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

• Published in: Journal of molecular and cellular cardiology 2017-07, Vol.108, p.8-16
• Main Authors: Pereira, Laëtitia, Bare, Dan J., Galice, Samuel, Shannon, Thomas R., Bers, Donald M.
• Format: Article
• Language: English
• Subjects: Calcium calmodulin-dependent protein kinase; Epac; Excitation-contraction coupling; Nitric oxide synthase; Ryanodine receptor; Sarcoplasmic reticulum
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2017.04.005

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. [Display omitted] •Beta-adrenergic receptors (B-AR) induce arrhythmogenic SR Ca leak in adult rabbit and mouse ventricular myocytes.•Previously proposed parallel pathways involving Epac or NOS signals are actually in series.•This SR Ca leak is via a B1AR-cAMP-Epac2-PI3K-Akt-NOS1-CaMKIIδ-RyR2 phosphorylation cascade.•This parallels B-AR/cAMP inotropic and lusitropic PKA effects on Ca current and SR Ca uptake.