PMID- 32683896 OWN - NLM STAT- MEDLINE DCOM- 20210831 LR - 20211204 IS - 1524-4539 (Electronic) IS - 0009-7322 (Print) IS - 0009-7322 (Linking) VI - 142 IP - 12 DP - 2020 Sep 22 TI - Loss of SPEG Inhibitory Phosphorylation of Ryanodine Receptor Type-2 Promotes Atrial Fibrillation. PG - 1159-1172 LID - 10.1161/CIRCULATIONAHA.120.045791 [doi] AB - BACKGROUND: Enhanced diastolic calcium (Ca(2+)) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca(2+) leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca(2+)/calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. METHODS: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca(2+) leak in atrial cardiomyocytes was assessed using confocal Ca(2+) imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9. RESULTS: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca(2+) spark frequency in atrial cardiomyocytes with Ca(2+) imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca(2+) leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. CONCLUSIONS: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca(2+) release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment. FAU - Campbell, Hannah M AU - Campbell HM AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Molecular Physiology & Biophysics (H.M.C., A.P.Q., D.Y.C., F.K., T.A.W., M.H., K.M.A., H.-L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. FAU - Quick, Ann P AU - Quick AP AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Molecular Physiology & Biophysics (H.M.C., A.P.Q., D.Y.C., F.K., T.A.W., M.H., K.M.A., H.-L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. FAU - Abu-Taha, Issam AU - Abu-Taha I AD - Institute of Pharmacology (I.A.-T., D.D.), University Duisburg-Essen, Germany. FAU - Chiang, David Y AU - Chiang DY AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Molecular Physiology & Biophysics (H.M.C., A.P.Q., D.Y.C., F.K., T.A.W., M.H., K.M.A., H.-L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Medicine (Cardiovascular Division), Brigham and Women's Hospital, Harvard Medical School, Boston, MA (D.Y.C.). FAU - Kramm, Carlos F AU - Kramm CF AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. FAU - Word, Tarah A AU - Word TA AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Molecular Physiology & Biophysics (H.M.C., A.P.Q., D.Y.C., F.K., T.A.W., M.H., K.M.A., H.-L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. FAU - Brandenburg, Soren AU - Brandenburg S AD - Heart Research Center Gottingen, Department of Cardiology & Pneumology, University Medical Center Gottingen, Germany (S.B., D.U., S.E.L.). FAU - Hulsurkar, Mohit AU - Hulsurkar M AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Molecular Physiology & Biophysics (H.M.C., A.P.Q., D.Y.C., F.K., T.A.W., M.H., K.M.A., H.-L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. FAU - Alsina, Katherina M AU - Alsina KM AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Molecular Physiology & Biophysics (H.M.C., A.P.Q., D.Y.C., F.K., T.A.W., M.H., K.M.A., H.-L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. FAU - Liu, Hui-Bin AU - Liu HB AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Molecular Physiology & Biophysics (H.M.C., A.P.Q., D.Y.C., F.K., T.A.W., M.H., K.M.A., H.-L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Institute of Clinical Pharmacy, the Second Affiliated Hospital of Harbin Medical University, China (H.-B.L.). FAU - Martin, Brian AU - Martin B AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Molecular Physiology & Biophysics (H.M.C., A.P.Q., D.Y.C., F.K., T.A.W., M.H., K.M.A., H.-L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. FAU - Uhlenkamp, Dennis AU - Uhlenkamp D AD - Institute of Pharmacology (I.A.-T., D.D.), University Duisburg-Essen, Germany. AD - Heart Research Center Gottingen, Department of Cardiology & Pneumology, University Medical Center Gottingen, Germany (S.B., D.U., S.E.L.). FAU - Moore, Oliver M AU - Moore OM AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Neuroscience (O.M.M., X.H.T.W.), Baylor College of Medicine, Houston, TX. FAU - Lahiri, Satadru K AU - Lahiri SK AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. FAU - Corradini, Eleonora AU - Corradini E AD - Biomolecular Mass Spectrometry & Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands (E.C., A.J.R.H.). FAU - Kamler, Markus AU - Kamler M AD - Department of Thoracic and Cardiovascular Surgery Huttrop (M.K.), University Duisburg-Essen, Germany. FAU - Heck, Albert J R AU - Heck AJR AD - Biomolecular Mass Spectrometry & Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands (E.C., A.J.R.H.). FAU - Lehnart, Stephan E AU - Lehnart SE AD - Heart Research Center Gottingen, Department of Cardiology & Pneumology, University Medical Center Gottingen, Germany (S.B., D.U., S.E.L.). FAU - Dobrev, Dobromir AU - Dobrev D FAU - Wehrens, Xander H T AU - Wehrens XHT AD - Cardiovascular Research Institute (H.M.C., A.P.Q., D.Y.C., C.F.K., T.A.W., M.H., K.MA., H.-B.L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Molecular Physiology & Biophysics (H.M.C., A.P.Q., D.Y.C., F.K., T.A.W., M.H., K.M.A., H.-L., B.M., O.M.M., S.K.L., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Neuroscience (O.M.M., X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Medicine (Cardiology) (X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Department of Pediatrics (X.H.T.W.), Baylor College of Medicine, Houston, TX. AD - Center for Space Medicine (X.H.T.W.), Baylor College of Medicine, Houston, TX. LA - eng GR - F30 HL140782/HL/NHLBI NIH HHS/United States GR - T32 HL139430/HL/NHLBI NIH HHS/United States GR - T32 HL007676/HL/NHLBI NIH HHS/United States GR - R01 HL136389/HL/NHLBI NIH HHS/United States GR - R01 HL091947/HL/NHLBI NIH HHS/United States GR - R01 HL131517/HL/NHLBI NIH HHS/United States GR - R01 HL089598/HL/NHLBI NIH HHS/United States GR - R01 HL147108/HL/NHLBI NIH HHS/United States GR - R01 HL117641/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20200720 PL - United States TA - Circulation JT - Circulation JID - 0147763 RN - 0 (Muscle Proteins) RN - 0 (RyR2 protein, human) RN - 0 (Ryanodine Receptor Calcium Release Channel) RN - 0 (ryanodine receptor 2. mouse) RN - EC 2.7.11.1 (Protein Serine-Threonine Kinases) RN - EC 2.7.11.1 (SPEG protein, human) RN - EC 2.7.11.1 (Speg protein, mouse) RN - EC 2.7.11.18 (Myosin-Light-Chain Kinase) SB - IM CIN - Circulation. 2020 Sep 22;142(12):1173-1175. PMID: 32955933 MH - Animals MH - Atrial Fibrillation/genetics/*metabolism MH - *Calcium Signaling MH - Female MH - Humans MH - Male MH - Mice MH - Mice, Knockout MH - Muscle Proteins/genetics/*metabolism MH - Myocardium/*metabolism MH - Myosin-Light-Chain Kinase/genetics/*metabolism MH - Phosphorylation MH - Protein Serine-Threonine Kinases/genetics/*metabolism MH - Ryanodine Receptor Calcium Release Channel/genetics/*metabolism MH - Sarcoplasmic Reticulum/genetics/metabolism PMC - PMC7508800 MID - NIHMS1621219 OTO - NOTNLM OT - SPEG protein, human OT - atrial fibrillation OT - excitation contraction coupling OT - ion channels OT - ryanodine receptor calcium release channel COIS- DISCLOSURES XHTW is a founding partner of Elex Biotech, a start-up company that developed drug molecules to target ryanodine receptors for treatment of cardiac arrhythmias. Other authors have no conflicts related to this study. EDAT- 2020/07/21 06:00 MHDA- 2021/09/01 06:00 PMCR- 2021/09/22 CRDT- 2020/07/21 06:00 PHST- 2020/07/21 06:00 [pubmed] PHST- 2021/09/01 06:00 [medline] PHST- 2020/07/21 06:00 [entrez] PHST- 2021/09/22 00:00 [pmc-release] AID - 10.1161/CIRCULATIONAHA.120.045791 [doi] PST - ppublish SO - Circulation. 2020 Sep 22;142(12):1159-1172. doi: 10.1161/CIRCULATIONAHA.120.045791. Epub 2020 Jul 20.