Regulation of cardiac calcium release channels during acute beta-adrenergic stimulation

During β-adrenergic stimulation of the heart, ryanodine receptors (RyRs) Ca²⁺ release channels in the SR can be phosphorylated at residues S2808, S2814 and S2030 causing an increase in RyR activity. The project is to investigate how acute β-adrenergic stimulation of the heart alters regulation of RyRs by intracellular Ca00B²⁺ and Mg²⁺ and the role of these changes in SR Ca²⁺ release and pacemaking. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 minute and subject to 1 minute perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N₂ to capture their phosphorylation state. Western Blots showed that under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60s of β-adrenergic stimulation. S2030 phosphorylation was not detected. 1 minute β-adrenergic stimulation significantly altered Ca²⁺/Mg²⁺ regulation of RyR2 activity: #1) a 3- to 5-fold increase in RyR2 activation by luminal Ca²⁺ and decreased RyR2 inhibition by luminal Mg²⁺; both actions being attributable to changes in the luminal Ca²⁺ binding site (L-site), #2) diminished Mg²⁺ inhibition at mM concentrations attributable to decreased affinity of the I₁-site and possibly the A-site, and #3) increased RyR2 mean open durations, attributable to a decreased rate of cytoplasmic Ca²⁺ inactivation (I₂-site). RyR2 gating model was fitted to the single channel data. It predicted that in diastole, the main effects of 1 minute β-adrenergic stimulation are 1) increasing the activating potency of Ca²⁺ binding to the luminal Ca²⁺ site and decreasing its affinity for luminal Mg²⁺ and 2) decreasing affinity of the low affinity Ca²⁺/Mg²⁺ cytoplasmic inhibition site. However in systole, the main effect is the latter. SAN cell model revealed the additive contributions from increased SERCA2a and RyR2 activity to the sarcolemmal pacemaking current, which determines heart rate in a near proportional manner. In early diastole, increased SERCA2a activity leads to an increase in I_f and late in diastole, increased RyR2 activation increases the NCX current.