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PRE-CLINICAL RESEARCH

Dantrolene, a Therapeutic Agent for Malignant Hyperthermia, Markedly Improves the Function of Failing Cardiomyocytes by Stabilizing Interdomain Interactions Within the Ryanodine Receptor

Shigeki Kobayashi, MD, PhD^_*, Masafumi Yano, MD, PhD^_*,_*, Takeshi Suetomi, MD^_*, Makoto Ono, MD^_*, Hiroki Tateishi, MD^_*, Mamoru Mochizuki, MD, PhD^_*, Xiaojuan Xu, MD^_*, Hitoshi Uchinoumi, MD^_*, Shinichi Okuda, MD, PhD^_*, Takeshi Yamamoto, MD, PhD^_*, Noritaka Koseki, BS, Hiroyuki Kyushiki, PhD, Noriaki Ikemoto, PhD^, and Masunori Matsuzaki, MD, PhD^_*

^_* Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
First Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan
Boston Biomedical Research Institute, Watertown, Massachusetts
Department of Neurology, Harvard Medical School, Boston, Massachusetts

Manuscript received June 23, 2008; revised manuscript received January 12, 2009, accepted January 19, 2009.

^_* Reprint requests and correspondence: Dr. Masafumi Yano, Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan (Email: yanoma{at}yamaguchi-u.ac.jp).

Objectives: We sought to investigate the effect of dantrolene, a drug generally used to treat malignant hyperthermia, on the Ca²⁺ release and cardiomyocyte function in failing hearts.

Background: The N-terminal (N: 1–600) and central (C: 2000–2500) domains of the ryanodine receptor (RyR) harbor many mutations associated with malignant hyperthermia in skeletal muscle RyR (RyR1) and polymorphic ventricular tachycardia in cardiac RyR (RyR2). There is strong evidence that interdomain interaction between these regions plays an important role in the mechanism of channel regulation.

Methods: Sarcoplasmic reticulum vesicles and cardiomyocytes were isolated from the left ventricular muscles of dogs (normal or rapid ventricular pacing for 4 weeks), for Ca²⁺ leak, transient, and spark assays. To assess the zipped or unzipped state of the interacting domains, the RyR was labeled fluorescently with methylcoumarin acetate in a site-directed manner. We used a quartz-crystal microbalance technique to identify the dantrolene binding site within the RyR2.

Results: Dantrolene specifically bound to domain 601–620 in RyR2. In the sarcoplasmic reticulum isolated from pacing-induced failing dog hearts, the defective interdomain interaction (domain unzipping) had already occurred, causing spontaneous Ca²⁺ leak. Dantrolene suppressed both domain unzipping and the Ca²⁺ leak, demonstrating identical drug concentration-dependence (IC₅₀ = 0.3 µmol/l). In failing cardiomyocytes, both diastolic Ca²⁺ sparks and delayed afterdepolarization were observed frequently, but 1 µmol/l dantrolene inhibited both events.

Conclusions: Dantrolene corrects defective interdomain interactions within RyR2 in failing hearts, inhibits spontaneous Ca²⁺ leak, and in turn improves cardiomyocyte function in failing hearts. Thus, dantrolene may have a potential to treat heart failure, specifically targeting the RyR2.

Key Words: heart failure • calcium • sarcoplasmic reticulum

Abbreviations and Acronyms   ARVC2 = arrhythmogenic right ventricular cardiomyopathy type 2   CPVT = catecholaminergic polymorphic ventricular tachycardia   DAD = delayed afterdepolarization   LV = left ventricle/ventricular   MH = malignant hyperthermia   QCM = quartz crystal microbalance   RyR = ryanodine receptor   SR = sarcoplasmic reticulum

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