Both PKA and CaMKII phosphorylation drive pacemaker cell automaticity

National Institute On Aging

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Abstract

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 beta-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 Ca2+ 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 Ca2+ load, SR Ca2+ cycling in skinned SANC is more robust than in skinned VM. The unique ability of cardiac pacemaker cells to sustain elevated RyR Ca2+ releases is explained, in part at least, by increased expression of SERCA and decreased expression of PLB proteins compared to VM. Because of this, SANC have more efficient SERCA function able to provide elevated Ca2+ supply to SR. Increase 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 phosphorylation, and increase in RyR phosphorylation at PKA and CaMKII-dependent Ser2809 site makes RyR more leaky. Elevation of cytosolic Ca2+ increases RyR phosphorylation at Ser2809 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 of Ca2+ to SR in SANC, sustains robust basal SR Ca2+ cycling in SANC to drive primary pacemaker cell automaticity.