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Beta-Arrestin-dependent activation of Ca(2+)/calmodulin kinase II after beta(1)-adrenergic receptor stimulation.

ABSTRACT: Ca(2+)/calmodulin kinase II (CaMKII) plays an important role in cardiac contractility and the development of heart failure. Although stimulation of beta(1)-adrenergic receptors (ARs) leads to an increase in CaMKII activity, the molecular mechanism by which beta(1)-ARs activate CaMKII is not completely understood. In this study, we show the requirement for the beta(1)-AR regulatory protein beta-arrestin as a scaffold for both CaMKII and Epac (exchange protein directly activated by cAMP). Stimulation of beta(1)-ARs induces the formation of a beta-arrestin-CaMKII-Epac1 complex, allowing its recruitment to the plasma membrane, whereby interaction with cAMP leads to CaMKII activation. beta-Arrestin binding to the carboxyl-terminal tail of beta(1)-ARs promotes a conformational change within beta-arrestin that allows CaMKII and Epac to remain in a stable complex with the receptor. The essential role for beta-arrestin and identification of the molecular mechanism by which only beta(1)-ARs and not beta(2)-ARs activate CaMKII significantly advances our understanding of this important cellular pathway.

SUBMITTER: Mangmool S

PROVIDER: S-EPMC2867304 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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beta-Arrestin-dependent activation of Ca(2+)/calmodulin kinase II after beta(1)-adrenergic receptor stimulation.

Mangmool Supachoke S Shukla Arun K AK Rockman Howard A HA

The Journal of cell biology 20100426 3

Ca(2+)/calmodulin kinase II (CaMKII) plays an important role in cardiac contractility and the development of heart failure. Although stimulation of beta(1)-adrenergic receptors (ARs) leads to an increase in CaMKII activity, the molecular mechanism by which beta(1)-ARs activate CaMKII is not completely understood. In this study, we show the requirement for the beta(1)-AR regulatory protein beta-arrestin as a scaffold for both CaMKII and Epac (exchange protein directly activated by cAMP). Stimulat ...[more]

PMID: 20421423

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Project description:BACKGROUND:Sustained ?-adrenergic receptor (?-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. OBJECTIVE:The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained ?-AR stimulation and associated functional implications on IKs. METHODS:KCNQ1 phosphorylation was assessed using liquid chromatography-tandem mass spectrometry after sustained ?-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral ?CaMKII overexpression. RESULTS:Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained ?-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest ?CaMKII phosphorylation. Sustained ?-AR stimulation reduced IKs activation (P = .02 vs control) similar to the phosphorylated mimic (P = .62 vs sustained ?-AR). Individual phosphorylated mimics at S484 (P = .04) but not at T482 (P = .17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P < .01). ?CaMKII overexpression reduced IKs similar to ISO treatment in wild type (P < .01) but not in the dephosphorylated S484 mimic (P = .99). CONCLUSION:CaMKII regulates KCNQ1 at S484 during sustained ?-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.

Project description:AIMS:The EMPA-REG OUTCOME study showed reduced mortality and hospitalization due to heart failure (HF) in diabetic patients treated with empagliflozin. Overexpression and Ca2+ -dependent activation of Ca2+ /calmodulin-dependent kinase II (CaMKII) are hallmarks of HF, leading to contractile dysfunction and arrhythmias. We tested whether empagliflozin reduces CaMKII- activity and improves Ca2+ -handling in human and murine ventricular myocytes. METHODS AND RESULTS:Myocytes from wild-type mice, mice with transverse aortic constriction (TAC) as a model of HF, and human failing ventricular myocytes were exposed to empagliflozin (1 μmol/L) or vehicle. CaMKII activity was assessed by CaMKII-histone deacetylase pulldown assay. Ca2+ spark frequency (CaSpF) as a measure of sarcoplasmic reticulum (SR) Ca2+ leak was investigated by confocal microscopy. [Na+ ]i was measured using Na+ /Ca2+ -exchanger (NCX) currents (whole-cell patch clamp). Compared with vehicle, 24 h empagliflozin exposure of murine myocytes reduced CaMKII activity (1.6 ± 0.7 vs. 4.2 ± 0.9, P < 0.05, n = 10 mice), and also CaMKII-dependent ryanodine receptor phosphorylation (0.8 ± 0.1 vs. 1.0 ± 0.1, P < 0.05, n = 11 mice), with similar results upon TAC. In murine myocytes, empagliflozin reduced CaSpF (TAC: 1.7 ± 0.3 vs. 2.5 ± 0.4 1/100 μm-1 s-1 , P < 0.05, n = 4 mice) but increased SR Ca2+ load and Ca2+ transient amplitude. Importantly, empagliflozin also significantly reduced CaSpF in human failing ventricular myocytes (1 ± 0.2 vs. 3.3 ± 0.9, P < 0.05, n = 4 patients), while Ca2+ transient amplitude was increased (F/F0 : 0.53 ± 0.05 vs. 0.36 ± 0.02, P < 0.05, n = 3 patients). In contrast, 30 min exposure with empagliflozin did not affect CaMKII activity nor Ca2+ -handling but significantly reduced [Na+ ]i . CONCLUSIONS:We show for the first time that empagliflozin reduces CaMKII activity and CaMKII-dependent SR Ca2+ leak. Reduced Ca2+ leak and improved Ca2+ transients may contribute to the beneficial effects of empagliflozin in HF.

Dataset Information

Beta-Arrestin-dependent activation of Ca(2+)/calmodulin kinase II after beta(1)-adrenergic receptor stimulation.

Publications

beta-Arrestin-dependent activation of Ca(2+)/calmodulin kinase II after beta(1)-adrenergic receptor stimulation.

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