title: The RyR2-P2328S mutation downregulates Nav1.5 producing arrhythmic substrate in murine ventricles.
abstract: Catecholaminergic polymorphic ventricular tachycardia (CPVT) predisposes to ventricular arrhythmia due to altered Ca(2+) homeostasis and can arise from ryanodine receptor (RyR2) mutations including RyR2-P2328S. Previous reports established that homozygotic murine RyR2-P2328S (RyR2 (S/S)) hearts show an atrial arrhythmic phenotype associated with reduced action potential (AP) conduction velocity and sodium channel (Nav1.5) expression. We now relate ventricular arrhythmogenicity and slowed AP conduction in RyR2 (S/S) hearts to connexin-43 (Cx43) and Nav1.5 expression and Na(+) current (I Na). Stimulation protocols applying extrasystolic S2 stimulation following 8 Hz S1 pacing at progressively decremented S1S2 intervals confirmed an arrhythmic tendency despite unchanged ventricular effective refractory periods (VERPs) in Langendorff-perfused RyR2 (S/S) hearts. Dynamic pacing imposing S1 stimuli then demonstrated that progressive reductions of basic cycle lengths (BCLs) produced greater reductions in conduction velocity at equivalent BCLs and diastolic intervals in RyR2 (S/S) than WT, but comparable changes in AP durations (APD90) and their alternans. Western blot analyses demonstrated that Cx43 protein expression in whole ventricles was similar, but Nav1.5 expression in both whole tissue and membrane fractions were significantly reduced in RyR2 (S/S) compared to wild-type (WT). Loose patch-clamp studies similarly demonstrated reduced I Na in RyR2 (S/S) ventricles. We thus attribute arrhythmogenesis in RyR2 (S/S) ventricles resulting from arrhythmic substrate produced by reduced conduction velocity to downregulated Nav1.5 reducing I Na, despite normal determinants of repolarization and passive conduction. The measured changes were quantitatively compatible with earlier predictions of linear relationships between conduction velocity and the peak I Na of the AP but nonlinear relationships between peak I Na and maximum Na(+) permeability.
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, No. 277 West Yanta Road, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Feifei
authors.initials: F
authors.lastname: Ning
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, No. 277 West Yanta Road, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Ling
authors.initials: L
authors.lastname: Luo
authors.affiliation: Physiological Laboratory, University of Cambridge, Cambridge, CB2 3EG, UK.
authors.forename: Shiraz
authors.initials: S
authors.lastname: Ahmad
authors.affiliation: Physiological Laboratory, University of Cambridge, Cambridge, CB2 3EG, UK.
authors.forename: Haseeb
authors.initials: H
authors.lastname: Valli
authors.affiliation: Faculty of Health and Medical Science, Duke of Kent Building, University of Surrey, Guildford, GU2 7TE, UK.|Perdana University-Royal College of Surgeons Ireland, 43400 Serdang, Selangor, Darul Ehsan, Malaysia.
authors.forename: Kamalan
authors.initials: K
authors.lastname: Jeevaratnam
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, No. 277 West Yanta Road, Xi'an, Shaanxi, 710061, People's Republic of China.|Key Laboratory of Molecular Cardiology, Shaanxi Province, People's Republic of China.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education, Xi'an, People's Republic of China.
authors.forename: Tingzhong
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK.|Niche Science and Technology, Falstaff House, Bardolph Road, Richmond, UK.
authors.forename: Laila
authors.initials: L
authors.lastname: Guzadhur
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, No. 277 West Yanta Road, Xi'an, Shaanxi, 710061, People's Republic of China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Yang
authors.affiliation: Physiological Laboratory, University of Cambridge, Cambridge, CB2 3EG, UK.
authors.forename: James A
authors.initials: JA
authors.lastname: Fraser
authors.affiliation: Physiological Laboratory, University of Cambridge, Cambridge, CB2 3EG, UK.|Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK.
authors.forename: Christopher L-H
authors.initials: CL
authors.lastname: Huang
authors.affiliation: Department of Cardiovascular Medicine, First Affiliated Hospital of Xi'an Jiaotong University, No. 277 West Yanta Road, Xi'an, Shaanxi, 710061, People's Republic of China. maaiqun@medmail.com.cn.|Key Laboratory of Molecular Cardiology, Shaanxi Province, People's Republic of China. maaiqun@medmail.com.cn.|Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education, Xi'an, People's Republic of China. maaiqun@medmail.com.cn.
authors.forename: Aiqun
authors.initials: A
authors.lastname: Ma
authors.affiliation: Physiological Laboratory, University of Cambridge, Cambridge, CB2 3EG, UK. ss2148@cam.ac.uk.
authors.forename: Samantha C
authors.identifier: http://orcid.org/0000-0002-5793-2349
authors.initials: SC
authors.lastname: Salvage
mesh_terms: D000200:Action Potentials; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000378:metabolism*; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D005260:Female; D006352:Heart Ventricles / Q000378:metabolism* / Q000503:physiopathology; D008297:Male; D051379:Mice; D020125:Mutation, Missense; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000235:genetics / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*; D016276:Ventricular Function
chemical_list: D018031:Connexin 43; D062554:NAV1.5 Voltage-Gated Sodium Channel; D019837:Ryanodine Receptor Calcium Release Channel; C568321:Scn5a protein, mouse
keywords: Arrhythmogenicity; CPVT; Ca2+ homeostasis; Conduction velocity; Nav1.5; RyR2