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Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1 dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft

Journal Contribution - Journal Article

In cardiac myocytes, β-adrenergic stimulation enhances Ca(2+) cycling through an integrated signalling cascade modulating L-type Ca(2+) channels (LTCC), phospholamban, and ryanodine receptors (RyRs). CaMKII and nitric oxide synthase1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca(2+) microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole-cell voltage-clamp and confocal line-scan imaging with Fluo-4 as a [Ca(2+) ]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca(2+) sparks, which are assigned to coupled and non-coupled RyR clusters. ISO (10 nM) increases Ca(2+) spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces ICaL and local Ca(2+) transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca(2+) transient. In contrast, PKA inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca(2+) content, Ca(2+) transient amplitude and relaxation. In conclusion, CaMKII activation during β-adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC-triggered local Ca(2+) release as well as spontaneous diastolic Ca(2+) release whilst PKA is the major pathway increasing global Ca(2+) cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy. This article is protected by copyright. All rights reserved.
Journal: Journal of Physiology
ISSN: 0022-3751
Issue: 20
Volume: 594
Pages: 5923 - 5939
Number of pages: 17
Publication year:2016