β-Adrenergic induced SR Ca2+ leak is mediated by an Epac-NOS pathway

Abstract

Cardiac β-adrenergic receptors (β-AR) and Ca²⁺-Calmodulin dependent protein kinase (CaMKII) regulate both physiological and pathophysiological Ca²⁺ signaling. Elevated diastolic Ca²⁺ leak from the sarcoplasmic reticulum (SR) contributes to contractile dysfunction in heart failure and to arrhythmogenesis. β-AR activation is known to increase SR Ca²⁺ leak via CaMKII-dependent phosphorylation of the ryanodine receptor. Two independent and reportedly parallel pathways have been implicated in this β-AR-CaMKII cascade, one involving exchange protein directly activated by cAMP (Epac2) and another involving nitric oxide synthase 1 (NOS1). Here we tested whether Epac and NOS function in a single series pathway to increase β-AR induced and CaMKII-dependent SR Ca²⁺ leak. Leak was measured as both Ca²⁺ spark frequency and tetracaine-induced shifts in SR Ca²⁺, in mouse and rabbit ventricular myocytes. Direct Epac activation by 8-CPT (8-(4-chlorophenylthio)-2'-O-methyl-cAMP) mimicked β-AR-induced SR Ca²⁺ leak, and both were blocked by NOS inhibition. The same was true for myocyte CaMKII activation (assessed via a FRET-based reporter) and ryanodine receptor phosphorylation. Inhibitor and phosphorylation studies also implicated phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) downstream of Epac and above NOS activation in this pathway. We conclude that these two independently characterized parallel pathways function mainly via a single series arrangement (β-AR-cAMP-Epac-PI3K-Akt-NOS1-CaMKII) to mediate increased SR Ca²⁺ leak. Thus, for β-AR activation the cAMP-PKA branch effects inotropy and lusitropy (by effects on Ca²⁺ current and SR Ca²⁺-ATPase), this cAMP-Epac-NOS pathway increases pathological diastolic SR Ca²⁺leak. This pathway distinction may allow novel SR Ca²⁺ leak therapeutic targeting in treatment of arrhythmias in heart failure that spare the inotropic and lusitropic effects of the PKA branch.

Keywords: Calcium calmodulin-dependent protein kinase; Epac; Excitation-contraction coupling; Nitric oxide synthase; Ryanodine receptor; Sarcoplasmic reticulum.