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.

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.

Propranolol prevents the development of heart failure by restoring FKBP12.6-mediated stabilization of ryanodine receptor.

BACKGROUND: In heart failure, protein kinase A-mediated hyperphosphorylation of ryanodine receptors (RyRs) in sarcoplasmic reticulum (SR) causes dissociation of FKBP12.6 from RyRs. This results in an abnormal Ca2+ leak through RyRs, possibly leading to cardiac dysfunction. In the present study, we assess whether beta-blockers can correct this defect in RyR in tachycardia-induced heart failure and thereby improve cardiac function. METHODS AND RESULTS: SRs were isolated from dog left ventricular muscles (normal group, 4 weeks of rapid right ventricular pacing with or without propranolol [P(+) or P(-)]). End-diastolic and end-systolic diameters both increased less in P(+) than P(-), associated with a smaller decrease in fractional shortening in P(+). In SR from P(-), a prominent Ca2+ leak was observed, and FK506 (which dissociates FKBP12.6 from RyR) did not induce an additional Ca2+ leak. However, there was no appreciable Ca2+ leak in SR from P(+), although FK506 induced a Ca2+ leak as in normal SRs. In SR from P(+), an FK506-induced conformational change in RyR, which was virtually absent in SR from P(-), was observed as in normal SRs. Both the stoichiometry of FKBP12.6 versus RyR, assessed by [3H]FK506 and [3H]ryanodine binding assays, and the protein expression of FKBP12.6, assessed by Western blot analysis, were restored by propranolol toward the levels seen in normal SRs. CONCLUSIONS: Low-dose propranolol corrects the defective interaction of FKBP12.6 with RyR (restoration of RyR conformational change and prevention of Ca2+ leak from RyR), apparently resulting in an attenuation of intracellular Ca2+ overload and hence preventing the development of left ventricular remodeling in heart failure.

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.

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 beta-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 beta-receptors and concurrently restored SR function without improving resting cardiac function.

AT1 receptor antagonist restores cardiac ryanodine receptor function, rendering isoproterenol-induced failing heart less susceptible to Ca2+ -leak induced by oxidative stress.

BACKGROUND: The Ca(2+) regulatory proteins in the sarcoplasmic reticulum (SR) play a key role in the pathogenesis of heart failure. In the present study the effect of chronic beta-receptor-stimulation on cardiac and SR functions was assessed, with or without angiotensin-II receptor antagonist treatment recently reported to have anti-beta-adrenergic activity. METHODS AND RESULTS: Rats were treated with isoproterenol with (+) or without (-) candesartan (CAN) and then SR vesicles were isolated from the left ventricular muscle. Both Ca(2+)-uptake and the amount of SR Ca(2+)-ATPase were significantly lower in the CAN (-) group than in the shams, but those were almost normally restored in the CAN (+). Although the level of the protein kinase A (PKA)-phosphorylation of the SR Ca(2+) release channel, known as the ryanodine receptor (RyR2), was elevated in the CAN (-), no Ca(2+)-leak was detected. However, SIN-1 (O(2) (-) donor) induced Ca(2+)-leak in the CAN (-) at a 10-fold lower dose than in the sham and CAN (+). In cardiomyocytes, SIN-1 decreased cell shortening and the peak Ca(2+) transient and prolonged time from peak to 70% decline in CAN (-), again at 10-fold lower dose than in the sham and CAN (+). CONCLUSION: Chronic beta-receptor-stimulation did not induce any Ca(2+)-leak from the SR, whereas Ca(2+)-leak was easily induced when oxidative stress was applied to the PKA-phosphorylated RyR2. Candesartan not only improved Ca(2+)-uptake, but also prevented PKA-phosphorylation, rendering the SR less susceptible to Ca(2+)-leak.

A new cardioprotective agent, JTV519, improves defective channel gating of ryanodine receptor in heart failure.

Defective interaction between FKBP12.6 and ryanodine receptors (RyR) is a possible cause of cardiac dysfunction in heart failure (HF). Here, we assess whether the new cardioprotective agent JTV519 can correct it in tachycardia-induced HF. HF was induced in dogs by 4-wk rapid ventricular pacing, and sarcoplasmic reticulum (SR) was isolated from left ventricular muscles. In failing SR, JTV519 increased the rate of Ca(2+) release and [(3)H]ryanodine binding. RyR were then labeled in a site-directed fashion with the fluorescent conformational probe methylcoumarin acetamide. In failing SR, the polylysine induced a rapid change in methylcoumarin acetamide fluorescence, presumably because the channel opening preceding the Ca(2+) release was smaller than in normal SR (consistent with a decreased rate of Ca(2+) release in failing SR), and JTV519 increased it. In conclusion, JTV519, a new 1,4-benzothiazepine derivative, corrected the defective channel gating in RyR (increase in both the rapid conformational change and the subsequent Ca(2+) release rate) in HF.