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J Thorac Cardiovasc Surg 1994;108:1100-1114
© 1994 Mosby, Inc.

CARDIOPULMONARY BYPASS, MYOCARDIAL MANAGEMENT, AND SUPPORT TECHNIQUES

The direct effects of protamine sulfate on myocyte contractile processes

Charleston, S.C.

This work 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 of the American Heart Association.

Received for publication Jan. 27, 1994. Accepted for publication June 16, 1994. Address for reprints: Francis G. Spinale, MD, PhD, Division of Cardiothoracic Surgery, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425

Abstract

The use of protamine sulfate in patients has been associated with circulatory collapse and is suspected to directly depress left ventricular function. However, the cellular basis for these changes that occur after protamine administration are unknown. Accordingly, the first objective of this study was to determine the direct effects of protamine on isolated myocyte contractile function. Myocytes were isolated from porcine hearts and contractile function was examined at baseline and then after the administration of protamine in concentrations of 20, 40, or 80µg/ml. These concentrations were chosen because they reflect the serum concentrations of protamine commonly obtained in patients. The presence of protamine resulted in a dose-dependent decline in myocyte contractile function. For example, in the presence of a 20µg/ml concentration of protamine myocyte contractile function did not change significantly from baseline values, whereas an 80µg/ml protamine concentration caused myocyte percent and velocity of shortening to fall by more than 35% from baseline values. In light of the fact that protamine directly depressed myocyte contractile function, a second objective of this study was to examine potential cellular mechanisms responsible for this effect. Accordingly, in the next series of experiments, the effects of protamine on the myocyte sarcolemmal ß-adrenergic receptor system were examined by measuring myocyte contractile function with the ß-adrenergic agonist isoproterenol (25 nmol/L), as well as with the concomitant addition of protamine and isoproterenol. In the presence of protamine, myocyte ß-adrenergic responsiveness was significantly reduced. For example, in the presence of an 80µg/ml dose of protamine, both myocyte percent and velocity of shortening fell by greater that 50% when compared with isoproterenol alone values (p < 0.05). To determine the reversibility of these protamine effects, we performed additional experiments in the presence of heparin. Incubation with heparin before protamine addition prevented the negative effects of protamine on myocyte function. However, the addition of heparin after protamine incubation failed to reverse the negative effects of protamine on myocyte function. In a final set of experiments, the effects of protamine on isolated myocyte electrophysiologic properties were examined using microelectrode techniques at baseline and with either 40 or 80µg/ml doses of protamine. Myocyte resting membrane potential changed from baseline with the addition of a 40µg/ml dose of protamine (-79.2 ± 0.5 versus -75.2 ± 0.8 mV (p < 0.05), with no further change at an 80µg/ml dose of protamine (-73.0 ± 1.3 mV). Myocyte action potential duration increased by 35% from baseline values with the addition of a 40µg/ml dose of protamine (p < 0.05), with no further change at an 80µg/ml dose of protamine. Additionally, maximum upstroke velocity of the myocyte action potential fell from baseline with the addition of an 80µg/ml dose of protamine (131 ± 3.1 versus 107 ± 3.5 V/sec; p < 0.05). Excitation-contraction results revealed that protamine selectively influenced myocyte relaxation properties. Summary: Protamine sulfate directly depressed isolated myocyte contractile function in dose-dependent manner. Potential contributory mechanisms for the depressant effects of protamine on myocyte contractile function include changes in myocyte sarcolemmal transduction systems, as well as alterations in basic myocyte electrophysiology. Thus this study for the first time provides a potential cellular mechanism for the depressed left ventricular function that has been observed clinically after the administration of protamine. (J THORACCARDIOVASCSURG1994;108:1100-14)

Anticoagulation with heparin is currently a fundamental requirement for patients undergoing cardiopulmonary bypass and is frequently used in patients undergoing vascular operations. The anticoagulant effects of heparin are routinely reversed by the administration of the polycationic peptide, protamine sulfate. However, the administration of protamine has been associated with circulatory collapse and cardiac arrhythmias. ^1-6 Furthermore, several previous studies have suggested that protamine directly depressed left ventricular function. ^1,2,7-10 For example, Wakefield and associates ⁷ reported in isolated rabbit hearts a dose-dependent decline in peak developed left ventricular pressure in the presence of protamine. Although these studies have suggested that protamine may directly depress left ventricular contractile function, the cellular basis for this effect is unknown.

The amino acid arginine comprises more than 60% of the protamine molecule, which results in a high net positive charge. ¹¹ These highly charged amino acids found in protamine may induce alterations in the function of cellular membranes, such as the myocyte sarcolemma. Two important functions of the myocyte sarcolemma include transduction of extracellular receptor signals and maintenance of ionic potentials. ¹² The ß-adrenergic receptor system contains a large number of extracellular receptors located within the myocyte sarcolemma. ¹² Thus, if the effects of protamine are mediated by altering sarcolemmal function, then the ß-adrenergic receptor system may be significantly influenced. Accordingly, one objective of the present study was to explore this possibility by examining the effects of protamine on the biologic responsiveness of isolated myocytes to ß-adrenergic receptor stimulation.

Protamine administration has been associated with cardiac arrhythmias in patients and animals. ^2-5 A possible explanation for these observations is that protamine may affect myocyte electrophysiologic properties. Specifically, protamine may alter the function of sarcolemma-based ion pumps and/or channels and therefore change myocyte resting membrane potential. Accordingly, a second objective of the present study was to examine the electrophysiologic properties of isolated myocytes in the presence of protamine.

In addition to the systemic effects of protamine, several studies have suggested that protamine may depress left ventricular contractile function. ^1,2,7-10 Furthermore, Lin and colleagues ⁸ reported that protamine influenced the electrophysiologic properties of Purkinje fibers. ⁸ One possible mechanism for the depressed left ventricular contractile function observed with protamine administration is an alteration in myocyte excitation-contraction coupling. However, this possibility has remained unexplored. Thus the final objective of this study was to determine if protamine directly affects the excitation-contraction coupling relationship in isolated ventricular myocytes.

METHODS

Myocyte isolation
Five age and weight matched pigs (Yorkshire strain, 6 mo, 25 to 30 kg) were anesthetized with isoflurane (0.5%/1.5 L/min) and their lungs 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. All animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. According to methods described by this laboratory previously, ^13,14 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 was then minced into 2 mm sections and added to an oxygenated trituration solution containing a 400 µmol/L concentration of calcium chloride 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, filtered, and the cells allowed to settle. The myocyte pellet was then resuspended in standard culture medium (M199, Ca2+ 2 mmol/L, Gibco Laboratories, Grand Island, N.Y.).

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, Mass.) where the polarity of the stimulating electrodes was alternated at every pulse. Myocyte contraction profiles were imaged with a charge-coupled device with a noninterlaced scan rate of 240 Hz (GPCD60, Panasonic, Secaucus, N.J.). 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, Mich.) for subsequent analysis. ¹⁴ Stimulated myocytes were allowed a 5-minute stabilization period after which contraction data for each myocyte was 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 lengthening (micrometers per second), and total contraction duration (milliseconds). Computation of the myocyte contractile parameters has been described previously. ¹³

Isolated myocyte electrophysiologic measurements
Microelectrodes were made from 1.2 mm thin-walled glass capillary tubing (1B120F-4; World Precision Instruments, Sarasota, Fla.) using a horizontal electrode puller (P-87; Sutter Instrument Inc., Novato, Calif.). Microelectrodes (tip resistances: 20 to 35 Mohm) were filled with potassium chloride 3 mol/L and attached to a high-input impedance negative capacitance amplifier (Axoclamp-2A; Axon Instruments, Burlingame, Calif.). A silver–silver chloride reference electrode was placed in the experimental chamber. Myocytes were then impaled by gently advancing the microelectrodes at a 45-degree angle onto the sarcolemmal surface with a micromanipulator (MO-103; Narshige Instruments, Tokyo, Japan). Impalements were considered stable if myocytes could be stimulated continuously for 10 minutes with stable resting membrane potential, action potential duration, and contraction amplitude. ¹⁵ Myocyte action potentials and contractions were elicited by current injection through the microelectrode using 1 msec pulses and at intervals of 1000 msec. Stimulation current was adjusted (3 to 5 nA) so that there was at least a 1 msec separation between the stimulation pulse and the upstroke of the action potential.

After a 15-minute stabilization period, myocyte action potential and contractile data were collected from a minimum of five consecutive contractions. Myocyte contraction profiles were imaged and converted into a voltage signal as described in the previous section. The amplified transmembrane membrane potential signal and the myocyte contractile signal were digitized simultaneously at 12-bit resolution by means of a sampling frequency of 10 kHz/signal (AT-MIO-16; National Instruments, Austin, Tex.), and the digitized signals were stored to disk on a 486 computer (Zeos International, Minneapolis, Minn.). During off-line analysis, the first-order differential of the myocyte contractile signal and the membrane potential signal were computed. Action potential parameters derived from the undifferentiated and differentiated membrane potential signals included resting membrane potential (millivolts), maximum action potential amplitude (millivolts), action potential durations at 50% (APD₅₀) and 90% (APD₉₀) repolarization (milliseconds), and the maximum action potential upstroke velocity (volts per second). Maximum action potential amplitude was computed as the difference between the peak membrane potential and the resting membrane potential. APD₅₀ and APD₉₀ were computed as the time required for the action potential to repolarize to 50% and 10% of the maximum action potential amplitude, respectively. Maximum action potential upstroke velocity was defined as the peak positive value of the differentiated membrane potential signal.

To determine the direct relationship between potential changes in myocyte action potential and contractile function that may have occurred with protamine, we derived excitation-contraction coupling parameters. The digitized and temporally aligned myocyte membrane potential and contractile profile signals were used for this purpose. To determine if a change in electrical/mechanical events occurred with protamine, we computed the myocyte action potential amplitude at peak velocity of shortening, at peak shortening, and at peak velocity of relengthening. To determine whether a change in the temporal relationships between myocyte excitation and contraction events occurred with protamine, we computed the time differences between the two following events: (1) the initiation of the action potential and peak myocyte contraction and (2) APD₉₀ and peak myocyte contraction. Excitation-contraction coupling parameters were computed from the membrane potential and myocyte contractile signals recorded simultaneously from 5 consecutive contractions.

Experimental protocol
In the first series of experiments, myocyte contractile measurements were obtained at baseline and then in the presence of protamine. Crystalline protamine sulfate suspended in 0.1N saline (Elkins-Sinn, Inc. Cherry Hill, N.J.) was added to the bath in final concentrations of 20, 40, or 80 µg/ml. Protamine concentrations of 20, 40, or 80 µg/ml are approximately equal to serum concentrations obtained in patients dosed with protamine at 1.25, 2.5, and 5 mg/kg, respectively. ^7,8 Myocytes were randomly assigned to receive protamine concentrations of 20, 40, or 80 µg/ml, after which contractions were again recorded.

The next series of experiments were performed to determine whether protamine influenced myocyte sarcolemmal ß-adrenergic receptor responsiveness. Myocyte contractile function was measured in the presence of protamine and isoproterenol, 25 nmol/L (Sigma Chemical Co., St. Louis, Mo.). This concentration of isoproterenol has been shown previously to elicit the 100% maximal response in contractile function of porcine myocytes. ¹⁴ First, myocyte contractile data were measured in cells treated with protamine concentrations of 20, 40, or 80 µg/ml; then isoproterenol, 25 nmol/L, was added and measurements were repeated.

In the clinical setting, the anticoagulant effects of heparin are reversed by subsequent protamine administration. Accordingly, in a separate series of experiments, the effects of heparin alone, protamine alone, and the interaction between heparin and protamine on myocyte contractile function and ß-adrenergic responsiveness were examined. In the first set of experiments, myocyte contractile function was measured in the presence of heparin, 40 µg/ml (beef lung heparin, Upjohn, Kalamazoo, Mich.). Myocyte ß-adrenergic responsiveness in the presence of this concentration of heparin was determined by the addition of isoproterenol, 25 nmol/L. This concentration of heparin (40 µg/ml) was selected because it is assumed that heparin and protamine bind in a 1:1 fashion and therefore a 40 µg/ml dose of heparin will neutralize a 40 µg/ml dose of protamine. ¹⁶ In the second series of experiments, myocytes were incubated with a 40 µg/ml concentration of heparin and then exposed to a 40 µg/ml concentration of protamine, after which myocyte contractile function and ß-adrenergic responsiveness were measured. A final subset of experiments was performed to determine whether the effects of protamine on myocyte contractile function and ß-adrenergic responsiveness could be reversed by subsequent administration of heparin. In this series of experiments, myocytes were incubated with protamine, 40 µg/ml, and then subsequently exposed to a 40 µg/ml dose of heparin, after which myocyte function and ß-adrenergic responsiveness were examined.

A final series of experiments were performed to investigate whether protamine affected the excitation-contraction coupling relationship in myocytes. First, simultaneous measurements of the action potential and contraction profile of an isolated myocyte at baseline (no protamine) were obtained. Subsequently, a 40 µg/ml dose of protamine was added to the experimental chamber and the same measurements were repeated. Furthermore, this series of experiments was repeated in the presence of an 80 µg/ml dose of protamine.

The pH and electrolyte concentrations of media in the experimental chamber were routinely checked to ascertain if any changes occurred after the addition of protamine. Chambers with standard media had Na⁺ = 130 mmol/L, K⁺ = 4.8 mmol/L, carbon dioxide tension = 36 torr, oxygen tension = 165 torr, and pH = 7.55; chambers with media plus protamine had Na⁺ = 127 carbon dioxide tension, K⁺ = 4.7 mmol/ L, oxygen tension = 208 torr, carbon dioxide tension = 34 torr, and pH = 7.57. There were no significant differences in these values between media without protamine and media with protamine.

Data analysis
Analysis of myocyte function data was performed by analysis of variance. If the analysis of variance revealed significant differences, pairwise tests of individual group means were compared by Bonferroni probabilities. ¹⁷ For the excitation contraction couplings results, the values were first subjected to a Welch analysis of variance to determine equality of variance among the three treatment groups. ¹⁷ If significant differences in variances were detected, then a Winsorized mean value was computed and then subjected to comparison by Bonferroni bounds. ¹⁸ If, on the other hand, the excitation contraction coupling data were not normally distributed according to analysis of variance, then the nonparametric Mann-Whitney test was used. ¹⁷ The statistical treatment and procedures used in this study are indicated in the respective tables. Results are presented as mean ± standard error of the mean. Values of p < 0.05 were considered statistically significant.

RESULTS

Myocyte contractile function: Effects of protamine sulfate
Indices of steady-state myocyte contractile function obtained at baseline (no protamine) are summarized in Table I. The measurements of myocyte contractile function obtained in the present study for normal porcine ventricular myocytes are similar to those reported previously. ^13,14 Results from the present study in which indices of myocyte contractile function were obtained in the presence of protamine are summarized in Table I. Myocytes incubated with a 20 µg/ml concentration of protamine had no significant change in indices of contractile function when compared with baseline values. In the presence of a 40 µg/ml concentration of protamine, both myocyte percent and velocity of shortening fell significantly from baseline values. Representative contraction profiles of an isolated myocyte at baseline and in the presence of a protamine concentration of 80 µg/ml are shown in Fig. 1. In the presence of an 80 µg/ml concentration of protamine, all indices of myocyte contractile function fell significantly from baseline values. For example, in the presence of an 80 µg/ml concentration of protamine, myocyte velocity of shortening fell by 38% from baseline values. A further decline in myocyte percent and velocity of shortening was observed in the presence of an 80 µg/ml concentration of protamine when compared with a 40 µg/ml concentration but did not reach statistical significance (p = 0.23). Thus, the present study demonstrated that a 20 µg/ml concentration of protamine had no effect on myocyte contractile function, whereas higher concentrations of protamine had a dose-dependent effect on myocyte function.

View larger version (19K): [in this window] [in a new window]   Fig. 1. A representative contraction profile of an isolated ventricular myocyteat baseline (no protamine) and in the presence of an 80 µg/ml concentration of protamine. In the presence of 80 µg/ml protamine, there was a significant reduction in myocyte shortening when compared with baseline values. Indices of myocyte contractile function at baseline and in the presence of protamine are summarized in Table I.

View larger version (25K): [in this window] [in a new window]   Fig. 2. The percent change in myocyte velocity of shortening from baseline values after the addition of a 25 nmol/L dose of isoproterenol alone (solid bar), a 25 nmol/L dose of isoproterenol with a 40µg/ml dose of protamine (dashed lines), and a 25 nmol/L dose of isoproterenol with an 80 µg/ml dose of protamine (squares). The addition of isoproterenol resulted in a significant increase in the percent change from baseline (p < 0.05). However, the response to isoproterenol in the presence of the 40 µg/ml dose of protamine was significantly reduced (p < 0.05) when compared with isoproterenol alone values. Furthermore, in the presence of an 80 µg/ml concentration of protamine, myocyte ß-adrenergic responsiveness was completely abolished.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Myocyte percent shortening after stimulation with a 25 nmol/L dose of isoproterenol in the absence of protamine (NO PROT) is shown as a solid bar. In the presence of a 40 µg/ml dose of protamine alone, ß-adrenergic responsiveness was reduced (PROT; dashed lines) and this effect could not be reversed by the subsequent addition of a 40 µg/ml dose of heparin (PROTHEP; dashed lines). Heparin alone (HEP; boxes) had no effect on myocyte ß-adrenergic responsiveness. Preincubation of myocytes with heparin followed by protamine (HEPPROT; boxes) also did not affect myocyte ß-adrenergic responsiveness.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Representative recording of an action potential in the presence of an 80 µg/ml dose of protamine. In this recording, a spontaneous subthreshold after depolarization was observed (arrow). This observation provided further evidence that protamine caused significant changes in myocyte electrophysiologic properties.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Representative simultaneous recordings of a myocyte action potential (top) and contraction profile (bottom) at baseline (no protamine) and in the presence of a 40 µg/ml concentration of protamine. Protamine caused prolonged action potential duration and a less negative membrane potential in conjunction with depressed myocyte contractile properties. Action potential data are summarized in Table IV and excitation-contraction data are summarized in Table VI.

Next, the effect of protamine on the temporal relationship between the myocyte action potential and myocyte contractile processes was examined. The baseline (no protamine) time difference between maximum upstroke velocity of the action potential and peak contraction (i.e., contraction phase) did not change in the presence of protamine. In contrast, in the presence of a 40 µg/ml concentration of protamine, the time difference between 90% repolarization and peak contraction (i.e., active relaxation phase) was significantly higher than baseline values. A similar directional increase in the time difference between 90% repolarization and peak contraction was observed in the presence of an 80 µg/ml concentration of protamine, but this increase did not reach statistical significance (p = 0.11). Thus, the presence of protamine significantly altered the temporal relationship between the myocyte action potential and active myocyte relengthening processes. Specifically, protamine prolonged the time between the onset of active relaxation and 90% repolarization of the myocyte action potential.

DISCUSSION

Protamine sulfate is routinely used to reverse the anticoagulant effects of heparin. However, significant left ventricular dysfunction has been associated with the administration of protamine in both human beings and animals. ^1,2,7-10 The exact cellular mechanisms responsible for this left ventricular dysfunction after protamine administration remain to be defined. A recent report from this laboratory demonstrated that protamine influenced myocyte function. ²¹ The present study builds on this past report by more carefully examining the dose-dependent effects of protamine on myocyte contractile function and ß-adrenergic responsiveness, as well as the interactive effects with heparin. More important, the present study examined fundamental contributory processes for changes in myocyte function in the presence of protamine; the myocyte action potential. Four conclusions are warranted from the results of the present study: (1) Isolated myocyte contractile function fell in a dose-dependent manner in the presence of protamine; (2) ß-adrenergic responsiveness of isolated myocytes was depressed with the intermediate concentration of protamine and completely abolished in the presence of the higher concentration of protamine used in the present study; (3) protamine significantly altered myocyte resting membrane potential, prolonged the total duration of the myocyte action potential, and at the higher concentration of protamine significantly blunted action potential maximum upstroke velocity; (4) finally, excitation-contraction coupling studies revealed that protamine significantly altered the relationship between myocyte membrane potential and active relaxation properties. Thus this study demonstrated that protamine directly depressed isolated myocyte contractile function in a dose-dependent manner and for the first time provides direct evidence that potential mechanisms responsible for this contractile deficit include alterations in the sarcolemmal transduction system as well as alterations in basic electrophysiologic properties of the myocyte.

Previous clinical and experimental studies have suggested that protamine has a depressive effect on left ventricular pump function. ^1,2,7-10 For example, Jastrzebski, Sykes, and Woods ¹ reported a decreased cardiac index in patients after protamine administration in the early postcardiopulmonary bypass period. However, there are significant changes in left ventricular loading conditions, heart rate, and circulating catecholamine levels that also occur in this early postcardiopulmonary bypass period. ²² Thus determining the direct effect of protamine on left ventricular contractile function in this setting is problematic. In experimental studies, Wakefield and colleagues ⁷ reported that protamine had a dose-dependent depressive effect on left ventricular peak developed dP/dt in isolated rabbit hearts. In an additional study by this group of investigators, protamine was found to have a toxic effect on cultured bovine pulmonary artery endothelium. ²³ Additionally, Morel, Costabella, and Pittet ²⁴ observed an increase in plasma concentrations of thromboxane A₂ after the administration of protamine to sheep. Thus from these clinical and experimental studies it remained unclear whether protamine directly depressed left ventricular contractile function or whether the effects were due to the release of cytokines or generation of oxygen free radicals from intact endothelium. The present study builds on these past studies by demonstrating that protamine has a direct depressive effect on isolated myocyte contractile function. Thus these results suggest that the left ventricular dysfunction observed after protamine administration in patients and animals may be due to a direct depressive effect on myocyte contractile function.

One potential mechanism for the depressive effect of protamine on myocyte contractile function is that this highly charged peptide may interfere with sarcolemmal function. In the present study the effects of protamine on the sarcolemmal ß-adrenergic receptor system were examined. The intermediate concentration of protamine significantly depressed myocyte ß-adrenergic responsiveness, whereas in the presence of the highest concentration of protamine myocyte ß-adrenergic responsiveness was completely abolished. Thus protamine significantly altered the physiologic responsiveness of myocytes to ß-adrenergic receptor stimulation. In a recent study, Hu and colleagues ²⁵ reported that protamine caused allosteric conformational changes in sarcolemmal muscarinic receptors. The results from this past report and the present study suggest that protamine may interfere with sarcolemmal transduction of extracellular receptor systems. Furthermore, in the presence of protamine, resting membrane potential was significantly decreased (i.e., less negative) when compared to control (no protamine) values. The determinants of myocyte resting membrane potential are a normal voltage-dependent outward rectifier along with operational ionic pumps and exchangers. ¹² These results suggest that protamine may inhibit sarcolemmal ionic pump processes and thereby cause the observed changes in resting membrane potential, action potential duration, ²⁶ and afterdepolarizations. ²⁷ Therefore, results from the present study provide putative evidence that protamine may interfere with the ability of the myocyte sarcolemma to transduce extracellular receptor signals (i.e., depressed ß-adrenergic responsiveness) and maintain ionic potentials (i.e., altered resting membrane potential).

Anticoagulation with heparin is a mainstay for most cardiovascular procedures. The effects of heparin are commonly reversed by the subsequent addition of protamine. In light of the fact that the use of heparin followed by subsequent protamine administration is the common scenario encountered clinically, the present study examined the interactive effects of heparin and protamine on myocyte contractile function and ß-adrenergic responsiveness. An important finding of the present study was that pretreatment of myocytes with heparin before protamine administration preserved myocyte contractile function and ß-adrenergic responsiveness. In contrast, when myocytes were pretreated with protamine, the subsequent addition of an equivalent concentration of heparin failed to reverse the reduction in myocyte contractile function and ß-adrenergic responsiveness caused by protamine. The basis for these findings may lie in the ionic interactions between heparin, protamine, and the sarcolemma. First, there were no effects on myocyte contractile function or ß-adrenergic responsiveness when myocytes were exposed to the negatively charged heparin molecule. ¹⁰ Subsequent addition of the positively charged protamine molecule presumably complexed with the heparin present in the myocyte media, which thereby protected the myocyte from the detrimental effects of the protamine molecule on myocyte contractile function and ß-adrenergic responsiveness. In contrast, when myocytes were initially exposed to the positively charged protamine molecule, protamine presumably interacted with the negatively charged glycocalyx of the sarcolemma ¹² and subsequent heparin addition was not effective in reversing the detrimental effects on myocyte contractile function and ß-adrenergic responsiveness. The results from this portion of the present study provide a unique insight into potential interactions between heparin, protamine, and the myocyte sarcolemma.

The present study examined the direct effects of 20, 40, and 80 µg/ml concentrations of protamine on myocyte contractile processes. These protamine concentrations reflect estimated serum levels that would be obtained after the clinically administered doses 1.25, 2.5, and 5 mg/kg, respectively. ^7,8 However, the concentrations of protamine used in the present study are dependent on several assumptions. First, the concentrations of protamine used in the current experimental design assume that protamine has an equivalent and predictable volume of distribution. However, it remains unclear whether protamine is equally distributed within the extracellular compartment. Thus it remains speculative as to whether the concentration of protamine used in the present in vitro study reflects actual protamine concentrations to which myocytes would be exposed in vivo. However, past experimental studies have provided evidence to suggest that protamine traverses from the vascular compartment to the extracellular space, thereby exposing the myocyte to significant concentrations of protamine. ^28,29 Specifically, it has been demonstrated that the negative charges localized to the endothelium and interstitium can act as a cationic exchange gel. ²⁸ Thus, on the basis of these electrochemical conditions, it would be predicted that cationic molecules such as protamine would be freely exchanged with other cationic molecules in the interstitium. Second, protamine may neutralize the negative charges of the endothelium and thereby increase vascular permeability. ^28,29 Furthermore, it has been suggested that a sequela of hypothermic cardioplegic arrest and cardiopulmonary bypass is altered endothelial integrity with subsequently increased microvascular permeability. ³⁰ In light of the fact that protamine is commonly administered after hypothermic cardioplegic arrest and subsequent rewarming, then enhanced influx of protamine into the extracellular space may occur as a result of alterations in endothelial integrity. Finally, Delucia and associates ³¹ demonstrated that protamine is preferentially distributed to the kidneys, lung, and heart. Taken together, the results from these past reports suggest that protamine readily traverses into the interstitium surrounding myocytes and is preferentially distributed to the myocardium. The present study demonstrated that significant effects in myocyte function occurred with protamine concentrations that reflect a volume of distribution that may be encountered clinically. In light of these findings, a future study that directly addresses the extent to which myocytes are exposed to protamine administered in vivo would be appropriate.

Past clinical and experimental reports have suggested an association between the administration of protamine and the development of cardiac arrhythmias. ^2-5 There are three important determinants of the myocyte action potential that may contribute to a proarrhythmic substrate: (1) a less negative resting membrane potential, (2) decreased maximum upstroke velocity, and (3) prolongation of action potential duration. ¹² In the present study these specific alterations in the myocyte action potential were observed in the presence of protamine and suggest that protamine may have a potential proarrhythmic effect. It is known that prolongation of the myocyte action potential duration may lead to the development of afterdepolarizations, which in turn can lead to the development of sustained arrhythmias. ³² In the present study myocyte action potential afterdepolarizations were observed to develop in several myocytes after the addition of protamine. A representative action potential afterdepolarization after the administration of protamine is shown in Fig. 4. In a past report, Lin and coworkers ⁸ demonstrated action potential afterdepolarizations in human atrial tissue after protamine administration. Thus results from the present study, as well as this past report, suggest that protamine alters myocyte action potential morphology, which may be a potential contributory factor for the genesis of arrhythmias. However, although the present study demonstrated alterations in myocyte action potential in vitro, it remains unclear whether protamine causes a proarrhythmic substrate in vivo. Future in vivo studies that measure conduction velocities and use provocative electrical stimulation protocols in the presence of protamine may help clarify this issue.

This study for the first time examined the direct effects of protamine on the myocyte excitation-contraction relationship (i.e., relationship between ionic and mechanical events). During the myocyte contraction phase, protamine had no effect on the excitation-contraction relationship. Specifically, the time from action potential maximum upstroke velocity (phase 0) to myocyte peak contraction did not change with protamine. In contrast, protamine altered the temporal relationship between myocyte action potential repolarization processes and myocyte active relengthening (i.e., active relaxation phase). Specifically, there was a prolongation in the time from myocyte peak contraction to 90% repolarization of the action potential. Thus the results from this study provide new evidence to suggest that protamine selectively depresses myocyte active relaxation processes. Fundamental mechanisms by which protamine may depress myocyte contractile function remain speculative but include (1) alterations in sarcolemmal function, (2) changes in Ca²⁺ homeostasis, and (3) fundamental changes in the myofilament contractile apparatus. ¹² The present study provides evidence for the first of these proposed mechanisms; alterations in sarcolemmal function. Specifically, myocyte responsiveness to ß-adrenergic receptor stimulation was depressed with protamine. Thus protamine may have influenced transduction processes of this sarcolemmal based receptor system. Second, protamine caused alterations in the myocyte action potential. These findings suggest that protamine may influence the function of sarcolemmal based ion pumps and/or channels. To directly address this issue, the present study examined the direct effects of protamine on myocyte electrophysiology in the presence of the specific fast Na⁺ channel inhibitor, tetrodotoxin. Tetrodotoxin was chosen for this portion of the study in light of the similar compositional characteristics to the protamine molecule. ^11,20 However, results from this portion of the present study demonstrated that tetrodotoxin failed to inhibit the effects of protamine on myocyte action potential characteristics. Specifically, myocyte maximum upstroke velocity, which is a direct reflection of fast Na⁺ channel opening, ¹² declined with protamine administration despite pretreatment with tetrodotoxin. Thus the effects of protamine on the myocyte action potential are probably not mediated by specific inhibition of ion channels, but rather reflect nonspecific effects on sarcolemmal conformation and ionic homeostasis. The second potential mechanism by which protamine may depress myocyte contractile function is through alterations in Ca²⁺ homeostasis. Myocyte active relaxation is a process by which Ca²⁺ is sequestered via Ca²⁺-adenosinetriphosphatase into the sarcoplasmic reticulum. ¹² Results from the present study provide evidence that protamine preferentially affected myocyte active relaxation processes. Further, Wakefield and coworkers ²³ reported that protamine caused decreased adenosine triphosphate production in endothelial cells. Thus the alterations in myocyte active relaxation with protamine may be due to reduced adenosine triphosphate and diminished Ca²⁺-adenosine triphosphatase activity. A final possible mechanism for the effect of protamine on myocyte processes is basic alterations in myofilament contractile function. In the present study, it was observed that a 40 µg/ml dose of protamine caused no change in the action potential maximum upstroke velocity. However, this same concentration of protamine caused a significant depression in myocyte contractile function. The influx of Na⁺ (of which maximum upstroke velocity is a rough index) is expected to be proportional to the flux of Ca²⁺ via the Na⁺/Ca²⁺ exchanger. ³³ Thus the depressed myocyte contractile function that occurred in the presence of protamine despite the absence of change in the maximum upstroke velocity suggests changes in myofilament sensitivity to Ca²⁺. In light of the fact that protamine had profound consequences on myocyte contractile function, future studies focused on defining basic mechanisms responsible for these effects are warranted.

Several limitations to the present study must be recognized. First, this study examined the effects of protamine on myocyte preparations that were independent of extracellular influences or nonmyocyte cell populations. The effects of protamine that may be modulated by these extracellular or nonmyocyte cell influences could not be addressed in the present study. A second limitation of the present study is that myocytes were directly exposed to protamine. However, in vivo, there are several determinants that can influence the amount and duration of protamine to which the myocytes are exposed. These determinants include myocardial blood flow, capillary permeability, and extracellular matrix buffering and diffusion. Furthermore, in vivo protamine administration has a wide range of systemic effects that could not be addressed through the experimental design used in the present study. Specifically, protamine administration is associated with the release of cytokines and other neurohormonal mediators ²² which, in turn, may influence myocyte contractile processes. A fourth limitation of the present study is that the effects of protamine on myocyte function were examined acutely and at only one time. Future studies that examine the effects of protamine on myocyte contractile function over longer periods may be warranted. Thus, although the limitations described herein must be recognized, the results from the present study clearly demonstrated that unbound protamine caused alterations in myocyte contractile processes, as well as abnormalities in myocyte electrophysiologic properties.

Each year in the United States over 0.5 million patients will receive heparin and require subsequent reversal with protamine. A number of clinical studies have reported adverse sequelae of protamine administration. ^1-3,6,11 These adverse effects of protamine include hemodynamic collapse, left ventricular dysfunction, pulmonary hypertension, and anaphylactic reactions. However, it has remained unclear whether the effects of protamine are mediated exclusively via systemic alterations or if protamine directly affects myocyte contractile function. The present study provides evidence that unbound protamine has profound consequences on myocyte contractile processes. In addition, the present study demonstrated that protamine interferes with myocyte responsiveness to ß-adrenergic receptor stimulation. These results suggest that protamine may blunt left ventricular responsiveness to stimulation with ß-adrenergic receptor agonists. Thus, if hemodynamic compromise does occur after protamine administration, depressed myocyte ß-adrenergic responsiveness may explain why ß-adrenergic receptor agonists are not totally successful in restoring the hemodynamic state. This suggests that if hemodynamic compromise does occur after protamine administration, consideration of an alternative method of inotropic support may be prudent. Further studies investigating the mechanisms by which protamine depresses myocyte contractile function and ß-adrenergic responsiveness are warranted.

Footnotes

From the Divisions of Cardiothoracic Surgery ^a and Pediatric Cardiology, ^b Medical University of South Carolina, Charleston, S.C.

J THORAC CARDIOVASC SURG 1994;108:1100-14

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