To establish links between physiological regulation of the LCR period and kinetics of SR Ca2+ refilling we employed, as indexes of SR pumping rate and LCR period, phosphorylation of phospholamban (PLB) and the time to 90% decay of the AP-initiated global cytosolic Ca2+ transient (T-90). Graded PLB phosphorylation by -AR stimulation (ISO), a broad-spectrum phosphodiesterase inhibitor (IBMX), specific phosphodiesterase-3 inhibitor (milrinone), or by specific PKA inhibitor peptide (PKI) were paralleled by proportional changes in T-90. Concurrent changes in T-90 and LCR period were highly correlated with concurrent changes in the spontaneous cycle length. The comparison of SR Ca loading in permeabilized SANC and VM demonstrated that it is similar or larger in VM than in SANC over a wide range of free Ca2+: 50 - 250 nmol/L. However, SANC, compared to VM, spontaneously released 2-3 fold more Ca2+, suggesting that at a given SR Ca load, SR Ca2+ cycling in skinned SANC is more robust than in skinned VM. Elevation of cytosolic Ca2+ from 0 to 150 nmol/L increased PLB phosphorylation 80% at PKA-dependent site and 37% at CaMKII-dependent site in SANC, but not in VM. While phosphorylation of the CaMKII-dependent site in SANC is Ca2+-dependent, the phosphorylation of PKA-dependent site is due to an increase in Ca2+-activated adenylyl cyclase activity which is also activated by an increase in Ca2+. It is known that RyR Ca release is dependent not only from SR Ca2+ content but also from RyR Ca2+ release, and increase in RyR phosphorylation at 2809 site makes RyR more leaky elevating RyR Ca2+ release. Increase in cytosolic Ca2+ increased RyR phosphorylation at 2809 site and this effect is reversed by either inhibition of PKA or CaMKII. Thus, a unique combination of PKA-and CaMKII-dependent phosphorylation of Ca2+ cycling proteins, responsible for release and reuptake in SANC, sustain robust basal SR Ca cycling in SANC to drive primary pacemaker cell automaticity.
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