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The inotropic peptide ?ARKct improves ?AR responsiveness in normal and failing cardiomyocytes through G(??)-mediated L-type calcium current disinhibition.

ABSTRACT: The G(??)-sequestering peptide ?-adrenergic receptor kinase (?ARK)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 ?-adrenergic receptor (?AR) responsiveness. However, molecular targets mediating improved cardiac contractile performance by ?ARKct and its impact on G(??)-mediated signaling have yet to be fully elucidated.We sought to identify G(??)-regulated targets and signaling mechanisms conveying ?ARKct-mediated enhanced ?AR responsiveness in normal (NC) and failing (FC) adult rat ventricular cardiomyocytes.Assessing viral-based ?ARKct gene delivery with electrophysiological techniques, analysis of contractile performance, subcellular Ca²(+) handling, and site-specific protein phosphorylation, we demonstrate that ?ARKct enhances the cardiac L-type Ca²(+) channel (LCC) current (I(Ca)) both in NCs and FCs on ?AR stimulation. Mechanistically, ?ARKct augments I(Ca) by preventing enhanced inhibitory interaction between the ?1-LCC subunit (Cav1.2?) and liberated G(??) subunits downstream of activated ?ARs. Despite improved ?AR contractile responsiveness, ?ARKct neither increased nor restored cAMP-dependent protein kinase (PKA) and calmodulin-dependent kinase II signaling including unchanged protein kinase (PK)C?, extracellular signal-regulated kinase (ERK)1/2, Akt, ERK5, and p38 activation both in NCs and FCs. Accordingly, although ?ARKct significantly increases I(Ca) and Ca²(+) transients, being susceptible to suppression by recombinant G(??) protein and use-dependent LCC blocker, ?ARKct-expressing cardiomyocytes exhibit equal basal and ?AR-stimulated sarcoplasmic reticulum Ca²(+) load, spontaneous diastolic Ca²(+) leakage, and survival rates and were less susceptible to field-stimulated Ca²(+) waves compared with controls.Our study identifies a G(??)-dependent signaling pathway attenuating cardiomyocyte I(Ca) on ?AR as molecular target for the G(??)-sequestering peptide ?ARKct. Targeted interruption of this inhibitory signaling pathway by ?ARKct confers improved ?AR contractile responsiveness through increased I(Ca) without enhancing regular or restoring abnormal cAMP-signaling. ?ARKct-mediated improvement of I(Ca) rendered cardiomyocytes neither susceptible to ?AR-induced damage nor arrhythmogenic sarcoplasmic reticulum Ca²(+) leakage.

SUBMITTER: Volkers M

PROVIDER: S-EPMC4013502 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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The inotropic peptide βARKct improves βAR responsiveness in normal and failing cardiomyocytes through G(βγ)-mediated L-type calcium current disinhibition.

Völkers Mirko M Weidenhammer Christian C Herzog Nicole N Qiu Gang G Spaich Kristin K Wegner Frederic V FV Peppel Karsten K Müller Oliver J OJ Schinkel Stefanie S Rabinowitz Joseph E JE Hippe Hans-Jorg HJ Brinks Henriette H Katus Hugo A HA Koch Walter J WJ Eckhart Andrea D AD Friedrich Oliver O Most Patrick P

Circulation research 20101124 1

<h4>Rationale</h4>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 signa ...[more]

PMID: 21106943

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Project description:Aims:Abnormal intracellular Ca2+ cycling contributes to triggered activity and arrhythmias in the heart. We investigated the properties and underlying mechanisms for systolic triggered Ca2+ waves in left atria from normal and failing dog hearts. Methods and results:Intracellular Ca2+ cycling was studied using confocal microscopy during rapid pacing of atrial myocytes (36?°C) isolated from normal and failing canine hearts (ventricular tachypacing model). In normal atrial myocytes (NAMs), Ca2+ waves developed during rapid pacing at rates???3.3?Hz and immediately disappeared upon cessation of pacing despite high sarcoplasmic reticulum (SR) load. In heart failure atrial myocytes (HFAMs), triggered Ca2+ waves (TCWs) developed at a higher incidence at slower rates. Because of their timing, TCW development relies upon action potential (AP)-evoked Ca2+ entry. The distribution of Ca2+ wave latencies indicated two populations of waves, with early events representing TCWs and late events representing conventional spontaneous Ca2+ waves. Latency analysis also demonstrated that TCWs arise after junctional Ca2+ release has occurred and spread to non-junctional (cell core) SR. TCWs also occurred in intact dog atrium and in myocytes from humans and pigs. ?-adrenergic stimulation increased Ca2+ release and abolished TCWs in NAMs but was ineffective in HFAMs making this a potentially effective adaptive mechanism in normals but potentially arrhythmogenic in HF. Block of Ca-calmodulin kinase II also abolished TCWs, suggesting a role in TCW formation. Pharmacological manoeuvres that increased Ca2+ release suppressed TCWs as did interventions that decreased Ca2+ release but these also severely reduced excitation-contraction coupling. Conclusion:TCWs develop during the atrial AP and thus could affect AP duration, producing repolarization gradients and creating a substrate for reentry, particularly in HF where they develop at slower rates and a higher incidence. TCWs may represent a mechanism for the initiation of atrial fibrillation particularly in HF.

Project description:This study investigated the hypothesis whether S100A1 gene therapy can improve pathological key features in human failing ventricular cardiomyocytes (HFCMs).Depletion of the Ca²?-sensor protein S100A1 drives deterioration of cardiac performance toward heart failure (HF) in experimental animal models. Targeted repair of this molecular defect by cardiac-specific S100A1 gene therapy rescued cardiac performance, raising the immanent question of its effects in human failing myocardium.Enzymatically isolated HFCMs from hearts with severe systolic HF were subjected to S100A1 and control adenoviral gene transfer and contractile performance, calcium handling, signaling, and energy homeostasis were analyzed by video-edge-detection, FURA2-based epifluorescent microscopy, phosphorylation site-specific antibodies, and mitochondrial assays, respectively.Genetically targeted therapy employing the human S100A1 cDNA normalized decreased S100A1 protein levels in HFCMs, reversed both contractile dysfunction and negative force-frequency relationship, and improved contractile reserve under beta-adrenergic receptor (?-AR) stimulation independent of cAMP-dependent (PKA) and calmodulin-dependent (CaMKII) kinase activity. S100A1 reversed underlying Ca²? handling abnormalities basally and under ?-AR stimulation shown by improved SR Ca²? handling, intracellular Ca²? transients, diastolic Ca²? overload, and diminished susceptibility to arrhythmogenic SR Ca²? leak, respectively. Moreover, S100A1 ameliorated compromised mitochondrial function and restored the phosphocreatine/adenosine-triphosphate ratio.Our results demonstrate for the first time the therapeutic efficacy of genetically reconstituted S100A1 protein levels in HFCMs by reversing pathophysiological features that characterize human failing myocardium. Our findings close a gap in our understanding of S100A1's effects in human cardiomyocytes and strengthen the rationale for future molecular-guided therapy of human HF.

Dataset Information

The inotropic peptide ?ARKct improves ?AR responsiveness in normal and failing cardiomyocytes through G(??)-mediated L-type calcium current disinhibition.

Publications

The inotropic peptide βARKct improves βAR responsiveness in normal and failing cardiomyocytes through G(βγ)-mediated L-type calcium current disinhibition.

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