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CARDIOVASCULAR ANESTHESIA

Myocyte Endothelin Exposure During Cardioplegic Arrest Exacerbates Contractile Dysfunction After Reperfusion

B. Hugh Dorman, MD, PhD^*, R. Brent New, MD, Brian R. Bond, PhD^*, Rupak Mukherjee, PhD, Y. V. Mukhin, PhD, James H. McElmurray, BS, and Francis G. Spinale, MD, PhD

Departments of *Anesthesia & Perioperative Medicine, Cardiothoracic Surgery, and Medicine, Medical University of South Carolina, Charleston, South Carolina

Address correspondence and reprint requests to B. Hugh Dorman, MD, PhD, Department of Anesthesia & Perioperative Medicine, Medical University of South Carolina, 165 Ashley Ave., Suite 525, PO Box 250912, Charleston, SC 29425. Address e-mail to dormanhb{at}musc.edu

	Abstract

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Transient left ventricular (LV) dysfunction can occur after cardioplegic arrest. The contributory mechanisms for this phenomenon are not completely understood. We tested the hypothesis that exposure of LV myocytes to endothelin (ET) during simulated cardioplegic arrest would have direct effects on contractile processes with subsequent reperfusion. LV porcine myocytes were randomly assigned to three groups: 1) Control: normothermic (37°C) cell media (n = 204); 2) Cardioplegia: simulated cardioplegic arrest (K⁺ 24 mEq/L, 4°C x 2 h) followed by reperfusion and rewarming with cell media (n = 164); and 3) Cardioplegia/

ET: simulated cardioplegic arrest in the presence of ET (200 pM) followed by reperfusion with cell media containing ET (n = 171). Myocyte contractility was measured by computer-assisted video microscopy. In a subset of experiments, myocyte intracellular calcium was determined after Fluo-3 (Molecular Probes, Eugene, OR) loading by digital fluorescence image analysis. Myocyte shortening velocity was reduced after cardioplegic arrest compared with controls (52 ± 2 vs 84 ± 3 µm/s, respectively; P < 0.05) and was further reduced with cardioplegic arrest and ET exposure (43 ± 2 µm/s, P < 0.05). Intracellular calcium was significantly increased in myocytes exposed to cardioplegia compared with normothermic control myocytes and was further augmented by cardioplegia with ET supplementation (P < 0.05). Exposure of the LV myocyte to ET during cardioplegic arrest directly contributed to contractile dysfunction after reperfusion. Moreover, alterations in intracellular calcium may play a role in potentiatiing the myocyte contractile dysfunction associated with ET exposure during cardioplegic arrest.

Implications: Exposure of the left ventricular myocyte to endothelin during cardioplegic arrest directly contributed to contractile dysfunction after reperfusion. Moreover, alterations in intracellular calcium may play a role in potentiating the myocyte contractile dysfunction associated with endothelin exposure during cardioplegic arrest.

	Introduction

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Hypothermic, hyperkalemic cardioplegic arrest is a common means of providing a quiescent heart during cardiac surgery. Although transient left ventricular (LV) dysfunction can occur after cardioplegic arrest, the contributory mechanisms for this phenomenon are not completely understood. A number of hemodynamic and neurohormonal changes occur during cardioplegic arrest and after separation from cardiopulmonary bypass (CPB) which can in turn influence LV pump function (1–3). Specifically, the induction of cardioplegic arrest and institution of CPB has been associated with increased levels of the potent vasoactive peptide, endothelin (ET) (3,4). ET causes increased vascular resistance and has been implicated in influencing LV myocyte contractility (5–8). For example, several studies have demonstrated that ET caused a negative inotropic effect in animal models of chronic LV pump failure (8). However, whether and to what degree changes in ET levels can specifically influence myocyte contractile properties in the setting of cardioplegic arrest has not been defined. Therefore, we tested the central hypothesis that exposure of LV myocytes to ET during simulated cardioplegic arrest would result in negative effects on contractile performance with subsequent reperfusion and rewarming.

The early reperfusion and rewarming period after cardioplegic arrest is associated with changes in LV loading conditions and neurohormonal system activation (1–3). These multiple systemic effects, which are operative in vivo, preclude the ability to specifically identify how ET may influence LV myocardial contractility in the setting of cardioplegic arrest. This laboratory has previously described an isolated LV myocyte system which simulates cardioplegic arrest and rewarming (9). This in vitro system of cardioplegic arrest is well suited to define specific mechanisms that may contribute to LV myocyte contractile dysfunction after reperfusion and rewarming. Accordingly, we used this isolated myocyte system to determine whether ET exposure during cardioplegic arrest would influence contractile performance and ß-adrenergic receptor responsiveness after reperfusion and rewarming. Alterations in intracellular calcium were also measured during cardioplegic arrest to better define mechanisms of myocyte contractile dysfunction.

	Methods

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All study 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), and the study was approved by the Institutional Animal Investigation Committee. LV myocytes were isolated from 13 Yorkshire pigs by using methods described previously (10). By using this technique, a high yield of myocytes was routinely obtained for study, and the characteristics of these isolated LV myocytes have been described in detail previously (10).

The isolated LV myocytes were randomly assigned to one of three treatment groups: 1) Cardioplegia: simulated cardioplegic arrest was performed by incubating myocytes in a crystalloid cardioplegic solution (lactated Ringer’s solution, 24 mEq/L K⁺, 30 mEq/L HCO₃^-, PO₂ >300 mm Hg) for 2 h at 4°C (n = 164) (9); 2) Cardioplegia/ET: myocytes were exposed to simulated cardioplegic arrest in which the solutions contained 200 pM ET (ET-1; Peninsula Laboratories, Belmont, CA) (n = 171); and 3) Normothermic Control: normothermic control myocytes were maintained in 37°C cell media and used for comparison (n = 204). A separate control group examining the effects of hypothermia alone was not included because a previous study by this laboratory demonstrated that myocyte contractile function after hypothermic conditions was unchanged compared with normothermic control values (9). After the treatment interval, myocytes were reperfused and rewarmed with normothermic standard cell culture media for 5 min, and contractile function was then examined. In the Cardioplegia/ET group, all reperfusion solutions contained 200 pM ET. After baseline measurements of contractility, myocytes were then exposed to the ß-adrenergic receptor agonist, isoproterenol (25 nM) (Sigma Chemical Co, St. Louis, MO), and myocyte contractile function measurements were repeated. The concentration of isoproterenol used in the present study was based on previously performed dose-response studies (8). Previous studies performed by this laboratory have also demonstrated dose-dependent (10–500 pM) contractile effects mediated by ET on isolated porcine myocytes (8). Two hundred picomolar ET was chosen because this dose induced maximal percent shortening and velocity of shortening in normothermic myocytes.

Myocyte contractions were elicited by field stimulation, imaged by using a charge-coupled device, digitized, and input into a computer for subsequent analysis, as previously described (9,10). Variables computed from the digitized contraction profiles included percent shortening (%), velocity of shortening (µm/s), velocity of relengthening (µm/s), and total contraction duration (ms). All variables were calculated for a minimum of 20 consecutive contractions, and the results were averaged.

To better understand mechanisms involved in myocyte contractile dysfunction associated with cardioplegic arrest and ET exposure, intracellular calcium was measured for 90 min in normothermic control myocytes, in myocytes undergoing cardioplegic arrest, and in myocytes undergoing cardioplegic arrest with 200 pM ET. Porcine myocytes were isolated as previously described for the contractile function measurements. The isolated myocytes were resuspended in a dye-loading buffer (1 x Hanks, 20 mM HEPES, 1% bovine serum albumin) at a concentration of 134 x 106 cells/mL with Fluo-3 (1 µg/ml; Molecular Probes, Eugene, OR) and probenecid (1 mM; Sigma Chemical Co.). These myocytes were then plated onto a matrigel-coated (Collaberative Research, Inc., Bedford, MA) 96-well plate (20 x 104 cells/well) and allowed to incubate for 45 min (37°C, 95% O₂/5% CO₂). After the incubation period, the myocytes were washed with the loading buffer and placed in a FLIPR (Fluorometric Imaging Plate Reader; Molecular Devices Corp., Sunnyvale, CA). Myocytes were then randomly assigned to one of the three treatment groups for 90 min. FLIPR measurements of intracellular calcium were performed every minute for 90 min and expressed as fluorescent counts after subtracting values for control myocytes. Fluorescent counts were averaged in eight wells for each treatment group per experiment, with four separate experiments performed. For analysis, fluorescent counts were normalized to 100% for myocytes exposed to cardioplegia only. Viable myocytes included those that retained a rod shape, excluded trypan blue, and responded to electrical stimulation. The yield of viable myocytes was greater than 80% in all preparations, and the myocytes remained quiescent with normal rod-shaped morphology for the 90-min incubation period. Because the myocytes were attached to a basement membrane substrate, the myocyte environment could be altered without disturbing myocyte position or adhesion (11).

The initial data analysis was performed on 13 independent experiments by using a multi-way analysis of variance (ANOVA), constructed by using a randomized, split-plot design, to identify any differences in indices of baseline myocyte contractility among the treatment groups. The results from myocyte contractile function studies were pooled from each experiment (n = 13). Each experiment was thus considered a complete block for the purposes of the ANOVA design, and the number of myocytes studied under each protocol were considered repeated observations within each treatment block. A multi-way ANOVA was then performed to identify the specific treatment effect (ß-adrenergic receptor stimulation) on contractile performance under both normothermic control conditions and after simulated cardioplegic arrest in the presence and absence of ET. If the multi-way ANOVA revealed significant differences, pair-wise tests of individual group means were compared by using Bonferroni’s probabilities. For the intracellular Ca⁺² measurements, a nonparametric ANOVA was performed. Specifically, Friedman’s test was used, in which the response variable for intracellular calcium levels at discrete time intervals was ranked for the main treatment effects of cardioplegia and cardioplegia/ET. Individual group means were compared by using the Kruskal-Wallis procedure. All statistical analysis was performed by using BMDP statistical software programs (BMDP Statistical Software, Inc., Los Angeles, CA). Results were presented as mean ± SEM. Values P < 0.05 were considered to be statistically significant.

	Results

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Indices of LV myocyte contractile function are shown in Table 1. Myocyte contractile performance was significantly reduced after 2 h of hyperkalemic cardioplegic arrest and subsequent rewarming (Cardioplegia group). Specifically, myocyte velocity of shortening after cardioplegic arrest was decreased by >40% compared with normothermic control myocytes. Myocyte velocity of relengthening was also decreased by approximately 50% in myocytes after hyperkalemic cardioplegic arrest.

Isoproterenol (25 nM) was added to Normothermic Control, Cardioplegia, and Cardioplegia/ET myocytes to determine the effects of ET exposure on ß-adrenergic responsiveness after cardioplegic arrest (Table 1). Contractile function was significantly increased in all myocyte groups after ß-adrenergic receptor stimulation with isoproterenol. Myocyte contractile response to ß-adrenergic receptor stimulation was significantly blunted after cardioplegic arrest. There were significant decreases in all contractile function indices in the presence of isoproterenol in myocytes after cardioplegic arrest relative to normothermic control myocytes. The inclusion of ET to hyperkalemic cardioplegia further reduced ß-adrenergic responsiveness after cardioplegic arrest compared with values observed with cardioplegia alone. For example, significant decreases in percent shortening (27%) and velocity of shortening (32%) were observed after cardioplegic arrest and isoproterenol administration in myocytes from the Cardioplegia/ET group relative to myocytes from the Cardioplegia group (Table 1).

Repeated measurements of intracellular calcium were performed under normothermic control conditions, during hyperkalemic cardioplegic arrest, and during cardioplegic arrest in the presence of ET to better understand the mechanism underlying the observed changes in myocyte contractile function. A representative intracellular calcium concentration plot is shown in Figure 1. Intracellular calcium significantly increased with the onset of hyperkalemic cardioplegic arrest and remained elevated relative to normothermic control myocytes for the 90-min period of incubation. Hyperkalemic cardioplegic arrest in the presence of ET also caused an immediate increase in intracellular calcium, which initially was similar in magnitude to myocytes undergoing cardioplegia without ET supplementation. However, after 50 min of incubation, intracellular calcium was significantly greater in myocytes in the Cardioplegia/ET group (103.8% ± 0.53%; P < 0.05) relative to myocytes in the Cardioplegia group and remained significantly elevated through the 90-min period of incubation (108.6% ± 0.92%; P < 0.05). Thus, ET potentiated the increase in intracellular calcium observed during hyperkalemic cardioplegic arrest.

View larger version (17K): [in this window] [in a new window]   Figure 1. Representative recording of intracellular calcium measurements in normothermic myocytes, in myocytes exposed to hyperkalemic cardioplegia (Cardioplegia; K+ 24 mEq/L), and in myocytes exposed to hyperkalemic cardioplegia with 200 pM endothelin (Cardioplegia/ET). Intracellular calcium measurements were performed every minute for a 90-min incubation period and were expressed as fluorescent counts after subtracting values for normothermic control myocytes. Hyperkalemic cardioplegia caused a significant increase in intracellular calcium compared with normothermic control myocytes, which was further augmented by the addition of endothelin to the cardioplegic solution.

	Discussion

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Several studies have documented an increase in circulating plasma ET levels during and after cardiac surgical procedures (3,4). ET is produced by a number of different cell types including myocytes, vascular smooth muscle cells, endothelial and endocardial cells, thus providing several different sources of ET to which myocytes may be exposed (12,13). Bugajski et al. (14) and Fogelson et al. (15) have reported that coronary sinus plasma ET levels are increased during reperfusion when compared with precardioplegic levels, which indicates increased myocardial production of ET and/or elevated ET levels in the blood perfusing the myocardium. Therefore, these studies suggest that myocytes are exposed to elevated ET during CPB. Increased plasma ET that occurs during and after hypothermic cardioplegic arrest and CPB may also be caused by spillover from the interstitial compartment (3,8). Other studies have suggested that this interstitial spillover of ET can occur when ET receptor binding is at maximum or as a result of cold-induced displacement of ET from binding sites (4). Other vasoactive peptides, such as angiotensin II, are compartmentalized within the myocardial interstitium at more than 100-fold higher than plasma concentrations (16). In the present study, myocytes were exposed to an ET concentration approximately 10-fold higher than plasma levels reported after cardioplegic arrest and CPB (3). Therefore, while remaining speculative, the ET concentration used in this in vitro study may likely reflect myocardial interstitial concentrations present during and after cardioplegic arrest and CPB. However, the direct quantification of interstitial ET levels during CPB has yet to be performed. In light of our findings in which myocyte exposure to ET during cardioplegic arrest exacerbated contractile dysfunction, future studies which more carefully determine myocardial interstitial ET content and the amount of ET released from myocytes in the setting of cardioplegic arrest and CPB are warranted.

In the present study, a significant reduction in myocyte contractile processes occurred after hyperkalemic cardioplegic arrest. The addition of ET to hyperkalemic cardioplegia caused further reductions in myocyte contractile function. This laboratory has previously reported dose-dependent positive inotropic effects of ET in normal myocytes and a negative inotropic effect in myocytes from a pig model of congestive heart failure (8). We demonstrated that ET exerts a negative inotropic effect in the myocyte after simulated cardioplegic arrest. Therefore, the results of these past and present studies suggest that prolonged exposure of ET may negatively influence myocardial performance in certain conditions. The reductions in myocardial contractile processes that occurred after cardioplegic arrest were associated with increases in intracellular calcium. Increased intracellular calcium appears to play an important role in myocardial stunning and reperfusion injury with decreased mechanical function and is associated with diastolic dysfunction, which may contribute to the reduction in myocyte contractile function observed after cardioplegic arrest in our study (17–20). The addition of ET to the hyperkalemic cardioplegia caused additional accumulation of intracellular calcium during cardioplegic arrest and further reductions in myocyte contractile function after reperfusion. A well characterized intracellular event after ET receptor stimulation is activation of the Na⁺/H⁺ exchanger, which subsequently modifies intracellular pH, and thereby directly affects myofilament Ca²⁺ sensitivity (21). Another end result of ET receptor binding is activation of phospholipase C and protein kinase C, which can cause alterations of intracellular Ca²⁺ content and Ca²⁺ homeostatic mechanisms (6,21,22). In a previous study performed by this laboratory, intracellular myocyte calcium levels were shown to be increased during rewarming and reperfusion as well as cardioplegic arrest (11). Thus, although intracellular calcium levels during rewarming and reperfusion were not measured in the present study, alterations in calcium with reperfusion likely contributed to myocardial contractile dysfunction after cardioplegic arrest. A more complete examination of intracellular Ca²⁺ levels and the effect of calcium channel antagonists during cardioplegia and reperfusion are warranted and should provide a better understanding of the mechanisms underlying the observed changes in contractile function.

A blunted ß-adrenergic receptor response occurs after cardioplegic arrest and CPB (1,9). A key intracellular process necessary for ß-adrenergic receptor-mediated events is the formation of cyclic adenosine monophosphate (cAMP). In vitro, ET receptor stimulation can reduce cAMP formation (22), and therefore attenuate cAMP-dependent contractile processes. For example, ET has been shown to reduce the ß-adrenergic receptor augmented change in L-type Ca²⁺ currents (7,8,23). Independent of cAMP production, ET may alter several downstream processes critical to the translation of ß-adrenergic receptor stimulation into an inotropic response (7). Specifically, ET can alter myofilament Ca²⁺ sensitivity secondary to changes in pH, as well as modulate Ca²⁺ handling mechanisms (19,21,23). Whereas the interactions between ET and ß-adrenergic receptor transduction are worthy of further investigation, our results suggest that the increased ET levels in the postcardioplegic arrest period directly contribute to reduced ß-adrenergic responsiveness.

There are limitations of the in vitro system and experimental design we used. First, this myocyte system excludes extrinsic factors present in vivo, including changes in systemic loading conditions and neurohormonal influences. In past reports, however, it has been demonstrated that changes in steady-state isolated myocyte contractile function directly reflect changes in the intrinsic LV contractile performance, as well as the capacity of the LV myocyte to function against a given load (24). Thus, while the isolated myocyte function studies we describe were performed under equivalent unloaded conditions, it is likely that these findings can be translated into intrinsic myocardial contractile capacity. Second, only one ET concentration was used; a dose-response study of the effects of ET on myocyte contractile function after cardioplegic arrest is warranted. Third, the contractile variables were only evaluated at one point after reperfusion, and the study design does not allow for separating the effect of ET exposure during cardioplegia versus exposure during reperfusion only. Our focus was to define the effect of ET on contractile recovery during reperfusion after simulated cardioplegic arrest. It has been demonstrated that ET is increased at the induction of CPB and appears to peak six hours post-CPB (25). Therefore, future studies determining the complete temporal relationship between post-CPB ET levels and myocyte contractility are warranted. Finally, ET receptor antagonist studies may further define mechanisms involved in myocyte contractile dysfunction after reperfusion. Nevertheless, our results suggest that ET contributes to the LV dysfunction and reduced ß-adrenergic responsiveness, which can be encountered after cardioplegic arrest and CPB, by exerting a direct negative inotropic effect on the myocyte, the fundamental contractile unit of the heart.

	Acknowledgments

 
Supported by a National American Heart Association Grant-in-Aid and National Institute of Health Grants HL-56603 and HL-45024. FGS is an Established Investigator of the AHA.

The authors wish to express their appreciation to Mark Clair, Stephen Krombach, Jennifer Hendrick, Terry Heslin, Laura Finklea, and Gregory Austin for technical assistance during this project.

	References

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