DIRECT EFFECTS OF PROTAMINE SULFATE ON MYOCYTE CONTRACTILE PROCESSES - CELLULAR AND MOLECULAR MECHANISMS

Background Administration of the arginine-rich, highly charged protami ne (PROT) molecule has been associated with episodes of acute left ven tricular (LV) dysfunction. The objective of the present study was to t est the hypothesis that PROT has direct effects on isolated LV myocyte contractile processes and sarcolemmal transduction systems. Methods a nd Results Exposure of porcine LV myocytes (n=305) to 40 mu g/mL PROT (reflecting a dose of 2.5 mg/kg) decreased basal contractile function and beta-adrenergic responsiveness. For example, myocyte percent short ening was 4.3+/-0.1% in control myocytes and decreased to 2.8+/-0.2% i n the presence of 40 mu g/mL PROT (P<.05). Myocyte percent shortening was 9.3+/-0.7% after beta-adrenergic receptor stimulation (isoproteren ol; 25 nmol/L) and was significantly reduced in the presence of 40 mu g/mL PROT (5.7+/-0.7%, P<.05). PROT reduced myocyte responsiveness to forskolin (100 mu mol/L), which directly activates adenylate cyclase, by >40% from forskolin. In addition, PROT abolished the inotropic effe cts of ouabain on myocyte contractile function. To determine contribut ory mechanisms for the effects of PROT on myocyte sarcolemmal systems, beta-receptor- and cardiac glycoside-binding characteristics were det ermined in sarcolemmal preparations. P-receptor binding was 175+/-10 f mol/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 blunte d in the presence of PROT. Variants of the PROT molecule were construc ted 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 eff ects on myocyte contractile processes; despite identical overall charg e (21+). However, a PROT variant with an 18+ charge but different amin o acid sequence induced significant negative effects on myocyte functi on and inotropic responsiveness. Thus, the effects of PROT on myocyte contractile processes are not due simply to the high positive charge o f the molecule. To further establish that PROT can contribute to chang es in LV function in the clinical setting, fluorescein-labeled PROT wa s circulated in antegradely perfused rabbit hearts. Microscopic examin ation revealed that PROT could traverse the vascular compartment of th e 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 p rotamine administration may be the direct effect of unbound protamine on myocyte contractile processes.