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Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII.

ABSTRACT: Enhanced cardiac late Na current (late INa) and increased sarcoplasmic reticulum (SR)-Ca(2+)-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late INa and SR-Ca(2+)-leak in atrial cardiomyocytes (CMs).In murine atrial CMs, SR-Ca(2+)-leak was increased by the late INa enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca(2+)/calmodulin-dependent protein kinase II (Autocamide-2-related inhibitory peptide), protein kinase A (H89), or late INa (Ranolazine or Tetrodotoxin) all prevented ATX-II-dependent SR-Ca(2+)-leak. The SR-Ca(2+)-leak induction by ATX-II was not detected when either the Na(+)/Ca(2+) exchanger was inhibited (KBR) or in CaMKII?c-knockout mice. FRET measurements revealed increased cAMP levels upon ATX-II stimulation, which could be prevented by inhibition of adenylyl cyclases (ACs) 5 and 6 (NKY 80) but not by inhibition of phosphodiesterases (IBMX), suggesting PKA activation via an AC-dependent increase of cAMP levels. Western blots showed late INa-dependent hyperphosphorylation of CaMKII as well as PKA target sites at ryanodine receptor type-2 (-S2814 and -S2808) and phospholamban (-Thr17, -S16). Enhancement of late INa did not alter Ca(2+)-transient amplitude or SR-Ca(2+)-load. However, upon late INa activation and simultaneous CaMKII inhibition, Ca(2+)-transient amplitude and SR-Ca(2+)-load were increased, whereas PKA inhibition reduced Ca(2+)-transient amplitude and load and additionally slowed Ca(2+) elimination. In atrial CMs from patients with atrial fibrillation, inhibition of late INa, CaMKII, or PKA reduced the SR-Ca(2+)-leak.Late INa exerts distinct effects on Ca(2+) homeostasis in atrial myocardium through activation of CaMKII and PKA. Inhibition of late INa represents a potential approach to attenuate CaMKII activation and decreases SR-Ca(2+)-leak in atrial rhythm disorders. The interconnection with the cAMP/PKA system further increases the antiarrhythmic potential of late INa inhibition.

SUBMITTER: Fischer TH

PROVIDER: S-EPMC4476413 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII.

Fischer Thomas H TH Herting Jonas J Mason Fleur E FE Hartmann Nico N Watanabe Saera S Nikolaev Viacheslav O VO Sprenger Julia U JU Fan Peidong P Yao Lina L Popov Aron-Frederik AF Danner Bernhard C BC Schöndube Friedrich F Belardinelli Luiz L Hasenfuss Gerd G Maier Lars S LS Sossalla Samuel S

Cardiovascular research 20150518 1

<h4>Aims</h4>Enhanced cardiac late Na current (late INa) and increased sarcoplasmic reticulum (SR)-Ca(2+)-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late INa and SR-Ca(2+)-leak in atrial cardiomyocytes (CMs).<h4>Methods and results</h4>In murine atrial CMs, SR-Ca(2+)-leak was increased by the late INa enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca(2+)/calmodulin-dependent protein kinase II (Autocamide-2-related inhibito ...[more]

PMID: 25990311

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Project description:Delayed afterdepolarizations (DADs) carried by Na(+)-Ca(2+)-exchange current (I(NCX)) in response to sarcoplasmic reticulum (SR) Ca(2+) leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined.Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca(2+)](i) (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca(2+)](i) and SR Ca(2+) content (integrated I(NCX) during caffeine-induced Ca(2+) transient) were unchanged, whereas diastolic SR Ca(2+) leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ?50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca(2+) leak. Calmodulin expression (60%), Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca(2+) leak, the frequency of spontaneous Ca(2+) release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca(2+) sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca(2+)](i) and I(NCX) density revealed I(NCX) upregulation in cAF. Spontaneous Ca(2+) release events accompanied by inward I(NCX) currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca(2+)](i) (diastolic [Ca(2+)](i)-voltage coupling gain) was higher in cAF compared with control.Enhanced SR Ca(2+) leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I(NCX) for a given SR Ca(2+) release and increased diastolic [Ca(2+)](i)-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

Dataset Information

Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII.

Publications

Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII.

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