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

Abstract

Rationale The Gβγ-sequestering peptide βARKct derived from the G-protein coupled receptor kinase 2 (GRK2) carboxy-terminus has emerged as a promising target for gene-based heart failure (HF) 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 Gβγ-mediated signaling have yet to be fully elucidated. Objective We sought to identify Gβγ-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 Ca2+ handling and site-specific protein phosphorylation, we demonstrate that βARKct enhances the cardiac L-type Ca2+ channel (LCC) current (Ica) both in NCs and FCs upon βAR stimulation. Mechanistically, βARKct augments Ica by preventing enhanced inhibitory interaction between the α1-LCC subunit (Cav1.2α) and liberated Gβγ subunits downstream of activated βARs. Despite improved βAR contractile responsiveness, βARKct neither increased nor restored cAMP-dependent protein kinase A (PKA) and calmodulin-dependent kinase II (CaMKII) signaling including unchanged protein kinase Cε (PKCε), ERK1/2, Akt, ERK5 and p38 activation both in NCs and FCs. Accordingly, though βARKct significantly increases Ica and Ca2+ transients being susceptible to suppression by recombinant Gβγ protein and use-dependent LCC blocker, βARKct-expressing cardiomyocytes exhibit equal basal and βAR-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 Gβγ-dependent signaling pathway attenuating cardiomyocyte Ica upon βAR as molecular target for the Gβγ-sequestering peptide βARKct. Targeted interruption of this inhibitory signaling pathway by βARKct confers improved βAR contractile responsiveness through increased Ica without enhancing regular or restoring abnormal cAMP-signaling. βARKct-mediated improvement of Ica rendered cardiomyocytes neither susceptible to βAR-induced damage nor arrhythmogenic SR Ca2+ leakage.

Pathological hypertrophy amelioration by PRAS40-mediated inhibition of mTORC1

“…In vivo cardiac-targeted PRAS40 expression in normal mouse hearts was obtained by using tail vein injection of an AAV9 harboring the PRAS40 gene or PRAS40TA mutant driven by a cardiomyocyte-specific CMV-MLC2v0.8 promoter as previously described (34).…”

Dissecting the Role of G-Protein–Coupled Receptor Kinase 2 for Excitation-Contraction Coupling

“…For instance, why does the phenotype of GRK2 deletion differ profoundly from the recently characterized EC coupling phenotype after ␤ARKct overexpression 25 ? In the latter, the only change of EC coupling that could be observed was ␤ARKct-mediated relief of G ␤␥ -induced inhibition of LTCC, supporting the earlier idea that ␤ARKct mediates its beneficial effects via G ␤␥ scavenging rather than GRK2 inhibition.…”

Targeting GRK2 by gene therapy for heart failure: benefits above β-blockade

“…Most studies-including studies with bARKct expression in HF pigs-have shown a reversal of bAR dysfunction including receptor upregulation and a normalization of signaling; however, no doubt, there are effects of the bARKct that go beyond resensitization of cardiac bARs and these effects are currently being explored by us and others. 54,55 Adrenal GRK2 as a novel HF gene therapy target As discussed above, HF is associated with enhanced catecholamine secretion from the adrenal glands because of a 2c AR desensitization by GRK2. This central role in the critical elevation of plasma catecholamine levels contributing to HF development and progression renders adrenal GRK2 an interesting target for HF treatment.…”

“…59 This observation suggests that the contractile effects of bARKct might be because of inhibition of additional G bg pathways other than a bAR-mediated effect, although this has yet to be proven experimentally. In addition, Völkers et al 54 recently demonstrated that a part of the positive inotropic effects of bARKct is attributable to the disruption of G bg -mediated inhibition of cardiac L-type Ca 2+ channels, as bARKct expression in myocytes significantly improved Ca 2+ cycling via augmentation of the L-type Ca 2+ channel current. Another bAR-independent effect of the bARKct in the heart appears to be reversal of GRK2-mediated insulin resistance in myocytes, 55 which definitely needs to be explored as contributing to the therapeutic effects of GRK2 inhibition in HF.…”