Qingping Feng

Development of Heart Failure and Congenital Septal Defects in Mice Lacking Endothelial Nitric Oxide Synthase

Background—Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in the regulation of cell growth, apoptosis, and tissue perfusion. Recent studies showed that mice deficient in eNOS developed abnormal aortic bicuspid valves. The aim of the present study was to additionally investigate the role of eNOS in heart development. Methods and Results—We examined postnatal mortality, cardiac function, and septum defects in eNOS−/−, eNOS+/−, and wild-type mice. Postnatal mortality was significantly increased in eNOS−/− (85.1%) and eNOS+/− (38.3%) compared with wild-type mice (13.3%, P <0.001). Postmortem examination found severe pulmonary congestion with focal alveolar edema in mice deficient in eNOS. Heart shortening determined by ultrasound crystals was significantly decreased in eNOS−/− compared with wild-type mice (P <0.05). Congenital atrial and ventricular septal defects were found in neonatal hearts. The incidence of atrial or ventricular septal defects was significantly increased in eNOS−/− (75%) and eNOS+/− (32.4%) neonates compared with those of the wild-type mice (4.9%). At embryonic days 12.5 and 15.5, cardiomyocyte apoptosis and myocardial caspase-3 activity were increased in the myocardium of eNOS−/− compared with wild-type embryos (P <0.01), and increases in apoptosis persisted to neonatal stage in eNOS−/− mice. Conclusions—Deficiency in eNOS results in heart failure and congenital septal defects during cardiac development, which is associated with increases in cardiomyocyte apoptosis. Our data demonstrate that eNOS plays an important role in normal heart development.

Tumor necrosis factor-α induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes

OBJECTIVE It has been demonstrated that tumor necrosis factor-alpha (TNF alpha) induces apoptosis in cardiac myocytes. However, its mechanism of action is still not well understood. In the present study, we hypothesized that TNF alpha induces myocardial apoptosis by induction of inducible nitric oxide synthase (iNOS). METHODS Neonatal cardiac myocytes were isolated from iNOS (-/-) mutant and C57BL6 wild type mice. Cells were cultured for 3 days before treatment with an NO donor or TNF alpha. Following treatment with S-nitroso-N-acetyl-penicillamine (SNAP) or TNF-alpha, cells were tested for apoptosis by terminal deoxynucleotidyl transfer-mediated end labeling (TUNEL) staining and cell death detection ELISA. NO production was measured by nitrite concentration in the culture medium. Cardiomyocyte expression of iNOS and TNF type 1 receptor (TNFR1) mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS SNAP (0.01-100 microM) induced apoptosis of cardiac myocytes in a concentration-dependent manner in the wild type mice (n = 5, P < 0.01). TNFR1 mRNA was expressed in neonatal cardiomyocytes from both wild type and iNOS (-/-) mutant mice. TNF alpha induced a concentration-dependent increase in iNOS mRNA expression and nitrite production as well as significant apoptosis of cardiomyocytes in the wild type mice (n = 4, P < 0.01). However, without iNOS expression, the apoptotic effects of TNF-alpha were significantly attenuated in cardiomyocytes from iNOS (-/-) mutant mice (n = 4, P < 0.05). CONCLUSION TNF alpha induces apoptosis via iNOS expression and NO production in neonatal mouse cardiomyocytes.

miR133a regulates cardiomyocyte hypertrophy in diabetes

Diabetic cardiomyopathy, characterized by cardiac hypertrophy and contractile dysfunction, eventually leads to heart failure. We have previously shown that alterations of a number of key molecules are involved in producing cardiomyocyte hypertrophy in diabetes. The aim of the present study was to determine whether microRNAs (miRNA) play a role in mediating altered gene expression and structural/functional deficits in the heart in diabetes.

Rac1 is required for cardiomyocyte apoptosis during hyperglycemia

OBJECTIVE Hyperglycemia induces reactive oxygen species (ROS) and apoptosis in cardiomyocytes, which contributes to diabetic cardiomyopathy. The present study was to investigate the role of Rac1 in ROS production and cardiomyocyte apoptosis during hyperglycemia. RESEARCH DESIGN AND METHODS Mice with cardiomyocyte-specific Rac1 knockout (Rac1-ko) were generated. Hyperglycemia was induced in Rac1-ko mice and their wild-type littermates by injection of streptozotocin (STZ). In cultured adult rat cardiomyocytes, apoptosis was induced by high glucose. RESULTS The results showed a mouse model of STZ-induced diabetes, 7 days of hyperglycemia-upregulated Rac1 and NADPH oxidase activation, elevated ROS production, and induced apoptosis in the heart. These effects of hyperglycemia were significantly decreased in Rac1-ko mice or wild-type mice treated with apocynin. Interestingly, deficiency of Rac1 or apocynin treatment significantly reduced hyperglycemia-induced mitochondrial ROS production in the heart. Deficiency of Rac1 also attenuated myocardial dysfunction after 2 months of STZ injection. In cultured cardiomyocytes, high glucose upregulated Rac1 and NADPH oxidase activity and induced apoptotic cell death, which were blocked by overexpression of a dominant negative mutant of Rac1, knockdown of gp91phox or p47phox, or NADPH oxidase inhibitor. In type 2 diabetic db/db mice, administration of Rac1 inhibitor, NSC23766, significantly inhibited NADPH oxidase activity and apoptosis and slightly improved myocardial function. CONCLUSIONS Rac1 is pivotal in hyperglycemia-induced apoptosis in cardiomyocytes. The role of Rac1 is mediated through NADPH oxidase activation and associated with mitochondrial ROS generation. Our study suggests that Rac1 may serve as a potential therapeutic target for cardiac complications of diabetes.

Pivotal Role of gp91phox-Containing NADH Oxidase in Lipopolysaccharide-Induced Tumor Necrosis Factor-α Expression and Myocardial Depression

Background—Lipopolysaccharide (LPS) induces cardiomyocyte tumor necrosis factor-α (TNF-α) production, which is responsible for myocardial depression during sepsis. The aim of this study was to investigate the role of gp91phox-containing NADH oxidase signaling in cardiomyocyte TNF-α expression and myocardial dysfunction induced by LPS. Methods and Results—In cultured mouse neonatal cardiomyocytes, LPS increased NADH oxidase (gp91phox subunit) expression and superoxide generation. Deficiency of gp91phox or inhibition of NADH oxidase blocked TNF-α expression stimulated by LPS. TNF-α induction was also inhibited by tempol, N-acetylcysteine, or 1,3-dimethyl-2-thiourea. NADH oxidase activation by LPS increased ERK1/2 and p38 phosphorylation, and inhibition of ERK1/2 and p38 phosphorylation blocked the effect of NADH oxidase on TNF-α expression. Isolated mouse hearts were perfused with LPS (5 μg/mL) alone or in the presence of apocynin for 1 hour. Myocardial TNF-α production was decreased in gp91phox-deficient or apocynin-treated hearts compared with those of wild type (P<0.05). To investigate the role of gp91phox-containing NADH oxidase in endotoxemia, mice were treated with LPS (4 mg/kg IP) for 4 and 24 hours, and their heart function was measured with a Langendorff system. Deficiency of gp91phox significantly attenuated LPS-induced myocardial depression (P<0.05). Conclusions—gp91phox-Containing NADH oxidase is pivotal in LPS-induced TNF-α expression and cardiac depression. Effects of NADH oxidase activation are mediated by ERK1/2 and p38 MAPK pathway. The present results suggest that gp91phox-containing NADH oxidase may represent a potential therapeutic target for myocardial dysfunction in sepsis.

Fasudil, a Rho-Kinase Inhibitor, Attenuates Angiotensin II–Induced Abdominal Aortic Aneurysm in Apolipoprotein E–Deficient Mice by Inhibiting Apoptosis and Proteolysis

Background—Angiotensin II (Ang II) accelerates atherosclerosis and induces abdominal aortic aneurysm (AAA) in an experimental mouse model. Agonism of a G protein–coupled receptor by Ang II activates Rho-kinase and other signaling pathways and results in activation of proteolysis and apoptosis. Enhanced proteolysis and smooth muscle cell apoptosis are important mechanisms associated with AAA. In this study, we tested the hypothesis that fasudil, a Rho-kinase inhibitor, could attenuate Ang II–induced AAA formation by inhibiting vascular wall apoptosis and extracellular matrix proteolysis. Methods and Results—Six-month-old apolipoprotein E–deficient mice were infused with Ang II (1.44 mg · kg−1 · d−1) for 1 month. Animals were randomly assigned to treatment with fasudil (136 or 213 mg · kg−1 · d−1 in drinking water) or tap water. Ang II infusion induced AAA formation in 75% of the mice, which was accompanied by an increase in proteolysis detected by zymographic analysis and quantified by active matrix metalloproteinase-2 activity, as well as apoptosis detected by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling and quantified by both caspase-3 activity and histone-associated DNA fragmentation. The level of DNA fragmentation in the suprarenal aorta correlated with AAA diameter. Ang II also increased atherosclerotic lesion area and blood pressure. Fasudil treatment resulted in a dose-dependent reduction in both the incidence and severity of AAA. At the higher dose, fasudil decreased AAA by 45% while significantly inhibiting both apoptosis and proteolysis, without affecting atherosclerosis or blood pressure. Conclusions—These data demonstrate that inhibition of Rho-kinase by fasudil attenuated Ang II–induced AAA through inhibition of both apoptosis and proteolysis pathways.

Elevation of an endogenous inhibitor of nitric oxide synthesis in experimental congestive heart failure

OBJECTIVE NG,NG-dimethylarginine (asymmetric dimethylarginine, ADMA) is an important endogenous substance with potent inhibitory actions on nitric oxide (NO) synthesis. The present study was designed to determine circulating ADMA levels and endothelium-dependent, NO mediated vasodilation in a rat model of congestive heart failure (CHF). METHODS CHF was induced in rats by coronary artery ligation. Sham-operated rats served as normal controls. Plasma ADMA was determined by high performance liquid chromatography with fluorescence detection. Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured by the clearance of inulin and p-aminohippuric acid, respectively. Endothelial function of the aorta was assessed in an organ bath. RESULTS Plasma levels of ADMA in rats with CHF (0.94 +/- 0.05 mumol/l) were significantly increased compared with sham-operated controls (0.75 +/- 0.06 mumol/l, p < 0.05). Plasma levels of ADMA was negatively correlated with GFR (r = -0.65, p < 0.05). Decreased endothelium-dependent relaxation to acetylcholine in the aorta of CHF was completely restored by L-arginine (300 microM) (p < 0.01) while endothelium-independent relaxation to nitroprusside was not altered. ADMA potently inhibited endothelium-dependent relaxation in thoracic aorta of normal and CHF rats. The effect of ADMA was completely antagonized by L-arginine in both groups (p < 0.01). Moreover, L-arginine improved endothelium-dependent relaxation in CHF rats in the presence of ADMA. CONCLUSIONS An endogenous NO synthesis inhibitor ADMA is increased in the circulation of rats with CHF. The increased plasma levels of ADMA may contribute to the decreased endothelium-dependent relaxation in CHF, which is restored by L-arginine, possibly by competitive antagonism of ADMA.

Neuronal Nitric Oxide Synthase Protects Against Myocardial Infarction-Induced Ventricular Arrhythmia and Mortality in Mice

Background— Neuronal nitric oxide synthase (nNOS) is expressed in cardiomyocytes and plays a role in regulating cardiac function and Ca2+ homeostasis. However, the role of nNOS in cardiac electrophysiology after myocardial infarction (MI) is unclear. We hypothesized that nNOS deficiency increases ventricular arrhythmia and mortality after MI. Methods and Results— MI was induced in wild-type (WT) or nNOS−/− mice by ligation of the left coronary artery. Thirty-day mortality was significantly higher in nNOS−/− compared with WT mice. Additionally, nNOS−/− mice had impaired cardiac function 2 days after MI. Telemetric ECG monitoring showed that compared with WT, nNOS−/− mice had significantly more ventricular arrhythmias and were more likely to develop ventricular fibrillation after MI. Treatment with the L-type Ca2+ channel blocker verapamil reduced the incidence of arrhythmia and ventricular fibrillation in nNOS−/− mice after MI. To assess the role of nNOS in Ca2+ handling, patch-clamp and Ca2+ fluorescence techniques were used. Ca2+ transients and L-type Ca2+ currents were higher in nNOS−/− compared with WT cardiomyocytes. Additionally, nNOS−/− cardiomyocytes exhibited significantly higher systolic and diastolic Ca2+ over a range of pacing frequencies. Treatment with the NO donor S-nitroso N-acetyl-penicillamine decreased Ca2+ transients and L-type Ca2+ current in both nNOS−/− and WT cardiomyocytes. Furthermore, S-nitrosylation of Ca2+ handling proteins was significantly decreased in nNOS−/− myocardium after MI. Conclusions— Deficiency in nNOS increases ventricular arrhythmia and mortality after MI in mice. The antiarrhythmic effect of nNOS involves inhibition of L-type Ca2+ channel activity and regulation of Ca2+ handling proteins via S-nitrosylation.

Inhibition of p38 MAPK decreases myocardial TNF-alpha expression and improves myocardial function and survival in endotoxemia

OBJECTIVES The role of p38 mitogen-activated protein kinase (MAPK) activation in lipopolysaccharide (LPS)-induced myocardial dysfunction has not been clearly defined. Our aim was to investigate the contribution of p38 MAPK in myocardial tumor necrosis factor-alpha (TNF-alpha) expression, cardiac function and survival during acute endotoxemia in mice. METHODS Acute endotoxemia was induced by LPS (10 mg/kg, i.p.) in mice. Two hours after LPS treatment, left ventricular (LV) function was assessed. Phosphorylation of p38 MAPK was measured by Western blotting. TNF-alpha mRNA and protein levels were determined by semi-quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS LPS rapidly increased phosphorylation of p38 MAPK, followed by TNF-alpha mRNA expression and protein expression in the LV myocardium. Pre-treatment of the p38 MAPK inhibitor SB202190 (2 mg/kg, i.p.) decreased TNF-alpha mRNA and protein by 65 and 36%, respectively (P<0.05). Immunohistochemical staining confirmed that cardiomyocytes were the major source of TNF-alpha production in the myocardium and blocking p38 MAPK activation inhibited TNF-alpha expression in response to LPS. Pre-treatment of SB202190 or a TNF-alpha antagonist etanercept (2 mg/kg, i.p) significantly reversed LPS-induced LV depression (P<0.05). LPS (20 mg/kg, i.p.) induced 94% mortality in mice within 72 h and pre-treatment with SB202190 and etanercept decreased LPS-induced mortality to 65 and 40%, respectively (P<0.01). CONCLUSION p38 MAPK activation represents an important mechanism leading to myocardial TNF-alpha production and cardiac dysfunction during acute endotoxemia in mice. Our data suggest that p38 MAPK is a potential therapeutic target of endotoxemia.

Calpain activation contributes to hyperglycaemia-induced apoptosis in cardiomyocytes

AIMS Cardiomyocyte apoptosis contributes to cardiac complications of diabetes. The aim of this study was to investigate the role of calpain in cardiomyocyte apoptosis induced by hyperglycaemia. METHODS AND RESULTS In cultured adult rat ventricular cardiomyocytes, high glucose (33 mM) increased calpain activity and induced apoptosis, concomitant with the impairment of Na+/K+ ATPase activity. These effects of high glucose on cardiomyocytes were abolished by various pharmacological calpain inhibitors, knockdown of calpain-1 but not calpain-2 using siRNA, or over-expression of calpastatin, a specific endogenous calpain inhibitor. The effect of calpain inhibition on cardiomyocyte apoptosis was abrogated by ouabain, a selective inhibitor of Na+/K+ ATPase. Furthermore, blocking gp91(phox)-NADPH oxidase activation, L-type calcium channels, or ryanodine receptors prevented calpain activation and apoptosis in high glucose-stimulated cardiomyocytes. In a mouse model of streptozotocin-induced diabetes, administration of different calpain inhibitors blocked calpain activation, increased the Na+/K+ ATPase activity, and decreased apoptosis in the heart. CONCLUSION Calpain-1 activation induces apoptosis through down-regulation of the Na+/K+ ATPase activity in high glucose-stimulated cardiomyocytes and in vivo hyperglycaemic hearts. High glucose-induced calpain-1 activation is mediated through the NADPH oxidase-dependent pathway and associated with activation of L-type calcium channels and ryanodine receptors. Our data suggest that calpain activation may be important in the development of diabetic cardiomyopathy and thus may represent a potential therapeutic target for diabetic heart diseases.