Guan-Sheng Liu1, Ana Morales2, Elizabeth Vafiadaki3, Chi Keung Lam1, Wen-Feng Cai4, Kobra Haghighi1, George Adly1, Ray E Hershberger5, Evangelia G Kranias6. 1. Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, PO Box 670575, 231 Albert Sabin Way, Cincinnati, OH, USA. 2. Division of Human Genetics, Ohio State University College of Medicine, Columbus, OH, USA Dorothy M. Davis Heart and Lung Research Institute, Ohio State University College of Medicine, Columbus, OH 45267-0575, USA. 3. Molecular Biology Division, Biomedical Research Foundation, Academy of Athens, Greece. 4. Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA. 5. Division of Human Genetics, Ohio State University College of Medicine, Columbus, OH, USA Dorothy M. Davis Heart and Lung Research Institute, Ohio State University College of Medicine, Columbus, OH 45267-0575, USA Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University College of Medicine, Columbus, OH, USA. 6. Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, PO Box 670575, 231 Albert Sabin Way, Cincinnati, OH, USA Molecular Biology Division, Biomedical Research Foundation, Academy of Athens, Greece litsa.kranias@uc.edu.
Abstract
AIMS: Depressed sarcoplasmic reticulum (SR) Ca(2+) cycling, a universal characteristic of human and experimental heart failure, may be associated with genetic alterations in key Ca(2+)-handling proteins. In this study, we identified a novel PLN mutation (R25C) in dilated cardiomyopathy (DCM) and investigated its functional significance in cardiomyocyte Ca(2+)-handling and contractility. METHODS AND RESULTS: Exome sequencing identified a C73T substitution in the coding region of PLN in a family with DCM. The four heterozygous family members had implantable cardiac defibrillators, and three developed prominent ventricular arrhythmias. Overexpression of R25C-PLN in adult rat cardiomyocytes significantly suppressed the Ca(2+) affinity of SR Ca(2+)-ATPase (SERCA2a), resulting in decreased SR Ca(2+) content, Ca(2+) transients, and impaired contractile function, compared with WT-PLN. These inhibitory effects were associated with enhanced interaction of R25C-PLN with SERCA2, which was prevented by PKA phosphorylation. Accordingly, isoproterenol stimulation relieved the depressive effects of R25C-PLN in cardiomyocytes. However, R25C-PLN also elicited increases in the frequency of Ca(2+) sparks and waves as well as stress-induced aftercontractions. This was accompanied by increased Ca(2+)/calmodulin-dependent protein kinase II activity and hyper-phosphorylation of RyR2 at serine 2814. CONCLUSION: The findings demonstrate that human R25C-PLN is associated with super-inhibition of SERCA2a and Ca(2+) transport as well as increased SR Ca(2+) leak, promoting arrhythmogenesis under stress conditions. This is the first mechanistic evidence that increased PLN inhibition may impact both SR Ca(2+) uptake and Ca(2+) release activities and suggests that the human R25C-PLN may be a prognostic factor for increased ventricular arrhythmia risk in DCM carriers. Published on behalf of the European Society of Cardiology. All rights reserved.
AIMS: Depressed sarcoplasmic reticulum (SR) Ca(2+) cycling, a universal characteristic of human and experimental heart failure, may be associated with genetic alterations in key Ca(2+)-handling proteins. In this study, we identified a novel PLN mutation (R25C) in dilated cardiomyopathy (DCM) and investigated its functional significance in cardiomyocyte Ca(2+)-handling and contractility. METHODS AND RESULTS: Exome sequencing identified a C73T substitution in the coding region of PLN in a family with DCM. The four heterozygous family members had implantable cardiac defibrillators, and three developed prominent ventricular arrhythmias. Overexpression of R25C-PLN in adult rat cardiomyocytes significantly suppressed the Ca(2+) affinity of SR Ca(2+)-ATPase (SERCA2a), resulting in decreased SR Ca(2+) content, Ca(2+) transients, and impaired contractile function, compared with WT-PLN. These inhibitory effects were associated with enhanced interaction of R25C-PLN with SERCA2, which was prevented by PKA phosphorylation. Accordingly, isoproterenol stimulation relieved the depressive effects of R25C-PLN in cardiomyocytes. However, R25C-PLN also elicited increases in the frequency of Ca(2+) sparks and waves as well as stress-induced aftercontractions. This was accompanied by increased Ca(2+)/calmodulin-dependent protein kinase II activity and hyper-phosphorylation of RyR2 at serine 2814. CONCLUSION: The findings demonstrate that human R25C-PLN is associated with super-inhibition of SERCA2a and Ca(2+) transport as well as increased SR Ca(2+) leak, promoting arrhythmogenesis under stress conditions. This is the first mechanistic evidence that increased PLN inhibition may impact both SR Ca(2+) uptake and Ca(2+) release activities and suggests that the human R25C-PLN may be a prognostic factor for increased ventricular arrhythmia risk in DCM carriers. Published on behalf of the European Society of Cardiology. All rights reserved.
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