Kiyoshi Nobori

Nitric oxide inhibits ischemia/reperfusion-induced myocardial apoptosis by modulating cyclin A-associated kinase activity

OBJECTIVE Ischemia/reperfusion in the heart causes myocardial apoptosis and increase nitric oxide (NO) production. We have reported that myocardial apoptosis is related to activation of cell cycle regulatory proteins. However, the role of nitric oxide (NO) in ischemia/reperfusion-induced apoptosis is still unclear. This study was designated to elucidate novel apoptosis mechanisms induced by ischemia/reperfusion, especially the interaction between NO and cell cycle regulators. METHODS AND RESULTS Neonatal cardiomyocytes from 1- or 2-day-old Wistar rats were subjected to 1-h ischemia and then to reperfusion. The rate of cardiomyocyte apoptosis increased significantly after 24 h of reperfusion as evaluated by TUNEL analysis. NO increased 1.8-fold after 15 min of reperfusion in cardiomyocytes. After 36 h of reperfusion, the apoptosis rate was greatly increased by the NO synthetase inhibitor, Nitro-L-arginine methyl ester (L-NAME), and decreased by the NO donor of S-nitroso-N-acetylpenicillamine (SNAP). Immunoblot analysis showed that the protein levels of cyclin A accumulated in a time-dependent manner in response to ischemia/reperfusion, and L-NAME inhibited this response. Ischemia/reperfusion also increased the activity of cyclin A-associated kinase, and the apoptosis was inhibited by infection of dominant-negative cdk2 adenovirus. To clarify the involvement of p21(cip1/waf1) protein, which is the suppressor of cyclin A-associated kinase, we performed immunoblot analysis and examined its kinase activity. Treatment of cardiomyocytes with L-NAME suppressed the p21(cip1/waf1) protein level and increased the cyclin A-associated kinase activity. The addition of SNAP showed inverse results. CONCLUSION Our data indicates that NO released from cardiomyocytes under condition of ischemia/reperfusion exerts an antiapoptotic effect by modulating cyclin A-associated kinase activity via p21(cip1/waf1) accumulation.

ATF3 Protects against Renal Ischemia-Reperfusion Injury

Oxidative stress-induced cell death plays a major role in the progression of ischemic acute renal failure. Using microarrays, we sought to identify a stress-induced gene that may be a therapeutic candidate. Human proximal tubule (HK2) cells were treated with hydrogen peroxide (H2O2) and RNA was applied to an Affymetrix gene chip. Five genes were markedly induced in a parallel time-dependent manner by cluster analysis, including activating transcription factor 3 (ATF3), p21(WAF1/CiP1) (p21), CHOP/GADD153, dual-specificity protein phosphatase, and heme oxygenase-1. H2O2 rapidly induced ATF3 approximately 12-fold in HK2 cells and approximately 6.5-fold in a mouse model of renal ischemia-reperfusion injury. Adenovirus-mediated expression of ATF3 protected HK2 cells against H2O2-induced cell death, and this was associated with a decrease of p53 mRNA and an increase of p21 mRNA. Moreover, when ATF3 was overexpressed in mice via adenovirus-mediated gene transfer, ischemia-reperfusion injury was reduced. In conclusion, ATF3 plays a protective role in renal ischemia-reperfusion injury and the mechanism of the protection may involve suppression of p53 and induction of p21.

Down-regulation of p27Kip1 promotes cell proliferation of rat neonatal cardiomyocytes induced by nuclear expression of cyclin D1 and CDK4: Evidence for impaired Skp2-dependent degradation of p27 in terminal differentiation

Mammalian cardiomyocytes lose their capacity to proliferate during terminal differentiation. We have previously reported that the expression of nuclear localization signal-tagged cyclin D1 (D1NLS) and its partner cyclin-dependent kinase 4 (CDK4) induces proliferation of rat neonatal cardiomyocytes. Here we show that the D1NLS/CDK4 cells, after their entry into the cell cycle, accumulated cyclin-dependent kinase inhibitor p27 in the nuclei and decreased the cyclin-dependent kinase 2 (CDK2) activity, leading to early cell cycle arrest. Biochemical analysis demonstrated that Skp2-dependent p27 ubiquitylation was remarkably suppressed in cardiomyocytes, whereas Skp2, a component of Skp1-Cullin-F-box protein ubiquitin ligase, was more actively ubiquitylated compared with proliferating rat fibroblasts. Specific degradation of p27 by co-expressing Skp2 or p27 small interfering RNA caused an increase of CDK2 activity and overrode the limited cell cycle. These data altogether indicate that the impaired Skp2-dependent p27 degradation is causally related to the loss of proliferation in cardiomyocytes. This provides a novel insight in understanding the molecular mechanism by which mammalian cardiomyocytes cease to proliferate during terminal differentiation.

Adaptive servo-ventilation improves renal function in patients with heart failure

BACKGROUND Impaired cardiac function and sleep-disordered breathing (SDB) are associated with progression of chronic kidney disease (CKD) in heart failure (HF) patients. Adaptive servo-ventilation (ASV) therapy improves cardiac function in HF patients regardless of the SDB severity through hemodynamic support and prevention of repetitive hypoxic stress. This study was designed to test the hypothesis that ASV therapy improves renal function in HF patients with SDB. METHODS AND RESULTS Of 59 consecutively enrolled HF patients, 43 with moderate-to-severe SDB underwent ASV therapy. HF patients were divided into the ASV-treated group (n = 27) and the non-ASV-treated group (n = 16). Estimated glomerular filtration rate (eGFR), echocardiographic parameters, and inflammatory biomarkers were measured before and 12 months after ASV initiation. Improvement in the eGFR was found in the ASV-treated group, but not in the non-ASV-treated group. There was a positive correlation between the increases in eGFR and left ventricular ejection fraction (r = 0.488, p = 0.001). The changes in high-sensitivity C-reactive protein were negatively correlated with change in the eGFR (r = -0.416, p = 0.006). CONCLUSIONS ASV therapy could improve renal dysfunction in HF patients through hemodynamic support. Additionally, prevention of SDB with the use of ASV therapy could exert anti-inflammatory effects, which could contribute to the improvement of renal function in HF patients.

Expression of cyclin D1 and CDK4 causes hypertrophic growth of cardiomyocytes in culture: A possible implication for cardiac hypertrophy

Differentiated cardiomyocytes have little capacity to proliferate and show the hypertrophic growth in response to alpha1-adrenergic stimuli via the Ras/MEK pathway. In this study, we investigated a role of cyclin D1 and CDK4, a positive regulator of cell cycle, in cultured neonatal rat cardiomyocyte hypertrophy. D-type cyclins including cyclin D1 were induced in cells stimulated by phenylephrine. This induction was inhibited by MEK inhibitor PD98059 and the dominant negative RasN17, but mimicked by expression of the constitutive active Ras61L. Over-expression of cyclin D1 and CDK4 using adenovirus gene transfer caused the hypertrophic growth of cardiomyocytes, as evidenced by an increase of the cell size as well as the amount of cellular protein and its rate of synthesis. However, the cyclin D1/CDK4 kinase activity was not up-regulated in cells treated by hypertrophic stimuli or in cells over-expressing the cyclin D1 and CDK4. Furthermore, a CDK inhibitor, p16, did not inhibit the hypertrophic growth of cardiomyocytes. These results clearly indicated that cyclin D1 and CDK4 have a role in hypertrophic growth of cardiomyocytes through a novel mechanism(s) which appears not to be related to its activity required for cell cycle progression.

Comparison of anti-inflammatory effects and high-density lipoprotein cholesterol levels between therapy with quadruple-dose rosuvastatin and rosuvastatin combined with ezetimibe

BackgroundStatins are frequently administered to reduce low-density lipoprotein cholesterol (LDL-C) and vascular inflammation, because LDL-C and high sensitive C-reactive protein (hs-CRP) are associated with high risk for cardiovascular events. When statins do not reduce LDL-C to desired levels in high-risk patients with coronary artery disease (CAD), ezetimibe can be added or the statin dose can be increased. However, which strategy is more effective for treating patients with CAD has not been established. The present study compares anti-inflammatory effects and lipid profiles in patients with CAD and similar LDL-C levels who were treated by increasing the statin dose or by adding ezetimibe to the original rosuvastatin dose to determine the optimal treatment for such patients.Methods46 patients with high-risk CAD and LDL-C and hs-CRP levels of >70 mg/dL and >1.0 mg/L, respectively, that were not improved by 4 weeks of rosuvastatin (2.5 mg/day) were randomly assigned to receive 10 mg (R10, n = 24) of rosuvastatin or 2.5 mg/day of rosuvastatin combined with 10 mg/day of ezetimibe (R2.5/E10, n = 22) for 12 weeks. The primary endpoint was a change in hs-CRP.ResultsBaseline characteristics did not significantly differ between the groups. At 12 weeks, LDL-C and inflammatory markers (hs-CRP, interleukin-6, tumour necrosis factor-alpha and pentraxin 3) also did not significantly differ between the two groups (LDL-C: R10 vs. R2.5/E10: -19.4 ± 14.2 vs. -22.4 ± 14.3 mg/dL). However, high-density lipoprotein cholesterol (HDL-C) was significantly improved in the R10, compared with R2.5/E10 group (4.6 ± 5.9 vs. 0.0 ± 6.7 mg/dL; p < 0.05).ConclusionBoth enhanced therapies exerted similar anti-inflammatory effects under an equal LDL-C reduction in patients with high-risk CAD despite 2.5 mg/day of rosuvastatin. However, R10 elevated HDL-C more effectively than R2.5/E10.Trial registrationUMIN000003746

Increased circulating CD3+/CD31+ T cells in patients with acute coronary syndrome

The number of circulating endothelial progenitor cells (EPCs) is considered to be a surrogate marker for coronary artery disease (CAD). Recent studies have identified a novel T-cell subset labeled with CD3+/CD31+, which is necessary for EPC colony formation and constitutes the central cluster. However, the clinical relevance of the CD3+/CD31+ T cells in CAD remains unclear. We sought to clarify whether circulating CD3+/CD31+ T cells are increased in patients with acute coronary syndrome (ACS). Circulating CD3+/CD31+ T cells were determined in 16 ACS patients undergoing emergency percutaneous coronary intervention (PCI) and in 16 control subjects with angiographically normal coronary arteries. Although no differences between the groups were found in baseline patient characteristics, the ratio of circulating CD3+/CD31+ T cells before PCI was higher in ACS patients as compared with that in control subjects (51.8 % ± 7.8 % vs 31.8 % ± 9.6 %, respectively; P < 0.001). The increased ratio of CD3+/CD31+ T cells in ACS patients was not altered 24 h after PCI, but became comparable with that in control subjects within 6 months after PCI. These results suggest that mobilization of CD3+/CD31+ T cells occurs in ACS, but is no longer detectable at 6 months after PCI.

A free-floating left atrial thrombus develops intermittent entrapment in the mid-ventricle during diastole

Free-floating left atrial thrombi are rare. Here we report a case of a 75-year-old woman with atrial fibrillation who was admitted for treatment of acute myocardial infarction. A free-floating left atrial thrombus was found incidentally on echocardiography. Ten days after percutaneous coronary intervention, the patient had mild faintness with transient hypotension, and it was found that the left atrial thrombus had developed intermittent entrapment in the mid-ventricle during diastole, with abrupt rebound back to the left atrial cavity during systole. Urgent removal of the thrombus was performed successfully. Although the free-floating thrombus had appeared to be spherical, like a ball thrombus, on echocardiography, the excised thrombus was pedunculated. A cut section revealed a laminated thrombus with an onion-skin-like appearance.

P-350 Double Transgenic Mice Crossing Alpha MyHC-mCherry-LC3 Mice with CAG-GFP-LC3 Mice is a New Method to Analyze Autophagy in Cardiomyocytes

P-350 Double Transgenic Mice Crossing Alpha MyHC-mCherry-LC3 Mice with CAG-GFP-LC3 Mice is a New Method to Analyze Autophagy in Cardiomyocytes Mai Terada, Kiyoshi Nobori, Yoshiko Munehisa, Manabu Kakizaki, Takayoshi Ohba, Takashi Koyama, Yoichiro Takahashi, Masaru Ishida, Kenji Iino, Toshimitsu Kosaka, Hiroyuki Watanabe, Hitoshi Hasegawa, Hiroshi Ito. Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Akita University School of Medicine, Japan