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Adenylyl cyclase 6 deletion increases mortality during sustained ?-adrenergic receptor stimulation.

ABSTRACT: Sustained ?-adrenergic receptor stimulation is associated with cardiomyopathy, an affect thought to result from cAMP-associated cardiac injury. Using a murine line with adenylyl cyclase 6 gene deletion (AC6KO), we tested the hypothesis that AC6 deletion, by limiting cAMP production, would attenuate cardiomyopathy in the setting of sustained ?-adrenergic receptor stimulation. During 7d isoproterenol infusion, there was unexpected higher mortality in AC6KO mice compared to wild type control mice (p<0.0001). However, left ventricular function was similarly impaired in isoproterenol-infused control and AC6KO mice. There were no group differences in left ventricular hypertrophy, apoptosis, and fibrosis. Telemetric electrocardiography showed progressive prolongation of PR interval (p<0.0001), QRS duration (p<0.0005), and QTc (p<0.0001), as well as reduction in heart rate (p<0.0001), in AC6KO mice during isoproterenol infusion. These defective electrophysiological properties in isoproterenol-infused AC6KO mice were associated with decreased longitudinal ventricular conduction velocity (p<0.05) and reduced phosphorylation of connexin 43 at S368 in left ventricular samples (p=0.006). Taken together, these data demonstrate that limiting cAMP production does not prevent sustained ?-adrenergic receptor stimulation-induced cardiomyopathy. Moreover, AC6 deletion impairs electrophysiological properties and increases mortality during sustained ?-adrenergic receptor stimulation. Decreased connexin 43 phosphorylation and impaired ventricular conduction may be of mechanistic importance for the defective electrophysiological properties.

SUBMITTER: Tang T

PROVIDER: S-EPMC3987812 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Adenylyl cyclase 6 deletion increases mortality during sustained β-adrenergic receptor stimulation.

Tang Tong T Lai N Chin NC Wright Adam T AT Gao Mei Hua MH Lee Paul P Guo Tracy T Tang Ruoying R McCulloch Andrew D AD Hammond H Kirk HK

Journal of molecular and cellular cardiology 20130412

Sustained β-adrenergic receptor stimulation is associated with cardiomyopathy, an affect thought to result from cAMP-associated cardiac injury. Using a murine line with adenylyl cyclase 6 gene deletion (AC6KO), we tested the hypothesis that AC6 deletion, by limiting cAMP production, would attenuate cardiomyopathy in the setting of sustained β-adrenergic receptor stimulation. During 7d isoproterenol infusion, there was unexpected higher mortality in AC6KO mice compared to wild type control mice ( ...[more]

PMID: 23587598

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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:Background and purposeDespite the view that only ?2- as opposed to ?1-adrenoceptors (?ARs) couple to G(i), some data indicate that the ?1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Experimental approachWe used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate ?2ARs (?1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.Key resultsPTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.Conclusions and implicationsTogether, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

Project description:BackgroundThe type VI adenylyl cyclase (AC6) is a main contributor of cAMP production in the heart. The amino acid (aa) sequence of AC6 is highly homologous to that of another major cardiac adenylyl cyclase, AC5, except for its N-terminus (AC6-N, aa 1-86). Activation of AC6, rather than AC5, produces cardioprotective effects against heart failure, while the underlying mechanism remains to be unveiled. Using an AC6-null (AC6-/-) mouse and a knockin mouse with AC6-N deletion (AC6 ?N/?N), we aimed to investigate the cardioprotective mechanism of AC6 in the heart.MethodsWestern blot analysis and immunofluorescence staining were performed to determine the intracellular distribution of AC6, AC6-?N (a truncated AC6 lacking the first 86 amino acids), and STAT3 activation. Activities of AC6 and AC6-?N in the heart were assessed by cAMP assay. Apoptosis of cardiomyocytes were evaluated by the TUNEL assay and a propidium iodine-based survival assay. Fibrosis was examined by collagen staining.ResultsImmunofluorescence staining revealed that cardiac AC6 was mainly anchored on the sarcolemmal membranes, while AC6-?N was redistributed to the sarcoplasmic reticulum. AC6?N/?N and AC6-/- mice had more apoptotic myocytes and cardiac remodeling than WT mice in experimental models of isoproterenol (ISO)-induced myocardial injury. Adult cardiomyocytes isolated from AC6?N/?N or AC6-/- mice survived poorly after exposure to ISO, which produced no effect on WT cardiomyocytes under the condition tested. Importantly, ISO treatment induced cardiac STAT3 phosphorylation/activation in WT mice, but not in AC6?N/?N and AC6-/- mice. Pharmacological blockage of PKA-, Src-, or STAT3- pathway markedly reduced the survival of WT myocytes in the presence of ISO, but did not affect those of AC6?N/?N and AC6-/- myocytes, suggesting an important role of AC6 in mediating cardioprotective action through the activation of PKA-Src-STAT3-signaling.ConclusionsCollectively, AC6-N controls the anchorage of cardiac AC6 on the sarcolemmal membrane, which enables the coupling of AC6 with the pro-survival PKA-STAT3 pathway. Our findings may facilitate the development of novel therapies for heart failure.

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Adenylyl cyclase 6 deletion increases mortality during sustained ?-adrenergic receptor stimulation.

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Adenylyl cyclase 6 deletion increases mortality during sustained β-adrenergic receptor stimulation.

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