanisms by which cross-bridge cycling rates are translated into overall myocyte shortening. It has been demonstrated previously that PKC activation influences the phosphorylation state and orientation of a number of contractile and cytoskeletal proteins. 21,22,25 Thus, in the present study, PKC pretreatment may have altered cytoskeletal architecture within the myocyte, which in turn would influence sarcomere shortening to be translated to cell shortening. The present study demonstrated that an index of myocyte active relaxation, the time to 50% relaxation, remained prolonged with PMA pretreatment. The time to 50% relaxation reflects the period of cross-bridge release and Ca+2 resequestration by the sarcoplasmic reticulum. The findings of the present study suggest that the time necessary for cross-bridge release and the return of the myofilament array to a resting position was altered after cardioplegic arrest and could not be reversed by PMA pretreatment. PKC activation influences a number of Ca+2 homeostatic mechanisms within the myocyte. 21,22,25,26 However, whether the persistent abnormalities in active relaxation with PMA pretreatment followed by cardioplegic arrest that were observed in the present study were due to temporal changes in myocyte Ca+2 homeostatic properties remains unclear. In light of the findings from the present study and the spectrum of intracellular events that occur after PKC activation, future studies that more carefully examine Ca+2 homeostatic processes with PKC activation and cardioplegic arrest are warranted.
In the present study, PKC activation under normothermic steady-state conditions reduced indices of myocyte contractile function. Karmazyn, Watson, and Moffat 27 demonstrated that PMA treatment in rat heart preparations reduced indices of LV contractile performance. In studies of ischemic preconditioning in which a period of myocardial protection is conferred, the interval before the prolonged ischemic insult is often associated with a condition described as "myocardial stunning." 1-3 Several past reports have suggested that a contributory intracellular mechanism for this "myocardial stunning" phenomenon is PKC activation. 7-13 Thus the reduction in myocyte contractile performance that was observed in the present study after the acute administration of the phorbol ester PMA, with subsequent PKC activation, was not unexpected. In fact, the dose of PMA that caused a decline in steady-state function was used as a criterion that significant PKC activation had occurred within the myocyte. 11,19,27 However, it may be possible to develop a protocol by which adequate PKC activation can be achieved to confer myocardial protection without a concomitant fall in contractile performance. Future studies with the myocyte model of simulated cardioplegic arrest described in the present study and in past reports, 14-16 but which more carefully address this issue, would be appropriate.
Intracellular activation of PKC in most cell types is normally through the production of diacylglycerol, which is produced by a number of receptor-mediated events. 12,21,22,26,28,29 Tumor-promoting phorbol esters such as PMA are membrane permeable and have structures similar to diacylglycerol, which provide a means to activate PKC directly. 21,22,29 In addition to phorbol esters being described as tumor-promoting agents, these compounds are not efficiently degraded by intrinsic enzymatic pathways and can cause a number of systemic effects. 29 Thus the clinical application of phorbol esters such as PMA to induce PKC activation within the LV myocardium is problematic. However, several sarcolemmal receptor transduction systems have been demonstrated to induced PKC activation. 21,22 For example, activation of the adenosine receptor system(s) that are located within the myocyte sarcolemma cause activation of PKC. 3,12 Furthermore, Armstrong, Downey, and Ganote 12 demonstrated that the protective effects of adenosine-receptor activation in the setting of global ischemia could be abolished through inhibition of PKC activation. 12 Future studies that directly determine the relationship between the adenosine receptor and PKC activational states in the setting of cardioplegic arrest are warranted.
The isolated myocyte model used in the present study provides an uncomplicated means by which to determine the direct effects of pretreatment strategies with simulated cardioplegic arrest on contractile performance. Although this myocyte system has some advantages over an in vivo preparation, it has important limitations that must be recognized. The isolated myocyte system removes differences in loading conditions, neurohormonal activity, and the influence of nonmyocyte cell populations that would be encountered in vivo after cardioplegic arrest and rewarming. Another important consideration is that this isolated myocyte system differs from in vivo preparations in which capillary diffusion distances are affected by coronary artery disease, hypertrophy, and nonuniform maintenance and control of temperature. The limitations of the isolated myocyte system must be recognized, and extrapolation of the results from these in vitro studies to in vivo conditions should be performed with caution. In the present study, a qualitative morphometric examination was performed to determine the relative PKC activational state after PMA treatment. Although this portion of the study demonstrated that PMA treatment was associated with PKC translocation and that this brief interval of PKC activation before cardioplegic arrest improved myocyte function with subsequent rewarming, quantitation of the degree and nature of PKC activation within the myocytes was not addressed. PKC is not a single entity but comprises a large family of kinases encoded by different genes. 21,22 PKC has been broadly categorized into three separate classes, based primarily on Ca+2 sensitivity of the amino-terminal regulatory region. 22 At present, more than 10 isoforms of PKC have been identified, and the relative expression of these isoforms within the myocardium is an area of active investigation. 21 Therefore, on the basis of the findings of the present study, future studies that more carefully examine the specific isoforms of PKC which can be recruited to provide protective effects in the setting of cardioplegic arrest would be appropriate. Nevertheless, the results from this study demonstrated that an endogenous means of providing improved myocardial protection during prolonged hypothermic, hyperkalemic cardioplegic arrest can be achieved through a brief period of PKC activation.
Acknowledgments
We appreciate the assistance of Latha Hebbar and Melissa Doscher in the execution of this project.
References
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isozyme. Circ Res 1995;76:654-63. This article has been cited by other articles:
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  A. M. Deschamps, J. Zavadzkas, R. L. Murphy, C. N. Koval, J. E. McLean, L. Jeffords, S. M. Saunders, N. J. Sheats, R. E. Stroud, and F. G. Spinale Interruption of endothelin signaling modifies membrane type 1 matrix metalloproteinase activity during ischemia and reperfusion Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H875 - H883. [Abstract] [Full Text] [PDF]
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M. L. Coker, J. R. Jolly, C. Joffs, T. Etoh, J. R. Holder, B. R. Bond, and F. G. Spinale Matrix metalloproteinase expression and activity in isolated myocytes after neurohormonal stimulation Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H543 - H551. [Abstract] [Full Text] [PDF]
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 C. Stamm, I. Friehs, D. B. Cowan, H. Cao-Danh, S. Noria, M. Munakata, F. X. McGowan Jr., and P. J. del Nido Post-ischemic PKC inhibition impairs myocardial calcium handling and increases contractile protein calcium sensitivity Cardiovasc Res, July 1, 2001; 51(1): 108 - 121. [Abstract] [Full Text] [PDF]
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 A. T. Goldberg, B. R. Bond, R. Mukherjee, R. B. New, J. L. Zellner, F. A. Crawford Jr, and F. G. Spinale Endothelin receptor pathway in human left ventricular myocytes: relation to contractility Ann. Thorac. Surg., March 1, 2000; 69(3): 711 - 715. [Abstract] [Full Text] [PDF]
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F. G. Spinale Cellular and molecular therapeutic targets for treatment of contractile dysfunction after cardioplegic arrest Ann. Thorac. Surg., November 1, 1999; 68(5): 1934 - 1941. [Abstract] [Full Text] [PDF]
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B. Z. Simkhovich, K. Przyklenk, and R. A. Kloner Role of protein kinase C as a cellular mediator of ischemic preconditioning: a critical review Cardiovasc Res, October 1, 1998; 40(1): 9 - 22. [Abstract] [Full Text] [PDF]
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p < 0.05 vs [PMA] 10-10 mol/L; 
p < 0.05 vs [PMA] 10-9 mol/L).
