title: Calcium-dependent Nedd4-2 upregulation mediates degradation of the cardiac sodium channel Nav1.5: implications for heart failure.
abstract: OBJECTIVE
  Reductions in voltage-gated sodium channel (Nav1.5) function/expression provide a slowed-conduction substrate for cardiac arrhythmias. Nedd4-2, which is activated by calcium, post-translationally modulates Nav1.5. We aim to investigate whether elevated intracellular calcium ([Ca2+]i) reduces Nav1.5 through Nedd4-2 and its role in heart failure (HF).
  
  
  METHODS
  Using a combination of biochemical, electrophysiological, cellular and in vivo methods, we tested the effect and mechanism of calcium on Nedd4-2 and in turn Nav1.5.
  
  
  RESULTS
  Increased [Ca2+]i, following 24-h ionomycin treatment, decreased sodium current (INa) density and Nav1.5 protein without altering its mRNA in both neonatal rat cardiomyocytes (NRCMs) and HEK 293 cells stably expressing Nav1.5. The calcium chelator BAPTA-AM restored the reduced Nav1.5 and INain NRCMs pre-treated by ionomycin. Nav1.5 was decreased by Nedd4-2 transfection and further decreased by 6-h ionomycin treatment. These effects were not observed in cells transfected with the catalytically inactive mutant, Nedd4-2 C801S, or with Y1977A-Nav1.5 mutant containing the impaired Nedd4-2 binding motif. Furthermore, elevated [Ca2+]iincreased Nedd4-2, the interaction between Nedd4-2 and Nav1.5, and Nav1.5 ubiquitination. Nav1.5 protein is decreased, whereas Nedd4-2 is increased in volume-overload HF rat hearts, with increased co-localization of Nav1.5 with ubiquitin or Nedd4-2 as indicated by immunofluorescence staining. BAPTA-AM rescued the reduced Nav1.5 protein, INaand increased Nedd4-2 in hypertrophied NRCMs induced by isoproterenol or angiotensin II.
  
  
  CONCLUSIONS
  Calcium-mediated increases in Nedd4-2 downregulate Nav1.5 by ubiquitination. Nav1.5 is downregulated and co-localizes with Nedd4-2 and ubiquitin in failing rat heart. These data suggest a role of Nedd4-2 in Nav1.5 downregulation in HF.
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
authors.forename: L
authors.initials: L
authors.lastname: Luo
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
authors.forename: F
authors.initials: F
authors.lastname: Ning
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Du
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
authors.forename: B
authors.initials: B
authors.lastname: Song
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
authors.forename: D
authors.initials: D
authors.lastname: Yang
authors.affiliation: Physiological Laboratory, University of Cambridge, Cambridge, UK.
authors.forename: S C
authors.initials: SC
authors.lastname: Salvage
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Physiological Laboratory, University of Cambridge, Cambridge, UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Fraser
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.|Key Laboratory of Molecular Cardiology, Xi'an, Shaanxi Province, China.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education, Xi'an, China.
authors.forename: A
authors.initials: A
authors.lastname: Ma
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.|Key Laboratory of Molecular Cardiology, Xi'an, Shaanxi Province, China.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education, Xi'an, China.
authors.forename: T
authors.initials: T
authors.lastname: Wang
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D057809:HEK293 Cells; D006333:Heart Failure / Q000378:metabolism*; D006801:Humans; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology; D051381:Rats; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation
chemical_list: D062554:NAV1.5 Voltage-Gated Sodium Channel; D000075702:Nedd4 Ubiquitin Protein Ligases; D002118:Calcium
keywords: Nav1.5; Nedd4-2; arrhythmia; calcium homoeostasis; heart failure