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Hyposmotic challenge modulates function of L-type calcium channel in rat ventricular myocytes through protein kinase C.

ABSTRACT: AIM: To study the effects and mechanisms by which hyposmotic challenge modulate function of L-type calcium current (I(Ca,L)) in rat ventricular myocytes. METHODS: The whole-cell patch-clamp techniques were used to record I(Ca,L) in rat ventricular myocytes. RESULTS: Hyposmotic challenge(∼220 mosmol/L) induced biphasic changes of I(Ca,L), a transient increase followed by a sustained decrease. I(Ca,L) increased by 19.1%±6.1% after short exposure (within 3 min) to hyposmotic solution. On the contrary, long hyposmotic challenge (10 min) decreased I(Ca,L) to 78.1%±11.0% of control, caused the inactivation of I(Ca,L), and shifted the steady-state inactivation curve of I(Ca,L) to the right. The decreased I(Ca,L) induced by hyposmotic swelling was reversed by isoproterenol or protein kinase A (PKA) activator foskolin. Hyposmotic swelling also reduced the stimulated I(Ca,L) by isoproterenol or foskolin. PKA inhibitor H-89 abolished swelling-induced transient increase of I(Ca,L), but did not affect the swelling-induced sustained decrease of I(Ca,L). NO donor SNAP and protein kinase G (PKG) inhibitor Rp-8-Br-PET-cGMPS did not interfere with swelling-induced biphasic changes of I(Ca,L). Protein kinase C (PKC) activator PMA decreased I(Ca,L) and hyposmotic solution with PMA reverted the decreased I(Ca,L) by PMA. PKC inhibitor BIM prevented the swelling-induced biphasic changes of I(Ca,L). CONCLUSION: Hyposmotic challenge induced biphasic changes of I(Ca,L), a transient increase followed by a sustained decrease, in rat ventricular myocytes through PKC pathway, but not PKG pathway. PKA system could be responsible for the transient increase of I(Ca,L) during short exposure to hyposmotic solution.

SUBMITTER: Luo AT

PROVIDER: S-EPMC4003327 | biostudies-other | 2010 Nov

REPOSITORIES: biostudies-other

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Project description:Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca2+ signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca2+ ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca2+ signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na+ and Ca2+ free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca2+ content, respectively. In isolated rabbit cardiomyocytes bathed in Na+ and Ca2+ free solution, we found an increased decay of the cytosolic Ca2+ concentration [Ca2+]i and increased SR Ca2+ content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca2+ leak through SR TRPC channels increased the systolic and diastolic [Ca2+]i with only minor effects on the action potential and SR Ca2+ content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca2+ leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will guide us to an understanding of TRPC1 channels as modulators of [Ca2+]i and contractility in cardiomyocytes.

Project description:BACKGROUND:Heart failure (HF) is characterized by electrophysiological remodeling resulting in increased risk of cardiac arrhythmias. Previous reports suggest that elevated inward ionic currents in HF promote action potential (AP) prolongation, increased short-term variability of AP repolarization, and delayed afterdepolarizations. However, the underlying changes in late Na+ current (INaL), L-type Ca2+ current, and NCX (Na+/Ca2+ exchanger) current are often measured in nonphysiological conditions (square-pulse voltage clamp, slow pacing rates, exogenous Ca2+ buffers). METHODS:We measured the major inward currents and their Ca2+- and ?-adrenergic dependence under physiological AP clamp in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched control). RESULTS:AP duration and short-term variability of AP repolarization were increased in HF, and importantly, inhibition of INaL decreased both parameters to the control level. INaL was slightly increased in HF versus control even when intracellular Ca2+ was strongly buffered. But under physiological AP clamp with normal Ca2+ cycling, INaL was markedly upregulated in HF versus control (dependent largely on CaMKII [Ca2+/calmodulin-dependent protein kinase II] activity). ?-Adrenergic stimulation (often elevated in HF) further enhanced INaL. L-type Ca2+ current was decreased in HF when Ca2+ was buffered, but CaMKII-mediated Ca2+-dependent facilitation upregulated physiological L-type Ca2+ current to the control level. Furthermore, L-type Ca2+ current response to ?-adrenergic stimulation was significantly attenuated in HF. Inward NCX current was upregulated at phase 3 of AP in HF when assessed by combining experimental data and computational modeling. CONCLUSIONS:Our results suggest that CaMKII-dependent upregulation of INaL in HF significantly contributes to AP prolongation and increased short-term variability of AP repolarization, which may lead to increased arrhythmia propensity, and is further exacerbated by adrenergic stress.

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Hyposmotic challenge modulates function of L-type calcium channel in rat ventricular myocytes through protein kinase C.

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