Fu-Li Xiang

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.

Endothelial Nitric Oxide Synthase Promotes Bone Marrow Stromal Cell Migration to the Ischemic Myocardium via Upregulation of Stromal Cell‐Derived Factor‐1α

The aim of this study was to investigate the role of endothelial nitric oxide synthase (eNOS) in the host myocardium on bone marrow mesenchymal stromal cells (MSC) migration to the ischemic myocardium and whether stromal cell‐derived factor‐1α (SDF‐1α) contributes to eNOS‐mediated MSC migration. MSCs and coronary microvascular endothelial cells were isolated from adult wild‐type (WT) mouse bone marrow and hearts, respectively. Cultured neonatal cardiomyocytes from WT, eNOS−/−, and eNOS overexpressing transgenic (Tg) mice were subjected to anoxia and reoxygenation (A/R), and the conditioned medium was used as a chemoattractant for in vitro transendothelial migration assay. MSC migration was decreased in the presence of conditioned medium derived from eNOS−/− cardiomyocytes but increased in the presence of eNOS‐Tg conditioned medium. SDF‐1α expression was decreased in eNOS−/− but increased in eNOS‐Tg cardiomyocytes following A/R and in the myocardium following ischemia/reperfusion (I/R). SDF‐1α expression was cGMP‐dependent as inhibition of soluble guanylyl cyclase decreased SDF‐1α expression in WT cardiomyocytes. MSCs expressed very low levels of eNOS proteins compared with the adult myocardium. To examine MSC migration in vivo, MSCs derived from mice expressing enhanced green fluorescence protein (EGFP+) were intravenously administered to WT mice subjected to myocardial I/R. EGFP+ cells in the ischemic region were decreased in eNOS−/− but increased in eNOS‐Tg compared with WT hearts. MSC treatment improved cardiac function following I/R in WT but not in eNOS−/− mice. In conclusion, eNOS in the host myocardium promotes MSC migration to the ischemic myocardium and improves cardiac function through cGMP‐dependent increases in SDF‐1α expression. STEM CELLS 2009;27:961–970

Cardiomyocyte-Specific Overexpression of Human Stem Cell Factor Improves Cardiac Function and Survival After Myocardial Infarction in Mice

Background— Soluble stem cell factor (SCF) has been shown to mobilize bone marrow stem cells and improve cardiac repair after myocardial infarction (MI). However, the effect of membrane-associated SCF on cardiac remodeling after MI is not known. The present study investigated the effects of cardiomyocyte-specific overexpression of the membrane-associated isoform of human SCF (hSCF) on cardiac function after MI. Methods and Results— A novel mouse model with tetracycline-inducible and cardiac-specific overexpression of membrane-associated hSCF was generated. MI was induced by left coronary artery ligation. Thirty-day mortality after MI was decreased in hSCF/tetracycline transactivator (tTA) compared with wild-type mice. In vivo cardiac function was significantly improved in hSCF/tTA mice at 5 and 30 days after MI compared with wild-type mice. Endothelial progenitor cell recruitment and capillary density were increased and myocardial apoptosis was decreased in the peri-infarct area of hSCF/tTA mice. Myocyte size was decreased in hSCF/tTA mice 30 days after MI compared with WT mice. Furthermore, hSCF overexpression promoted de novo angiogenesis as assessed by matrigel implantation into the left ventricular myocardium. Conclusions— Cardiomyocyte-specific overexpression of hSCF improves myocardial function and survival after MI. These beneficial effects of hSCF may result from increases in endothelial progenitor cell recruitment and neovascularization and decreases in myocardial apoptosis and cardiac remodeling.

Role of heme oxygenase-1 in the cardioprotective effects of erythropoietin during myocardial ischemia and reperfusion.

We have recently demonstrated that erythropoietin (EPO) protects cardiomyocytes from apoptosis during myocardial ischemia-reperfusion (I/R). The objective of the present study was to investigate the role of heme oxygenase (HO)-1 in the antiapoptotic effects of EPO. Primary cultures of neonatal mouse cardiomyocytes were subjected to anoxia-reoxygenation (A/R). Pretreatment with EPO significantly reduced apoptosis in A/R-treated cells. This reduction in apoptosis was preceded by an increase in the mRNA and protein expression of HO-1. Selective inhibition of HO-1 using chromium mesoporphyrin (CrMP) significantly diminished the ability of EPO to inhibit apoptosis. Cotreatment of EPO with SB-202190, an inhibitor of p38 activation, blocked the EPO-mediated HO-1 expression and antiapoptotic effects, suggesting a p38-dependent mechanism. The in vivo significance of p38 and HO-1 as mediators of EPOs cardioprotection was investigated in mice subjected to myocardial I/R. Pretreatment with EPO decreased infarct size as well as I/R-induced apoptosis in wild-type mice. However, these effects were significantly diminished in HO-1(-/-) mice. Furthermore, EPO given during ischemia reduced infarct size in mice subjected to I/R, and this effect was blocked by CrMP treatment in wild-type mice. Moreover, inhibition of p38 diminished the cardioprotective effects of EPO. We conclude that upregulation of HO-1 expression via p38 signaling contributes to EPO-mediated cardioprotection during myocardial I/R.

Longitudinal Follow-up of Cardiac Structure and Functional Changes in an Infarct Mouse Model Using Retrospectively Gated Micro-Computed Tomography

Objectives:Mouse models of myocardial infarction are valuable in studying the effect of genetic modifications on structural and functional remodeling of the heart. Our group recently developed a method for acquiring three-dimensional images of the beating mouse heart using micro-computed tomography (micro-CT) and retrospective gating. In this study, we evaluated cardiac function in sham and infarcted mice longitudinally, using this novel technique. Materials and Methods:Thirteen mice (7 sham-operated, 6 infarcted; male, C57BL/6) were imaged at baseline and at weeks 1, 2, 3, and 4 postligation of the left anterior descending coronary artery. Animals were anesthetized with 1.5% isoflurane; mechanical ventilation was not used. Contrast between blood and tissue was provided by an iodinated blood-pool contrast agent (0.01 mL/g Fenestra VC). The cardiac and respiratory waveforms were recorded during the 50-second scan time, to enable retrospective gating. Once scanning was completed on week 4 postsurgery, hemodynamic measurements were performed using a Millar pressure conductance catheter. Results:There were significant differences in systolic and diastolic volumes, and ejection fraction, between sham and myocardial infarction groups (P < 0.0001). A comparison of ejection fraction derived from both CT and hemodynamic measurements was not significantly different (P > 0.1). Conclusions:We have demonstrated the first use of dynamic micro-CT for monitoring cardiac remodeling, resulting from myocardial infarction, over time. The fast scan times (<1 minute) and ability to track individual animals over an entire study make this quantitative noninvasive technique a promising tool for in vivo studies of cardiac disease in mouse models.

Erythropoietin Protects the Heart from Ventricular Arrhythmia during Ischemia and Reperfusion via Neuronal Nitric-Oxide Synthase

Erythropoietin (EPO) is a potent cardioprotective agent in models of myocardial ischemia and reperfusion (I/R). It has been suggested recently that EPO may also reduce ventricular arrhythmia after I/R. The present study investigated the role of neuronal nitric oxide synthase (nNOS) on the antiarrhythmic effects of EPO. EPO treatment increased nNOS expression in isolated neonatal mouse ventricular myocytes. Cotreatment with the phosphatidylinositol 3 (PI3)-kinase inhibitor, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], or treatment of cardiomyocytes infected with a dominant negative adenovirus targeted to Akt1 (ADV-dnAkt1) blocked the effects of EPO on nNOS expression, suggesting that EPO regulates nNOS expression via PI3-kinase and Akt. To examine the in vivo antiarrhythmic effects of EPO, wild-type (WT) and nNOS(-/-) mice were anesthetized and, after a baseline measurement, subjected to myocardial I/R to provoke ventricular arrhythmias. Pretreatment with EPO 24 h before ischemia increased nNOS expression and significantly reduced the number of premature ventricular contractions (PVCs) and the incidence of ventricular tachycardia (VT) in WT mice. In contrast, treatment with EPO had no effect on PVCs or the incidence of VT in nNOS(-/-) mice. Furthermore, EPO treatment after ischemia significantly reduced the threshold dose of cesium chloride (CsCl) to induce VT. We conclude that EPO via nNOS protects the heart from spontaneous and CsCl-induced ventricular arrhythmia during myocardial I/R.

North American ginseng protects the heart from ischemia and reperfusion injury via upregulation of endothelial nitric oxide synthase

Emerging evidence suggests ginseng has therapeutic potential in cardiovascular disease. The aim of this study was to investigate the role of endothelial nitric oxide synthase (eNOS) in the cardioprotective effects of ginseng during myocardial ischemia and reperfusion (I/R). Treatment with ginseng extract significantly increased Akt phosphorylation and eNOS protein levels in cultured neonatal cardiomyocytes. Upregulation of eNOS was blocked by LY294002, a PI3-kinase inhibitor, suggesting a PI3-kinase/Akt-dependent mechanism. To simulate I/R, cultured neonatal cardiomyocytes from eNOS(-/-) and wild-type (WT) mice were subjected to anoxia and reoxygenation (A/R). Ginseng treatment inhibited A/R-induced apoptosis in WT, but not in either eNOS(-/-) cardiomyocytes or WT cardiomyocytes treated with LY294002. To further study the cardioprotective effects of ginseng in vivo, WT and eNOS(-/-) mice were pretreated with ginseng extract (50mg/kg/day, oral gavage) for 7 days before they were subjected to myocardial I/R. Treatment with ginseng significantly increased Akt phosphorylation and eNOS protein levels in the myocardium. Furthermore, ginseng-induced myocardial eNOS expression was inhibited by LY294002. Strikingly, ginseng treatment significantly decreased infarct size and myocardial apoptosis following I/R in WT mice, but not in either eNOS(-/-) mice or WT mice treated with LY294002. We conclude that ginseng treatment protects the heart from I/R injury via upregulation of eNOS expression. Our study suggests that ginseng may serve as a potential therapeutic agent to limit myocardial I/R injury.

Nitric oxide synthase-3 deficiency results in hypoplastic coronary arteries and postnatal myocardial infarction

AIMS Hypoplastic coronary artery disease is a rare congenital abnormality that is associated with sudden cardiac death. However, molecular mechanisms responsible for this disease are not clear. The aim of the present study was to assess the role of nitric oxide synthase-3 (NOS3) in the pathogenesis of hypoplastic coronary arteries. METHODS AND RESULTS Wild-type (WT), NOS3(-/-), and a novel cardiac-specific NOS3 overexpression mouse model were employed. Deficiency in NOS3 resulted in coronary artery hypoplasia in foetal mice and spontaneous myocardial infarction in postnatal hearts. Coronary artery diameters, vessel density, and volume were significantly decreased in NOS3(-/-) mice at postnatal day 0. In addition, NOS3(-/-) mice showed a significant increase in the ventricular wall thickness, myocardial volume, and cardiomyocyte cell size compared with WT mice. Lack of NOS3 also down-regulated the expression of Gata4, Wilms tumour-1, vascular endothelial growth factor, basic fibroblast growth factor and erythropoietin, and inhibited migration of epicardial cells. These abnormalities and hypoplastic coronary arteries in the NOS3(-/-) mice were completely rescued by the cardiac-specific overexpression of NOS3. CONCLUSION Nitric oxide synthase-3 is required for coronary artery development and deficiency in NOS3 leads to hypoplastic coronary arteries.

Cardiac-Specific Overexpression of Human Stem Cell Factor Promotes Epicardial Activation and Arteriogenesis After Myocardial Infarction

Background—The adult epicardium is a potential source of cardiac progenitors after myocardial infarction (MI). We tested the hypothesis that cardiomyocyte-specific overexpression of membrane-associated human stem cell factor (hSCF) enhances epicardial activation, epicardium-derived cells (EPDCs) production, and myocardial arteriogenesis post MI. Methods and Results—Wild-type and the inducible cardiac-specific hSCF transgenic (hSCF/tetracycline transactivator) mice were subjected to MI. Wilms tumor-1 (Wt1)–positive epicardial cells were higher in hSCF/tetracycline transactivator compared with wild-type mice 3 days post MI. Arteriole density was significantly higher in the peri-infarct area of hSCF/tetracycline transactivator mice compared with wild-type mice 5 days post MI. In cultured EPDCs, adenoviral hSCF treatment significantly increased cell proliferation and growth factor expression. Furthermore, adenoviral hSCF treatment in wild-type cardiomyocytes significantly increased EPDC migration. These effects of hSCF overexpression on EPDC proliferation and growth factor expression were all abrogated by ACK2, a neutralizing antibody against c-kit. Finally, lineage tracing using ROSAmTmG;Wt1CreER mice showed that adenoviral hSCF treatment increased Wt1+ lineage–derived EPDC migration into the infarcted myocardium 5 days post MI, which was inhibited by ACK2. Conclusions—Cardiomyocyte-specific overexpression of hSCF promotes epicardial activation and myocardial arteriogenesis post MI.

Nitric Oxide Synthase-3 Promotes Embryonic Development of Atrioventricular Valves

Nitric oxide synthase-3 (NOS3) has recently been shown to promote endothelial-to-mesenchymal transition (EndMT) in the developing atrioventricular (AV) canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT) and NOS3−/− mice at postnatal day 0. Our data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3−/− compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3−/− mice. These phenotypes were all rescued by cardiac specific NOS3 overexpression. To assess EndMT, immunostaining of Snail1 was performed in the embryonic heart. Both total mesenchymal and Snail1+ cells in the AV cushion were decreased in NOS3−/− compared with WT mice at E10.5 and E12.5, which was completely restored by cardiac specific NOS3 overexpression. In cultured embryonic hearts, NOS3 promoted transforming growth factor (TGFβ), bone morphogenetic protein (BMP2) and Snail1expression through cGMP. Furthermore, mesenchymal cell formation and migration from cultured AV cushion explants were decreased in the NOS3−/− compared with WT mice. We conclude that NOS3 promotes AV valve formation during embryonic heart development and deficiency in NOS3 results in AV valve insufficiency.