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Ann Thorac Surg 1995;59:288-293
© 1995 The Society of Thoracic Surgeons

Direct Effects of Thrombin on Myocyte Contractile Function

Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, South Carolina

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Cardiopulmonary bypass activates the clotting cascade, resulting in elevated circulating levels of thrombin. In light of the fact that the function of a wide variety of cell types is modulated by thrombin, we hypothesized that thrombin may have a direct effect on myocyte function. Isolated left ventricular myocyte contractile function was measured from 6 adult dogs using videomicroscopy at baseline and after increasing concentrations of thrombin (1 to 10 U/mL). Indices of myocyte contractile function were reduced in a dose-dependent manner in the presence of increasing concentrations of thrombin. For example, myocyte percent shortening fell by 18% with 1 U/mL thrombin and by 43% with 2 U/mL thrombin. The addition of hirudin, a highly selective thrombin inhibitor, completely blocked the effects of thrombin on myocyte contractile function. ß-Adrenergic agonists are commonly used in the early post–cardiopulmonary bypass period. Accordingly, a final set of experiments examined the effects of thrombin on myocyte ß-adrenergic responsiveness using isoproterenol (25 nmol/L). In myocytes preincubated with 1 U/mL thrombin, myocyte ß-adrenergic responsiveness was significantly reduced. For example, in the presence of 1 U/mL thrombin, myocyte velocity of shortening fell by 25% from isoproterenol alone values. The results from the present study provide evidence that thrombin has a direct negative effect on steady-state contractile function and ß-adrenergic responsiveness in adult mammalian ventricular myocytes. These findings suggest that thrombin may be an additional contributory factor toward the transient left ventricular dysfunction that has been observed after cardiopulmonary bypass.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
See also page 293.

Each year in the United States alone, more than one-half million patients undergo cardiac operations that require cardiopulmonary bypass [1]. Extracorporeal circulatory support and the immediate postoperative period are associated with significant activation of the clotting cascade [2–5]. One of the important mediators of the clotting cascade is the proteolytic enzyme thrombin, also known as factor IIA [6]. The major substrate for activated thrombin is fibrinogen. However, it has been demonstrated clearly that thrombin can modulate the function of a wide variety of cell types via specific thrombin receptors localized to the cell membrane [7]. For example, thrombin has been shown to have a mitogenic effect on fibroblasts and smooth muscle cells [7]. More recently, several studies have demonstrated that thrombin influences inotropic properties of embryonal and neonatal myocytes [8, 9]. These past studies have suggested that thrombin may have specific and significant effects on myocyte contractile processes. This issue would have particular clinical relevance in the setting of significant thrombin activation, such as with cardiopulmonary bypass and myocardial ischemia. However, determining the direct effects of thrombin on myocyte contractile processes in vivo can be problematic. Specifically, thrombin mediates the release of vasoactive substances, which in turn may cause significant alterations in loading conditions and neurohormonal status [7]. Accordingly, the overall goal of the present study was to examine the direct effects of thrombin on ventricular myocyte contractile processes.

Past studies that have examined the effects of thrombin on myocyte function have been performed in both embryonal or neonatal culture preparations [8, 9]. Specifically, it has been reported that the number of spontaneously beating chick embryonal myocytes increased after the addition of thrombin [9]. However, there are significant differences in myocyte receptor systems, transduction properties, and contractile events between these embryonal preparations and adult mammalian ventricular myocytes [10, 11]. Thus, this project examined the direct effects of thrombin on adult mammalian myocytes. We hypothesized that in adult mammalian myocytes thrombin would influence myocyte contractile processes directly, potentially via a thrombin specific receptor. To test this hypothesis the current project had the following specific objectives: (1) to examine the dose-dependent effects of thrombin on myocyte contractile function, (2) to examine the effects of thrombin on myocyte ß-adrenergic responsiveness, and (3) to examine whether a highly selective thrombin inhibitor, hirudin, would modulate any effect of thrombin on myocyte function.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Myocyte Isolation
In the present study myocytes were isolated from the left ventricular free wall of 6 adult mongrel dogs of either sex (12 months of age; 15 to 25 kg; Hazelton, Kalamazoo, MI). All animals were treated and cared for in accordance with the National Institutes of Health ``Guide for the Care and Use of Laboratory Animals'' (NIH publication 85-23, revised 1985). All animals were anesthetized with 50 mg/mL pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL; 2 mL/kg) and ventilated through a nonrecirculating anesthesia circuit. The heart was quickly extirpated and placed in an oxygenated Krebs solution. The region of the left ventricular free wall comprising the left circumflex coronary artery was dissected free, and the artery was cannulated and prepared for myocyte isolation. Using methods described by this laboratory previously [12, 13], an oxygenated modified Krebs solution containing aerobic substrates and collagenase (0.5 mg/mL; Worthington, type II; 146 U/mg) was infused and recirculated through the cannulated circumflex artery for 20 minutes. The tissue then was minced into 2-mm sections and added to an oxygenated trituration solution containing 400 µmol/L CaCl₂ and collagenase (0.5 mg/mL). The tissue and trituration solution were transferred to a centrifuge tube and gently agitated. At 5-minute intervals, the supernatant was removed and filtered, and the cells were allowed to settle. The myocyte pellet then was resuspended in standard culture medium (Medium 199; 2 mmol/L Ca²⁺; Gibco BRL, Grand Island, NY).

Isolated Myocyte Function
Isolated myocytes were placed in a thermostatically controlled chamber (37°C) fitted with a coverslip on the bottom for imaging on an inverted microscope (Axiovert IM35; Zeiss Inc, Oberkochen, Germany). Myocyte contractions were elicited by field stimulation at 1 Hz (S11; Grass Instruments, Quincy, MA), where the polarity of the stimulating electrodes was alternated at every pulse. Myocyte contraction profiles were imaged using a charge-coupled device with a noninterlaced scan rate of 240 Hz (GPCD60; Panasonic, Secaucus, NJ). The distance between the left and right myocyte edges was converted into a voltage signal, digitized, and input to a computer (80286 ZBV2526; Zenith Data Systems, St. Joseph, MI) for subsequent analysis [13]. Stimulated myocytes were allowed a 5-minute stabilization period, after which contraction data for each myocyte were recorded from a minimum of 20 consecutive contractions. Parameters computed from the undifferentiated and differentiated myocyte contractile profile included percentage shortening, peak velocities of shortening and relengthening, time to 50% relaxation, and total contraction duration. Computation of the myocyte contractile parameters has been described previously [12, 13].

Experimental Protocol
In the first series of experiments, the dose-dependent effects of thrombin on myocyte function were examined using increasing concentrations of thrombin (1.0 to 10 U/mL; Sigma, St. Louis, MO). The dose-dependent effects of thrombin on myocyte percent and velocity of shortening are shown in Figure 1. A dose-dependent effect of thrombin on myocyte contractile function was observed with a significant fall in myocyte percent shortening beginning at 1 U/mL. Accordingly, the following series of experiments were performed using this concentration of thrombin.

View larger version (14K): [in this window] [in a new window]   Fig 1. . Dose-dependent effect of thrombin on indices of myocyte contractile function. With increasing concentrations of thrombin myocyte percent shortening fell in a dose-dependent manner (top). Similarly, myocyte velocity of shortening fell in a dose-dependent manner with the addition of increasing concentrations of thrombin (bottom).

The final series of experiments examined whether the effects of thrombin on myocyte contractile function could be blocked by the addition of the specific thrombin inhibitor hirudin (1 U/mL; Sigma) [7]. In these experiments, myocyte contractile function was measured at baseline, after the addition of 1 U/mL hirudin, and then after the subsequent addition of 1 U/mL thrombin.

Data Analysis
The effects of thrombin on indices of myocyte contractile function for the dose–response studies were examined using analysis of variance. Comparisons between ß-adrenergic stimulation in the presence and absence of thrombin was performed using Student's t test [15]. The interactive effects of hirudin and thrombin were examined by analysis of variance. All statistical procedures were performed using BMDP statistical software [16]. Results are presented as mean ± standard error of the mean. Values of p less than 0.05 were considered statistically significant.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Myocytes were harvested successfully from each of the 6 dogs used in the present study. A high yield (>80%) of viable myocytes was obtained from each isolation. Myocytes that maintained a rod shape and were quiescent in culture were deemed viable. Baseline (ie, no thrombin) steady-state indices of myocyte contractile function are summarized in Table 1. Indices of baseline myocyte contractile function obtained in the present study are similar to what has been reported previously for canine myocytes [13]. The dose-dependent effects of thrombin on myocyte contractile function are summarized in Table 1. Indices of myocyte function were reduced in a dose-dependent manner with the addition of increasing concentrations of thrombin. Figure 1 illustrates the dose-dependent effects of thrombin on myocyte percent and velocity of shortening. In the presence of 1 U/mL thrombin, myocyte percent shortening fell by 18% from baseline values (p < 0.05). The velocity of myocyte shortening fell by 15% from baseline values but did not reach statistical significance. Indices of myocyte contractile function fell with increasing concentrations of thrombin but appeared to plateau with concentrations higher than 5 U/mL. Interestingly, the time to 50% relaxation, an index of myocyte active relaxation properties, was significantly prolonged at a higher thrombin concentration (2 U/mL). Thus, thrombin significantly influenced myocyte contractile and relaxation properties.

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Cardiopulmonary bypass with hypothermic arrest and subsequent rewarming is associated with a wide spectrum of changes in neurohormonal systems as well as significant activation of the clotting cascade [2–5, 17]. Thrombin is a major component of the clotting cascade and is a potent proteolytic enzyme, which has been demonstrated to influence physiologic processes in a wide variety of cell types [7–9]. However, the direct effects of thrombin on adult mammalian ventricular myocytes were unknown. The present study determined the dose-dependent effects of thrombin on myocyte contractile function and ß-adrenergic responsiveness. Two important findings were made in the present study. First, thrombin caused a dose-dependent reduction in steady-state myocyte contractile function. Second, thrombin had a direct and negative interactive effect on myocyte ß-adrenergic responsiveness. Thus, this study provided direct evidence that thrombin modulated myocyte contractile processes in adult mammalian myocytes. The potential clinical implications from the present study are twofold. First, the left ventricular dysfunction observed in the setting of significant activation of the clotting cascade may be due in part to the direct effects of thrombin on myocyte contractile function. Second, thrombin may be an additional contributory factor toward the transient left ventricular dysfunction that has been observed in the immediate post–cardiopulmonary bypass period [18].

Significant alterations occur in homeostatic processes after cardiopulmonary bypass [2–5]. Specifically, extracorporeal circulation has been associated with platelet dysfunction and increased fibrinogen breakdown [5]. Furthermore, recent studies have demonstrated increased thrombin production during cardiopulmonary bypass [3, 4]. For example, Bosclair and associates [3] measured a tenfold increase in thrombin generation in patients undergoing cardiopulmonary bypass. In addition, blood products may be administered in the immediate postbypass period, and many of these products are rich in thrombin. Thus, the immediate post–cardiopulmonary bypass period is associated with significantly increased levels of thrombin. The present study demonstrated that thrombin had a direct and negative effect on myocyte contractile function. Furthermore, the negative effects of thrombin on myocyte contractile processes occurred in a dose-dependent manner. Thus, the significantly increased thrombin levels that occur after cardiopulmonary bypass may influence myocyte contractile function, and in turn may modulate left ventricular pump performance.

Thrombin is known to regulate the activity of a wide variety of cell types via specific receptors [7–9]. For example, thrombin receptors have been localized to fibroblasts, smooth muscle cells, and endothelial cells [7–9], all of which are derived from the mesenchymal layer during embryonic development [19]. The present study examined both the dose-dependent effects of thrombin on myocyte function and the effects of the specific thrombin inhibitor hirudin. Indices of myocyte function were reduced in a dose-dependent manner in the presence of thrombin. Furthermore, these effects of thrombin on myocyte contractile function were abolished by a specific thrombin inhibitor. Myocytes also are derived from a mesenchymal origin and therefore have the potential to express thrombin receptors on the sarcolemma [19]. Thus, results from the present study provide evidence that a functional thrombin receptor exists on adult mammalian myocytes.

During cardiac surgical procedures, ß-adrenergic agonists frequently are required to augment left ventricular pump function [20]. A significant temporal relationship exists between administration of ß-adrenergic agonists and high circulating levels of thrombin in the immediate postbypass period. To examine whether thrombin plays an interactive role in myocyte ß-adrenergic receptor responsiveness, the present study measured myocyte contractile function in the presence of isoproterenol (a potent ß-adrenergic agonist) and thrombin. The results from the present study demonstrated that thrombin directly interfered with myocyte ß-adrenergic responsiveness. Potential mechanisms for the interactive effects of thrombin on myocyte ß-adrenergic responsiveness include alterations in ß-adrenergic receptor binding and transduction as well as interference with downstream intracellular events. Thrombin is a member of the serine protease family and therefore has the potential for interacting with a wide variety of substrates [7]. Thus the proteolytic activity of thrombin may cause alterations in the myocyte sarcolemma, which in turn would reduce ß-adrenergic receptor binding and transduction. Thrombin receptor activation causes an increase in phospholipase C activity, which in turn alters intracellular calcium homeostasis [8]. ß-Adrenergic receptor stimulation causes an increase in cyclic adenosine monophosphate, which results in increased availability of intracellular calcium for actin-myosin crossbridging [14]. Therefore, the reduction in myocyte contractile function that occurred in the presence of thrombin after ß-adrenergic receptor stimulation may be due to alterations in intracellular calcium availability to the myofilament contractile apparatus. Thus, although the mechanisms for the effects of thrombin on myocyte contractile processes and ß-adrenergic responsiveness remain speculative, results from the present study clearly demonstrated that thrombin had a direct and negative effect on myocyte contractile function after ß-adrenergic receptor stimulation.

The present study examined the effects of physiologic concentrations of thrombin on myocyte function. Specifically, the concentrations of thrombin that were used in the present study are similar to clinical concentrations observed during coagulation [21]. However, the relative in vivo distribution of thrombin within the vascular and extravascular compartments remains unclear. Thus, it remains speculative as to whether the concentrations of thrombin used in the present in vitro study reflect actual thrombin concentrations to which myocytes would be exposed in vivo. However, past reports clearly have demonstrated that hypothermic cardioplegic arrest and cardiopulmonary bypass causes significant damage to endothelial integrity [22, 23]. Specifically, Laks and associates [22] demonstrated increased extravascular fluid accumulation after cardiopulmonary bypass. Furthermore, Harjula and colleagues [23] observed that hypothermic cardioplegic arrest caused damage to capillary endothelial cells. Thus, the immediate postbypass period may be associated with a significant flux of vascular constituents into the extravascular space. In light of the fact that significant levels of thrombin are present after hypothermic cardioplegic arrest and subsequent rewarming, enhanced influx of thrombin into the extravascular space may occur secondary to alterations in endothelial integrity. Results from the present study demonstrated that thrombin, in concentrations that are encountered clinically, significantly reduced myocyte contractile function and ß-adrenergic responsiveness. In light of these findings, future studies that directly address the extent to which myocytes are exposed to thrombin administered in vivo would be appropriate.

There are limitations to the present study that must be recognized. First, in vivo thrombin has a wide range of systemic effects that could not be addressed through the experimental design employed in the present study. Specifically, thrombin causes the release of cytokines and other neurohormonal mediators, which in turn may influence myocyte contractile processes [7, 8]. Furthermore, this study examined the effects of thrombin on myocyte preparations that were independent of in vivo influences such as non–myocyte cell populations, extracellular matrix buffering and diffusion, and myocardial blood flow. Therefore, any effects of thrombin that may be modulated by these in vivo factors could not be addressed in the present study. Thus, although the limitations described above must be recognized, the results from the present study clearly demonstrated that thrombin caused alterations in steady-state myocyte contractile function and ß-adrenergic responsiveness.

In summary, the immediate post–cardiopulmonary bypass period is associated with significant activation of the clotting cascade and has been associated with transient left ventricular dysfunction [3, 4, 18]. The present study examined whether a significant constituent of the clotting cascade, thrombin, would directly modulate contractile processes of adult ventricular mammalian myocytes. Thrombin depressed myocyte contractile function in a dose-dependent manner and interfered with ß-adrenergic responsiveness. These results suggest that a contributory factor for the left ventricular dysfunction observed in the setting of significant activation of the clotting cascade may be due to the direct effects of thrombin on myocyte contractile function.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This work was supported by National Institutes of Health grant HL45024 (F.G.S.) and MUSC research funds (R.B.H.). F.G.S. is an Established Investigator with the American Heart Association.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Presented at the Forty-first Annual Meeting of the Southern Thoracic Surgical Association, Marco Island, FL, Nov 10–12, 1994.

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

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments 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 Hird, R. B. Articles by Spinale, F. G. Search for Related Content PubMed PubMed Citation Articles by Hird, R. B. Articles by Spinale, F. G. Related Collections Related Article