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Ca2+-activated adenylyl cyclase 1 introduces Ca2+-dependence to beta-adrenergic stimulation of HCN2 current.

ABSTRACT: Previous observations show that ?-adrenergic modulation of pacemaker current (I(f)) in sinoatrial node (SAN) cells is impaired by disruption of normal Ca(2+)-homeostasis with ryanodine or BAPTA. Recently, the presence of Ca(2+)-activated adenylyl cyclase (AC) 1 was reported in SAN, and was proposed as a possible mechanism of Ca(2+)-dependence of ?-adrenergic modulation. However, direct evidence that pacemaker (HCN) channels can be regulated by Ca(2+)-activated AC and that such regulation introduces Ca(2+) dependence, is lacking. Here we co-expressed AC1 or AC6 with HCN2 in neonatal rat ventricular myocytes, which lack AC1. Although both isoforms have equivalent expression level and ability to interact with HCN2, only AC1 increases intracellular cAMP content, accelerates spontaneous beating rate and modifies HCN2 biophysics. Measured HCN2 current in the AC1 group activated ~10mV more positive than in GFP or AC6. The ?-adrenergic agonist isoproterenol induced a further positive shift under control conditions, but failed to do so after pretreatment with the Ca(2+) chelator BAPTA. In the AC6 group, isoproterenol shifted the HCN2 activation relation to a similar extent in the absence and presence of BAPTA. Thus, AC1 but not AC6 over-expression introduces Ca(2+)-sensitivity to the ?-adrenergic response of HCN2. These results demonstrate physical and functional interaction between AC isoforms and the HCN2 pacemaker channel and support a key role of Ca(2+) activated AC1 as a molecular mechanism in Ca(2+)-dependent modulation of ?-adrenergic response of heart rate.

SUBMITTER: Kryukova YN

PROVIDER: S-EPMC3348422 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Ca2+-activated adenylyl cyclase 1 introduces Ca2+-dependence to beta-adrenergic stimulation of HCN2 current.

Kryukova Yelena N YN Protas Lev L Robinson Richard B RB

Journal of molecular and cellular cardiology 20120329 6

Previous observations show that β-adrenergic modulation of pacemaker current (I(f)) in sinoatrial node (SAN) cells is impaired by disruption of normal Ca(2+)-homeostasis with ryanodine or BAPTA. Recently, the presence of Ca(2+)-activated adenylyl cyclase (AC) 1 was reported in SAN, and was proposed as a possible mechanism of Ca(2+)-dependence of β-adrenergic modulation. However, direct evidence that pacemaker (HCN) channels can be regulated by Ca(2+)-activated AC and that such regulation introdu ...[more]

PMID: 22484253

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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.

Dataset Information

Ca2+-activated adenylyl cyclase 1 introduces Ca2+-dependence to beta-adrenergic stimulation of HCN2 current.

Publications

Ca2+-activated adenylyl cyclase 1 introduces Ca2+-dependence to beta-adrenergic stimulation of HCN2 current.

Similar Datasets

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