title: A novel human R25C-phospholamban mutation is associated with super-inhibition of calcium cycling and ventricular arrhythmia.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, PO Box 670575, 231 Albert Sabin Way, Cincinnati, OH, USA.
authors: Guan-Sheng
authors: GS
authors: Liu
authors: 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.
authors: Ana
authors: A
authors: Morales
authors: Molecular Biology Division, Biomedical Research Foundation, Academy of Athens, Greece.
authors: Elizabeth
authors: E
authors: Vafiadaki
authors: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, PO Box 670575, 231 Albert Sabin Way, Cincinnati, OH, USA.
authors: Chi Keung
authors: CK
authors: Lam
authors: Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
authors: Wen-Feng
authors: WF
authors: Cai
authors: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, PO Box 670575, 231 Albert Sabin Way, Cincinnati, OH, USA.
authors: Kobra
authors: K
authors: Haghighi
authors: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, PO Box 670575, 231 Albert Sabin Way, Cincinnati, OH, USA.
authors: George
authors: G
authors: Adly
authors: 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.
authors: Ray E
authors: RE
authors: Hershberger
authors: 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.
authors: Evangelia G
authors: EG
authors: Kranias
mesh_terms: D000368:Aged; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000209:etiology*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002311:Cardiomyopathy, Dilated / Q000235:genetics; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol; D019837:Ryanodine Receptor Calcium Release Channel; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
keywords: Calcium cycling; Dilated cardiomyopathy; Mutation