Both PKA and CaMKII phosphorylation drive pacemaker cell automaticity

National Institute On Aging

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Abstract

(1)First, we studied regulation of intrinsic SR Ca2+ cycling in saponin-permeabilized rabbit SANC and VM without interference of ionic channels. At similar physiological intracellular Ca2+ concentrations LCRs were large and rhythmic in permeabilized SANC but were small and random in permeabilized VM. SANC spontaneously released more Ca2+ from the sarcoplasmic reticulum than did VM, despite comparable sarcoplasmic reticulum Ca2+ content in both cell types. This ability of SANC to generate more robust and rhythmic LCRs was associated with increased abundance of sarcoplasmic reticulum Ca2+-ATPase (SERCA), reduced abundance of the SERCA inhibitor phospholamban (PLB), and increased Ca2+-regulated PKA- and CaMKII-dependent phosphorylation of PLB and RyR. The increased phosphorylation of RyR in SANC may facilitate Ca2+ release from the sarcoplasmic reticulum, whereas Ca2+-dependent increase in phosphorylation of PLB relieves its inhibition of SERCA, augmenting the pumping rate of Ca2+ required to support robust, rhythmic LCRs. When PKA- or CaMKII-dependent phosphorylation was reduced with PKA or CaMKII inhibitor peptide PKI or AIP, respectively, there was marked decrease in LCR number, size, and robust rhythmic LCRs became stochastic Ca2+ releases that resembled Ca2+ sparks in VM. The differences in Ca2+ cycling between SANC and VM provide insights into the regulation of Ca2+ clock-like intracellular Ca2+-cycling that drives normal automaticity of cardiac pacemaker cells. (2) To test our second idea, we studied spontaneous Ca2+ release characteristics in permeabilized rabbit VM at physiological intracellular Ca2+ concentrations, prior to and following inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA. An increase in phosphorylation level of Ca2+-cycling proteins converted stochastic Ca2+ sparks into robust, periodic Ca2+ releases similar to ones observed in SANC. Thus, a Ca2+ clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca2+ cycling increases and spontaneous LCRs becomes partially synchronized. (3) Intact SANC had a high basal level of both PKA- and CaMKII-dependent protein phosphorylation, i.e. the basal level of activated (autophosphorylated) CaMKII in rabbit SANC surpassed that in VM by approximately 2-fold, and this was accompanied by high basal level of protein phosphorylation. Furthermore, phosphorylation of RyR at CaMKII-dependent Ser2815 site and PKA- and CaMKII-dependent Ser2809 site was 10-fold and 2-fold higher, respectively, in SANC than in VM. An increase in RyR phosphorylation facilitates RyR activation leading to generation of robust LCR in SANC. To support elevated RyR Ca2+ release the rate of Ca2+ pumping into SR in SANC should be higher compared to VM. Consistent with this idea SANC had 40% increase in expression of SR Ca2+-ATPase (SERCA) and 50% reduction in expression of PLB proteins compared to VM. Besides, phosphorylation of PLB at both PKA- (Ser16) and CaMKII-dependent (Thr17) sites was 10-fold and 3-fold higher, respectively, in SANC than in VM. The increased amount of SERCA protein, reduced amount of PLB protein and augmented PLB phosphorylation relieve SERCA inhibition to adjust SR Ca2+ pumping and support elevated RyR Ca2+ release in SANC. Suppression of PKA- or CaMKII-dependent phosphorylation with PKI or KN-93, respectively, markedly decreased PLB and RyR phosphorylation; reduced LCR periodicity, size and number per each spontaneous cycle; and prolonged the LCR period (time from AP-induced Ca2+ transient to the subsequent LCR), which predicted the concomitant increase in the cycle length. When CaMKII activity was inhibited with AIP or KN-93 there was marked decrease in the LCR number and size, while the LCR period was markedly prolonged. The prolongation of the LCR period was highly correlated with the concurrent increase in the spontaneous SANC cycle length. Thus, specific modifications in SR Ca2+ cycling protein expression and phosphorylation levels represent unique mechanisms that drive intracellular SR Ca2+-cycling (Ca2+ clock) in cardiac pacemaker cells to enable normal cardiac automaticity. (4) The signaling cascade that activates basal protein kinase activity in SANC is feedforward: Ca2+ release stimulates CaMKII and PKA (via Ca2+-calmodulin activated adenylyl cyclases (ACs)) and these kinases, by phosphorylation of membrane clock and SR Ca2+ cycling proteins, increase SR Ca2+ release, which further activates Ca2+-calmodulin activated ACs and CaMKII. Potent restraining mechanisms of this feed-forward signaling axis are required to maintain the basal spontaneous SANC firing rate, and PP activity could provide this potential brake on the feed-forward signaling axis. We discovered that rabbit SANC express both PP1, PP2A. Concurrent inhibition of PP1 and PP2A by Calyculin A in freshly isolated SANC: (a) significantly increased phospholamban (PLB) phosphorylation (by 23-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites; (b) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (c) substantially increased SR Ca2+ load; (d) augmented L-type Ca2+ current amplitude, leading to augmentation of LCRs characteristics and decrease of the LCR period in intact and permeabilized SANC, and thus increase in the spontaneous basal SANC firing rate. The selective PP2A inhibitor okadaic acid (100 nmol/L), however, had no significant effect on spontaneous SANC firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Thus, PP1, but not PP2A, modulates the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, L-type Ca2+ current, the SR Ca2+ load and LCRs. Insights from these studies may help in the design of gene- or cell-based biological pacemakers that could be used instead of electronic devices in individuals with sick sinus syndrome which is primarily a disease of the seniors and increases in an exponential manner with aging.