Ca ²⁺ -Dependent Phosphorylation of Ca ²⁺ Cycling Proteins Generates Robust Rhythmic Local Ca ²⁺ Releases in Cardiac Pacemaker Cells

Abstract

The spontaneous beating of the heart is governed by spontaneous firing of sinoatrial node cells, which, unlike primary contractile ventricular myocardial cells, generate action potentials due to spontaneous depolarization of the membrane potential, or diastolic depolarization. The spontaneous diastolic depolarization rate is determined by spontaneous local submembrane Ca2+ releases through ryanodine receptors (RyRs). We sought to identify specific mechanisms of intrinsic Ca2+ cycling by which sinoatrial node cells, but not ventricular myocytes, generate robust, rhythmic local Ca2+ releases. At similar physiological intracellular Ca2+ concentrations local Ca2+ releases were large and rhythmic in permeabilized sinoatrial node cells, but were small and random in permeabilized ventricular myocytes. Furthermore, sinoatrial node cells spontaneously released more Ca2+ from the sarcoplasmic reticulum than did ventricular myocytes, despite comparable sarcoplasmic reticulum Ca2+ content in both cell types. This ability of sinoatrial node cells 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, and increased Ca2+-dependent phosphorylation of phospholamban and RyR. The increased phosphorylation of RyR in sinoatrial node cells may facilitate Ca2+ release from the sarcoplasmic reticulum, whereas Ca2+-dependent increase in phosphorylation of phospholamban 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 sinoatrial node cells and ventricular myocytes provide insights into the regulation of “clock-like” intracellular Ca2+-cycling that drives normal automaticity of sinoatrial node cells.

Age-associated abnormalities of intrinsic automaticity of sinoatrial nodal cells are linked to deficient cAMP-PKA-Ca2+signaling

“…In addition, important age-associated changes in the mRNA profiles of different ion channel and exchanger genes have recently been described in the atrium and SAN (49). The ratio of PLB to SERCA in the present study increases in advanced age, suggesting the inhibition of SERCA by PLB could be higher in advanced age (44). In agreement with our study, mRNA levels of RyR2 were found to be significantly reduced in SAN tissue of rat aged 24 vs. 3 mo (62).…”

“…LCR characteristics are an important mechanism in determining the spontaneous AP firing rate. Prior studies showed that LCR characteristics are dependent on the SR Ca 2ϩ load (44). That the SR Ca 2ϩ load is reduced in single permeabilized SAN cells from aged heart (Fig.…”

“…Specifically, the basal pacemaker cell AP firing rate is regulated by intracellular Ca 2ϩ cycling and Ca 2ϩ -calmodulin activation of adenylyl cyclases (AC) and calmodulin kinase II (CaMKII), leading to an increase in cAMP and to an increase in cAMP/PKA-dependent and CaMKIIdependent phosphorylation of sarcoplasmic reticulum (SR) Ca 2ϩ cycling and surface membrane ion channel proteins (52,55). The kinetics of this intrinsic basal signaling are controlled by basal phosphodiesterase (PDE) and phosphatase activities (33,44,51). The ticking speed of this intrinsic clock system becomes further activated, or dampened, respectively, by adrenergic or cholinergic neurotransmitter stimulation.…”

Potassium channels in the sinoatrial node and their role in heart rate control

“…In SAN myocytes however these local calcium releases are larger involving a number of release sites [19–21]. In permeabilised cells the effects of membrane ion channels are removed and in these conditions these events are periodic in contrast to the stochastic nature of sparks in ventricular myocytes [22]. The SA node does not have t-tubules and calcium release is sub-sarcolemmal.…”

Unique Ca2+-Cycling Protein Abundance and Regulation Sustains Local Ca2+ Releases and Spontaneous Firing of Rabbit Sinoatrial Node Cells

“…Cell surface membrane permeabilization is a valuable tool for studying intracellular Ca 2+ cycling in the absence of ion channel interference in SANC [23,35] or ventricular myocytes [63,64,65,66]. This approach was employed to study mechanisms of CICR in cardiac cells [67].…”