BPS2026 – Preventing phosphorylation of rad and phospholamban abolishes the Ca2+-handling contribution to cardiac inotropy during adrenergic stimulation

In the heart, the proteins Rad and phospholamban (PLB) are phosphorylated during the β-adrenergic response, resulting in their unbinding from the L-type Ca 2+ channel (LTCC) and the sarco-(endo)-plasmic reticulum Ca 2+ ATPase 2 (SERCA2), respectively. This, along with phosphorylation of the ryanodine receptor 2 (RyR2), is believed to regulate contractile force (inotropy) during adrenergic stimulation via changes in Ca 2+ handling. However, the contribution of each of these components to the global adrenergic response is not fully understood. Therefore, we studied mouse models with ablation of phosphorylation sites in PLB (PLB-S16A/T17A, PLB-DM) and Rad (Rad-S25A/S38A/S272A/S300A, 4SA-Rad) that prevent their regulation by protein kinase A (PKA), the main effector downstream of the β-adrenergic receptor. We isolated individual cardiomyocytes and recorded calcium handling dynamics in basal conditions and in the presence of the β-adrenergic agonist isoproterenol. In WT cardiomyocytes, isoproterenol induced a significant increase in Ca 2+ transient amplitude, maximum rise velocity and rate of Ca 2+ decay, as well as a significant increase in the Ca 2+ load stored in the sarcoplasmic reticulum (SR), an intracellular store. As previously shown, both 4SA-Rad and PLB-DM cardiomyocytes showed a blunted, yet significant Ca 2+ -handling response to isoproterenol. Remarkably, cardiomyocytes from a double-mutant mouse (4SA-Rad/PLB-DM) did not show a discernible response and global Ca 2+ handling remained unchanged during isoproterenol treatment. Our data demonstrate that changes in global Ca 2+ -handling that mediate inotropy during β-adrenergic stimulation are fully mediated by phosphorylation of Rad and PLB, and their regulation of LTCC and SERCA2. Meanwhile, phosphorylation of RyR2, a major Ca 2+ -release channel of the SR, has limited contribution. Future studies are focused on elucidating how phosphorylation of RyR2 may regulate local, rather than global Ca 2+ handling. These studies are critical to understand pathophysiological regulation of cardiac function.