Abstract
The transient outward K+ current (Ito) modulates transmembrane Ca2+ influx into cardiomyocytes, which, in turn, might act on Ito. Here, we investigated whether Ca2+ modifies functional expression of Ito. Whole-cell Ito were recorded using the patch clamp technique in single right ventricular myocytes isolated from adult rats and incubated for 24 h at 37 degrees C in a serum-free medium containing various Ca2+ concentrations ([Ca2+]o). Increasing the [Ca2+]o from 0.5 to 1.0 and 2.5 mM produced a gradual decrease in Ito density without change in current kinetics. Quantitativereverse transcriptase-PCR showed that a decrease of the Kv4.2 mRNA could account for this decrease. In the acetoxymethyl ester form of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM)-loaded myocytes (a permeant Ca2+ chelator), Ito density increased significantly when cells were exposed for 24 h to either 1 or 2.5 mM [Ca2+]o. Moreover, 24-h exposure to the Ca2+ channel agonist, Bay K8644, in 1 mM [Ca2+]o induced a decrease in Ito density, whereas the Ca2+ channel antagonist, nifedipine, blunted Ito decrease in 2.5 mM [Ca2+]o. The decrease of Ito in 2.5 mM [Ca2+]o was also prevented by co-incubation with either the calmodulin inhibitor W7 or the calcineurin inhibitors FK506 or cyclosporin A. Furthermore, in myocytes incubated for 24 h with 2.5 mM [Ca2+]o, calcineurin activity was significantly increased compared with 1 mM [Ca2+]o. Our data suggest that modulation of [Ca2+]i via L-type Ca2+ channels, which appears to involve the Ca2+/calmodulin-regulated protein phosphatase calcineurin, down-regulates the functional expression of Ito. This effect might be involved in many physiological and pathological modulations of Ito channel expression in cardiac cells, as well other cell types.
Highlights
Disease, including acute or chronic diabetes mellitus, hypertrophy induced by pressure or volume overload, and cardiac failure [2]
In myocytes incubated for 24 h with 2.5 mM [Ca2؉]o, calcineurin activity was significantly increased compared with 1 mM [Ca2؉]o
Experimental manipulations of Ca2ϩ entry via in the L-type Ca2ϩ current (ICa), in adult rat ventricular myocytes incubated for 24 h, showed that increased cytosolic Ca2ϩ decreases Ito and down-regulates Kv4.2 mRNA expression, which involves Ca2ϩ/ calmodulin-regulated protein phosphatase calcineurin cascade
Summary
Cell Isolation and Incubation—Cardiac ventricular myocytes were isolated from adult male Wistar rats (250 –280 g) using an enzymatic. A, left panel, typical pattern of Ito generated by myocytes isolated from the right ventricle of the heart and incubated 24 h at 37 °C with 0.5 (up), 1 (medium), or 2.5 mM (down) [Ca2ϩ]o. Plots of averaged Ito densities (maximum current amplitude normalized to Cm) versus membrane potential obtained from cells incubated 24 h at 37 °C with 0.5 (gray shaded circles, n ϭ 19), 1 (solid black circles, n ϭ 16), or 2.5 mM [Ca2ϩ]o (white circles, n ϭ 22). B, quantitative real-time RT-PCR analysis of Kv4.2 after 24-h incubation with increased [Ca2ϩ]o. Data were normalized to the amount of mRNA coding for cyclophilin present in the same cell extracts and expressed as percentage of ratio observed in cells incubated with 1 mM [Ca2ϩ]o. A value of p Ͻ 0.05 was accepted as statistically significant
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
