Xujie Liu1,2, Suya Wang1, Xiaoling Guo1,3, Yifei Li4, Roza Ogurlu1, Fujian Lu1, Maksymilian Prondzynski1, Sofia de la Serna Buzon1, Qing Ma1, Donghui Zhang5, Gang Wang1, Justin Cotton1,6, Yuxuan Guo1, Ling Xiao7, David J Milan7, Yang Xu8, Michael Schlame8, Vassilios J Bezzerides1, William T Pu1,9. 1. Department of Cardiology, Boston Children's Hospital, MA (X.L., S.W., X.G., R.O., F.L., M.P., S.d.l.S.B., Q.M., G.W., J.C., Y.G., V.J.B., W.T.P.). 2. Department of Radiology, Basic Medical School, Chongqing Medical University, China (X.L.). 3. Center of Scientific Research, the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Zhejiang, China (X.G.). 4. Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu (Y.L.). 5. State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Science, Hubei University, Wuhan, China (D.Z.). 6. Harvard College, Cambridge, MA (J.C.). 7. Department of Cardiology, Massachusetts General Hospital, Boston (L.X., D.J.M.). 8. Department of Anesthesiology, New York University School of Medicine (Y.X., M.S.). 9. Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
Abstract
BACKGROUND: Mutations in tafazzin (TAZ), a gene required for biogenesis of cardiolipin, the signature phospholipid of the inner mitochondrial membrane, causes Barth syndrome (BTHS). Cardiomyopathy and risk of sudden cardiac death are prominent features of BTHS, but the mechanisms by which impaired cardiolipin biogenesis causes cardiac muscle weakness and arrhythmia are poorly understood. METHODS: We performed in vivo electrophysiology to define arrhythmia vulnerability in cardiac-specific TAZ knockout mice. Using cardiomyocytes derived from human induced pluripotent stem cells and cardiac-specific TAZ knockout mice as model systems, we investigated the effect of TAZ inactivation on Ca2+ handling. Through genome editing and pharmacology, we defined a molecular link between TAZ mutation and abnormal Ca2+ handling and contractility. RESULTS: A subset of mice with cardiac-specific TAZ inactivation developed arrhythmias, including bidirectional ventricular tachycardia, atrial tachycardia, and complete atrioventricular block. Compared with wild-type controls, BTHS-induced pluripotent stem cell-derived cardiomyocytes had increased diastolic Ca2+ and decreased Ca2+ transient amplitude. BTHS-induced pluripotent stem cell-derived cardiomyocytes had higher levels of mitochondrial and cellular reactive oxygen species than wild-type controls, which activated CaMKII (Ca2+/calmodulin-dependent protein kinase II). Activated CaMKII phosphorylated the RYR2 (ryanodine receptor 2) on serine 2814, increasing Ca2+ leak through RYR2. Inhibition of this reactive oxygen species-CaMKII-RYR2 pathway through pharmacological inhibitors or genome editing normalized aberrant Ca2+ handling in BTHS-induced pluripotent stem cell-derived cardiomyocytes and improved their contractile function. Murine Taz knockout cardiomyocytes also exhibited elevated diastolic Ca2+ and decreased Ca2+ transient amplitude. These abnormalities were ameliorated by Ca2+/calmodulin-dependent protein kinase II or reactive oxygen species inhibition. CONCLUSIONS: This study identified a molecular pathway that links TAZ mutation with abnormal Ca2+ handling and decreased cardiomyocyte contractility. This pathway may offer therapeutic opportunities to treat BTHS and potentially other diseases with elevated mitochondrial reactive oxygen species production.
BACKGROUND: Mutations in tafazzin (TAZ), a gene required for biogenesis of cardiolipin, the signature phospholipid of the inner mitochondrial membrane, causes Barth syndrome (BTHS). Cardiomyopathy and risk of sudden cardiac death are prominent features of BTHS, but the mechanisms by which impaired cardiolipin biogenesis causes cardiac muscle weakness and arrhythmia are poorly understood. METHODS: We performed in vivo electrophysiology to define arrhythmia vulnerability in cardiac-specific TAZ knockout mice. Using cardiomyocytes derived from human induced pluripotent stem cells and cardiac-specific TAZ knockout mice as model systems, we investigated the effect of TAZ inactivation on Ca2+ handling. Through genome editing and pharmacology, we defined a molecular link between TAZ mutation and abnormal Ca2+ handling and contractility. RESULTS: A subset of mice with cardiac-specific TAZ inactivation developed arrhythmias, including bidirectional ventricular tachycardia, atrial tachycardia, and complete atrioventricular block. Compared with wild-type controls, BTHS-induced pluripotent stem cell-derived cardiomyocytes had increased diastolic Ca2+ and decreased Ca2+ transient amplitude. BTHS-induced pluripotent stem cell-derived cardiomyocytes had higher levels of mitochondrial and cellular reactive oxygen species than wild-type controls, which activated CaMKII (Ca2+/calmodulin-dependent protein kinase II). Activated CaMKII phosphorylated the RYR2 (ryanodine receptor 2) on serine 2814, increasing Ca2+ leak through RYR2. Inhibition of this reactive oxygen species-CaMKII-RYR2 pathway through pharmacological inhibitors or genome editing normalized aberrant Ca2+ handling in BTHS-induced pluripotent stem cell-derived cardiomyocytes and improved their contractile function. Murine Taz knockout cardiomyocytes also exhibited elevated diastolic Ca2+ and decreased Ca2+ transient amplitude. These abnormalities were ameliorated by Ca2+/calmodulin-dependent protein kinase II or reactive oxygen species inhibition. CONCLUSIONS: This study identified a molecular pathway that links TAZ mutation with abnormal Ca2+ handling and decreased cardiomyocyte contractility. This pathway may offer therapeutic opportunities to treat BTHS and potentially other diseases with elevated mitochondrial reactive oxygen species production.
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