Both PKA and CaMKII phosphorylation drive pacemaker cell automaticity

(1)首先，我们研究了在不干扰离子通道的情况下，皂素渗透兔SANC和VM内禀SR Ca2+循环的调节。在相似的生理细胞内Ca2+浓度下，通透性SANC的lcr大且有节奏，而通透性VM的lcr小且随机。尽管两种细胞类型的肌浆网Ca2+含量相当，但与VM相比，SANC自发地从肌浆网释放更多的Ca2+。SANC产生更强健和有节奏的lcr的能力与肌浆网Ca2+- atp酶（SERCA）丰度的增加、SERCA抑制剂磷蛋白（PLB）丰度的降低以及Ca2+调节的PKA-和camkii依赖性PLB和RyR磷酸化的增加有关。在SANC中，RyR磷酸化的增加可能促进Ca2+从肌浆网释放，而PLB磷酸化的Ca2+依赖性增加减轻了其对SERCA的抑制，增加了支持稳健、有节奏的lcr所需的Ca2+泵送速率。当PKA或CaMKII抑制剂肽PKI或AIP分别减少PKA或CaMKII依赖性磷酸化时，LCR数量和大小显着减少，并且强劲的节律性LCR成为随机Ca2+释放，类似于VM中的Ca2+火花。SANC和VM之间Ca2+循环的差异为Ca2+时钟样细胞内Ca2+循环的调节提供了见解，Ca2+循环驱动心脏起搏器细胞的正常自动性。

(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. Basal level of PLB phosphorylation at both PKA-dependent Ser16 site and CaMKII-dependent Thr17 site was substantially higher in SANC than in VM. Basal phosphorylation of RyR at Ser2809 site, which is both PKA and CaMKII-dependent, and CaMKII-dependent Ser2815 site was also markedly higher in cardiac pacemaker than that in ventricular myocytes. 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. Compared to VM, SANC have adjusted Ca2+ cycling protein abundance (increased quantity of SERCA and reduced quantity of PLB) that supports elevated Ca2+ cycling in cardiac pacemaker cells. Moreover, Ca2+-regulated PKA- and CaMKII-dependent phosphorylation in cardiac pacemaker cells provides additional revival of Ca2+ cycling leading to stimulation of basal 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.