Takahiro Tokuhisa

FKBP12.6-Mediated Stabilization of Calcium-Release Channel (Ryanodine Receptor) as a Novel Therapeutic Strategy Against Heart Failure

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 Ca2+ 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 Ca2+ leak from RyR in normal SR and the spontaneous Ca2+ leak in failing SR. Third, there was no abnormal Ca2+ 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 Ca2+ leak through the RyR, prevented LV remodeling, and led to less severe heart failure.

Defective Regulation of Interdomain Interactions Within the Ryanodine Receptor Plays a Key Role in the Pathogenesis of Heart Failure

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 Gly2460-Pro2495 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 Ca2+ 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 Ca2+ 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 Ca2+ 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.

Correction of defective interdomain interaction within ryanodine receptor by antioxidant is a new therapeutic strategy against heart failure

Background— Defective interdomain interaction within the ryanodine receptor (RyR2) seems to play a key role in the pathogenesis of heart failure, as shown in recent studies. In the present study we investigated the effect of oxidative stress on the interdomain interaction, its outcome in the cardiac function in heart failure, and the possibility of preventing the problem with antioxidants. Methods and Results— Sarcoplasmic reticulum (SR) vesicles were isolated from dog left ventricular (LV) muscle (normal or rapid ventricular pacing for 4 weeks with or without the antioxidant edaravone). In the edaravone-treated paced dogs (EV+), but not in the untreated paced dogs (EV−), normal cardiac function was restored almost completely. In the SR vesicles isolated from the EV−, oxidative stress of the RyR2 (reduction in the number of free thiols) was severe, but it was negligible in EV+. The oxidative stress of the RyR2 destabilized interdomain interactions within the RyR2 (EV−), but its effect was reversed in EV+. Abnormal Ca2+ leak through the RyR2 was found in EV− but not in EV+. The amount of the RyR2-bound FKBP12.6 was less in EV− than in normal dogs, whereas it was restored almost to a normal amount in EV+. The NO donor 3-morpholinosydnonimine (SIN-1) reproduced, in normal SR, several abnormal features seen in failing SR, such as defective interdomain interaction and abnormal Ca2+ leak. Both cell shortening and Ca2+ transients were impaired by SIN-1 in isolated normal myocytes, mimicking the pathophysiological conditions in failing myocytes. Incubation of failing myocytes with edaravone restored the normal properties. Conclusions— During the development of heart failure, edaravone ameliorated the defective interdomain interaction of the RyR2. This prevented Ca2+ leak and LV remodeling, leading to an improvement of cardiac function and an attenuation of LV remodeling.

Valsartan Restores Sarcoplasmic Reticulum Function With No Appreciable Effect on Resting Cardiac Function in Pacing-Induced Heart Failure

Background—Although angiotensin II receptor blockade is considered to be useful for the treatment of human heart failure, little beneficial hemodynamic effect has been shown in some experimental failing hearts. In this study, we assessed the effect of an angiotensin II receptor blocker, valsartan, on sarcoplasmic reticulum (SR) function, defectiveness of which is a major pathogenic mechanism in heart failure. Methods and Results—SR vesicles were isolated from dog left ventricular muscle (normal or exposed to 4-week rapid ventricular pacing with or without valsartan). In the untreated and valsartan-treated paced dogs, cardiac function showed similar deterioration (compared with before pacing). However, both the density of &bgr;-receptors and the contractile response to dobutamine were greater in the valsartan-treated paced dogs than in the untreated paced dogs. In untreated paced hearts, the ryanodine receptor was protein kinase A–hyperphosphorylated, showed an abnormal Ca2+ leak, and had a decreased amount of ryanodine receptor–bound FKBP12.6. No such phenomena were seen in the valsartan-treated paced hearts. Both the SR Ca2+ uptake function and the amount of Ca2+-ATPase were decreased in the untreated failing SR, but both were restored in the valsartan-treated SR. Conclusions—During the development of pacing-induced heart failure, valsartan preserved the density of &bgr;-receptors and concurrently restored SR function without improving resting cardiac function.

Low dose of propranolol prevents the development of heart failure by restoring the defective interaction of FKBP12.6 with cardiac ryanodine receptor

Background. In head failure, hyperphosphorylation of ryanodine receptor (RyR) mediated through PKA has been shown to cause dissociation of FKBP12.6 from RyR, resulting in an abnormal Ca 2+ leak through RyR and possibly consequent cardiac dysfunction. Here, we assessed whether ~-blookede can restore this defective channel function of RyR and therefore improve cardiac function in heart failure. Methods and Results, Sarcoplasmic retioulum (SR) was isolated from dog LV muscles {normal (N), n=5; 4-weeks RV pacing with or without propranolol [P(+): n=4, P(-): n=5, respectively]}. In normal dogs, the dose of propranolol (0.05 mg/kg/day, iv) decreased head rate at baseline by 14 %, but did not attenuate the isoproterenol (0.8 pg/kg/min)-induced increase in peak +dP/ dt of LV pressure. 1) As compared with pre-RV pacing, both end-diastolic [36.2mm in P(+) versus 41.9mm in P(-), p<0.05] and end-systolic diameter [29.4mm in P(+) versus 37.5mm in P(-), p<0.05] were less increased in P(+) than P(-), associated with lesser decrease in fractional shortening [19.0% in P(+) versus 10.2% in P(-), p<0.05]. 2) In SR from P(-), a prominent Ca 2+ leak was observed and FK506 that dissociates FKBP12.6 from RyR did not induce further Ca 2+ leak because of a partial loss of FKBP12.6 from RyR. However, there was no appreciable Ca 2+ leak in SR from P(+), and FK506-inducad Ca 2+ leak was elicited like normal SR. 3) RyR was labeled in a sita-directed fashion with the fluorescent conformational probe methylcoumadn acetate (MCA). In SR from P(+), the FK506-inducad increase in MCA fluorescence, which was virtually absent in SR from P(-), was observed like in normal SR. 4) Indeed, both stoichiometry of FKBP12.6 versus RyR assessed by [3H]FKS06 and [3H]ryanodine-binding assays [3.6 : 1 in N, 1.1 : 1 in P(), 2.4 : 1 in P(+); p<0.05] and protein expression of FKBP12.6 assessed by Western Blot analysis were restored towards those in normal SR. Conclusions. Low dose of propranolol attenuated LV remodeling presumably by ameliorating the defective interaction of FKBP12.6 with RyR, presumably resulting in an inhibition of intracallular Ca 2+ overload and hence a prevention of the development of heart failure.