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(Circulation. 1995;92:433-446.)
© 1995 American Heart Association, Inc.

Articles

Direct Effects of Protamine Sulfate on Myocyte Contractile Processes

Cellular and Molecular Mechanisms

R. Barry Hird, MD; Thomas W. Wakefield, MD; Rupak Mukherjee, MS; Blanding U. Jones, BS; Fred A. Crawford, MD; Philip C. Andrews, PHD; James C. Stanley, MD; Francis G. Spinale, MD, PHD

From the Division of Cardiothoracic Surgery, Medical University of South Carolina (R.B.H., R.M., B.U.J., F.A.C., F.G.S.), Charleston, SC, and the Section of Vascular Surgery, Jobst Vascular Research Laboratory (T.W.W., J.C.S.), and the Department of Biochemistry (P.C.A.), University of Michigan, Ann Arbor.

Correspondence to Francis G. Spinale, MD, PhD, Division of Cardiothoracic Surgery, Medical University of South Carolina, 171 Ashley Ave, Charleston, SC 29425.

Background Administration of the arginine-rich, highly charged protamine (PROT) molecule has been associated with episodes of acute left ventricular (LV) dysfunction. The objective of the present study was to test the hypothesis that PROT has direct effects on isolated LV myocyte contractile processes and sarcolemmal transduction systems.

Methods and Results Exposure of porcine LV myocytes (n=305) to 40 µg/mL PROT (reflecting a dose of 2.5 mg/kg) decreased basal contractile function and ß-adrenergic responsiveness. For example, myocyte percent shortening was 4.3±0.1% in control myocytes and decreased to 2.8±0.2% in the presence of 40 µg/mL PROT (P<.05). Myocyte percent shortening was 9.3±0.7% after ß-adrenergic receptor stimulation (isoproterenol; 25 nmol/L) and was significantly reduced in the presence of 40 µg/mL PROT (5.7±0.7%, P<.05). PROT reduced myocyte responsiveness to forskolin (100 µmol/L), which directly activates adenylate cyclase, by >40% from forskolin. In addition, PROT abolished the inotropic effects of ouabain on myocyte contractile function. To determine contributory mechanisms for the effects of PROT on myocyte sarcolemmal systems, ß-receptor– and cardiac glycoside–binding characteristics were determined in sarcolemmal preparations. ß-receptor binding was 175±10 fmol/mg and was reduced to 140±6 fmol/mg in the presence of PROT (P<.05). Ouabain receptor binding was 7.1 pmol/mg and decreased to 2.6±0.4 pmol/mg in the presence of PROT. In addition, cAMP production after stimulation with isoproterenol and forskolin was significantly blunted in the presence of PROT. Variants of the PROT molecule were constructed by specific amino acid substitutions and deletions, which provided a means to vary charge as well as structure. Substitution of arginine with lysine in the PROT peptide sequence ameliorated the negative effects on myocyte contractile processes; despite identical overall charge (21+). However, a PROT variant with an 18+ charge but different amino acid sequence induced significant negative effects on myocyte function and inotropic responsiveness. Thus, the effects of PROT on myocyte contractile processes are not due simply to the high positive charge of the molecule. To further establish that PROT can contribute to changes in LV function in the clinical setting, fluorescein-labeled PROT was circulated in antegradely perfused rabbit hearts. Microscopic examination revealed that PROT could traverse the vascular compartment of the myocardium and come in direct contact with the myocyte.

Conclusions The unique findings from the present study suggest that a fundamental contributory mechanism for the changes in LV function observed after protamine administration may be the direct effect of unbound protamine on myocyte contractile processes.

Key Words: ventricles • proteins • surgery

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