Normal spontaneous firing of cardiac pacemaker cells is regulated by basal pkc delta activation

Abstract

Abstract The spontaneous beating rate of rabbit sinoatrial node cells (SANC) is regulated by local subsarcolemmal calcium releases (LCRs) from sarcoplasmic reticulum (SR). LCRs appear during diastolic depolarization (DD) and activate an inward sodium/calcium exchange current which increases DD rate and thus accelerates spontaneous SANC firing. High basal level of protein kinase A and calcium/calmodulin-dependent protein kinase II phosphorylation are required to sustain basal LCRs and normal spontaneous SANC firing. Recently we discovered that basal PKC activation is also obligatory for cardiac pacemaker function: inhibition of PKC activity by broad spectrum PKC inhibitors Bis I or calphostin C markedly suppressed SR calcium cycling and decreased or abolished spontaneous beating of freshly isolated rabbit SANC. Here we studied which PKC isoforms mediate PKC-dependent effects on cardiac pacemaker cell automaticity. The PKC superfamily consists of 3 major subgroups: conventional, novel and atypical. All PKC isoforms were detected at the RNA level (RT-qPCR) in the rabbit SA node and ventricle, and expression levels were comparable in both tissues. Expression of PKCβ, however, was markedly higher in the rabbit SA node, compared to other PKC isoenzymes in either tissue. We verified expression of conventional PKC (α, β) and novel PKC-delta at the protein level in SANC and ventricular myocytes (VM). Western blot confirmed RNA results, showing a 6-fold higher PKCβ protein abundance in SANC compared to VM. Expression of PKCα protein was similar in both cell types, while PKC-delta protein was more abundant in VM. To study whether PKCβ regulates spontaneous beating of SANC we employed selective inhibitor of conventional (α, β, gamma) PKC isoforms Go6976 (10 μmol/L), which had no effects on either LCR characteristics (confocal microscopy, calcium indicator Fluo-3AM) or spontaneous beating of freshly isolated rabbit SANC (perforated patch-clamp technique). Because selective PKC-delta inhibitors are not available, we explored effects of PKC-delta inhibition comparing effects of Go6976 (the inhibitor of conventional PKCs) and Go6983, which inhibits conventional PKCs and PKC-delta. In contrast to Go6976, Go6983 (5 μmol/L) markedly decreased the LCR size (from 7.1±0.4 to 4.5±0.3 μm) and number per each spontaneous cycle (from 1.3±0.1 to 0.8±0.1). It also markedly increased the LCR period (time from the prior AP-induced calcium transient to the subsequent LCR) which was paralleled by an increase in the spontaneous SANC cycle length. Rottlerin, another PKC-delta inhibitor, produced similar effects on LCR characteristics, and markedly and time-dependently decreased DD rate, leading to an increase in the spontaneous cycle length, and finally abrogated the spontaneous SANC firing. Thus, our data indicate that basal activity of PKC-delta, but not that of PKCβ, is essential for generation of LCRs and normal spontaneous firing of cardiac pacemaker cells. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Intramural Research Program, National Institute on Aging, National Institute of Health, USA