Effects of PP1/PP2A inhibitor calyculin A on the E-C coupling cascade in murine ventricular myocytes

1 Department of Biochemistry and Molecular Biology, and 2 Department of Physiology, University of Maryland School of Medicine, and 3 Medical Biotechnology Center, University of Maryland Biotechnology Institute, University of Maryland School of Medicine, Baltimore, Maryland 21201 Calyculin A was used to examine the importance of phosphatases in the modulation of cardiac contractile magnitude in the absence of any neural or humoral stimulation. Protein phosphatase (PP)1 and PP2A activity, twitch contractions, intracellular Ca 2+ concentration ([Ca 2+ ] i ) transients, action potentials, membrane currents, and myofilament Ca 2+ sensitivity were measured in isolated mouse ventricular myocytes. Calyculin A (125 nM) inhibited PP1 and PP2A by 50% and 85%, respectively, whereas it doubled the twitch magnitude and increased twitch duration by 50% in field-stimulated cells. Calyculin A-evoked increases in L-type Ca 2+ current (70%) and the resulting [Ca 2+ ] i transient (83%) explain the positive inotropic response. However, increases in twitch and action potential durations did not result from increased myofilament Ca 2+ sensitivity or K + current inhibition, respectively. Comparison of the effects of calyculin A and isoproterenol on [Ca 2+ ] i transients and twitch contractions revealed that calyculin A had a much smaller lusitropic effect than the -agonist, indicating that calyculin A did not significantly increase sarcoplasmic reticulum Ca 2+ reuptake. Thus while cardiac contractile magnitude is controlled by a steady-state kinase/phosphatase balance, this regulation is not equally operative at all of the steps in the excitation-contraction coupling pathway and may in fact be most important to the regulation of the L-type Ca 2+ channel. calcium current; calcium transient; phosphorylation; indo 1; fluo 3 * W. H. duBell and M. S. Gigena contributed equally to the data presented in this work.