Masateru Kohno

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.

Altered Stoichiometry of FKBP12.6 Versus Ryanodine Receptor as a Cause of Abnormal Ca2+ Leak Through Ryanodine Receptor in Heart Failure

BackgroundIn the pathogenesis of cardiac dysfunction in heart failure, a decrease in the activity of the sarcoplasmic reticulum (SR) Ca2+-ATPase is believed to be a major determinant. Here, we report a novel mechanism of cardiac dysfunction revealed by assessing the functional interaction of FK506–binding protein (FKBP12.6) with the cardiac ryanodine receptor (RyR) in a canine model of pacing-induced heart failure. Methods and ResultsSR vesicles were isolated from left ventricular muscles (normal and heart failure). The stoichiometry of FKBP12.6 per RyR was significantly decreased in failing SR, as assessed by the ratio of the Bmax values for [3H]dihydro-FK506 to those for [3H]ryanodine binding. In normal SR, the molar ratio was 3.6 (≈1 FKBP12.6 for each RyR monomer), whereas it was 1.6 in failing SR. In normal SR, FK506 caused a dose-dependent Ca2+ leak that showed a close parallelism with the conformational change in RyR. In failing SR, a prominent Ca2+ leak was observed even in the absence of FK506, and FK506 produced little or no further increase in Ca2+ leak and only a slight conformational change in RyR. The level of protein expression of FKBP12.6 was indeed found to be significantly decreased in failing SR. ConclusionsAn abnormal Ca2+ 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 Ca2+ leak might possibly cause Ca2+ overload and consequent diastolic dysfunction, as well as systolic dysfunction.

Altered interaction of FKBP12.6 with ryanodine receptor as a cause of abnormal Ca2+ release in heart failure

OBJECTIVE Little information is available as to the Ca(2+) release function of the sarcoplasmic reticulum (SR) in heart failure. We assessed whether the alteration in this function in heart failure is related to a change in the role of FK binding protein (FKBP), which is tightly coupled with the cardiac ryanodine receptor (RyR) and recently identified as a modulatory protein acting to stabilize the gating function of RyR. METHODS SR vesicles were isolated from dog LV muscles [normal (N), n=6; heart failure induced by 3-weeks pacing (HF), n=6]. The time course of the SR Ca(2+) release was continuously monitored using a stopped-flow apparatus, and [3H]ryanodine-binding and [3H]dihydro-FK506-binding assays were also performed. RESULTS FK506, which specifically binds to FKBP12.6 and dissociates it from RyR, decreased the polylysine-induced enhancement of [3H]ryanodine-binding by 38% in N (P<0.05) but it had no effect in HF. In HF, the rate constant for the polylysine-induced Ca(2+) release from the SR was 61% smaller than in N. FK506 decreased the rate constant for the polylysine-induced Ca(2+) release by 67% in N (P<0.05) but had no effect in HF. The [3H]dihydro-FK506-binding assay revealed that the number (B(max)) of FKBPs was decreased by 83% in HF (P<0.05), while the K(d) value was unchanged. FK506 did not significantly change SR Ca(2+.)-ATPase activity in either N or HF. CONCLUSIONS In HF, the number of FKBPs showed a tremendous decrease; this may underlie the RyR-channel instability and the impairment of the Ca(2+) release function of RyR seen in the failing heart.

Enhancement of Rho/Rho-kinase system in regulation of vascular smooth muscle contraction in tachycardia-induced heart failure

OBJECTIVE The Rho/Rho-kinase system regulates Ca(2+) sensitivity in vascular smooth muscle. A new drug, Y-27632, specifically inhibits Rho-kinase and hence decreases the phosphorylation of myosin light chain, thus reducing contraction. Here, we compare the effects of Y-27632 and nifedipine on the vasoconstrictor response of the femoral artery in heart failure. METHODS Heart failure (HF) was produced by chronic rapid RV pacing (250 bpm, 28 days, six dogs). Indo1-AM was loaded into endothelium-denuded femoral artery segments for measuring intracellular [Ca(2+)]. Tension and changes in intracellular [Ca(2+)] [the change in the ratio (418 nm/468 nm) of Indo1 fluorescence (F(ratio))] were simultaneously measured in Krebs-Ringer solution. RESULTS In HF: (i) norepinephrine (10 microM) produced greater tension (784+/-52 g/cm(2)) than in control (502+/-64 g/cm(2)) despite a similar increase in F(ratio), indicating increased Ca(2+) sensitivity in vascular smooth muscle; (ii) nifedipine attenuated this enhanced response by only a maximum of 27% at 1 micromol/l with a 56% reduction in F(ratio); (iii) Y-27632 attenuated it by a maximum of 80% at 100 micromol/l without a significant change in F(ratio); (iv) RhoA protein and mRNA expression levels in the femoral artery were up-regulated by +110% and +56%, respectively, while those of Rho-kinase were unchanged. CONCLUSIONS The Ca(2+)-sensitizing mechanism involving the Rho/Rho-kinase system may be deeply involved in the enhanced arterial vasoconstriction seen in HF. Since Y-27632 attenuated this response in small arteries, it shows potential as a novel, potent vasodilator for the treatment of HF.

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.

Impact of the Cross-Sectional Geometry of the Post-Deployment Coronary Stent on In-Stent Neointimal Hyperplasia

To establish the relationship between the cross-sectional geometry of the post-deployment stent and the degree of in-stent neointimal hyperplasia (INH), intravascular ultrasound (IVUS) was used to examine cross-sections of the coronary arteries from 23 patients with coronary stents 6 months after implantation. Stent cross-sectional area (Sa) and stent perimeter (Sp) from 200 stent cross-sections, and the stent radius (Sr) and thickness of INH (Id) of 2,880 radial axes, were measured, and the mean degree of roundness (Rd) of stent cross-section was calculated for each stent as Rd=4piSa/Sp2. The degree of deformity (Df) of the stent cross-section was also calculated by comparing it with a hypothetical circle (the area of this hypothetical circle was equal to the Sa): Df=Sr/R, where R is the radius of the hypothetical circle. The area of INH was significantly larger in the Rd<0.87 group (n=84) than in the Rd> or =0.87 group (n=116) (3.83+/-1.26 vs 3.16+/-1.32 mm2, p<0.0005). There were significant differences in the thickness of INH among the 3 groups classified by the value of Df (Df<0.95: n=425, 0.21+/-0.12mm; 0.95< or =Df<1.05: n=2008, 0.29+/-0.15mm; Df> or =1.05: n=447, 0.34+/-0.15mm, overall p<0.0001). These data suggest that in-stent neointimal proliferation is more likely to occur in stented coronary arteries with a more oval than rounded cross-section, and particularly within the more pronounced and curved portion of the oval.

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.