Posttranslational modifications of cardiac ryanodine receptors: Ca(2+) signaling and EC-coupling

Abstract

In cardiac muscle, a number of posttranslational protein modifications can alter the function of the Ca(2+) release channel of the sarcoplasmic reticulum (SR), also known as the ryanodine receptor (RyR). During every heartbeat RyRs are activated by the Ca(2+)-induced Ca(2+) release mechanism and contribute a large fraction of the Ca(2+) required for contraction. Some of the posttranslational modifications of the RyR are known to affect its gating and Ca(2+) sensitivity. Presently, research in a number of laboratories is focused on RyR phosphorylation, both by PKA and CaMKII, or on RyR modifications caused by reactive oxygen and nitrogen species (ROS/RNS). Both classes of posttranslational modifications are thought to play important roles in the physiological regulation of channel activity, but are also known to provoke abnormal alterations during various diseases. Only recently it was realized that several types of posttranslational modifications are tightly connected and form synergistic (or antagonistic) feed-back loops resulting in additive and potentially detrimental downstream effects. This review summarizes recent findings on such posttranslational modifications, attempts to bridge molecular with cellular findings, and opens a perspective for future work trying to understand the ramifications of crosstalk in these multiple signaling pathways. Clarifying these complex interactions will be important in the development of novel therapeutic approaches, since this may form the foundation for the implementation of multi-pronged treatment regimes in the future. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Fig. 1

Modulation of the ryanodine receptor (RyR) by Ca²⁺ and phosphorylation. Ca²⁺ influx via the L-type Ca channel (LTCC) activates the RyR and triggers Ca²⁺ release from the sarcoplasmic reticulum (SR), a process referred to as Ca²⁺ induced Ca²⁺ release or CICR, leading to myocyte contraction. The levels of free cytosolic Ca²⁺ are tightly regulated by the SR Ca²⁺ ATPase (SERCA) and the sarcolemmal Na⁺-Ca²⁺ exchanger (not indicated). After CICR and contraction, the Ca²⁺ store is refilled by pumping Ca²⁺ back into the SR thereby re-establishing diastolic Ca²⁺ levels. The sensitivity of RyR toward activating Ca²⁺ is modulated by phosphorylation. Stimulation of the β₁-adrenoreceptor (β-AR) leads to Gs-protein-mediated activation of adenylyl cyclase (AC) and further cAMP-dependent activation of PKA. PKA can directly phosphorylate RyR at several phosphorylation sites, presumably at S2808, possibly inducing dissociation of calstabin 2, and at S2030, but also modulates the LTCC and SERCA function, the latter by phosphorylation of phospholamban (PLB). Increased cytosolic Ca²⁺ levels activate CaMKII, which directly phosphorylates RyR at S2814. Similar to PKA, CaMKII also phosphorylates PLB and the LTCC leading to global changes in myocyte Ca²⁺ homeostasis.

Fig. 2

Redox-modifications of RyRs. Changes in the redox potential of the myocyte have been shown to have a serious influence on protein function, especially at the level of the RyR. The main sources for the production of reactive oxygen species (ROS) in cardiomyocytes are the sarcolemmal NADPH oxidase (NOX), the xanthine oxidase (XO) and the mitochondrial electron transport chain (complex I through IV). ROS can glutathionylate free cysteine residues on the RyR and also act in an indirect way via CaMKII activation and subsequent RyR phosphorylation. Nitric oxide synthases (NOS) are mainly responsible for the production of nitric oxide (NO) and reactive nitrogen species (RNS). In cardiomyocytes, sarcolemmal endothelial NOS (eNOS), which co-localizes with caveolin-3 (Cav3) in caveolae, and RyR-associated neuronal nNOS are primarily responsible for the production of NO, causing S-nitrosation at free thiol groups of the RyR and many other proteins. Most likely, these mechanisms work synergistically and induce parallel modifications of RyR function.