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

ABSTRACT: During physical exercise or stress, the sympathetic system stimulates cardiac contractility via ?-adrenergic receptor (?-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 ?-ARs stimulation with isoproterenol (Iso) and consistent SR Ca²⁺ loading and L-type Ca²⁺ current (I _Ca) 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 ?-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.

SUBMITTER: Potenza DM

PROVIDER: S-EPMC6363414 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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

Potenza Duilio M DM Janicek Radoslav R Fernandez-Tenorio Miguel M Camors Emmanuel E Ramos-Mondragón Roberto R Valdivia Héctor H HH Niggli Ernst E

The Journal of general physiology 20181212 2

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 ...[more]

PMID: 30541771

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Project description:ObjectiveThe inappropriate release of free fatty acids from obese adipose tissue stores has detrimental effects on metabolism, but key molecular mechanisms controlling FFA release from adipocytes remain undefined. Although obesity promotes systemic inflammation, we find activation of the inflammation-associated Mitogen Activated Protein kinase ERK occurs specifically in adipose tissues of obese mice, and provide evidence that adipocyte ERK activation may explain exaggerated adipose tissue lipolysis observed in obesity.Methods and resultsWe provide genetic and pharmacological evidence that inhibition of the MEK/ERK pathway in human adipose tissue, mice, and flies all effectively limit adipocyte lipolysis. In complementary findings, we show that genetic and obesity-mediated activation of ERK enhances lipolysis, whereas adipose tissue specific knock-out of ERK2, the exclusive ERK1/2 protein in adipocytes, dramatically impairs lipolysis in explanted mouse adipose tissue. In addition, acute inhibition of MEK/ERK signaling also decreases lipolysis in adipose tissue and improves insulin sensitivity in obese mice. Mice with decreased rates of adipose tissue lipolysis in vivo caused by either MEK or ATGL pharmacological inhibition were unable to liberate sufficient White Adipose Tissue (WAT) energy stores to fuel thermogenesis from brown fat during a cold temperature challenge. To identify a molecular mechanism controlling these actions, we performed unbiased phosphoproteomic analysis of obese adipose tissue at different time points following acute pharmacological MEK/ERK inhibition. MEK/ERK inhibition decreased levels of adrenergic signaling and caused de-phosphorylation of the β3-adrenergic receptor (β3AR) on serine 247. To define the functional implications of this phosphorylation, we showed that CRISPR/Cas9 engineered cells expressing wild type β3AR exhibited β3AR phosphorylation by ERK2 and enhanced lipolysis, but this was not seen when serine 247 of β3AR was mutated to alanine.ConclusionTaken together, these data suggest that ERK activation in adipocytes and subsequent phosphorylation of the β3AR on S247 are critical regulatory steps in the enhanced adipocyte lipolysis of obesity.

Dataset Information

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

Publications

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

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