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Ann Thorac Surg 1996;62:489-494
© 1996 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Direct and Interactive Effects of Cardioplegic Arrest and Protamine on Myocyte Contractility

Department of Surgery, Medical University of South Carolina, Charleston, South Carolina

Accepted for publication March 20, 1996.

Address correspondence to Dr Spinale, Division of Cardiothoracic Surgery, Medical University of South Carolina, 171 Ashley Ave, Charleston, SC 29425.

	Abstract

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Background. Cardioplegic arrest with rewarming and protamine administration have been implicated in causing transient left ventricular dysfunction perioperatively. However, whether interactive effects between cardioplegic arrest and rewarming with protamine occur with respect to myocyte contractile processes remains unclear. Accordingly, using an isolated myocyte model, the present study tested the hypothesis that simulated cardioplegic arrest with rewarming and protamine would have direct and interactive effects on myocyte contractile function.

Methods. Left ventricular isolated myocyte contractile function was examined using computer-aided videomicroscopy under normothermic conditions (37°C, cell medium; n = 183) and after simulated hypothermic, hyperkalemic cardioplegic arrest with rewarming (4°C, 24 mEq/L K⁺, 2 hours; then 37°C, cell medium, 5 minutes; n = 268). Myocyte function was then examined in the presence of protamine (10 to 40 µg/mL) under normothermic conditions (n = 102) and after cardioplegic arrest with rewarming (n = 175).

Results. Myocyte contractile function decreased by 43% from baseline after simulated cardioplegic arrest with rewarming. Under normothermic conditions, protamine (20 µg/mL) reduced myocyte contractile function by 43.9% ± 4.3%, whereas myocyte contractile function decreased by only 31.1% ± 2.7% with protamine (20 µg/mL) after cardioplegic arrest with rewarming. Thus, the negative effects of protamine on myocyte contractility were attenuated after cardioplegic arrest when compared with normothermic conditions.

Conclusions. The present study demonstrated that simulated cardioplegic arrest with rewarming and protamine have direct and interactive effects on myocyte contractile function, which are not additive or synergistic.

	Introduction

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More than 350,000 cardiac surgical procedures are performed in the United States annually requiring cardiopulmonary bypass and cardioplegic arrest [1]. However, transient left ventricular (LV) dysfunction can occur after cardioplegic arrest, particularly in the early reperfusion period [2, 3]. Using an isolated myocyte system, this laboratory has previously demonstrated that a contributory mechanism for the LV dysfunction that can occur after cardioplegic arrest and rewarming is abnormalities in myocyte contractile processes [4, 5]. Specifically, 2 hours of simulated cardioplegic arrest with subsequent reperfusion resulted in reduced basal myocyte contractile function and inotropic responsiveness [4]. A fundamental requirement for cardiopulmonary bypass is anticoagulation with heparin. After separation from cardiopulmonary bypass, the anticoagulant effect of heparin is routinely reversed with protamine [6]. However, administration of protamine has been implicated as a cause of LV pump dysfunction in this setting [7, 8]. This laboratory has previously demonstrated that protamine has direct and negative effects on isolated myocyte contractile processes [9–11]. Specifically, a dose-dependent decline in myocyte contractile function and reduced ß-adrenergic responsiveness occurs in the presence of protamine [9]. However, these previous basic studies did not examine whether interactive effects occur between cardioplegic arrest with rewarming and protamine on contractile function. In light of the fact that cardioplegic arrest and protamine administration are temporally related in the clinical setting, the goal of the present study was to examine the direct and interactive effects of cardioplegic arrest with rewarming and protamine on myocyte contractile processes.

	Material and Methods

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The present study was designed to test the hypothesis that differential effects on myocyte contractile processes would occur after simulated hypothermic, hyperkalemic cardioplegic arrest with rewarming in the presence of protamine. Accordingly, myocyte contractile function was examined in the presence of protamine under normothermic conditions, after simulated cardioplegic arrest, and after a combination of both treatments.

Myocyte Isolation and Contractile Function
Five age- and weight-matched Yorkshire pigs were the source of LV myocytes. All animals were cared for and treated in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (NIH publication 86-23). The animals were anesthetized with isoflurane (0.5%/1.5 L/min) and 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, the artery cannulated, and the tissue prepared for myocyte isolation, as described previously [12, 13]. Briefly, an oxygenated modified Krebs solution containing aerobic substrates and collagenase (0.5 mg/mL, type II; 146 U/mg; Worthington Biochemical Corp, Freehold, NJ) maintained at 37°C 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), 400 µmol/L CaCl₂, and collagenase (0.5 mg/mL; Worthington) and gently agitated. At 5-minute intervals, the supernatant was removed and filtered, and the cells were allowed to settle. The isolated myocytes were then suspended in standard culture medium (Media 199, 2 mmol/L Ca²⁺, pH 7.45; Gibco Laboratories, Grand Island, NY). A 2-mL aliquot of the isolated myocyte suspension (5 x 10⁴ cells/ml) was plated onto coverslips previously coated with a laminin/fibronectin matrix (Matrigel; Collaborative Research Inc, Bedford, MA) and incubated at 37°C for 1 hour in the presence of 95% O₂ and 5% CO₂. Previous studies have reported that viable myocytes included those that retained a rod shape, were calcium tolerant, remained quiescent in culture, responded to electrical stimulation, and excluded trypan blue [12, 13]. The yield of viable myocytes was greater than 75% from each preparation.

Myocyte contractile function was examined using video-assisted microscopy techniques described previously [12, 13]. Briefly, myocytes were imaged on an inverted microscope (Axiovert IM35; Zeiss Inc, Oberkochen, Germany) 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 using 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, 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, peak velocity of shortening, peak velocity of relengthening, and total contraction duration. Indices of contractile function at steady-state were examined in normothermic myocytes.

Simulated Hypothermic, Hyperkalemic Cardioplegic Arrest With Rewarming Protocol
A conventional crystalloid cardioplegic solution (lactated Ringer's, 24 mEq/L K⁺, 30 mEq/L HCO₃^-, oxygen tension > 300 mm Hg, pH 7.5) was used in the cardioplegic arrest protocol. Isolated myocytes were incubated in cardioplegic solution for 2 hours at 4°C. After cardioplegic arrest, the cardioplegic solution was rapidly changed with normothermic cell culture media and the myocytes were incubated for 5 minutes at 37°C. Baseline contractile function was then examined. For comparison, myocytes maintained under normothermic conditions in standard cell medium served as time-matched controls. This laboratory has previously shown that this simulated cardioplegic arrest with rewarming protocol results in myocyte contractile dysfunction [4, 5].

Protamine Sulfate Protocol
Isolated myocyte contractile function was examined at baseline and in the presence of protamine sulfate (Elkins-Sinn, Inc, Cherry Hill, NJ) suspended in a Krebs buffer solution. Protamine was added to the bath to achieve a final concentration of 10, 20, or 40 µg/mL, which approximates serum concentrations of patients dosed with protamine at 0.625, 1.25, or 2.5 mg/kg [14, 15]. Isolated myocytes were randomly assigned to receive 10, 20, or 40 µg/mL of protamine, and then indices of myocyte contractile function were examined. Protamine has been previously shown to reduce basal myocyte contractile function [9–11]. Isolated myocytes were subjected to the simulated hypothermic, hyperkalemic cardioplegic arrest protocol as described in the above paragraph. After a period of reperfusion and rewarming, isolated myocytes were incubated with 10, 20, or 40 µg/mL of protamine, and measurements of contractile function were then repeated.

Data Analysis
Indices of myocyte contractile function were compared using analysis of variance. If the analysis of variance detected significant differences with respect to treatment groups, mean separation was performed using Bonferroni bounds [16]. All statistical procedures were performed using the BMDP statistical software package (BMDP Statistical Software Inc, Los Angeles, CA). Results are presented as mean ± standard error of the mean. Values of p less than 0.05 were considered statistically significant.

	Results

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Myocyte Contractile Function: Effects of Cardioplegic Arrest With Rewarming
In the first series of experiments, myocyte contractile function was examined under normothermic conditions and after hypothermic, hyperkalemic cardioplegic arrest. The results are summarized in Table 1. Cardioplegic arrest and rewarming caused a decline in myocyte contractile function when compared with normothermic values. For example, myocyte velocity of shortening decreased by 43% after cardioplegic arrest with rewarming when compared with normothermia. The significant reduction in myocyte contractile function after cardioplegic arrest with rewarming is consistent with past reports from this laboratory [4, 5].

In light of the significant differences in basal myocyte contractile function between normothermic values and the values after cardioplegic arrest and rewarming, the differential effects of protamine can be difficult to interpret. Accordingly, the percent change from baseline in myocyte velocity of shortening was computed and shown in Figure 1. The percent change from baseline in myocyte velocity of shortening was significantly reduced with increasing concentrations of protamine after cardioplegic arrest with rewarming compared with normothermia. At all concentrations of protamine, the negative effects of protamine on myocyte contractile function were less pronounced after cardioplegic arrest with rewarming when compared with normothermic values.

View larger version (22K): [in this window] [in a new window]   Fig 1. . Percent change from baseline for myocyte shortening velocity in the presence of 10, 20, or 40 µg/mL of protamine was significantly decreased both in normothermia and after simulated cardioplegic arrest and rewarming. However, the relative negative effect of protamine on myocyte contractile function was less after simulated cardioplegic arrest compared with normothermic values (*p < 0.05 versus normothermia).

	Comment

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Previous clinical and experimental studies have reported that LV dysfunction can occur after cardioplegic arrest and rewarming [2, 3]. For example, Roberts and associates [2] reported a reduction in LV ejection fraction after cardioplegic arrest with rewarming in patients undergoing coronary artery bypass grafting procedures. Experimental studies have demonstrated that cardioplegic arrest with rewarming causes inherent defects in LV performance [3]. Specifically, Weinstein and colleagues [3] demonstrated that after cardioplegic arrest, LV peak positive rate of pressure change was reduced upon reperfusion in dogs. The effects of protamine administration on LV function have also been well documented [7, 8]. Del Re and colleagues [8] reported that the infusion of protamine resulted in a reduction in LV fractional shortening in patients undergoing cardiac surgical procedures. In an experimental study, Fadali and colleagues [7] reported a 38% decrease in LV peak positive rate of pressure change with protamine administration in dogs. However, cardioplegic arrest with rewarming and protamine administration in the intact animal can cause activation of neurohormonal systems and changes in loading conditions [2, 7, 8, 19, 20]. Accordingly, the present study employed an isolated myocyte model to examine the effects of cardioplegic arrest with rewarming and protamine on myocyte contractile function independent of loading conditions and neurohormonal influences. With these potentially confounding influences removed, the present study demonstrated interactive effects on myocyte contractile function between cardioplegic arrest with rewarming and protamine.

In a study by Davies and colleagues [21], myocytes were isolated from human nonfailing ventricles and myocyte percent shortening was reported as 3.9% ± 0.7%. In the present study, normal porcine myocyte percent shortening was 4.4% ± 0.1% and is comparable with this previous human myocyte study. Harding and associates [22] demonstrated a relationship between myocyte ß-adrenergic responsiveness and functional status in patients as defined by the New York Heart Association class. Specifically, in that report, myocyte ß-adrenergic response was reduced by approximately 30% in patients in New York Heart Association functional class III. In a report from this laboratory, myocyte ß-adrenergic responsiveness was reduced to a similar degree in pigs with severe congestive heart failure [23]. Taken together, the findings from previous studies suggest that porcine myocyte contractile function is comparable with that of human myocytes. Thus, the results obtained in the present study regarding the effects of cardioplegic arrest and protamine on porcine myocytes can likely be extended to human myocyte preparations.

Using an isolated myocyte model of simulated cardioplegic arrest, this laboratory has focused on identifying potential cellular and molecular mechanisms that contribute to the transient LV dysfunction that can occur after cardioplegic arrest with rewarming [4, 5]. Specifically, Handy and colleagues [4] demonstrated that 2 hours of simulated hypothermic, hyperkalemic cardioplegic arrest with rewarming resulted in reduced myocyte contractile function in the basal state and blunted ß-adrenergic responsiveness. Furthermore, that report suggested that the hyperkalemic environment during the period of cardioplegic arrest, not hypothermia alone, contributed to the abnormalities in myocyte contractile processes upon reperfusion with rewarming [4]. In addition to these previous studies using simulated cardioplegic arrest with rewarming, this laboratory has also examined the fundamental mechanisms for the effects of protamine on myocyte contractile processes [9–11]. Hird and colleagues [9] demonstrated that protamine caused a dose-dependent decline in myocyte contractile function and diminished inotropic responsiveness. The present study builds upon these previous reports by demonstrating that the combined effects of cardioplegic arrest with rewarming and protamine on myocyte contractile function are not synergistic. Specifically, the results from the present study suggest that simulated cardioplegic arrest and rewarming reduces the negative effects of protamine on myocyte contractile function when compared with normothermic conditions.

Because the negative effect of protamine on myocyte contractile function was reduced after cardioplegic arrest with rewarming, an interaction likely exists between these two treatments. Although the specific mechanisms responsible for this interactive effect remain speculative, a plausible explanation is that alterations in myocyte sarcolemmal structure or conformation occurred with hypothermic hyperkalemic cardioplegic arrest and rewarming, in turn reducing the interaction between the sarcolemma and protamine. Alterations in the structure and function of biological membrane systems due to hypothermia have been well described [24, 25]. Specifically, hypothermic conditions alter the structural and functional characteristics of the sarcolemmal proteins and lipid constituents [24, 25]. This phenomenon, known as thermotropic lateral phase separation, results in rearrangements of sarcolemmal lipids to form domains that exclude integral membrane proteins, such as sarcolemmal receptor systems [24, 25]. Previous studies from this laboratory and others have demonstrated that protamine can interact with the sarcolemma and modulate the activity of sarcolemmal transduction systems [11, 26]. In addition, a previous report [26] has demonstrated that protamine can allosterically modify sarcolemmal receptor systems. Therefore, alterations of the sarcolemma that may occur during cardioplegic arrest with rewarming may diminish the interaction between the sarcolemma and the protamine molecule, resulting in attenuation of the negative effects of protamine on myocyte contractile function. In light of these findings of the present study, future studies that more carefully examine the potential effects of hypothermia and rewarming on sarcolemmal structure and protamine binding characteristics may be appropriate.

Hird and colleagues [10] demonstrated that in the presence of equivalent concentrations of heparin and protamine, a protamine-heparin complex is formed, which has no effect on myocyte contractility. Thus, the negative effects of protamine on myocyte contractility are likely due to the unbound protamine molecule. In clinical practice, protamine is routinely administered in the presence of heparin, which favors the formation of a protamine-heparin complex. Therefore, the results from the present in vitro study with respect to the effects of the unbound protamine molecule should be viewed with caution.

In the present study, isolated myocyte contractile performance was examined independent of loading conditions and neurohormonal influences. This isolated myocyte system provided a means to identify the interactive effects of cardioplegic arrest with rewarming and protamine in the absence of these potentially confounding influences. However, this in vitro system prevents direct translation of these interactive effects to in vivo systems. Furthermore, in this isolated myocyte system, complete delivery of cardioplegic solution and protamine was achieved in the absence of any buffering capacity that may be present in vivo. In a previous experimental report, protamine was demonstrated to egress from the vascular space and occupy the interstitial space surrounding myocytes [11]. However, whether and to what extent protamine interacts with the myocyte after cardioplegic arrest and rewarming in the intact myocardium remains unclear. Nevertheless, this isolated myocyte system provided insights into the cellular basis for the combined effects of cardioplegic arrest with rewarming and protamine on contractile processes. In summary, the findings of the present study demonstrate that cardioplegic arrest with rewarming and protamine have direct and interactive effects on myocyte contractile function and provide a potential cellular mechanism for the transient LV dysfunction encountered in the early post–cardiac surgical setting.

	Acknowledgments

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This study was 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. Monty. H. Cox is a medical student research fellow of the American Heart Association. Doctor Spinale is an Established Investigator of the American Heart Association.

	References

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Fred A. Crawford, Jr Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by O, S.-J. Articles by Spinale, F. G. Search for Related Content PubMed PubMed Citation Articles by O, S.-J. Articles by Spinale, F. G.