Yasutaka Yamamoto

Tottori University, TTJ, Tottori, Japan

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Publications (43)138.56 Total impact

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    ABSTRACT: Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder caused by mutations in UMOD that encodes uromodulin. Topiroxostat, a novel non-purine analog, selectively inhibits xanthine oxidase and reduces the serum uric acid levels and the urinary albuminuria.
    Clinical and Experimental Nephrology 09/2014; · 1.71 Impact Factor
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    ABSTRACT: A KCNE1 polymorphism, D85N, causes long QT syndrome (LQTS) with a decrease in the slowly activating delayed-rectifier K(+) channel current (IKs ). We examined impacts of D85N polymorphism on KCNE1 protein stability and functions, and tested the ability of various drugs to modify them. KCNE1-D85N or the wild-type protein was coexpressed in COS7 cells with KCNQ1 to form K(+) channels. Expression, degradation, and intracellular localization of KCNE1 proteins, as well as the currents conferred by KCNQ1/KCNE1 complexes, were determined using immunoblots, immunofluorescence, and patch-clamp techniques. The protein level of KCNE1-D85N was lower than that of the wild-type, in spite of the comparable levels of their mRNA. KCNE1-D85N was highly ubiquitinated and rapidly degraded as compared to the wild-type; a proteasome inhibitor, MG132, inhibited its degradation and increased its steady-state level. Both KCNE1-D85N and the wild-type proteins were co-immunoprecipitated with KCNQ1. Immunofluorescent signals of KCNE1-D85N accumulated in the endoplasmic reticulum and Golgi apparatus, with reduced levels on the cell membrane. Patch-clamp experiments demonstrated that the membrane current corresponding to IKs was much smaller in cells expressing KCNE1-D85N than in those expressing the wild-type. Verapamil (0.5-10 μM) increased the protein level of KCNE1-D85N, decreased its ubiquitination, slowed its degradation, and enhanced KCNQ1/KCNE1-D85N channel currents. Pretreatment with amiodarone abolished these effects of verapamil. KCNE1-D85N is less stable than the wild-type protein, and is rapidly degraded through the ubiquitin-proteasome system. Verapamil may be of a therapeutic value in LQTS patients via preventing degradation of KCNE1-D85N.
    Pacing and Clinical Electrophysiology 02/2014; · 1.75 Impact Factor
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    ABSTRACT: Abstract Purpose: To examine effects of a long-acting calcium channel blocker (CCB) azelnidipine on uric acid metabolism in hypertensive patients. Methods: Azelnidipine was administered to 72 patients at a daily dose of 8 mg or 16 mg. In 22 cases out of the 72 patients, a different CCB was switched to azelnidipine. Blood pressure was measured and biochemical parameters of blood and urine were evaluated before and 2-3 months after the administration. Results: Azelnidipine significantly decreased both systolic and diastolic blood pressure and the heart rate. It decreased both serum urate levels and the urinary uric acid to creatinine ratio (Uur/Ucr), but did not affect the uric acid clearance to creatinine clearance ratio (Cur/Ccr). Azelnidipine decreased both Uur/Ucr and Cur/Ccr in patients with Uur/Ucr ≥0.5 or ≥0.34, although it did not change these clearance parameters in patients with Uur/Ucr <0.5 or <0.34. Azelnidipine decreased the serum urate levels and Uur/Ucr in hyperuricemic patients with uric acid levels ≥7.0 mg/dL in males and ≥6.0 mg/dL in females. It did not change these parameters in normouricemic patients with serum urate levels <7.0 mg/dL in males and <6.0 mg/dL in females. Azelnidipine decreased Uur/Ucr and Cur/Ccr in hyperuricemic patients with normal or overexcretion of uric acid, although it did not change these clearance parameters in hyperuricemic patients with uric acid hypoexcretion. Conclusions: Azelnidipine decreased the serum urate acid levels and Uur/Ucr, and this response was most prominent in hyperuricemic patients or patients with normal and overexcretion of uric acid.
    Clinical and Experimental Hypertension 01/2014; · 1.46 Impact Factor
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    ABSTRACT: We examined the role of Hsp90 in expression and maturation of wild-type and mutant HERG proteins by using Hsp90 inhibitors, geldanamycine (GA) and radicicol, and Hsp90 overexpression.Methods and ResultsThe proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analyzed by Western blotting, immunoprecipitation, immunofluorescence and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harboring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the wild-type; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for LQT2 caused by trafficking abnormalities of HERG proteins.
    Cardiovascular Research 08/2013; · 5.81 Impact Factor
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    ABSTRACT: Therapeutic angiogenesis has emerged as one of the most promising therapies for severe ischemic cardiovascular diseases with no optional therapy. Several investigators have reported that transplantation of cultured adipose-derived regenerative cells (cADRCs) to ischemic tissues promotes neovascularization and blood perfusion recovery; however, cell therapy using cultured cells has several restrictions. To resolve this problem, the angiogenic capacity of freshly isolated ADRCs (fADRCs) obtained from Lewis rats was compared with cADRCs, both in vivo and in vitro. Flow cytometric analysis showed that fADRCs contained several cell types such as endothelial progenitor cells and endothelial cells; however, these cells were present in a very small proportion in cADRCs. Transplantation of fADRCs in mice significantly improved blood perfusion, capillary density, and production of several angiogenic factors in transplanted ischemic limbs compared with a saline-injected group, whereas these effects were not observed in the cADRCs-injected group. fADRCs also showed significantly higher expression levels of angiogenic factors than cADRCs in the in vitro study. Furthermore, fADRC stimulated tube formation more remarkably than cADRC in an in vitro tube formation assay. These results suggested that fADRCs have an effective angiogenic capacity, and they would be more valuable as a source for cell-based therapeutic angiogenesis than cADRCs or other stem/progenitor cells.
    Biomedical Research 01/2013; 34(1):23-9. · 1.15 Impact Factor
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    ABSTRACT: It is unknown whether salicylate enhances the action of antiarrhythmic agents on human Na+ channels with state dependency and tissue specificity. We therefore investigated effects of salicylate on quinidine-induced block of human cardiac and skeletal muscle Na+ channels. Human cardiac wild-type (hH1), LQT3-related mutant (ΔKPQ), and skeletal muscle (hSkM1) Na+ channel α subunits were expressed in COS7 cells. Effects of salicylate on quinidine-induced tonic and use-dependent block of Na+ channel currents were examined by the whole-cell patch-clamp technique. Salicylate enhanced the quinidine-induced tonic and use-dependent block of both hH1 and hSkM1 currents at a holding potential (HP) of -100 mV but not at -140 mV. Salicylate decreased the IC50 value for the quinidine-induced tonic block of hH1 at an HP of -100 mV, and produced a negative shift in the steady-state inactivation curve of hH1 in the presence of quinidine. According to the modulated receptor theory, it is probable that salicylate decreases the dissociation constant for quinidine binding to inactivated-state channels. Furthermore, salicylate significantly enhanced the quinidine-induced tonic and use-dependent block of the peak and steady-state ΔKPQ channel currents. The results suggest that salicylate enhances quinidine-induced block of Na+ channels via increasing the affinity of quinidine to inactivated state channels.
    Biomedical Research 10/2011; 32(5):303-12. · 1.15 Impact Factor
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    ABSTRACT: Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K(v)1.5, Na(v)1.5, Hcn4, Ca(v)3.2, Ca(v)1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca(2+) transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca(2+) transients were recapitulated by a simulation model. Although a Ca(2+) antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca(2+) transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.
    Journal of Molecular Biology 09/2011; 413(4):857-78. · 3.91 Impact Factor
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    ABSTRACT: Nitric oxide (NO) is an important modulator of cardiovascular function. In this study, we examined whether cytosolic phospholipase A2α (cPLA2α), an initial enzyme in the arachidonic acid pathway, is involved in blood pressure (BP) elevation in a murine model of chronic NO inhibition. cPLA2α gene-deficient mice (cPLA2α-/-) and wild-type mice (WT) were administered the NO synthesis inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) for 4 weeks. Before treatment, BP was comparable in both groups; it increased significantly in the WT but not in the cPLA2α-/- after treatment. Bone marrow transplantation experiments showed that cPLA2α in blood cells and plasma eicosanoid concentrations were not involved in BP elevation by L-NAME treatment. Activation of cPLA2α and subsequent production of eicosanoids in the aortic endothelium but not in aortic smooth muscle cell, heart, or kidney was observed after L-NAME treatment. Aortic ring assays revealed that endothelial function was comparable in both groups of mice before treatment. L-NAME treatment disturbed endothelial function in WT but not in cPLA2α-/-. These results suggest that endothelial cPLA2α may play a principal role in L-NAME-induced hypertension and may be a target molecule for maintaining endothelial function under NO inhibition.
    Arteriosclerosis Thrombosis and Vascular Biology 02/2011; 31(5):1133-8. · 6.34 Impact Factor
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    ABSTRACT: The human ether-a-go-go-related gene (hERG) encodes the α subunit of the potassium current I(Kr). It is highly expressed in cardiomyocytes and its mutations cause long QT syndrome type 2. Heat shock protein (Hsp)70 is known to promote maturation of hERG. Hsp70 and heat shock cognate (Hsc70) 70 has been suggested to play a similar function. However, Hsc70 has recently been reported to counteract Hsp70. We investigated whether Hsc70 counteracts Hsp70 in the control of wild-type and mutant hERG stability. Coexpression of Hsp70 with hERG in HEK293 cells suppressed hERG ubiquitination and increased the levels of both immature and mature forms of hERG. Immunocytochemistry revealed increased levels of hERG in the endoplasmic reticulum and on the cell surface. Electrophysiological studies showed increased I(Kr). All these effects of Hsp70 were abolished by Hsc70 coexpression. Heat shock treatment of HL-1 mouse cardiomyocytes induced endogenous Hsp70, switched mouse ERG associated with Hsc70 to Hsp70, increased I(Kr), and shortened action potential duration. Channels with disease-causing missense mutations in intracellular domains had a higher binding capacity to Hsc70 than wild-type channels and channels with mutations in the pore region. Knockdown of Hsc70 by small interfering RNA or heat shock prevented degradation of mutant hERG proteins with mutations in intracellular domains. These results indicate reciprocal control of hERG stability by Hsp70 and Hsc70. Hsc70 is a potential target in the treatment of LQT2 resulting from missense hERG mutations.
    Circulation Research 02/2011; 108(4):458-68. · 11.09 Impact Factor
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    ABSTRACT: A combination therapy of a low-dose antihypertensive diuretic with an angiotensin II receptor blocker (ARB) may have unfavorable effects on serum urate levels. Forty-two hypertensive patients without hyperuricemia (18 men and 24 women, mean age 65 years) were randomly divided into three groups. Each of the group was allocated to a combination therapy with losartan (LOS; CAS 124750-99-8; 50 mg/day)/hydrochlorothiazide (HCTZ; CAS 58-93-5; 12.5 mg/day) (LOS/HCTZ group), telmisartan (TEL; CAS 144701-48-4; 40 mg/day)/HCTZ (12.5 mg/day) (TEL/HCTZ group), or candesartan (CND; CAS 145040-37-5; 8 mg/day)/HCTZ (12.5 mg/day) (CND/HCTZ group), respectively. Before and after the treatment, blood pressure and biochemical parameters of blood and urine were evaluated. Both systolic and diastolic blood pressures significantly decreased in all groups (p < 0.01) without any statistical differences. The LOS/HCTZ group showed no changes in serum urate levels (5.8 +/- 1.0 mg/dl to 5.8 +/- 1.4 mg/dl) and in % fractional excretion of urate (FEUA). In the TEL/HCTZ group, the serum urate level was significantly increased, from 5.5 +/- 0.9 mg/dl to 6.5 +/- 1.2 mg/dl (p < 0.01), whereas FEUA significantly decreased (p < 0.01). Similarly, the CND/HCTZ group showed a significant increase in the serum urate level from 5.4 +/- 0.9 mg/dl to 6.0 +/- 1.2 mg/dl (p < 0.01) and a significant decrease in FEUA (p < 0.01). No significant differences were found in fasting plasma glucose and electrolytes levels in any of the groups. A combination therapy with a low-dose HCTZ and ARBs resulted in reduced urate excretion and elevated serum urate levels. A combination therapy with the ARB losartan was not accompanied with these effects, likely because of its inhibitory action on urate transporter 1. The study limitations deserve mention in consideration of ethic restrictions, small size, short term examination and uncontrolled design.
    Arzneimittel-Forschung 01/2010; 60(2):71-5. · 0.51 Impact Factor
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    ABSTRACT: Cyclooxygenase inhibitors are often prescribed to relieve severe ischemic leg pain in critical ischemic limb patients. Prescription of high doses of aspirin and selective cyclooxygenase-2 inhibitors is reported to increase cardiovascular events through suppression of the vasodilative prostanoid prostaglandin I(2) in endothelium. Here, we evaluated the influence of aspirin and etodolac, a selective cyclooxygenase-2 inhibitor, on neovascularization using a murine ischemia hind limb model. C57BL/6J mice were treated with aspirin or etodolac for twenty-eight days after induction of ischemia. We exploited a concentration of the agents that suppressed cyclooxygenase activity efficiently, especially in prostaglandin I(2) production. Recovery of limb blood perfusion and capillary density in ischemic limbs was significantly suppressed by etodolac treatment when compared to the aspirin treated group and untreated group. Production of 6-keto prostaglandin F(1alpha) and prostaglandin E(2) was lower in the aspirin treated group when compared with the etodolac-treated group. Also, these concentrations were lower in both treatment groups compared with the untreated group. Immunohistochemical analysis suggested cyclooxygenase-2 was expressed in endothelium but not in inflammatory cells in ischemic tissue from the acute to chronic phase. Cyclooxygenase-1 was expressed strongly in inflammatory cells in the acute phase. Furthermore, bone marrow-derived mononuclear cell transplantation improved neovascularization, whereas aspirin and etodolac did not inhibit these effects. Production of arachidonic acid metabolites by transplanted cells was independent of the improvement of neovascularization. In conclusion, cyclooxygenase-2 inhibition reduces ischemia-induced neovascularization.
    European journal of pharmacology 10/2009; 627(1-3):223-8. · 2.59 Impact Factor
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    ABSTRACT: Autologous cell implantation and angiogenic gene therapy have been evaluated in critical limb ischemic patients. Here, we compared the features of these strategies individually and in combination. C57BL/6J mice with ischemic hindlimbs were injected with adherent mononuclear cells (aMNCs) from bone marrow or adenovirus encoding the hepatocyte growth factor (HGF) gene (Ad-HGF). Under comparable angiogenic conditions, 10 x 10(5) aMNCs produced significantly higher amounts of VEGF and FGF-2 and stimulated the number of arterioles in ischemic muscle compared with 1 x 10(8) plaque-forming units (pfu) of Ad-HGF. Ad-HGF produced 10 times more HGF in ischemic muscle compared with aMNCs. Injection of 0.3 x 10(5) aMNCs previously transfected with Ad-HGF (aMNC/Ad-HGF) increased blood flow and elevated the numbers of capillaries and arterioles to levels comparable with that seen with 10 x 10(5) aMNCs or 1 x 10(8) pfu of Ad-HGF. Hypoxic conditions induced the apoptotic death of aMNCs. However, coincubation with HGF or aMNC/Ad-HGF protected cells against apoptosis. HGF stimulated the migration of aMNCs, and the migration capacity of the aMNC/Ad-HGF group was significantly higher than that in the aMNC/Ad-LacZ group. In conclusion, cell-based HGF gene therapy decreased the number of cells required for neovascularization. This strategy can be an effective angiogenic therapy.
    AJP Heart and Circulatory Physiology 09/2009; 297(4):H1329-36. · 4.01 Impact Factor
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    ABSTRACT: Fenofibrate have been illustrated to stimulate nitric oxide (NO) pathway, which plays pivotal roles in neovascularization. Here, we evaluated the effect of fenofibrate on neovascularization using a murine ischemic hindlimb model. C57BL/6J mice were treated with fenofibrate and/or NG-nitro-l-arginine methyl ester hydrochloride (l-NAME) for 28 days after ischemia operation. We exploited a concentration of L-NAME that did not affect blood pressure levels but suppress NO activity. Limb blood perfusion and capillary density in ischemic limb, serum NO levels, and aortic NOS activity were significantly increased by fenofibrate treatment when compared with the untreatment group. And, these effects were abolished by coadministration of L-NAME. Fenofibrate treatment significantly lowered serum triglyceride levels. Cotreatment of L-NAME did not inhibit serum triglyceride level, lowering effect of fenofibrate. These results suggested that the lowering in serum triglyceride levels is not involved in the improvement of neovascularization. In an in vitro experiment, fenofibrate stimulated NOS activity in human umbilical vein endothelial cells. Also, fenofibrate stimulated in vitro angiogenesis, and this effect was abolished by coincubation with L-NAME. In conclusions, fenofibrate enhanced neovascularization in a murine hindlimb ischemia model. The mechanism is most likely through activation of NO pathway in endothelial cells.
    Journal of cardiovascular pharmacology 09/2009; 54(5):399-404. · 2.83 Impact Factor
  • CVD Prevention and Control 05/2009; 4.
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    ABSTRACT: We investigated the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the stability of Kv1.5 channel protein. The expression and function of Kv1.5 (Kv1.5-FLAG) in transfected African green monkey kidney fibroblast cells as well as rat atrium were estimated by immunoblotting, immunoprecipitation, immunofluorescence and patch-clamp techniques. Both EPA and DHA immediately blocked Kv1.5 channel current in a dose-dependent manner, accompanied by reduction of their phosphorylation. Chronic treatment (for 12 h) with EPA at lower concentrations (0.3-10 muM) increased the level of Kv1.5-FLAG protein as well as Kv1.5 channel current without changes in its gating kinetics, prolonging its half-life; in contrast, both EPA and DHA at higher concentrations (30-100 muM) decreased the expression of Kv1.5-FLAG. EPA at the higher concentrations also decreased mRNA of Kv1.5 and synapse-associated protein 97 expression. EPA at the lower concentrations increased Kv1.5 expression in the endoplasmic reticulum, Golgi apparatus and cell membrane. EPA-induced increase of Kv1.5 channel expression and current was abolished by pretreatment with the protein transport inhibitor brefeldin A or colchicines, and by the Kv1.5 channel blocker 4-aminopyridine. Oral administration of EPA (30 mg/kg) increased the level of endogenous Kv1.5 in rat atria. These results indicate that chronic treatment with EPA at lower concentrations stabilizes Kv1.5 channel protein in the endoplasmic reticulum and Golgi apparatus thereby enhancing the Kv1.5 channel current on the cell membrane.
    European journal of pharmacology 01/2009; 604(1-3):93-102. · 2.59 Impact Factor
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    ABSTRACT: Pendrin is an apical protein of thyroid follicular cells, responsible for the efflux of iodide into the follicular lumen via an iodide-chloride transport mechanism. It is unknown whether pendrin is recognized by autoantibodies. Our objective was to examine the prevalence of pendrin antibodies in autoimmune thyroid diseases and compare with that of thyroglobulin, thyroperoxidase, TSH receptor, and sodium iodide symporter antibodies. In a prevalent case-control study, we analyzed the sera of 140 autoimmune thyroid disease cases (100 with Graves' disease and 40 with Hashimoto's thyroiditis) and 80 controls (50 healthy subjects, 10 patients with papillary thyroid cancer, 10 with systemic lupus erythematosus, and 10 with rheumatoid arthritis). Pendrin antibodies were measured by immunoblotting using extract of COS-7 cells transfected with pendrin and a rabbit polyclonal pendrin antibody. Pendrin antibodies were found in 81% of the cases and 9% of controls (odds ratio = 44; P < 0.0001). Among cases, pendrin antibodies were more frequent and of higher titers in Hashimoto's thyroiditis than in Graves' disease. Pendrin antibodies correlated significantly with thyroglobulin, thyroperoxidase, and sodium iodide symporter antibodies but not with TSH receptor antibodies. Pendrin antibodies were equally effective as thyroglobulin and thyroperoxidase antibodies in diagnosis of autoimmune thyroid diseases, especially Hashimoto's thyroiditis. The study identifies pendrin as a novel autoantigen recognized by patients with autoimmune thyroid diseases and proposes the use of pendrin antibodies as an accurate diagnostic tool.
    Journal of Clinical Endocrinology &amp Metabolism 01/2009; 94(2):442-8. · 6.31 Impact Factor
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    ABSTRACT: The ubiquitin-proteasome system is responsible for the disappearance of truncated cardiac myosin-binding protein C, and the suppression of its activity contributes to cardiac dysfunction. This study investigated whether missense cardiac myosin-binding protein C gene (MYBPC3) mutation in hypertrophic cardiomyopathy (HCM) leads to destabilization of its protein, causes UPS impairment, and is associated with cardiac dysfunction. Mutations were identified in Japanese HCM patients using denaturing HPLC and sequencing. Heterologous expression was investigated in COS-7 cells as well as neonatal rat cardiac myocytes to examine protein stability and proteasome activity. The cardiac function was measured using echocardiography. Five novel MYBPC3 mutations -- E344K, DeltaK814, Delta2864-2865GC, Q998E, and T1046M -- were identified in this study. Compared with the wild type and other mutations, the E334K protein level was significantly lower, it was degraded faster, it had a higher level of polyubiquination, and increased in cells pretreated with the proteasome inhibitor MG132 (50 microM, 6 h). The electrical charge of its amino acid at position 334 influenced its stability, but E334K did not affect its phosphorylation. The E334K protein reduced cellular 20 S proteasome activity, increased the proapoptotic/antiapoptotic protein ratio, and enhanced apoptosis in transfected Cos-7 cells and neonatal rat cardiac myocytes. Patients carrying the E334K mutation presented significant left ventricular dysfunction and dilation. The conclusion is the missense MYBPC3 mutation E334K destabilizes its protein through UPS and may contribute to cardiac dysfunction in HCM through impairment of the ubiquitin-proteasome system.
    Journal of Molecular Biology 11/2008; 384(4):896-907. · 3.91 Impact Factor
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    ABSTRACT: The aim of this study was to elucidate the mechanisms for regulations of cardiac Kv1.5 channel expression. We particularly focused on the role of heat shock proteins (Hsps). We tested the effects of Hsps on the stability of Kv1.5 channels using biochemical and electrophysiological techniques: co-expression of Kv1.5 and Hsp family proteins in mammalian cell lines, followed by Western blotting, immunoprecipitation, pulse-chase analysis, immunofluorescence and whole-cell patch clamp. Hsp70 and heat shock factor 1 increased the expression of Kv1.5 protein in HeLa and COS7 cells, whereas either Hsp40, 27 or 90 did not. Hsp70 prolonged the half-life of Kv1.5 protein. Hsp70 was co-immunoprecipitated and co-localized with Kv1.5-FLAG. Hsp70 significantly increased the immunoreactivity of Kv1.5 in the endoplasmic reticulum, Golgi apparatus and on the cell membrane. Hsp70 enhanced Kv1.5 current of transfected cells, which was abolished by pretreatment with brefeldin A or colchicine. Thus, Hsp70, but not other Hsps, stabilizes functional Kv1.5 protein.
    Biochemical and Biophysical Research Communications 09/2008; 372(3):469-74. · 2.28 Impact Factor
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    ABSTRACT: Changes in the expression of hyperpolarization-activated cyclic nucleotide (HCN)-gated channels and I(f) currents during the differentiation of embryonic stem cells into cardiac cells remain unknown. We examined changes of HCN genes in expression and function during the differentiation of Nkx2.5-positive cardiac precursor cells derived from mouse ES cells using cell sorting, RTPCR, immunofluorescence and whole cell patch-clamp techniques. Cs(+)-induced inhibition of automaticity and transcription of HCN genes increased during differentiation. Expressions of Nkx2.5, a marker of cardiac progenitor cell, and Flk1, a marker of hemangioblast, were mutually exclusive. Messenger RNA and proteins encoded by HCN1 and 4 genes were predominantly observed in Nkx2.5-positive cells on day 15, although Flk1-positive cells did not express genes of the HCN family on that day. Cs(+)-induced prolongation of the cycle of spontaneous action potentials and I(f) currents were predominantly observed on day 15. These results suggested that a fraction of Nkx2.5-positive cardiac precursor cells was committed to pacemaking cells expressing I(f) channels predominantly encoded by HCN 1 and 4 genes.
    Biomedical Research 09/2008; 29(4):195-203. · 1.15 Impact Factor
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    ABSTRACT: The angiotensin receptor blocker losartan inhibited urate transporter 1 (URAT1) according to in vitro experiments. However, it is still unknown whether the inhibitory effect of losartan on URAT1 contributes to its uricosuric action in humans. Thirty-two patients with hypertension and nine patients with idiopathic renal hypouricemia (five with and four without hypertension) were enrolled for this study. Hypertensive patients were prescribed oral losartan (50 mg/day, n = 16) or candesartan (8 mg/day, n = 16). Before and after 1-month treatment, the serum concentration of urate (Sur) and creatinine (Scr), and the clearance value of urate (Cur) and creatinine (Ccr) were determined. Clearance studies using the URAT1 inhibitor benzbromarone (100 mg/day) or losartan (50 mg/day) loading test were also performed in these patients. Blood pressure (BP) significantly decreased in the patients treated with either losartan or candesartan. Losartan significantly reduced Sur, which was associated with a concomitant increase in the Cur/Ccr ratio, whereas candesartan did not alter these parameters. In hypertensive patients with loss-of-function mutation of URAT1, losartan did not alter either Sur or Cur/Ccr, nor did benzbromarone. The lack of effect of URAT1 inhibitors on renal excretion of urate was independent of the renal function of hypouricemic patients. On the other hand, both losartan and benzbromarone increased Cur/Ccr ratio in hypertensive patients harboring the wild URAT1 gene, regardless of the presence of hypouricemia. These findings suggested that losartan inhibited URAT1 and thereby it lowered Sur levels in hypertensive patients.
    American Journal of Hypertension 08/2008; 21(10):1157-62. · 3.40 Impact Factor

Publication Stats

367 Citations
138.56 Total Impact Points


  • 2002–2014
    • Tottori University
      • • Faculty of Medicine
      • • Department of Genetic Medicine and Regenerative Therapeutics
      TTJ, Tottori, Japan
  • 2007
    • National Cerebral and Cardiovascular Center
      • Department of Cardiovascular Medicine
      Ōsaka, Ōsaka, Japan