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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)

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