Xin Xu

Downregulation of Adipose Glutathione S-Transferase A4 Leads to Increased Protein Carbonylation, Oxidative Stress, and Mitochondrial Dysfunction

OBJECTIVE Peripheral insulin resistance is linked to an increase in reactive oxygen species (ROS), leading in part to the production of reactive lipid aldehydes that modify the side chains of protein amino acids in a reaction termed protein carbonylation. The primary enzymatic method for lipid aldehyde detoxification is via glutathione S-transferase A4 (GSTA4) dependent glutathionylation. The objective of this study was to evaluate the expression of GSTA4 and the role(s) of protein carbonylation in adipocyte function. RESEARCH DESIGN AND METHODS GSTA4-silenced 3T3-L1 adipocytes and GSTA4-null mice were evaluated for metabolic processes, mitochondrial function, and reactive oxygen species production. GSTA4 expression in human obesity was evaluated using microarray analysis. RESULTS GSTA4 expression is selectively downregulated in adipose tissue of obese insulin-resistant C57BL/6J mice and in human obesity-linked insulin resistance. Tumor necrosis factor-α treatment of 3T3-L1 adipocytes decreased GSTA4 expression, and silencing GSTA4 mRNA in cultured adipocytes resulted in increased protein carbonylation, increased mitochondrial ROS, dysfunctional state 3 respiration, and altered glucose transport and lipolysis. Mitochondrial function in adipocytes of lean or obese GSTA4-null mice was significantly compromised compared with wild-type controls and was accompanied by an increase in superoxide anion. CONCLUSIONS These results indicate that downregulation of GSTA4 in adipose tissue leads to increased protein carbonylation, ROS production, and mitochondrial dysfunction and may contribute to the development of insulin resistance and type 2 diabetes.

AMP Activated Protein Kinase-α2 Deficiency Exacerbates Pressure-Overload–Induced Left Ventricular Hypertrophy and Dysfunction in Mice

AMP activated protein kinase (AMPK) plays an important role in regulating myocardial metabolism and protein synthesis. Activation of AMPK attenuates hypertrophy in cultured cardiac myocytes, but the role of AMPK in regulating the development of myocardial hypertrophy in response to chronic pressure overload is not known. To test the hypothesis that AMPKα2 protects the heart against systolic overload–induced ventricular hypertrophy and dysfunction, we studied the response of AMPKα2 gene deficient (knockout [KO]) mice and wild-type mice subjected to 3 weeks of transverse aortic constriction (TAC). Although AMPKα2 KO had no effect on ventricular structure or function under control conditions, AMPKα2 KO significantly increased TAC-induced ventricular hypertrophy (ventricular mass increased 46% in wild-type mice compared with 65% in KO mice) while decreased left ventricular ejection fraction (ejection fraction decreased 14% in wild-type mice compared with a 43% decrease in KO mice). AMPKα2 KO also significantly exacerbated the TAC-induced increases of atrial natriuretic peptide, myocardial fibrosis, and cardiac myocyte size. AMPKα2 KO had no effect on total S6 ribosomal protein (S6), p70 S6 kinase, eukaryotic initiation factor 4E, and 4E binding protein-1 or their phosphorylation under basal conditions but significantly augmented the TAC-induced increases of p-p70 S6 kinaseThr389, p-S6Ser235, and p-eukaryotic initiation factor 4ESer209. AMPKα2 KO also enhanced the TAC-induced increase of p-4E binding protein-1Thr46 to a small degree and augmented the TAC-induced increase of p-AktSer473. These data indicate that AMPKα2 exerts a cardiac protective effect against pressure-overload–induced ventricular hypertrophy and dysfunction.

PGC-1α regulates expression of myocardial mitochondrial antioxidants and myocardial oxidative stress after chronic systolic overload

Mitochondria are a principal site for generation of reactive oxygen species (ROS) in the heart. Peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1 alpha) plays an important role in regulating mitochondrial biogenesis and myocardial metabolism, but whether PGC-1 alpha can simultaneously upregulate myocardial mitochondrial antioxidants has not been studied. In the present study, we examined the effect of PGC-1 alpha deficiency (PGC-1 alpha(-/-)) on oxidative stress and expression of a group of mitochondrial antioxidants in normal hearts and in hearts exposed to chronic systolic pressure overload produced by transverse aortic constriction (TAC). We found that PGC-1 alpha(-/-) caused moderate but significant decreases of myocardial mitochondrial antioxidant enzymes such as SOD2, and thioredoxin (Trx2), but had no effect on expression of myocardial oxidative stress markers and left ventricular (LV) function under basal conditions. However, in response to TAC for 6 weeks, PGC-1 alpha(-/-) mice showed greater increases of myocardial oxidative stress markers 3-nitrotyrosine and 4-hydroxynonenal, more severe LV hypertrophy and dilatation, pulmonary congestion, and a greater reduction of LV fractional shortening and dP/dt(max) than did wild-type hearts. SOD mimetic MnTMPyP treatment (6 mg/kg/day) significantly attenuated TAC-induced LV hypertrophy and dysfunction in PGC-1 alpha(-/-) mice. These data indicate that PGC-1 alpha plays an important role in regulating expression of myocardial mitochondrial antioxidants SOD2 and Trx2 and in protecting hearts against TAC-induced myocardial oxidative stress, hypertrophy, and dysfunction.

Inducible Nitric Oxide Synthase Deficiency Protects the Heart From Systolic Overload–Induced Ventricular Hypertrophy and Congestive Heart Failure

Inducible nitric oxide synthase (iNOS) protein is expressed in cardiac myocytes of patients and experimental animals with congestive heart failure (CHF). Here we show that iNOS expression plays a role in pressure overload–induced myocardial chamber dilation and hypertrophy. In wild-type mice, chronic transverse aortic constriction (TAC) resulted in myocardial iNOS expression, cardiac hypertrophy, ventricular dilation and dysfunction, and fibrosis, whereas iNOS-deficient mice displayed much less hypertrophy, dilation, fibrosis, and dysfunction. Consistent with these findings, TAC resulted in marked increases of myocardial atrial natriuretic peptide 4-hydroxy-2-nonenal (a marker of lipid peroxidation) and nitrotyrosine (a marker for peroxynitrite) in wild-type mice but not in iNOS-deficient mice. In response to TAC, myocardial endothelial NO synthase and iNOS was expressed as both monomer and dimer in wild-type mice, and this was associated with increased reactive oxygen species production, suggesting that iNOS monomer was a source for the increased oxidative stress. Moreover, systolic overload–induced Akt, mammalian target of rapamycin, and ribosomal protein S6 activation was significantly attenuated in iNOS-deficient mice. Furthermore, selective iNOS inhibition with 1400W (6 mg/kg per hour) significantly attenuated TAC induced myocardial hypertrophy and pulmonary congestion. These data implicate iNOS in the maladaptative response to systolic overload and suggest that selective iNOS inhibition or attenuation of iNOS monomer content might be effective for treatment of systolic overload-induced cardiac dysfunction.

Oxidative Stress Regulates Left Ventricular PDE5 Expression in the Failing Heart

Background— Phosphodiesterase type 5 (PDE5) inhibition has been shown to exert profound beneficial effects in the failing heart, suggesting a significant role for PDE5 in the development of congestive heart failure (CHF). The purpose of this study is to test the hypothesis that oxidative stress causes increased PDE5 expression in cardiac myocytes and that increased PDE5 contributes to the development of CHF. Methods and Results— Myocardial PDE5 expression and cellular distribution were determined in left ventricular samples from patients with end-stage CHF and normal donors and from mice after transverse aortic constriction (TAC)–induced CHF. Compared with donor human hearts, myocardial PDE5 protein was increased ≈4.5-fold in CHF samples, and the increase of myocardial PDE5 expression was significantly correlated with myocardial oxidative stress markers 3′-nitrotyrosine or 4-hydroxynonenal expression (P<0.05). Histological examination demonstrated that PDE5 was mainly expressed in vascular smooth muscle in normal donor hearts, but its expression was increased in both cardiac myocytes and vascular smooth muscle of CHF hearts. Myocardial PDE5 protein content and activity also increased in mice after TAC-induced CHF (P<0.05). When the superoxide dismutase (SOD) mimetic M40401 was administered to attenuate oxidative stress, the increased PDE5 protein and activity caused by TAC was blunted, and the hearts were protected against left ventricular hypertrophy and CHF. Conversely, increased myocardial oxidative stress in superoxide dismutase 3 knockout mice caused a greater increase of PDE5 expression and CHF after TAC. In addition, administration of sildenafil to inhibit PDE5 attenuated TAC-induced myocardial oxidative stress, PDE5 expression, and CHF. Conclusions— Myocardial oxidative stress increases PDE5 expression in the failing heart. Reducing oxidative stress by treatment with M40401 attenuated cardiomyocyte PDE5 expression. This and selective inhibition of PDE5 protected the heart against pressure overload-induced left ventricular hypertrophy and CHF.

Dimethylarginine Dimethylaminohydrolase-1 Is the Critical Enzyme for Degrading the Cardiovascular Risk Factor Asymmetrical Dimethylarginine

Objective—The objective of this study was to identify the role of dimethylarginine dimethylaminohydrolase-1 (DDAH1) in degrading the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and Ng-monomethyl-L-arginine (L-NMMA). Methods and Results—We generated a global-DDAH1 gene–deficient (DDAH1−/−) mouse strain to examine the role of DDAH1 in ADMA and L-NMMA degradation and the physiological consequences of loss of DDAH1. Plasma and tissue ADMA and L-NMMA levels in DDAH1−/− mice were several folds higher than in wild-type mice, but growth and development of these DDAH1−/− mice were similar to those of their wild-type littermates. Although the expression of DDAH2 was unaffected, DDAH activity was undetectable in all tissues tested. These findings indicate that DDAH1 is the critical enzyme for ADMA and L-NMMA degradation. Blood pressure was ≈20 mm Hg higher in the DDAH1−/− mice than in wild-type mice, but no other cardiovascular phenotype was found under unstressed conditions. Crossing DDAH1+/− male with DDAH1+/− female mice yielded DDAH1+/+, DDAH1+/−, and DDAH1−/− mice at the anticipated ratio of 1:2:1, indicating that DDAH1 is not required for embryonic development in this strain. Conclusion—Our findings indicate that DDAH1 is required for metabolizing ADMA and L-NMMA in vivo, whereas DDAH2 had no detectable role for degrading ADMA and L-NMMA.

Left Ventricular Failure Produces Profound Lung Remodeling and Pulmonary Hypertension in Mice: Heart Failure Causes Severe Lung Disease

Chronic left ventricular failure causes pulmonary congestion with increased lung weight and type 2 pulmonary hypertension. Understanding the molecular mechanisms for type 2 pulmonary hypertension and the development of novel treatments for this condition requires a robust experimental animal model and a good understanding of the nature of the resultant pulmonary remodeling. Here we demonstrate that chronic transverse aortic constriction causes massive pulmonary fibrosis and remodeling, as well as type 2 pulmonary hypertension, in mice. Thus, aortic constriction-induced left ventricular dysfunction and increased left ventricular end-diastolic pressure are associated with a ⩽5.3-fold increase in lung wet weight and dry weight, pulmonary hypertension, and right ventricular hypertrophy. Interestingly, the aortic constriction-induced increase in lung weight was not associated with pulmonary edema but resulted from profound pulmonary remodeling with a dramatic increase in the percentage of fully muscularized lung vessels, marked vascular and lung fibrosis, myofibroblast proliferation, and leukocyte infiltration. The aortic constriction-induced left ventricular dysfunction was also associated with right ventricular hypertrophy, increased right ventricular end-diastolic pressure, and right atrial hypertrophy. The massive lung fibrosis, leukocyte infiltration, and pulmonary hypertension in mice after transverse aortic constriction clearly indicate that congestive heart failure also causes severe lung disease. The lung fibrosis and leukocyte infiltration may be important mechanisms in the poor clinical outcome in patients with end-stage heart failure. Thus, the effective treatment of left ventricular failure may require additional efforts to reduce lung fibrosis and the inflammatory response.

Global Dimethylarginine Dimethylaminohydrolase-1 (DDAH1) Gene–Deficient Mice Reveal That DDAH1 Is the Critical Enzyme for Degrading the Cardiovascular Risk Factor Asymmetrical Dimethylarginine

Objective—The objective of this study was to identify the role of dimethylarginine dimethylaminohydrolase-1 (DDAH1) in degrading the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and Ng-monomethyl-L-arginine (L-NMMA). Methods and Results—We generated a global-DDAH1 gene–deficient (DDAH1−/−) mouse strain to examine the role of DDAH1 in ADMA and L-NMMA degradation and the physiological consequences of loss of DDAH1. Plasma and tissue ADMA and L-NMMA levels in DDAH1−/− mice were several folds higher than in wild-type mice, but growth and development of these DDAH1−/− mice were similar to those of their wild-type littermates. Although the expression of DDAH2 was unaffected, DDAH activity was undetectable in all tissues tested. These findings indicate that DDAH1 is the critical enzyme for ADMA and L-NMMA degradation. Blood pressure was ≈20 mm Hg higher in the DDAH1−/− mice than in wild-type mice, but no other cardiovascular phenotype was found under unstressed conditions. Crossing DDAH1+/− male with DDAH1+/− female mice yielded DDAH1+/+, DDAH1+/−, and DDAH1−/− mice at the anticipated ratio of 1:2:1, indicating that DDAH1 is not required for embryonic development in this strain. Conclusion—Our findings indicate that DDAH1 is required for metabolizing ADMA and L-NMMA in vivo, whereas DDAH2 had no detectable role for degrading ADMA and L-NMMA.

Indole Cytosolic Phospholipase A2 α Inhibitors: Discovery and in Vitro and in Vivo Characterization of 4-{3-[5-Chloro-2-(2-{[(3,4-dichlorobenzyl)sulfonyl]amino}ethyl)-1-(diphenylmethyl)-1H-indol-3-yl]propyl}benzoic Acid, Efipladib

The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.

Extracellular superoxide dismutase protects the heart against oxidative stress and hypertrophy after myocardial infarction

Extracellular superoxide dismutase (EC-SOD) contributes only a small fraction to total SOD activity in the heart but is strategically located to scavenge free radicals in the extracellular compartment. EC-SOD expression is decreased in myocardial-infarction (MI)-induced heart failure, but whether EC-SOD can abrogate oxidative stress or modify MI-induced ventricular remodeling has not been previously studied. Consequently, the effects of EC-SOD gene deficiency (EC-SOD KO) on left ventricular (LV) oxidative stress, hypertrophy, and fibrosis were studied in EC-SOD KO and wild-type mice under control conditions, and at 4 and 8 weeks after permanent coronary artery ligation. EC-SOD KO had no detectable effect on LV function in normal hearts but caused small but significant increases of LV fibrosis. At 8 weeks after MI, EC-SOD KO mice developed significantly more LV hypertrophy (LV mass increased 1.64-fold in KO mice compared to 1.35-fold in wild-type mice; p<0.01) and more fibrosis and myocyte hypertrophy which was more prominent in the peri-infarct region than in the remote myocardium. EC-SOD KO mice had greater increases of nitrotyrosine in the peri-infarct myocardium, and this was associated with a greater reduction of LV ejection fraction, a greater decrease of sarcoplasmic or endoplasmic reticulum calcium2+ ATPase, and a greater increase of atrial natriuretic peptide in the peri-infarct zone compared to wild-type mice. EC-SOD KO was associated with more increases of phosphorylated p38 (p-p38(Thr180/Tyr182)), p42/44 extracellular signal-regulated kinase (p-Erk(Thr202/Tyr204)), and c-Jun N-terminal kinase (p-JNK(Thr183/Tyr185)) both under control conditions and after MI, indicating that EC-SOD KO increases activation of mitogen-activated protein kinase signaling pathways. These findings demonstrate that EC-SOD plays an important role in protecting the heart against oxidative stress and infarction-induced ventricular hypertrophy.