Both PKA and CaMKII phosphorylation drive pacemaker cell automaticity

(1)首先，我们研究了在不受离子通道干扰的情况下，皂苷通透性兔SANC和Vm内源性SR钙循环的调节。在相似的生理细胞内钙离子浓度下，通透性SANC的LCR较大且有节律性，而通透性Vm的LCR较小且随机。SANC自发地从肌浆网释放比VM更多的钙，尽管两种细胞的肌浆网钙含量相似。SANC产生更强健和节律性LCRs的能力与肌浆网钙-ATPase(SERCA)的丰度增加、SERCA抑制剂磷蛋白(PLB)的减少以及钙调节的PKA和CaMKII依赖的PLB和RyR的磷酸化增加有关。SANC中RyR的磷酸化增加可能促进肌浆网的钙释放，而PLB的钙依赖的磷酸化增加解除了它对SERCA的抑制，增加了支持强健、有节奏的LCR所需的钙泵送速率。当依赖于PKA或CaMKII的磷酸化分别被PKA或CaMKII抑制肽PKI或AIP降低时，LCR的数量和大小显著减少，并且强健的节律性LCR变成类似于Vm中的钙火花的随机的钙释放。SANC和Vm之间在钙循环方面的差异为调控细胞内钙时钟样的钙循环提供了洞察力，从而驱动心脏起搏细胞的正常自律性。 (2)为了验证我们的第二个想法，我们提高了肌浆网相关蛋白PLB和RyR的磷酸化水平，并研究了通透性兔VM在生理细胞内钙浓度下、抑制蛋白磷酸酶(PP)和磷酸二酯酶(PDE)、加入外源性cAMP或在特异性抑制PLB与SERCA结合的抗体(2D12)存在下，通透性兔VM的自发钙释放特性。钙循环蛋白磷酸化水平的增加将随机的钙火花转化为与SANC中观察到的类似的强健的、周期性的钙释放。因此，钙时钟不是起搏细胞所特有的，但当肌浆网钙循环增加，自发的LCR部分同步时，也可以在Vm释放钙时钟。 (3)完整的SANC具有较高的依赖于PKA和CaMKII的基础蛋白磷酸化水平，即激活(自磷酸化)CaMKII的基础水平约为Vm的2倍，并伴随着较高的基础蛋白磷酸化水平。依赖于PKA的Ser16位点和依赖于CaMKII的Thr17位点的基础水平在SANC中显著高于在VM中。心脏起搏器中同时依赖PKA和CaMKII的Ser2809位和CaMKII依赖的Ser2815位的RyR基础磷酸化水平也显著高于心室肌细胞。L型钙通道既是膜时钟的一部分，又是钙时钟的一部分，因为它们分别在起搏细胞上产生动作电位上冲和为泵入SR提供钙供应。SANC中存在高度的基础PKA和CaMKII依赖性的L型钙通道的磷酸化，作为一种特异性的PKA抑制肽，PKI或CaMKII抑制剂KN-93或AIP分别抑制L型钙电流80%和50%。我们验证了依赖于PKA和CaMKII的基础蛋白磷酸化在完整兔心脏自发搏动中的作用。AIP或KN-93抑制CaMKII活性时，LCR数目和大小明显减少，LCR周期明显延长。CaMKII抑制后LCR周期的延长与自发SANC周期长度的增加高度相关。 与Vm相比，SANC调整了支持心脏起搏细胞内高钙循环的钙循环蛋白丰度(SERCA增加和PLB减少)。此外，钙离子调节的心脏起搏细胞中依赖的PKA和CaMKII依赖的磷酸化提供了额外的钙循环的复苏，从而刺激心脏起搏细胞的基础自发搏动。来自这些研究的见解可能有助于设计基于基因或细胞的生物起搏器，可用于病态窦房结综合征患者的电子设备，病态窦房结综合征主要是老年人的一种疾病，随着年龄的增长呈指数增长。

(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.