The Role of T-Tubule Organization in Regulation of Intracellular Ca2+ Handling by Muscarinic Receptor Stimulation in Ventricular Myocytes

Abstract

Muscarinic receptors expressed in cardiac myocytes play critical role in mediating regulation of heart function by the parasympathetic nervous system. How structural organization of cardiac myocytes affects muscarinic receptor mediated regulation of Ca2+ handling is not well defined. Using confocal Ca2+ imaging and patch-clamp techniques we studied the relationships between t-tubule density and cholinergic regulation of intracellular Ca2+ handling in normal murine ventricular myocytes and in myocytes in which the t-tubule system was disrupted by formamide treatment. Muscarinic receptor agonist CCh (10 μM) significantly inhibited amplitude of Ca2+ transients evoked by electrical field-stimulation in the presence of 100 nM isoproterenol (Iso), a beta-adrenergic agonist, in formamide-treated but not in control ventricular myocytes. Following Iso stimulation, formamide-treated cells exhibited reduced density of the inward Ca2+ current (ICa), decreased sarcoplasmic reticulum (SR) Ca2+ content, and enhanced fractional SR Ca2+ release when compared to control myocytes. CCh did not change SR Ca2+ content in Iso-stimulated formamide-treated myocytes but significantly reduced fractional release. This effect was associated with reduced Ca2+ release at coupled SR Ca2+ release sites and decreased Ca2+ sensitivity of the uncoupled release sites. CCh inhibited peak ICa recorded in a presence of Iso by ∼20 % in both control and detubulated ventricular myocytes. Reducing ICa amplitude by up to 40% by changing voltage step levels from 0 to −25 mV did not affect Ca2+ transients in control myocytes in the presence of Iso. In contrast, in formamide-treated myocytes similar decrease in ICa significantly diminished Ca2+ transients. We conclude that downregulation of the t-tubule system, as often observed in failing hearts, augments the inhibitory effects of muscarinic receptor agonists on Ca2+ transients by reducing functional reserve of excitation-contraction coupling.