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Aneurysm formation appears to be related to tumor size, and large aneurysms confer a higher probability of rupture.

Background-The development of heart failure is tightly correlated with a decrease in the stoichiometric ratio for FKBP12.6 binding to the ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR). We report that a new drug, the 1,4-benzothiazepine derivative JTV519, reverses this pathogenic process. JTV519 is known to have a protective effect against Ca 2ϩ overload-induced myocardial injury. Methods and Results-Heart failure was produced by 4 weeks of rapid right ventricular pacing, with or without JTV519; SR were then isolated from dog left ventricular (LV) muscles. First, in JTV519-treated dogs, no signs of heart failure were observed after 4 weeks of chronic right ventricular pacing, LV systolic and diastolic functions were largely preserved, and LV remodeling was prevented. Second, JTV519 acutely inhibited both the FK506-induced Ca 2ϩ leak from RyR in normal SR and the spontaneous Ca 2ϩ leak in failing SR. Third, there was no abnormal Ca 2ϩ leak in SR vesicles isolated from JTV519-treated hearts. Fourth, in JTV519-treated hearts, both the stoichiometry of FKBP12.6 binding to RyR and the amount of RyR-bound FKBP12.6 were restored toward the values seen in normal SR. Fifth, in JTV519-untreated hearts, RyR was PKA-hyperphosphorylated, whereas it was reversed in JTV519-treated hearts, returning the channel phosphorylation toward the levels seen in normal hearts. Conclusions-During the development of experimental heart failure, JTV519 prevented the amount of RyR-bound FKBP12.6 from decreasing. This in turn reduced the abnormal Ca 2ϩ leak through the RyR, prevented LV remodeling, and led to less severe heart failure.

Objectives To investigate the effect of dantrolene, a drug generally used to treat Malignant Hyperthermia (MH), 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 MH in skeletal muscle RyR (RyR1) and polymorphic ventricular tachycardia in cardiac RyR (RyR2). There is strong evidence that inter-domain interaction between these regions plays an important role in the mechanism of channel regulation. Methods Sarcoplasmic reticulum (SR) vesicles and cardiomyocytes were isolated from dog LV muscles (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 fluorescently labeled with methylcoumarin acetate in a site-directed manner. We employed 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 SR isolated from pacing-induced dog failing hearts, the defective inter-domain interaction_(domain unzipping) has already occurred, causing spontaneous Ca2+ leak. Dantrolene suppressed both domain unzipping and the Ca2+ leak, showing identical drug concentration-dependence (IC50=0.3 μmol/L). In failing cardiomyocytes, both diastolic Ca2+ sparks and delayed afterdepolarization were frequently observed, but 1 μmol/L dantrolene inhibited both events. Conclusions Dantrolene corrects defective inter-domain interactions within RyR2 in failing hearts, inhibits spontaneous Ca2+ leak, in turn improves cardiomyocyte function in failing hearts. Thus, dantrolene may have a potential to treat heart failure, specifically targeting the RyR2.

An abnormal Ca(2+) leak through the RyR is present in heart failure, and this leak is presumably caused by a partial loss of RyR-bound FKBP12.6 and the resultant conformational change in RyR. This abnormal Ca(2+) leak might possibly cause Ca(2+) overload and consequent diastolic dysfunction, as well as systolic dysfunction.

Background-According to our hypothesis, 2 domains within the ryanodine receptor (RyR) of sarcoplasmic reticulum (SR) (N-terminal [0 to 600] and central [2000 to 2500] domains), where many mutations have been found in patients with polymorphic ventricular tachycardia, interact with each other as a regulatory switch for channel gating. Here, we investigated whether the defective FKBP12.6-mediated stabilization of RyR in heart failure is produced by an abnormal interdomain interaction. Methods and Results-SR vesicles were isolated from dog left ventricular muscles, and then the RyR moiety of the SR was fluorescently labeled with methylcoumarin acetate (MCA) using DPc10, a synthetic peptide corresponding to Gly 2460 -Pro 2495 of RyR (one of the mutable domains in polymorphic ventricular tachycardia), as a site-directing carrier; the carrier was removed from the RyR after MCA labeling. Addition of DPc10 induced an unzipped state of the interacting N-terminal and central domains, as evidenced by an increase in the accessibility of the RyR-bound MCA fluorescence to a large fluorescence quencher. Domain unzipping resulted in Ca 2ϩ leak through the RyR and facilitated cAMP-dependent hyperphosphorylation of RyR and FKBP12.6 dissociation from RyR. When DPc10 was introduced into the isolated myocytes, the magnitude of intracellular Ca 2ϩ transient decreased, and its decay time was prolonged. In the SR isolated from pacing-induced dog failing hearts, the domain unzipping has already occurred, together with FKBP12.6 dissociation and Ca 2ϩ leak. Conclusions-The specific domain interaction within the RyR regulates the channel gating property, and the defectiveness in the mode of the interdomain interaction seems to be the initial critical step of the pathogenesis of heart failure.

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in a well-defined region of the cardiac type 2 ryanodine receptor (RyR)2. However, the underlying mechanism by which a single mutation in such a large molecule produces drastic effects on channel function remains unresolved. Objective: Using a knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we investigated the molecular mechanism by which CPVT is induced by a single point mutation within the RyR2. Methods and Results: The R2474S/؉ KI mice showed no apparent structural or histological abnormalities in the heart, but they showed clear indications of other abnormalities. Bidirectional or polymorphic ventricular tachycardia was induced after exercise on a treadmill. The interaction between the N-terminal (amino acids 1 to 600) and central (amino acids 2000 to 2500) domains of the RyR2 (an intrinsic mechanism to close Ca 2؉ channels) was weakened (domain unzipping). On protein kinase A-mediated phosphorylation of the RyR2, this domain unzipping further increased, resulting in a significant increase in the frequency of spontaneous Ca Key Words: ryanodine receptor Ⅲ calcium Ⅲ ventricular tachycardia Ⅲ sarcoplasmic reticulum T o date, more than 70 cardiac ryanodine receptor (RyR)2 missense mutations have been identified that are linked with 2 inherited forms of sudden cardiac death: catecholaminergic polymorphic ventricular tachycardia (CPVT) 1 and arrhythmogenic right ventricular cardiomyopathy type 2. 1 These mutations cluster in 3 well-defined regions of the RyR2 that correspond to malignant hyperthermia or the central core disease mutable regions, designated as the N-terminal domain (amino acids 1 to 600), central domain (amino acids 2000 to 2500), and the C-terminal transmembrane channel domain of the skeletal muscle-type ryanodine receptor (RyR1). 1 This suggests that the RyR2 shares a common domain-mediated channel regulation mechanism with RyR1. Mutations at different positions in each of these domains result in the nearly identical phenotype of channel dysfunctions such as hyperactivation of the Ca 2ϩ channel and hypersensitization to agonists. To account for these phenomena, Ikemoto et al 2,3 proposed the so-called "domain switch hypothesis" and stated that in the resting or nonactivated state, the N-terminal domain and the central domain make close contact at several subdomains (domain zipping). Then, on physiological or pharmacological stimulation, these critical interdomain contacts are weakened, resulting in the loss of conformational constraints (domain unzipping), thus lowering the energy barrier for Ca 2ϩ channel opening. Consistent with this hypothesis, single particle analysis of the 3D structure of the RyR2 molecule revealed that the N-terminal and central domains (located in domains 5 and 6 of the so-called clamp Original received September 15, 2009; revision received February 9, 2010; accepted February 26, 2010 region, respectively) are in a close apposition ...

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca 2؉ channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The SternVolmer quenching constant (K Q ) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22°C and was unaffected by calmodulin. [ 3 H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca 2؉ release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37°C, yet required the presence of calmodulin at 22°C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca 2؉ channel opening rather than the domain switch itself.Dantrolene (hydrated 1-(((5-(4-nitrophenyl)-2-furanyl)methylene)amino)-2,4-imidazolidinedione sodium salt) is an intracellularly acting skeletal muscle relaxant used for the treatment of malignant hyperthermia (MH). 1 MH is a potentially deadly, pharmacogenetically mediated, hypermetabolic response to volatile anesthetics that results from unregulated intramyoplasmic Ca 2ϩ release (1). The drug is known to inhibit excitation-contraction coupling of skeletal muscle (2) by suppressing depolarization-induced sarcoplasmic reticulum (SR) Ca 2ϩ release in normal and MH-susceptible skeletal muscle without affecting voltage sensor activation (3). In MH, the voltage dependence of contractile activation is shifted to lower voltages (4), whereas in the presence of clinical concentrations of dantrolene, i.e. 10 M (5), the voltage dependence of contractile activation is shifted to higher voltages (6, 7). Normalization of the voltage dependence of contractile activation may therefore be one of the important components of the therapeutic action of dantrolene. Dantrolene also confers a normal Mg 2ϩ sensitivity to MH-susceptible muscle fibers, which would otherwise show a considerably reduced sensitivity to the normal inhibitory action of myoplasmic Mg 2ϩ on the SR Ca 2ϩ release mechanism (...