Phosphorylation of the ryanodine receptor 2 at serine 2030 is required for a complete β-adrenergic response

Abstract

During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in protein kinase A (PKA)–mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent “hyperphosphorylation” of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation–contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch–clamp recordings and confocal Ca2+ imaging during β-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (ICa) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during β-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.

Unbalance Between Sarcoplasmic Reticulum Ca2 + Uptake and Release: A First Step Toward Ca2 + Triggered Arrhythmias and Cardiac Damage

“…Whereas in quiescent cardiac myocytes the RyR2 appears to be completely unphosphorylated (Huke and Bers, 2008), this site has been suggested as the major phosphorylation site in RyR2 responding to PKA activation upon β-ARS in normal and failing hearts (Xiao et al, 2006). In this context, it has recently been described that phosphorylation of RyR2 at Ser 2030 is required for a complete effect of β-ARS (Potenza et al, 2019) in mouse lines with genetic ablation of this site (RyR2-S2030A).…”

Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes

“…; Potenza et al . ). We hypothesized that RyR2 de‐phosphorylation might make the channels more Ca 2+ sensitive and thus shorten the Ca 2+ spark recovery time.…”

“…To evaluate this possibility, we performed the Ca 2+ spark recovery analysis as previously described (Potenza et al . ) (for detail, see Methods). Briefly, isolated intact myocytes were exposed to ryanodine at a low concentration of 50 nmol L −1 (see ‘Limitations’ below; Sobie et al .…”

“…Conversely, a major relevance has been ascribed to phosphorylation of RyR2, which may enhance the Ca 2+ release during the β‐adrenergic response, possibly contributing to positive inotropy (Bers, ; Potenza et al . ). Furthermore, ‘hyper‐phosphorylation’ of the RyR2s was also described as a major impairment that contributes to heart failure development (Marx et al .…”

“…; Potenza et al . ) recently reported that interventions enhancing RyR2 phosphorylation levels (e.g. isoproterenol treatment and high cyclic adenosine monophosphate concentration) lead to an increased Ca 2+ spark frequency in both intact and permeabilized myocytes.…”