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

• A Ca2 + clock emerges in permeabilized ventricular myocytes at normal [Ca2 +]i.
• The Ca2 + clock has phosphorylation-dependent mechanism.
• The Ca2 + clock is manifested by rhythmic local Ca2 + releases as in pacemaker cells.
• The Ca2 + clock can be activated by PP and PDE inhibition.
• The Ca2 + clock can be arrhythmogenic and also a candidate for biopacemaker design.

Basal phosphorylation of sarcoplasmic reticulum (SR) Ca2 + proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca2 + releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca2 + sparks. We tested the hypothesis that in a physiologic Ca2 + milieu, and independent of increased Ca2 + influx, an increase in basal phosphorylation of SR Ca2 + cycling proteins will convert stochastic Ca2 + sparks into periodic, high-power Ca2 + signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca2 + release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca2 +], prior to and during 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-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca2 + releases of low power and low amplitude, as assessed by confocal Ca2 + imaging and spectral analysis. When the kinetics of Ca2 + pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, “clock-like” local Ca2 + releases, partially synchronized in space and time (Ca2 + wavelets), emerged, and the ensemble of these rhythmic local Ca2 + wavelets generated a periodic high-amplitude Ca2 + signal. 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. This unleashed Ca2 + clock that emerges in a physiological Ca2 + milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature of novel bio-pacemaker designs.