Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G

Gonano, Luis Alberto; Aitken‐Buck, Hamish M.; Chakraborty, Akash Deep; Worthington, Luke P; Cully, Tanya R.; Lamberts, Regis R.; Petroff, Martín Gerardo Vila; Jones, Paula G.

doi:10.1016/j.crphys.2022.03.004

Cited by 6 publications

(23 citation statements)

References 36 publications

(81 reference statements)

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“…42 It has also been shown recently that RyR2-S2030 or S2808 is required for PKG-dependent modulation of RyR2-mediated spontaneous Ca 2+ release. 56 These observations raise a possibility that PKG may also modulate Ca 2+ release termination and Ca 2+ alternans. Further systematic and detailed studies will be required to determine whether RyR2-S2808 and/or -S2814 or PKG plays a role in Ca 2+ release termination and cardiac Ca 2+ alternans (Figure 8).…”

Section: Discussionmentioning

confidence: 99%

RyR2 Serine-2030 PKA Site Governs Ca ²⁺ Release Termination and Ca ²⁺ Alternans

Wei

Guo

Wang

et al. 2023

Circulation Research

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Background: PKA (protein kinase A)–mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca 2+ release and induction of cardiac Ca 2+ alternans. To determine the role of the PKA phosphorylation site RyR2-S2030 in Ca 2+ release termination in vitro and cardiac Ca 2+ alternans in intact hearts. Methods and Results: We performed single-cell endoplasmic reticulum Ca 2+ imaging to assess the impact of S2030 mutations on Ca 2+ release termination in HEK293 cells. We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca 2+ level at which Ca 2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM–wild-type increased, whereas Ca 2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca 2+ /CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM–wild-type, CaM-M, and PKA, but not CaMKII, in Ca 2+ release termination. To determine the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation. Using confocal Ca 2+ imaging, we found that isoproterenol and CaM-M suppressed pacing-induced Ca 2+ alternans and accelerated Ca 2+ transient recovery in intact working hearts, whereas CaM–wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor H89 and the CaMKII inhibitor KN93 individually and together, respectively. S2030L abolished the impact of CaM–wild-type, CaM-M, and H89-sensitive component, but not the KN93-sensitive component, of isoproterenol. Conclusions: These data demonstrate, for the first time, that the PKA phosphorylation site RyR-S2030 is an important determinant of PKA-regulated, CaM-dependent Ca 2+ release termination, and Ca 2+ alternans.

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Section: Discussionmentioning

confidence: 99%

RyR2 Serine-2030 PKA Site Governs Ca ²⁺ Release Termination and Ca ²⁺ Alternans

Wei

Guo

Wang

et al. 2023

Circulation Research

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“…Stable inducible human embryonic kidney (HEK 293) cells expressing wild-type RyR2 and S2808A were generated as previously described [5]. Cells were grown in grown in low (5.5 mM) or high glucose (25 mM) supplemented Dulbecco's Modi ed Eagle Medium (DMEM) (Life technologies).…”

Section: Maintenance Of Stable Inducible Hek 293 Cell Linesmentioning

confidence: 99%

“…Upon con rmation that RyR2 is directly O-GlcNAcylated, we next determined the impact of O-GlcNAcylation on the function of RyR2 using our well-established HEK 293 cell model. These cells have been extensively characterised, show spontaneous RyR2 activity comparable to cardiac cells and have served to investigate the impact of other post-translational modi cations of RyR2 [5,7]. To manipulate the level of O-GlcNAcylation, cells were grown in low (5.5 mM) or high (25 mM) glucose in the presence or absence of Thm-G or DON, a O-GlcNAcylation promotor and inhibitor, respectively.…”

Section: O-glcnacylation Of Ryr2 Increases the Propensity For Soicrmentioning

confidence: 99%

“…Although these data strongly implicate a direct modi cation of S2808 by O-GlcNAcylation, it does not rule out further indirect regulation by changes in kinase activity other than CaMKII. For example, we and others have shown that S2808 is a target of PKA and PKG [5,8], so it remains possible that the loss of effect of O-GlcNAcylation on the S2808A mutant might be via changes in the activity of one of these kinases. However, in our hands, phosphorylation of S2808 has a modest impact on SOICR compared to the changes mediated by O-GlcNAcylation seen here [25].…”

Section: O-glcnacylation Can Increase Soicr Independent Of Camkiimentioning

confidence: 99%

“…RyR2 can be regulated by post-translational modi cations such as phosphorylation and oxidation [4][5][6][7].…”

Section: Introductionmentioning

confidence: 99%

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Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation

Okolo

Khaing

Mereacre

et al. 2023

Preprint

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Background O-GlcNAcylation is the enzymatic addition of a sugar, O-linked β-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca2+ leak through the cardiac ryanodine receptor (RyR2) via Ca2+/calmodulin-dependent kinase II (CaMKII). However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca2+ leak. Methods O-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca2+ leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2. Results We found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in Ca2+ leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca2+ leak. Conclusions These data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808.

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Clustering properties of the cardiac ryanodine receptor in health and heart failure

Waddell,

Mereacre,

Alvarado

et al. 2023

Journal of Molecular and Cellular Cardiology

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Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G

Cited by 6 publications

References 36 publications

RyR2 Serine-2030 PKA Site Governs Ca ²⁺ Release Termination and Ca ²⁺ Alternans

RyR2 Serine-2030 PKA Site Governs Ca ²⁺ Release Termination and Ca ²⁺ Alternans

Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation

Clustering properties of the cardiac ryanodine receptor in health and heart failure

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Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G

Cited by 6 publications

References 36 publications

RyR2 Serine-2030 PKA Site Governs Ca 2+ Release Termination and Ca 2+ Alternans

RyR2 Serine-2030 PKA Site Governs Ca 2+ Release Termination and Ca 2+ Alternans

Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation

Clustering properties of the cardiac ryanodine receptor in health and heart failure

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RyR2 Serine-2030 PKA Site Governs Ca ²⁺ Release Termination and Ca ²⁺ Alternans

RyR2 Serine-2030 PKA Site Governs Ca ²⁺ Release Termination and Ca ²⁺ Alternans