Research Achievements - Original paper -
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Inhalation anesthesia is preferable for recording rat cardiac function using an electrocardiogram.
Manabu Murakami, Hidetoshi Niwa, Tetsuya Kushikata, Hiroyuki Watanabe, Kazuyoshi Hirota, Kyoichi Ono, Takayoshi Ohba
Biological & pharmaceutical bulletin 37 ( 5 ) 834 - 9 2014 [Refereed]
Research paper (journal)
The effects of inhalation anesthesia (2% isoflurane, sevoflurane, or enflurane) and intraperitoneal anesthesia with pentobarbital (65 mg/kg) were compared in rats using an electrocardiogram (ECG) and determination of blood oxygen saturation (SPO2) levels. Following inhalation anesthesia, heart rate (HR) and SPO2 were acceptable while pentobarbital anesthesia decreased HR and SPO2 significantly. This indicates that inhalation anesthesia is more preferable than pentobarbital anesthesia when evaluating cardiovascular factors. Additionally, pentobarbital significantly increased HR variability (HRV), suggesting a regulatory effect of pentobarbital on the autonomic nervous system, and resulted in a decreased response of the baro-reflex system. Propranolol or atropine had limited effects on ECG recording following pentobarbital anesthesia. Taken together, these data suggest that inhalation anesthesia is suitable for conducting hemodynamic analyses in the rat.
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Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy.
Hironori Kiso, Takayoshi Ohba, Kenji Iino, Kazuhiro Sato, Yutaka Terata, Manabu Murakami, Kyoichi Ono, Hiroyuki Watanabe, Hiroshi Ito
Biochemical and biophysical research communications 436 ( 3 ) 514 - 8 2013.07 [Refereed]
Research paper (journal)
BACKGROUND: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. METHODS AND RESULTS: In cultured neonatal rat cardiomyocytes, a 48 h treatment with 10nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. CONCLUSIONS: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression.
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The mechanism of increased postnatal heart rate and sinoatrial node pacemaker activity in mice.
Takeshi Adachi, Shigehiro Shibata, Yosuke Okamoto, Shinichi Sato, Susumu Fujisawa, Takayoshi Ohba, Kyoichi Ono
The journal of physiological sciences : JPS 63 ( 2 ) 133 - 46 2013.03 [Refereed]
Research paper (journal)
Heart rate (HR) of mammalian species changes postnatally, i.e., HR of large animals including humans decreases, while HR in small animals such as mice and rats increases. To clarify cellular mechanisms underlying the postnatal HR changes, we performed in vivo HR measurement and electrophysiological analysis on sinoatrial node (SAN) cells in mice. The in vivo HR was ~320 beats min(-1) (bpm) immediately after birth, and increased with age to ~690 bpm at postnatal day 14. Under blockage of autonomic nervous systems, HR remained constant until postnatal day 5 and then increased day by day. The spontaneous beating rate of SAN preparation showed a similar postnatal change. The density of the L-type Ca(2+) current (LCC) was smaller in neonatal SAN cells than in adult cells, accompanied by a positive shift of voltage-dependent activation. Thus, the postnatal increase in HR is caused by both the increased sympathetic influence and the intrinsic activity of SAN cells. The different conductance and kinetics of LCC may be involved in the postnatal increase in pacemaker activity.
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Cytosolic Ca2+-induced apoptosis in rat cardiomyocytes via mitochondrial NO-cGMP-protein kinase G pathway.
Kazuhiko Seya, Kyoichi Ono, Susumu Fujisawa, Ken Okumura, Shigeru Motomura, Ken-ichi Furukawa
The Journal of pharmacology and experimental therapeutics 344 ( 1 ) 77 - 84 2013.01 [Refereed]
Research paper (journal)
Previously, we showed that in adult rat cardiomyocytes, nitric oxide (NO) donors stimulate mitochondrial cGMP production, followed by cytochrome c release, independently of the mitochondrial permeable transition pore. We investigated whether mitochondrial cGMP-induced cytochrome c release from cardiac mitochondria is Ca(2+)-sensitive. Mitochondria and primary cultured cardiomyocytes were prepared from left ventricles of male Wistar rats. The cytosolic Ca(2+) concentration was adjusted with Ca(2+)-EGTA buffers. Cytochrome c released from mitochondria was measured by Western blotting. Cardiomyocyte apoptosis was assessed by Annexin V staining. Cytochrome c release from cardiac mitochondria was evoked by buffered Ca(2+) (1 μM); this was inhibited by NO-cGMP pathway inhibitors such as N(G)-monomethyl-l-arginine monoacetate (inhibitor of NO synthase), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NO scavenger), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, NO-sensitive guanylyl cyclase inhibitor) and voltage-dependent anion channel (VDAC) inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, but not by cyclosporin A (mitochondrial permeable transition pore inhibitor). Furthermore, this release was significantly and dose dependently inhibited by 0.3-3 μM KT5823 (protein kinase G inhibitor). At the cellular level, intracellular perfusion of cardiomyocytes with buffered Ca(2+) (1 μM) also induced apoptosis, which was inhibited in the presence of ODQ. A membrane-permeable cGMP analog, 8-Br-cGMP, but not cGMP itself, mimicked buffered Ca(2+) actions in both cardiac mitochondria and cardiomyocytes. We further confirmed an increase in protein kinase G activity by adding cGMP in mitochondrial protein fraction. Our results suggest that mitochondrial NO-cGMP pathway-induced cytochrome c release from cardiac mitochondria, triggered by increased cytosolic Ca(2+), occurs through VDAC via the stimulation of an undiscovered mitochondrial protein kinase G.
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Enhancement of liver regeneration by adenosine triphosphate-sensitive K⁺ channel opener (diazoxide) after partial hepatectomy.
Yasuhiko Nakagawa, Masato Yoshioka, Yuki Abe, Hiroshi Uchinami, Takayoshi Ohba, Kyoichi Ono, Yuzo Yamamoto
Transplantation 93 ( 11 ) 1094 - 100 2012.06 [Refereed]
Research paper (journal)
BACKGROUND: Enhancement of liver regeneration is a matter of importance after partial liver transplantation including small-for-size grafting. Mitochondrial adenosine triphosphate (ATP)-sensitive K⁺ (mitoKATP) channel plays an important role in mitochondrial bioenergetics, which is a prerequisite for liver regeneration. However, the ATP-sensitive K⁺ (KATP) channel in hepatocytes is incompletely understood. We investigated the KATP channel in hepatocytes and examined the effects of diazoxide, a potent KATP channel opener, on liver regeneration using a rat model. METHODS: Using rat primary hepatocytes, expression and localization of KATP channel subunits, Kir6.x and sulfonylurea receptor (SUR)x, were studied by polymerase chain reaction, Western blotting, and immunostaining. To investigate the role of KATP channel openers in liver regeneration, we allocated rats into four groups: control (vehicle) (n=24), diazoxide (n=24), vehicle plus channel blocker (n=6), and diazoxide plus channel blocker (n=6) groups. After 70% partial hepatectomy, hepatic tissue ATP levels, liver-to-body weight ratio, and proliferation rate of hepatocytes were examined. RESULTS: KATP channel subunits, Kir6.1 and SUR1, were detected on hepatic mitochondria. During liver regeneration, liver-to-body weight ratio, proliferation rate of hepatocytes, and the hepatic ATP level were significantly higher in the diazoxide group than the control group at 2 days after partial hepatectomy. These effects of diazoxide were neutralized by a KATP channel blocker. CONCLUSIONS: We demonstrated the existence of a mitoKATP channel in hepatocytes composed of Kir6.1 and SUR1. Diazoxide could enhance liver regeneration by keeping a higher ATP content of the liver tissue. These results suggest that diazoxide will sustain the mitochondrial energetics through the mitoKATP channel opening.
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Arrhythmogenic coupling between the Na+ -Ca2+ exchanger and inositol 1,4,5-triphosphate receptor in rat pulmonary vein cardiomyocytes.
Yosuke Okamoto, Makoto Takano, Takayoshi Ohba, Kyoichi Ono
Journal of molecular and cellular cardiology 52 ( 5 ) 988 - 97 2012.05 [Refereed]
Research paper (journal)
Atrial fibrillation, the most common sustained arrhythmia, is believed to be triggered by ectopic electrical activity originating in the myocardial sleeves surrounding the pulmonary veins (PVs). It has been reported that myocardial sleeves have the potential to generate automaticity in response to norepinephrine. This study investigated the cellular mechanisms underlying norepinephrine-induced automaticity in PV cardiomyocytes isolated from rats. Application of 10 μM norepinephrine to PV cardiomyocytes induced repetitive and transient increases in intracellular Ca(2+) concentrations. The Ca(2+) transient was accompanied by depolarization, and induced automatic rhythmic action potentials at approximately 4Hz in perforated patch clamp preparations in 27% of myocytes were observed. When the recording mode was switched from current-clamp to voltage-clamp mode during the continuous presence of automaticity, an oscillatory current was observed. The oscillatory current was always inward, irrespective of the membrane potential, indicating that the current was derived mainly from the Na(+)-Ca(2+) exchanger (NCX). The norepinephrine-induced automaticity was suppressed by blocking either the β(1)- or α(1)-adrenoceptor. Additionally, this automaticity was blocked by inhibitors of phospholipase C and the inositol 1,4,5-triphosphate receptor (IP(3)R) but not by a protein kinase C inhibitor. We observed that the transverse-tubule system was enriched in cardiomyocytes in the PV, in contrast to those of the atrium, and that the NCX and IP(3)R were co-localized along transverse tubules. These findings suggest that a functional coupling between the NCX and IP(3)R causes arrhythmic excitability of the PV during the presence of combined β(1)- and α(1)-adrenoceptor stimulation.
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Direct effects of esmolol and landiolol on cardiac function, coronary vasoactivity, and ventricular electrophysiology in guinea-pig hearts.
Shigehiro Shibata, Yosuke Okamoto, Shigeatsu Endo, Kyoichi Ono
Journal of pharmacological sciences 118 ( 2 ) 255 - 65 2012 [Refereed]
Research paper (journal)
The ultra-short acting, selective β(1)-adrenergic antagonists landiolol and esmolol are widely used perioperatively; however, little is known about their acute direct actions on the heart. The current study utilized the Langendorff perfused heart system to measure changes in cardiac function and hemodynamics in response to each drug. Furthermore, electrophysiological analysis was performed on isolated ventricular myocytes. Direct application of esmolol significantly decreased systolic left ventricular pressure and heart rate at concentrations > 10 µM, while it dose-dependently increased coronary perfusion pressure. Esmolol also shortened the action potential duration (APD) in a concentration-dependent manner, an action maintained even when the delayed rectifier K(+) current or ATP sensitive K(+) current was blocked. Moreover, esmolol inhibited both the inward rectifier K(+) current (I(K1)) and the L-type Ca(2+) current (I(CaL)) and increased the outward current dose-dependently. In contrast, landiolol had minimal cardiac effects. In the Kyoto Model computer simulation, inhibition of either I(K1) or I(CaL) alone failed to shorten the APD; however, an additional increase in the time-independent outward current caused shortening of the APD, equal to that induced by esmolol. In conclusion, esmolol directly inhibits cardiac performance significantly more so than landiolol, an effect revealed to be at least in part mediated by esmolol-induced APD shortening.
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Cardiac T-type Ca(2+) channels in the heart.
Kyoichi Ono, Toshihiko Iijima
Journal of molecular and cellular cardiology 48 ( 1 ) 65 - 70 2010.01 [Refereed] [Invited]
Research paper (journal)
Two different Ca(2+) channels exist in cardiac myocytes. While the L-type Ca(2+) channel is ubiquitous and the main source of Ca(2+) for excitation-contraction coupling and pacemaker activity, the functional role of the T-type Ca(2+) channel is diverse and depends on mammalian species, heart region, age and various cardiac diseases. Two isoforms of T-type Ca(2+) channel proteins in the heart, Ca(V)3.1 and Ca(V)3.2, are functionally expressed in embryonic hearts, but markedly diminish during development. In the adult heart, the T-type Ca(2+) channel is almost undetectable in ventricular myocytes and is most prevalent in the conduction system, playing a functional role in facilitating pacemaker depolarization of the sinoatrial node. Interestingly, the T-type Ca(2+) channel is re-expressed in atrial and ventricular myocytes under various pathological conditions such as hypertrophy and heart failure, and contributes to abnormal electrical activity and excitation-contraction coupling, but the T-type channel provides a smaller contribution to the trigger for Ca(2+) release than does the L-type Ca(2+) channel. Instead, the T-type Ca(2+) channel has been shown to play a crucial role in the process of pathological cardiac hypertrophy. Increased Ca(2+) influx via Ca(V)3.2, the T-type Ca(2+) channel, induces calcineurin/NFAT (nuclear factor of activated T-cell) hypertrophic signaling. Furthermore, new evidence has been accumulating on the regulatory mechanism of T-type Ca(2+) channel expression, including the neuron restrictive silencer element-neuron restrictive silencer factor (NRSE-NRSF) system, mitogen activated protein (MAP) kinases and cardiac homeobox transcription factor Csx/Nkx2.5. This review summarizes our present knowledge regarding cardiac T-type Ca(2+) channels, and discusses their pathophysiological significance in the heart.
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Assessment of a new piezoelectric transducer sensor for noninvasive cardiorespiratory monitoring of newborn infants in the NICU.
Shinichi Sato, Wako Ishida-Nakajima, Akira Ishida, Masanari Kawamura, Shinobu Miura, Kyoichi Ono, Nobuya Inagaki, Goro Takada, Tsutomu Takahashi
Neonatology 98 ( 2 ) 179 - 90 2010 [Refereed]
Research paper (journal)
BACKGROUND: Electrocardiogram (ECG) and impedance pneumography (IPG), the most widely used techniques for cardiorespiratory monitoring in the neonatal intensive care unit (NICU), have the disadvantage of causing skin damage when used for very premature newborn infants. To prevent skin damage, we designed a new piezoelectric transducer (PZT) sensor. OBJECTIVE: To assess the potential of the PZT sensor for cardiorespiratory monitoring in the NICU. METHODS: The PZT sensor was placed under a folded towel under a neonate to detect an acoustic cardiorespiratory signal, from which heart rate (HR) and breathing rate (BR) were calculated, together with simultaneous ECG/IPG recording for 1-9 days for long and brief (1-min) assessment. RESULTS: The brief assessment showed average correlation coefficients of 0.92 +/- 0.12 and 0.95 +/- 0.02 between instantaneous HRs/BRs detected by the PZT sensor and ECG/IPG in 27 and 11 neonates examined. During the long assessment, the HR detection rate by the PZT sensor was approximately 10% lower than that by ECG (82.6 +/- 12.9 vs. 91.8 +/- 4.1%; p = 0.001, n = 27), although comparable (90.3 +/- 4.1 vs. 92.5 +/- 3.4%, p = 0.081) in approximately 70% (18/27) of neonates examined; BR detection rate was comparable between the PZT sensor and IPG during relatively stable signal conditions (95.9 +/- 4.0 vs. 95.3 +/- 3.5%; p = 0.38, n = 11). The PZT sensor caused neither skin damage nor body movement increase in all neonates examined. CONCLUSION: The PZT sensor is noninvasive and does not cause skin irritation, and we believe it does provide a reliable, accurate cardiorespiratory monitoring tool for use in the NICU, although the issue of mechanical-ventilation noise remains to be solved.
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Rapid increase to double breathing rate appears during REM sleep in synchrony with REM - a higher CNS control of breathing? -.
Shinichi Sato, Takashi Kanbayashi, Hideaki Kondo, Namiko Matsubuchi, Kyoichi Ono, Tetsuo Shimizu
Advances in experimental medicine and biology 669 249 - 52 2010 [Refereed]
Research paper (journal)
Breathing rate (BR) during rapid eye movement (REM) sleep is known to fluctuate largely, while increases in BR during REM sleep reported were small. In our mice experiments, we found that mice exhibit a rapid increase in instantaneous BR (RIBR) of >2 fold during natural sleep with accompanying atonia, laying their sides down. The RIBR was further found in a sleeping mouse attached with EEG electrodes when the EEG amplitude and delta wave power were lower. Therefore, it is likely that mice show RIBRs during REM sleep. Interestingly, similar RIBRs accompanied by atonia and REM burst during REM sleep were also found in humans by standard polysomnographic studies in 11 healthy volunteers (age: 22.3 +/- 2.8) with BR measurement by nasal/oral airflow sensors and chest/abdomen belt sensors. All subjects underwent RIBR of doubled BR at least once a night. As SpO(2) before RIBRs was a level not effective to be a respiratory stimulant (96.7 +/- 1.6 %, n = 63), the RIBR seems to be controlled by higher central nervous system rather than autonomic nervous system control on response to central and peripheral chemical sensors. In fact, tachypnea with suppressed amplitude during RIBR resulted in a slight fall in SpO(2) (96.4 +/- 1.7 %, p = 0.0007). In the present study, RIBRs accompanied by atonia and REM were not necessarily consistent in change in rate and/or amplitude, therefore, these various pattern of RIBRs may be potential indices of dreams with various emotional contents. Analysis of instantaneous BR, thus, may be a helpful tool for understanding the neural control of breathing during REM sleep.
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Essential role of STIM1 in the development of cardiomyocyte hypertrophy.
Takayoshi Ohba, Hiroyuki Watanabe, Manabu Murakami, Takako Sato, Kyoichi Ono, Hiroshi Ito
Biochemical and biophysical research communications 389 ( 1 ) 172 - 6 2009.11 [Refereed]
Research paper (journal)
Store-operated Ca(2+) entry (SOCE) through transient receptor potential (TRP) channels is important in the development of cardiac hypertrophy. Recently, stromal interaction molecule 1 (STIM1) was identified as a key regulator of SOCE. In this study, we examined whether STIM1 is involved in the development of cardiomyocyte hypertrophy. RT-PCR showed that cultured rat cardiomyocytes constitutively expressed STIM1. Endothelin-1 (ET-1) treatment for 48h enhanced TRPC1 expression, SOCE, and nuclear factor of activated T cells activation without upregulating STIM1. However, the knockdown of STIM1 suppressed these effects, thereby preventing a hypertrophic response. These results suggest that STIM1 plays an essential role in the development of cardiomyocyte hypertrophy.
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The pathological role of transient receptor potential channels in heart disease.
Hiroyuki Watanabe, Manabu Murakami, Takayoshi Ohba, Kyoichi Ono, Hiroshi Ito
Circulation journal : official journal of the Japanese Circulation Society 73 ( 3 ) 419 - 27 2009.03 [Refereed]
Research paper (journal)
Transient receptor potential (TRP) channels are expressed in almost every human tissue, including the heart and vasculature. Most are permeable to Ca(2+) and play unique roles as multifunctional cellular sensors. Their involvement in many fundamental cell functions (eg, contraction, proliferation, and cell death) has made investigating their roles in human disease an urgent priority for medical science. This review presents an overview of current knowledge about the pathological role of TRP channels in heart disease and highlights some TRP channels with anticipated roles in disease. Evidence suggests that (a) upregulation of TRPC channels is involved in the development of cardiac hypertrophy and heart failure; (b) TRPC1, TRPC6, and TRPV2 play a role in the pathogenesis of cardiomyopathy associated with muscular dystrophy; (c) TRPC6 or TRPM4 is involved in the delayed after-depolarization; (d) TRPP2 is involved in the normal development of the interventricular and interatrial septa; and (e) neuronal TRPV1 acts as a detector of pain-producing stimuli. Ultimately, TRP channels might become novel pharmacological targets in the treatment of human heart disease.
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Molecular and electrical remodeling of L- and T-type Ca(2+) channels in rat right atrium with monocrotaline-induced pulmonary hypertension.
Takashi Koyama, Kyoichi Ono, Hiroyuki Watanabe, Takayoshi Ohba, Manabu Murakami, Kenji Iino, Hiroshi Ito
Circulation journal : official journal of the Japanese Circulation Society 73 ( 2 ) 256 - 63 2009.02 [Refereed]
Research paper (journal)
BACKGROUND: Atrial arrhythmia is often encountered in chronic pulmonary disease with pulmonary hypertension (PH), but few studies have investigated the electrical remodeling of atrial Ca(2+) channels under PH. METHODS AND RESULTS: Wistar rats were injected with monocrotaline (MCT), resulting in PH with right atrial and ventricular hypertrophy. The L-type Ca(2+) channel current density was significantly decreased in right atrial cells of MCT-treated rats, accompanied by a significant reduction in mRNA expression of the CaV1.2 (alpha(1C)) subunit and accessory beta(2) subunit. Conversely, the low voltage-activated Ca(2+) current was more marked in the right atrial cells of MCT-treated rats than in those of control rats. The current-voltage relationship and the time course of inactivation closely resembled those of T-type Ca(2+) channels, although the current was only slightly inhibited by 10-100 micromol/L Ni(2+). No significant differences were observed in the mRNA expression levels of CaV3.1 (alpha(1G)) and CaV3.2 (alpha(1H)) or the protein level of the CaV3.1 subunit. In left atrial cells, the electrophysiological molecular properties of Ca(2+) channels were unaffected by MCT treatment. CONCLUSIONS: PH causes right atrial hypertrophy, associated with alteration of the electrophysiological molecular properties of Ca(2+) channels.
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Identification and physiological activity of survival factor released from cardiomyocytes during ischaemia and reperfusion.
Yoichi Mizukami, Kyoichi Ono, Cheng-Kun Du, Toshihiko Aki, Naoya Hatano, Yosuke Okamoto, Yasuhiro Ikeda, Hiroshi Ito, Kimikazu Hamano, Sachio Morimoto
Cardiovascular research 79 ( 4 ) 589 - 99 2008.09 [Refereed]
Research paper (journal)
AIMS: We carried out a screening of survival factors released from cells exposed to simulated ischaemia and reperfusion (sI/R) using the embryonic rat heart-derived cell line, H9c2 cells, and examined the physiological role of the identified factor. METHOD AND RESULTS: The culture medium supernatant of H9c2 cells exposed to sI/R was separated by column chromatography and the fractions examined for survival activity. The protein with survival activity was identified by mass spectrometry, and its physiological role was examined in the models of ischaemia. Cell survival activity was detected in at least three fractions of the cell supernatant collected during sI/R and subjected to a series of column chromatographic steps. Among the proteins measured by mass spectrometry and western blotting, a p36 protein identified as a glycolytic enzyme, lactate dehydrogenase muscle subunit (M-LDH), showed strong survival activity. H(2)O(2)-induced intracellular calcium overload in H9c2 cells and irregular Ca(2+) transients in adult rat cardiomyocytes were both found to be inhibited by pretreatment with M-LDH. M-LDH also lowered the frequency and amplitude of early afterdepolarizations induced by H(2)O(2) in adult rat cardiomyocytes and suppressed the ischaemia-reperfusion-induced reduction of cardiac output from mouse working heart preparations. M-LDH was found to increase the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), which plays a role in H9c2 cell survival. CONCLUSION: M-LDH released from cardiomyocytes after hypoxia and reoxygenation has a role in protecting the heart from oxidative stress-induced injury through an intracellular signal transduction pathway involving ERK1/2.
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Modified sympathetic nerve system activity with overexpression of the voltage-dependent calcium channel beta3 subunit.
Manabu Murakami, Takayoshi Ohba, Feng Xu, Eisaku Satoh, Ichiro Miyoshi, Takashi Suzuki, Yoichirou Takahashi, Eiki Takahashi, Hiroyuki Watanabe, Kyoichi Ono, Hironobu Sasano, Noriyuki Kasai, Hiroshi Ito, Toshihiko Iijima
The Journal of biological chemistry 283 ( 36 ) 24554 - 60 2008.09 [Refereed]
Research paper (journal)
N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory beta3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a beta3-overexpressing transgenic (beta3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of beta3-null and CaV2.2 (alpha1B)-null mice. The beta3-Tg mice showed increased expression of the beta3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The beta3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the beta3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the beta3-Tg mice, whereas beta3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the beta3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of beta3-Tg mice revealed increased calcium channel currents, especially N- and L-type currents. These results identify a determining role for the beta3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.
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Amlodipine inhibits cell proliferation via PKD1-related pathway.
Takayoshi Ohba, Hiroyuki Watanabe, Manabu Murakami, Milena Radovanovic, Kenji Iino, Masaru Ishida, Shinya Tosa, Kyoichi Ono, Hiroshi Ito
Biochemical and biophysical research communications 369 ( 2 ) 376 - 81 2008.05 [Refereed]
Research paper (journal)
Human coronary artery smooth muscle cell (hCASMC) proliferation is involved in the progression of coronary artery disease. Amlodipine, a widely used antihypertensive drug, exerts antiproliferative effects by increasing the expression of p21((Waf1/Cip1)). Polycystic kidney disease 1 (PKD1) is also involved in cell cycle inhibition via p21((Waf1/Cip1)) up-regulation. We clarified the involvement of PKD1-related signaling on hCASMCs. Cultured hCASMCs, which constitutively express PKD1, were stimulated with 5% serum. Amlodipine increased p21((Waf1/Cip1)) expression in a dose- and time-dependent manner, resulting in reduced hCASMC proliferation. The inhibitory effect of amlodipine was mimicked by overexpression of PKD1 and was reversed by a dominant-negative version of PKD1 (R4227X). Immunoblot analysis showed that phosphorylated JAK2 was increased by amlodipine treatment or PKD1 overexpression. A luciferase assay revealed that the overexpression of PKD1 induced STAT1 enhancer activity. These data suggest that PKD1 contributes to the antiproliferative effect of amlodipine on hCASMCs via JAK/STAT signaling and p21((Waf1/Cip1)) up-regulation.
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Involvement of transient receptor potential canonical 1 (TRPC1) in angiotensin II-induced vascular smooth muscle cell hypertrophy.
Yoichiro Takahashi, Hiroyuki Watanabe, Manabu Murakami, Takayoshi Ohba, Milena Radovanovic, Kyoichi Ono, Toshihiko Iijima, Hiroshi Ito
Atherosclerosis 195 ( 2 ) 287 - 96 2007.12 [Refereed]
Research paper (journal)
Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) hypertrophy as one of the major events leading to atherosclerosis. Increased Ca(2+) entry is an important stimulus for VSMC hypertrophy, but the association with Ang II remains to be determined. Transient receptor potential canonical 1 (TRPC1) forms store-operated Ca(2+) (SOC) channels that are involved in Ca(2+) homeostasis. Our aim was to ascertain the potential involvement of TRPC1 in Ang II-induced VSMC hypertrophy. For this purpose, we used cultured human coronary artery smooth muscle cells (hCASMCs). Store-operated Ca(2+) entry (SOCE) increased in the Ang II-induced hypertrophied cells, and SOC channel blocker inhibited the Ang II-induced hypertrophic response. Although hCASMCs constitutively expressed TRPC1, C3, C4, C5, and C6, only TRPC1 increased in response to Ang II stimulation. TRPC1 siRNA decreased SOCE and prevented Ang II-induced hypertrophy. We found NF-kappaB binding sites in the 5'-regulatory region of the human TRPC1 gene. An electrophoretic mobility shift assay showed that Ang II increased the TRPC1 promoter's NF-kappaB binding activity. Co-treatment with NF-kappaB decoy oligonucleotides not only reduced TRPC1 expression, but also inhibited the hypertrophic responses. In conclusion, our data suggest that Ang II and subsequent NF-kappaB activation induces hCASMC hypertrophy through an enhancement of TRPC1 expression.
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Characterization of SN-6, a novel Na+/Ca2+ exchange inhibitor in guinea pig cardiac ventricular myocytes.
Chun-Feng Niu, Yasuhide Watanabe, Kyoichi Ono, Takahiro Iwamoto, Kanna Yamashita, Hiroshi Satoh, Tuyoshi Urushida, Hideharu Hayashi, Junko Kimura
European journal of pharmacology 573 ( 1-3 ) 161 - 9 2007.11 [Refereed]
Research paper (journal)
We examined the effect of SN-6, a new benzyloxyphenyl Na(+)/Ca(2+) exchange (NCX) inhibitor on the Na(+)/Ca(2+) exchange current (I(NCX)) and other membrane currents in isolated guinea pig ventricular myocytes using the whole-cell voltage-clamp technique. SN-6 suppressed I(NCX) in a concentration-dependent manner. The IC(50) values of SN-6 were 2.3 microM and 1.9 microM for the outward and inward components of the bi-directional I(NCX), respectively. On the other hand, SN-6 suppressed the outward uni-directional I(NCX) more potently (IC(50) value of 0.6 microM) than the inward uni-directional I(NCX). SN-6 at 10 microM inhibited the uni-directional inward I(NCX) by only 22.4+/-3.1%. SN-6 and KB-R7943 suppressed I(NCX) more potently when intracellular Na(+) concentration was higher. Thus, both drugs inhibit NCX in an intracellular Na(+) concentration-dependent manner. Intracellular application of trypsin via a pipette solution did not change the blocking effect of SN-6 on I(NCX). Therefore, SN-6 is categorized as an intracellular-trypsin-insensitive NCX inhibitor. SN-6 at 10 microM inhibited I(Na), I(Ca), I(K) and I(K1) by about 13%, 34%, 33% and 13%, respectively. SN-6 at 10 microM shortened the action potential duration at 50% repolarization (APD(50)) by about 34%, and that at 90% repolarization (APD(90)) by about 25%. These results indicate that SN-6 inhibits NCX in a similar manner to that of KB-R7943. However, SN-6 at 10 microM affected other membrane currents less potently than KB-R7943.
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Functional role of stromal interaction molecule 1 (STIM1) in vascular smooth muscle cells.
Yoichiro Takahashi, Hiroyuki Watanabe, Manabu Murakami, Kyoichi Ono, Yoshiko Munehisa, Takashi Koyama, Kiyoshi Nobori, Toshihiko Iijima, Hiroshi Ito
Biochemical and biophysical research communications 361 ( 4 ) 934 - 40 2007.10 [Refereed]
Research paper (journal)
We investigated the functional role of STIM1, a Ca(2+) sensor in the endoplasmic reticulum (ER) that regulates store-operated Ca(2+) entry (SOCE), in vascular smooth muscle cells (VSMCs). STIM1 was mainly localized at the ER and plasma membrane. The knockdown of STIM1 expression by small interfering (si) RNA drastically decreased SOCE. In contrast, an EF-hand mutant of STIM1, STIM1(E87A), produced a marked increase in SOCE, which was abolished by co-transfection with siRNA to transient receptor potential canonical 1 (TRPC1). In addition, transfection with siRNA against STIM1 suppressed phosphorylation of cAMP-responsive element binding protein (CREB) and cell growth. These results suggest that STIM1 is an essential component of SOCE and that it is involved in VSMC proliferation.
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Essential role of the N-terminus of murine Orai1 in store-operated Ca2+ entry.
Yoichiro Takahashi, Manabu Murakami, Hiroyuki Watanabe, Hitoshi Hasegawa, Takayoshi Ohba, Yoshiko Munehisa, Kiyoshi Nobori, Kyoichi Ono, Toshihiko Iijima, Hiroshi Ito
Biochemical and biophysical research communications 356 ( 1 ) 45 - 52 2007.04 [Refereed]
Research paper (journal)
Store-operated Ca(2+) entry (SOCE) is a physiologically important process that is triggered by intracellular Ca(2+) depletion. Recently, human Orai1 (the channel-forming subunit) and STIM1 (the calcium sensor) were identified as essential molecules for SOCE. Here, we report the cloning and functional analysis of three murine orthologs of Orai1, termed Orai1, 2, and 3. Among the genes identified, Orai1 contains a distinctive proline- and arginine-rich N-terminal cytoplasmic sequence. Co-expression of STIM1 with Orai1 produced a marked effect on SOCE, while co-expression with Orai2 or Orai3 had little effect. Expression of Orai1 without its N-terminal tail had a marginal effect on SOCE, while chimeric Orai2 containing the Orai1 N-terminus produced a marked increase in SOCE. In addition, a truncated version of Orai1 containing the N-terminus without the pore-forming transmembrane domain had a dominant negative effect on SOCE. These results reveal the essential role of Orai1 and its N-terminal sequence in SOCE.