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 elevated phosphorylation of sarcoplasmic reticulum-associated proteins, PLB and RyR and 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. L-type Ca2+ channels are part of both Membrane clock and Ca2+ clock, since they generate action potential upstroke in primary cardiac pacemaker cells and provide Ca2+ supply for pumping into SR, respectively. There is a high basal PKA- and CaMKII-dependent phosphorylation of L-type Ca2+ channels in SANC, as a specific PKA inhibitor peptide, PKI, or CaMKII inhibitors, KN-93 or AIP, suppressed L-type Ca2+ current by 80% and 50%, respectively. We verified role of basal PKA- and CaMKII-dependent protein phosphorylation for spontaneous beating of intact rabbit SANC. 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 in response to CaMKII inhibition 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 Ca2+-cycling, Ca2+ clock, in cardiac pacemaker cells to enable normal cardiac automaticity. (4) In dormant guinea-pig SANCs membrane potential was depolarized, and LCRs were small and disorganized. The phosphorylation state of SANC was estimated using ratio of phosphorylated PLB at PKA-dependent Ser16 site to total PLB (P-PLB/PLB) which has been quantified in the same cells following Ca2+ measurements. Compared to beating SANC, dormant SANC had lower P-PLB/PLB ratio. -AR stimulation or application of permeable cAMP (CPT-cAMP) led to generation of spontaneous AP in 44% and 46% of dormant SANC, respectively. Following -adrenoceptor stimulation dormant SANC start to generate APs and this was accompanied by an increased P-PLB/PLB ratio, confirming critical role of PKA-dependent protein phosphorylation for spontaneous beating of cardiac pacemaker cells. 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.