title: Enhanced binding of calmodulin to RyR2 corrects arrhythmogenic channel disorder in CPVT-associated myocytes.
abstract: OBJECTIVE
  Calmodulin (CaM) plays a key role in modulating channel gating in ryanodine receptor (RyR2). Here, we investigated (a) the pathogenic role of CaM in the channel disorder in CPVT and (b) the possibility of correcting the CPVT-linked channel disorder, using knock-in (KI) mouse model with CPVT-associated RyR2 mutation (R2474S).
  
  
  RESULTS
  Transmembrane potentials were recorded in whole cell current mode before and after pacing (1-5 Hz) in isolated ventricular myocytes. CaM binding was assessed by incorporation of exogenous CaM fluorescently labeled with HiLyte Fluor(®) in saponin-permeabilized myocytes. In the presence of cAMP (1 μM) the apparent affinity of CaM binding to the RyR decreased in KI cells (Kd: 140-400 nM), but not in WT cells (Kd: 110-120 nM). Gly-Ser-His-CaM (GSH-CaM that has much higher RyR-binding than CaM) restored normal binding to the RyR of cAMP-treated KI cells (140 nM). Neither delayed afterdepolarization (DAD) nor triggered activity (TA) were observed in WT cells even at 5Hz pacing, whereas both DAD and TA were observed in 20% and 12% of KI cells, respectively. In response to 10nM isoproterenol, only DAD (but not TA) was observed in 11% of WT cells, whereas in KI cells the incidence of DAD and TA further increased to 60% and 38% of cells, respectively. Addition of GSH-CaM (100 nM) to KI cells decreased both DADs and TA (DAD: 38% of cells; TA: 10% of cells), whereas CaM (100 nM) had no appreciable effect. Addition of GSH-CaM to saponin-permeabilized KI cells decreased Ca(2+) spark frequency (+33% of WT cells), which otherwise markedly increased without GSH-CaM (+100% of WT cells), whereas CaM revealed much less effect on the Ca(2+) spark frequency (+76% of WT cells). Then, by incorporating CaM or GSH-CaM to intact cells (with protein delivery kit), we assessed the in situ effect of GSH-CaM (cytosolic [CaM]=~240 nM, cytosolic [GSH-CaM]=~230 nM) on the frequency of spontaneous Ca(2+) transient (sCaT, % of total cells). Addition of 10nM isoproterenol to KI cells increased sCaT after transient 5 Hz pacing (37%), whereas it was much more attenuated by GSH-CaM (9%) than by CaM (26%) (P<0.01 vs CaM).
  
  
  CONCLUSIONS
  Several disorders in the RyR channel function characteristic of the CPVT-mutant cells (increased spontaneous Ca(2+) leak, delayed afterdepolarization, triggered activity, Ca(2+) spark frequency, spontaneous Ca(2+) transients) can be corrected to a normal function by increasing the affinity of CaM binding to the RyR.
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Fukuda
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan. Electronic address: kenyama@yamaguchi-u.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Shigehiko
authors.initials: S
authors.lastname: Nishimura
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Kato
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Wakako
authors.initials: W
authors.lastname: Murakami
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Hino
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ono
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tateishi
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Testuro
authors.initials: T
authors.lastname: Oda
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Okuda
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kobayashi
authors.affiliation: First Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan.
authors.forename: Noritaka
authors.initials: N
authors.lastname: Koseki
authors.affiliation: First Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kyushiki
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yano
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D055879:Gene Knock-In Techniques; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*; D017180:Tachycardia, Ventricular / Q000188:drug therapy* / Q000235:genetics / Q000503:physiopathology; D000098850:Polymorphic Catecholaminergic Ventricular Tachycardia
chemical_list: C000589340:CaM-(Gly-Ser-His); D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D007545:Isoproterenol
keywords: CPVT; Calcium; Calmodulin; Ryanodine receptor; Sarcoplasmic reticulum