The inotropic peptide ßARKct improves ßAR responsiveness in normal and failing cardiomyocytes through G(ßγ)-mediated L-type calcium current disinhibition.

ABSTRACT

RATIONALE The G(ßγ)-sequestering peptide ß-adrenergic receptor kinase (ßARK)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 ß-adrenergic receptor (ßAR) responsiveness. However, molecular targets mediating improved cardiac contractile performance by ßARKct and its impact on G(ßγ)-mediated signaling have yet to be fully elucidated. OBJECTIVE: We sought to identify G(ßγ)-regulated targets and signaling mechanisms conveying ßARKct-mediated enhanced ßAR responsiveness in normal (NC) and failing (FC) adult rat ventricular cardiomyocytes. METHODS AND RESULTS: Assessing viral-based ßARKct gene delivery with electrophysiological techniques, analysis of contractile performance, subcellular Ca²(+) handling, and site-specific protein phosphorylation, we demonstrate that ßARKct enhances the cardiac L-type Ca²(+) channel (LCC) current (I(Ca)) both in NCs and FCs on ßAR stimulation. Mechanistically, ßARKct augments I(Ca) by preventing enhanced inhibitory interaction between the α1-LCC subunit (Cav1.2α) and liberated G(ßγ) subunits downstream of activated ßARs. Despite improved ßAR contractile responsiveness, ßARKct 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 ßARKct significantly increases I(Ca) and Ca²(+) transients, being susceptible to suppression by recombinant G(ßγ) protein and use-dependent LCC blocker, ßARKct-expressing cardiomyocytes exhibit equal basal and ßAR-stimulated sarcoplasmic reticulum Ca²(+) load, spontaneous diastolic Ca²(+) leakage, and survival rates and were less susceptible to field-stimulated Ca²(+) waves compared with controls. CONCLUSION: Our study identifies a G(ßγ)-dependent signaling pathway attenuating cardiomyocyte I(Ca) on ßAR as molecular target for the G(ßγ)-sequestering peptide ßARKct. Targeted interruption of this inhibitory signaling pathway by ßARKct confers improved ßAR contractile responsiveness through increased I(Ca) without enhancing regular or restoring abnormal cAMP-signaling. ßARKct-mediated improvement of I(Ca) rendered cardiomyocytes neither susceptible to ßAR-induced damage nor arrhythmogenic sarcoplasmic reticulum Ca²(+) leakage.