To study intrinsic SR Ca2+ cycling in SANC and VM without interference of ionic channels and to test our hypothesis we employed saponin-permeabilized rabbit SANC and VM. (1) At similar physiological intracellular Ca2+ concentrations local Ca2+ releases 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 local Ca2+ releases 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 local Ca2+ releases. 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 local Ca2+ release becomes partially synchronized. (3) Finally, we verified role of basal PKA- and CaMKII-dependent protein phosphorylation for spontaneous beating of intact rabbit SANC. Cardiac pacemaker cells had a high basal level of both PKA- and CaMKII-dependent protein phosphorylation, which was replicated in basal phosphorylation of Ca2+ cycling proteins PLB and RyR. Specifically, basal level of PLB phosphorylation at PKA-dependent Ser16 site was substantially higher in SANC than in VM; phosphorylation of PLB at CaMKII-dependent Thr17 site was also markedly elevated in SANC compared to VM, and ratio of P-PLB/total PLB exceeded that in VM by 3-fold. Basal phosphorylation of RyR at CaMKII-dependent Ser2815 site in the rabbit SA node was markedly higher than that in ventricular tissue, and ratio of P-Ser2815/total RyR in the SA node surpassed that in the ventricle by 10-fold. 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 syndrom which is primarily a disease of the seniors and increases in an exponential manner with aging.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000260-08
Application #
9147257
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Aging
Department
Type
DUNS #
City
State
Country
Zip Code
Vinogradova, Tatiana M; Tagirova Sirenko, Syevda; Lakatta, Edward G (2018) Unique Ca2+-Cycling Protein Abundance and Regulation Sustains Local Ca2+ Releases and Spontaneous Firing of Rabbit Sinoatrial Node Cells. Int J Mol Sci 19:
Sirenko, Syevda G; Maltsev, Victor A; Yaniv, Yael et al. (2016) Electrochemical Na+ and Ca2+ gradients drive coupled-clock regulation of automaticity of isolated rabbit sinoatrial nodal pacemaker cells. Am J Physiol Heart Circ Physiol 311:H251-67
Li, Yue; Sirenko, Syevda; Riordon, Daniel R et al. (2016) CaMKII-dependent phosphorylation regulates basal cardiac pacemaker function via modulation of local Ca2+ releases. Am J Physiol Heart Circ Physiol 311:H532-44
Sirenko, Syevda; Maltsev, Victor A; Maltseva, Larissa A et al. (2014) Sarcoplasmic reticulum Ca2+ cycling protein phosphorylation in a physiologic Ca2+ milieu unleashes a high-power, rhythmic Ca2+ clock in ventricular myocytes: relevance to arrhythmias and bio-pacemaker design. J Mol Cell Cardiol 66:106-15
Sirenko, Syevda; Yang, Dongmei; Li, Yue et al. (2013) Ca²?-dependent phosphorylation of Ca²? cycling proteins generates robust rhythmic local Ca²? releases in cardiac pacemaker cells. Sci Signal 6:ra6
Lakatta, Edward G; Maltsev, Victor A (2012) Rebuttal: what I(f) the shoe doesn't fit? ""The funny current has a major pacemaking role in the sinus node"". Heart Rhythm 9:459-60
Zahanich, Ihor; Sirenko, Syevda G; Maltseva, Larissa A et al. (2011) Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling. J Mol Cell Cardiol 50:66-76
Vinogradova, Tatiana M; Brochet, Didier X P; Sirenko, Syevda et al. (2010) Sarcoplasmic reticulum Ca2+ pumping kinetics regulates timing of local Ca2+ releases and spontaneous beating rate of rabbit sinoatrial node pacemaker cells. Circ Res 107:767-75
Vinogradova, Tatiana M; Lakatta, Edward G (2009) Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP-mediated PKA-dependent Ca2+ cycling with surface membrane channels. J Mol Cell Cardiol 47:456-74
Lyashkov, Alexey E; Vinogradova, Tatiana M; Zahanich, Ihor et al. (2009) Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca(2+) cycling and I(KACh). Am J Physiol Heart Circ Physiol 297:H949-59

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