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. 2020 Aug 14;295(33):11720-11728.
doi: 10.1074/jbc.RA120.014054. Epub 2020 Jun 24.

Muscarinic-dependent phosphorylation of the cardiac ryanodine receptor by protein kinase G is mediated by PI3K-AKT-nNOS signaling

Stephen Baine  1 , Justin Thomas  1 , Ingrid Bonilla  2 , Marina Ivanova  2 , Andriy Belevych  2 , Jiaoni Li  3 , Rengasayee Veeraraghavan  3 , Przemyslaw B Radwanski  1 , Cynthia Carnes  1 , Sandor Gyorke  4
Affiliations

Muscarinic-dependent phosphorylation of the cardiac ryanodine receptor by protein kinase G is mediated by PI3K-AKT-nNOS signaling

Stephen Baine et al. J Biol Chem. .

Abstract

Post-translational modifications of proteins involved in calcium handling in myocytes, such as the cardiac ryanodine receptor (RyR2), critically regulate cardiac contractility. Recent studies have suggested that phosphorylation of RyR2 by protein kinase G (PKG) might contribute to the cardioprotective effects of cholinergic stimulation. However, the specific mechanisms underlying these effects remain unclear. Here, using murine ventricular myocytes, immunoblotting, proximity ligation as-says, and nitric oxide imaging, we report that phosphorylation of Ser-2808 in RyR2 induced by the muscarinic receptor agonist carbachol is mediated by a signaling axis comprising phosphoinositide 3-phosphate kinase, Akt Ser/Thr kinase, nitric oxide synthase 1, nitric oxide, soluble guanylate cyclase, cyclic GMP (cGMP), and PKG. We found that this signaling pathway is compartmentalized in myocytes, as it was distinct from atrial natriuretic peptide receptor-cGMP-PKG-RyR2 Ser-2808 signaling and independent of muscarinic-induced phosphorylation of Ser-239 in vasodilator-stimulated phosphoprotein. These results provide detailed insights into muscarinic-induced PKG signaling and the mediators that regulate cardiac RyR2 phosphorylation critical for cardiovascular function.

Keywords: AKT; AKT PKB; NOS1; NOS3; PI3K; cardiac RyR2; cyclase; guanylate cyclase (guanylyl cyclase); nitric oxide; nitric oxide synthase; protein kinase G (PKG); ryanodine receptor; soluble guanylate; vasodilator stimulatory phosphoprotein.

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.
Parasympathetic regulation of RyR2 Ser-2808 occurs through muscarinic acetylcholine receptor 2. A, 10 μm CCh does not significantly increase RyR2 Ser-2808 phosphorylation in M2R KO mice. B, plot of optical density (OD) normalized for phosphorylated RyR2 Ser-2808/total RyR2 levels. Data shown are median ± S.D. C, representative Western blot of WT ventricular cardiomyocytes treated with 10 μm CCh significantly increases RyR2 Ser-2808 phosphorylation (*, p < 0.05 versus control (CTL)), Student's t test. Additionally, cardiomyocytes treated with M2R allosteric agonist 10 μm LY2119620 (LY) and 10 μm CCh show enhanced RyR2 2808 phosphorylation versus CCh alone (*, p < 0.01 versus CTL; †, p < 0.01 versus CCh), Student's t test. D, plot of OD normalized for phosphorylated RyR2 Ser-2808/total RyR2 levels. Data shown are median ± S.D. WT, n = 5–7 mice; minimum of 3 experiments/group. M2R KO, n = 3-4 mice, minimum of 3 experiments/group; AU, arbitrary units.
Figure 2.
Figure 2.
Carbachol-mediated parasympathetic stimulation is facilitated by nitric oxide. A, representative ventricular myocyte (CTL) treated with 10 μm CCh exhibits increased NO-sensitive DAF-FM fluorescence. Myocytes were treated with 1–5 mm spermine NONOate (SP) to elicit maximal NO response at the conclusion of each scan. NO release was measured as the slope of the increase in DAF-FM fluorescence over time (s). All slope values were normalized to maximal NO release. B, representative line trace demonstrating 10 μm CCh increases DAF-FM fluorescence over time. Arrows, addition of CCh and spermine NONOate, respectively. C, plot of normalized slope values for each treatment group (*, p < 0.001 versus CTL; †, p < 0.001 versus CCh), Student's t test. Data shown are median ± S.D. D, representative ventricular myocyte pretreated with NOS1 inhibitor NP abrogates CCh-mediated increase in DAF-FM fluorescence (N + P). E, representative line trace indicating that selective NOS1 inhibition prevents a CCh-mediated increase in DAF-FM fluorescence. Arrows represent treatment with 1 μm NP, 1 μm NP + 10 μm CCh, and 1–5 mm spermine NONOate, respectively. F, plot of normalized slope values for each treatment group (*, p < 0.001 versus CTL; †, p < 0.001 versus CCh), Student's t test. Data shown are median ± S.D.; n = 5–10 WT mice, 25–50 cells/treatment condition, 4–7 experiments/group. Scale bar, 50 μm.
Figure 3.
Figure 3.
Nitric oxide synthase 1 mediates CCh-induced RyR2 Ser-2808 phosphorylation. A, representative Western blot showing that 10 μm CCh increases RyR Ser-2808 phosphorylation. Inhibition of NOS1 with specific inhibitor 1 μm NP abolishes CCh-induced RyR2 Ser-2808 phosphorylation. Inhibition of NOS3 with 1 μm LN failed to inhibit CCh-mediated RyR2 2808 phosphorylation (*, p < 0.05 versus CTL; †, p < 0.05 versus CCh + NP), Student's t test. B, plot of OD normalized for phosphorylated RyR2 Ser-2808/total RyR2 levels. Data shown are median ± S.D. C, in isolated ventricular NOS1 KO myocytes, CCh fails to increase RyR2 2808 phosphorylation, and NOS3 inhibition fails to inhibit CCh-induced RyR Ser-2808 phosphorylation (*, p < 0.05 versus CTL), Student's t test. D, plot of OD normalized for phosphorylated RyR2 Ser-2808/total RyR2 levels. Data shown are median ± S.D. E, representative Western blot analysis demonstrating that in NOS3 KO myocytes, CCh increases RyR2 Ser-2808 phosphorylation (*, p < 0.05 versus CTL), Student's t test. F, plot of OD normalized for phosphorylated RyR2 Ser-2808/total RyR2 levels. Data shown are median ± S.D. n = 3–4 WT mice, 3–4 experiments/group; n = 3 NOS1 KO mice, 3–4 experiments/group; n = 3 NOS3 KO mice, 3–4 experiments/group.
Figure 4.
Figure 4.
Carbachol mediates phosphorylation of RyR2 Ser-2808 via M2R–PI3K–Akt signaling axis. A, representative Western blot demonstrating that 10 μm CCh increases Akt Ser-473 phosphorylation in WT ventricular cardiomyocytes (*, p < 0.05 versus CTL), Student's t test. Treatment with specific PI3K inhibitor wortmannin (WM) (100 nm) +10 μm CCh significantly abrogates Akt Ser-473 phosphorylation (†‡, p < 0.001 versus CCh; †, p < 0.05 versus CCh), Student's t test. B, plot of OD normalized for phosphorylated Akt Ser-473/total Akt levels. Data shown are median ± S.D. C, representative Western blotting indicating that 10 μm CCh significantly increases phosphorylation of RyR2 Ser-2808 phosphorylation in WT ventricular cardiomyocytes (*, p < 0.05 versus CTL), Student's t test. However, treatment with WM (100 nm) in the presence of 10 μm CCh attenuates RyR2 Ser-2808 phosphorylation (†, p < 0.05 versus CCh), Student's t test. D, plot of OD normalized for phosphorylated RyR2 Ser-2808/total RyR2 levels. Data shown are median ± S.D. WT, n = 3-5 mice, minimum of 3 experiments/group.
Figure 5.
Figure 5.
Parasympathetic regulation of RyR2 Ser-2808 occurs through protein kinase G. A, representative Western blot indicating that RyR2 Ser-2808 phosphorylation can be promoted via soluble guanylyl cyclase activator 100 nm BAY 58-2667(Cinaciguat) (*, p < 0.05 versus CTL), Student's t test. B, plot of OD normalized for phosphorylated RyR2 Ser-2808/total RyR2 levels. Data shown are median ± S.D. C, Western blot illustrating that RyR2 2808 phosphorylation can be promoted with PKG activator 1 mm 8-Br-cGMP in WT ventricular myocytes (*, p < 0.05 versus CTL), Student's t test. D, plot of OD normalized for phosphorylated RyR2 Ser-2808/total RyR2 levels. Data shown are median ± S.D. WT, n = 3–5 mice, minimum of 3 experiments/group.
Figure 6.
Figure 6.
Cholinergic-induced phosphorylation of VASP Ser-239 is NOS1/NOS3-independent. A, Western blot demonstrating that 10 μm CCh induced phosphorylation of VASP Ser-239 (*, p < 0.05 versus CTL), Student's t test. B, plot of OD normalized for phosphorylated VASP Ser-239/total VASP levels. Data shown are median ± S.D. C, representative Western blot from isolated ventricular myocytes of WT mice demonstrating that CCh increases phosphorylation of VASP Ser-239 independent of NOS1 and NOS3 (*, p < 0.05 versus CTL), Student's t test. D, plot of OD normalized for phosphorylated VASP Ser-239/total VASP levels. Data shown are median ± S.D. E, representative Western blotting demonstrating 100 nm atrial natriuretic peptide increases RyR2 Ser-2808 phosphorylation in isolated ventricular cardiomyocytes (*, p < 0.05 versus CTL), Student's t test. F, plot of OD normalized for phosphorylated RyR2 Ser-2808/total RyR2 levels. Data shown are median ± S.D. WT, n = 3–4 mice, 3–4 experiments/group.
Figure 7.
Figure 7.
Muscarinic-induced phosphorylation targets revealed via proximity ligation assay. A, PLA demonstrates that 10 μm CCh significantly increases phospho-RyR2 2808 puncta in WT ventricular myocytes (*, p < 0.05 versus CTL), Student's t test. B, plot of punctae/µm2. Data shown are median ± S.D. C, PLA analysis reveals a significant increase in phosphorylated VASP Ser-239 puncta in myocytes treated with 10 μm CCh (*, p < 0.05 versus CTL). D, plot of punctae/µm2. Data shown are median ± S.D. E, PLA indicates that 10 μm CCh significantly increases Akt Ser-473 puncta in WT ventricular cardiomyocytes (*, p < 0.05 versus CTL), Student's t test. F, plot of punctae/µm2. Data shown are median ± S.D. WT mice (labeled CTL), n = 3–4 mice, 3–4 experiments/group.
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