Direct effects of protamine sulfate on myocyte contractile processes. Cellular and molecular mechanisms

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.

Exposure of porcine LV myocytes (n = 305) to 40 micrograms/mL PROT (reflecting a dose of 2.5 mg/kg) decreased basal contractile function and beta-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 micrograms/mL PROT (P < .05). Myocyte percent shortening was 9.3 +/- 0.7% after beta-adrenergic receptor stimulation (isoproterenol; 25 nmol/L) and was significantly reduced in the presence of 40 micrograms/mL PROT (5.7 +/- 0.7%, P < .05). PROT reduced myocyte responsiveness to forskolin (100 mumol/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, beta-receptor- and cardiac glycoside-binding characteristics were determined in sarcolemmal preparations. beta-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 moelcule 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.

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