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

KEY POINTS: The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+ /calmodulin-dependent kinase II (CaMKII) activation during high-frequency stimulation. Sympathetic stimulation through ß-adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII. Here we report that CaMKII activation during ß-adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs. In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A-dependent. The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. ABSTRACT: In cardiac myocytes, ß-adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L-type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+ /calmodulin-dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ 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 [Ca2+ ]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non-coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ 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 Ca2+ transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca2+ transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and relaxation. In conclusion, CaMKII activation during ß-adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC-triggered local Ca2+ release as well as spontaneous diastolic Ca2+ release whilst PKA is the major pathway increasing global Ca2+ cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy.