The Inotropic Peptide βARKct Improves βAR Responsiveness in Normal and Failing Cardiomyocytes Through G βγ -Mediated L-Type Calcium Current Disinhibition

Abstract

Rationale: The G βγ -sequestering peptide β-adrenergic receptor kinase (βARK)ct derived from the G-protein–coupled receptor kinase (GRK)2 carboxyl terminus has emerged as a promising target for gene-based heart failure therapy. Enhanced downstream cAMP signaling has been proposed as the underlying mechanism for increased β-adrenergic receptor (βAR) responsiveness. However, molecular targets mediating improved cardiac contractile performance by βARKct and its impact on G βγ -mediated signaling have yet to be fully elucidated. Objective: We sought to identify G βγ -regulated targets and signaling mechanisms conveying βARKct-mediated enhanced βAR responsiveness in normal (NC) and failing (FC) adult rat ventricular cardiomyocytes. Methods and Results: Assessing viral-based βARKct gene delivery with electrophysiological techniques, analysis of contractile performance, subcellular Ca 2+ handling, and site-specific protein phosphorylation, we demonstrate that βARKct enhances the cardiac L-type Ca 2+ channel (LCC) current ( I Ca ) both in NCs and FCs on βAR stimulation. Mechanistically, βARKct augments I Ca by preventing enhanced inhibitory interaction between the α1-LCC subunit (Cav1.2α) and liberated G βγ subunits downstream of activated βARs. Despite improved βAR contractile responsiveness, βARKct neither increased nor restored cAMP-dependent protein kinase (PKA) and calmodulin-dependent kinase II signaling including unchanged protein kinase (PK)Cε, extracellular signal-regulated kinase (ERK)1/2, Akt, ERK5, and p38 activation both in NCs and FCs. Accordingly, although βARKct significantly increases I Ca and Ca 2+ transients, being susceptible to suppression by recombinant G βγ protein and use-dependent LCC blocker, βARKct-expressing cardiomyocytes exhibit equal basal and βAR-stimulated sarcoplasmic reticulum Ca 2+ load, spontaneous diastolic Ca 2+ leakage, and survival rates and were less susceptible to field-stimulated Ca 2+ waves compared with controls. Conclusion: Our study identifies a G βγ -dependent signaling pathway attenuating cardiomyocyte I Ca on βAR as molecular target for the G βγ -sequestering peptide βARKct. Targeted interruption of this inhibitory signaling pathway by βARKct confers improved βAR contractile responsiveness through increased I Ca without enhancing regular or restoring abnormal cAMP-signaling. βARKct-mediated improvement of I Ca rendered cardiomyocytes neither susceptible to βAR-induced damage nor arrhythmogenic sarcoplasmic reticulum Ca 2+ leakage.