Abstract
Muscarinic receptors expressed in cardiac myocytes play a critical role in the regulation of heart function by the parasympathetic nervous system. How the structural organization of cardiac myocytes affects the regulation of Ca2+ handling by muscarinic receptors is not well-defined. Using confocal Ca2+ imaging, patch-clamp techniques, and immunocytochemistry, the relationship between t-tubule density and cholinergic regulation of intracellular Ca2+ in normal murine ventricular myocytes and myocytes with acute disruption of the t-tubule system caused by formamide treatment was studied. The inhibitory effect of muscarinic receptor agonist carbachol (CCh, 10 μM) on the amplitude of Ca2+ transients, evoked by field-stimulation in the presence of 100 nM isoproterenol (Iso), a β-adrenergic agonist, was directly proportional to the level of myocyte detubulation. The timing of the maximal rate of fluorescence increase of fluo-4, a Ca2+-sensitive dye, was used to classify image pixels into the regions functionally coupled or uncoupled to the sarcolemmal Ca2+ influx (ICa). CCh decreased the fraction of coupled regions and suppressed Ca2+ propagation from sarcolemma inside the cell. Formamide treatment reduced ICa density and decreased sarcoplasmic reticulum (SR) Ca2+ content. CCh did not change SR Ca2+ content in Iso-stimulated control and formamide-treated myocytes. CCh inhibited peak ICa recorded in the presence of Iso by ∼20% in both the control and detubulated myocytes. Reducing ICa amplitude up to 40% by changing the voltage step levels from 0 to –25 mV decreased Ca2+ transients in formamide-treated but not in control myocytes in the presence of Iso. CCh inhibited CaMKII activity, whereas CaMKII inhibition with KN93 mimicked the effect of CCh on Ca2+ transients in formamide-treated myocytes. It was concluded that the downregulation of t-tubules coupled with the diminished efficiency of excitation–contraction coupling, increases the sensitivity of Ca2+ release and propagation to muscarinic receptor-mediated inhibition of both ICa and CaMKII activity.
Highlights
The autonomic nervous system exerts its effects on the mechanical and electrical activity of the heart in part via β-adrenergic and muscarinic receptors expressed in ventricular myocytes. β-Adrenergic receptors mediate stimulatory effects of the sympathetic nervous system via cAMP-dependent activation of Ca2+ handling proteins, such as the L-type Ca2+ channels, sarcoplasmic reticulum (SR) Ca2+ ATPase, and RyR2, SR Ca2+ release channels (Bers, 2001)
We studied the effects of the muscarinic receptor agonist, CCh, on Ca2+ transients evoked by 1 Hz electrical field stimulation in ventricular myocytes isolated from WT mouse heart
To test whether the regulation of Ca2+ handling by muscarinic receptors in ventricular myocytes depends on the spatial organization of t-tubules, the effects of CCh in myocytes with reduced t-tubule density resulting from formamide treatment were studied (Brette et al, 2002)
Summary
The autonomic nervous system exerts its effects on the mechanical and electrical activity of the heart in part via β-adrenergic and muscarinic receptors expressed in ventricular myocytes. β-Adrenergic receptors mediate stimulatory effects of the sympathetic nervous system via cAMP-dependent activation of Ca2+ handling proteins, such as the L-type Ca2+ channels, sarcoplasmic reticulum (SR) Ca2+ ATPase, and RyR2, SR Ca2+ release channels (Bers, 2001). Muscarinic receptors contribute to the inhibitory effects of the parasympathetic system mostly by antagonizing β-adrenergic responses (Löffelholz and Pappano, 1985; Harvey and Belevych, 2003). In cardiac diseases, such as heart failure, the abnormal autonomic regulation of cardiac function is usually accompanied by the structural remodeling of the heart that at the cellular level is often associated with downregulation of the t-tubule system (Guo et al, 2013; Poláková and Sobie, 2013). Little is known about the role of t-tubules in muscarinic receptor–mediated regulation of intracellular Ca2+ handling in ventricular myocytes
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