Abstract-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 AMPK␣2 protects the heart against systolic overload-induced ventricular hypertrophy and dysfunction, we studied the response of AMPK␣2 gene deficient (knockout [KO]) mice and wild-type mice subjected to 3 weeks of transverse aortic constriction (TAC). Although AMPK␣2 KO had no effect on ventricular structure or function under control conditions, AMPK␣2 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). AMPK␣2 KO also significantly exacerbated the TAC-induced increases of atrial natriuretic peptide, myocardial fibrosis, and cardiac myocyte size. AMPK␣2 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 kinase Thr389 , p-S6 Ser235 , and p-eukaryotic initiation factor 4E Ser209 . AMPK␣2 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-Akt Ser473 . These data indicate that AMPK␣2 exerts a cardiac protective effect against pressure-overloadinduced ventricular hypertrophy and dysfunction. Key Words: hypertrophy Ⅲ congestive heart failure Ⅲ mTOR I ncreases of cardiac work resulting from systolic overload necessitate an increase of ATP use in proportion to the increase in left ventricular (LV) systolic wall stress. 1,2 In response to chronic systolic overload, cardiac myocyte hypertrophy occurs, characterized by increased protein synthesis, whereas myocardial oxygen consumption and carbon substrate use are increased to accommodate the need for increased energy availability. This initially occurs with no change in high energy phosphate levels, but with the development of pathological hypertrophy and congestive heart failure, ATP levels fall and cytosolic free ADP levels increase (as indicated by a decrease of the myocardial phosphocreatine:ATP ratio). 2,3 In this situation, the adenylate kinase reaction can catalyze the reaction of 2 molecules of ADP to produce 1 molecule of ATP and 1 molecule of AMP. An increased AMP:ATP ratio results in activation of the energy stress sensor known as AMP activated protein kinase (AMPK).AMPK is composed of 1 catalytic ␣ subunit (either ␣1 or ␣2) and 2 regulatory subunits (␤ and ␥). AMPK␣2 is the dominant catalytic subunit in the heart, 3,4 where it is predominantly expressed in cardiac myocytes. AMPK is activated by metabolic stres...

Objectives The objective of this study was to identify the role of dimethylarginine dimethylaminohydrolase-1 (DDAH1) in degrading the endogenous NOS inhibitors ADMA and 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 fold higher than in wild type mice, but growth and development of these DDAH1−/− mice was similar to 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 mmHg 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+/+ mice, DDAH1+/− mice and DDAH1−/− mice at anticipated ratios of 1:2:1, indicating that DDAH1 is not required for embryonic development in this strain. Conclusions Our findings indicate that DDAH1 is required for metabolizing ADMA and L-NMMA in vivo, while DDAH2 had no detectable role for degrading ADMA and L-NMMA.

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 Ϸ4.5-fold in CHF samples, and the increase of myocardial PDE5 expression was significantly correlated with myocardial oxidative stress markers 3Ј-nitrotyrosine or 4-hydroxynonenal expression (PϽ0.05). 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 (PϽ0.05). 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. (Circulation. 2010;121:1474-1483.)Key Words: heart failure Ⅲ oxidative stress Ⅲ cyclic nucleotide phosphodiesterases, type 5 C ongestive heart failure (CHF) is the leading cause of mortality in developed countries and continues to increase in prevalence. Phosphodiesterase type 5 (PDE5) selectively hydrolyzes cyclic 3Ј,5Ј-guanosine monophosphate (cGMP), and selective inhibition of PDE5 can increase cGMP bioavailability. It is generally believed that PDE5 is not present in normal cardiac myocytes, so that selective PDE5 inhibition has no direct inotropic effect in normal hearts. 1 However, recent work by Kass et al demonstrated that selective inhibition of PDE5 with sildenafil markedly attenuated the left ventricular (LV) hypertrophy and dysfunction produced by chronic pressure overload secondary to transverse aortic constriction (TAC) in mice. 2 Thus PDE5 inhibition has also been reported to attenuate myocardial infarctinduced LV remodeling 3 and LV hypertrophy produc...

Mitochondria are a principal site for generation of reactive oxygen species (ROS) in the heart. Peroxisome proliferator activated receptor g coactivator 1a (PGC-1a) plays an important role in regulating mitochondrial biogenesis and myocardial metabolism, but whether PGC-1a can simultaneously upregulate myocardial mitochondrial antioxidants has not been studied. In the present study, we examined the effect of PGC-1a deficiency (PGC-1a -=-) 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-1a -=-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-1a -=-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-1a -=-mice. These data indicate that PGC-1a 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. Antioxid.

Abstract-Extracellular superoxide dismutase (SOD) contributes only a small fraction to total SOD activity in the normal heart but is strategically located to scavenge free radicals in the extracellular compartment. To examine the physiological significance of extracellular SOD in the response of the heart to hemodynamic stress, we studied the effect of extracellular SOD deficiency on transverse aortic constriction (TAC)-induced left ventricular remodeling. Under unstressed conditions extracellular SOD deficiency had no effect on myocardial total SOD activity, the ratio of glutathione:glutathione disulfide, nitrotyrosine content, or superoxide anion production but resulted in small but significant increases in myocardial fibrosis and ventricular mass. In response to TAC for 6 weeks, extracellular SOD-deficient mice developed more severe left ventricular hypertrophy (heart weight increased 2.56-fold in extracellular SOD-deficient mice as compared with 1.99-fold in wild-type mice) and pulmonary congestion (lung weight increased 2.92-fold in extracellular SOD-deficient mice as compared with 1.84-fold in wild-type mice). Extracellular SOD-deficient mice also had more ventricular fibrosis, dilation, and a greater reduction of left ventricular fractional shortening and rate of pressure development after TAC. TAC resulted in greater increases of ventricular collagen I, collagen III, matrix metalloproteinase-2, matrix metalloproteinase-9, nitrotyrosine, and superoxide anion production. TAC also resulted in a greater decrease of the ratio of glutathione:glutathione disulfide in extracellular SOD-deficient mice. The finding that extracellular SOD deficiency had minimal impact on myocardial overall SOD activity but exacerbated TAC induced myocardial oxidative stress, hypertrophy, fibrosis, and dysfunction indicates that the distribution of extracellular SOD in the extracellular space is critically important in protecting the heart against pressure overload. Key Words: extracellular SOD Ⅲ hypertrophy Ⅲ congestive heart failure Ⅲ oxidative stress Ⅲ ventricular fibrosis Ⅲ MMP C ongestive heart failure (CHF) because of a variety of conditions is associated with depressed antioxidant reserves and increased products of oxygen free radical reactions, suggesting that oxidative stress might contribute to contractile dysfunction in the failing heart. 1 Superoxide dismutase (SOD) is the first line of defense against free radical attack. Three SOD isozymes have been identified, including a copper/zinc-containing SOD (SOD1), which is primarily cytosolic in location, a mitochondrial manganese SOD (SOD2), and an extracellular SOD (SOD3). SOD3 is a glycoprotein secreted into the extracellular fluid by fibroblasts that bind to sulfated polysaccharides, such as heparin and heparan sulfate, 2,3 as well as to other matrix components. 4,5 As a result, SOD3 binds to the surface of endothelial cells and the extracellular matrix, which has a high abundance of heparan sulfate. 6 Several recent studies have demonstrated that SOD3 expression is decre...

Substantial evidence suggests that cyclin D1 plays a pivotal role in the control of the hepatocyte cell cycle in response to mitogenic stimuli, whereas the closely related protein cyclin D3 has not been extensively evaluated. In the current study, we examined the regulation of cyclins D1 and D3 during hepatocyte proliferation in vivo after 70% partial hepatectomy (PH) and in culture. In contrast to cyclin D1, which was nearly undetectable in quiescent liver and substantially up-regulated after PH, cyclin D3 was constitutively expressed and induced only modestly. In the regenerating liver, the concentration of cyclin D3 was only about 10% of that of cyclin D1. Cyclin D1 formed complexes primarily with cyclin-dependent kinase 4 (cdk4), which were markedly activated in the regenerating liver and readily sequestered the cell cycle inhibitory proteins, p21 and p27. Cyclin D3 bound to both cdk4 and cdk6. Cyclin D3/cdk6 activity was readily detectable in quiescent liver and changed little after PH, and this complex appeared to play a minor role in sequestering p21 and p27. In cultured hepatocytes, epidermal growth factor or insulin had little effect, but the combination of these agents substantially induced cyclin D1 and cell cycle progression. Inhibition of Mek1 or phosphoinositide 3-kinase markedly inhibited cyclin D1 expression and replication. In contrast, cyclin D3 was expressed in the absence of mitogens and was only modestly affected by these manipulations. In addition, growth-inhibitory extracellular matrix conditions inhibited cyclin D1 but not cyclin D3 expression. In conclusion, these results support the concept that cyclin D1 is critically regulated by extracellular stimuli that control proliferation, whereas cyclin D3 is regulated through different pathways and plays a distinct role in the liver. (HEPATOLOGY 2002;36:30-38.) P rogression through the cell cycle is regulated by the activity of protein kinase complexes consisting of cyclins and cyclin-dependent kinases (cdk), which are activated at distinct stages of proliferation. 1-3 During G1 phase, mitogens up-regulate the D-type cyclins, which form complexes with cdk4 and cdk6 that phosphorylate the retinoblastoma protein (Rb) and the related p107 and p130 proteins. The three D-type cyclins (D1, D2, and D3) display significant homology, suggesting that they may perform overlapping functions. 4,5 This concept is supported by studies showing that cyclin D1 or D2 knockout mice are viable and show relatively limited phenotypic effects. 6-8 On the other hand, the expression patterns of the D-type cyclins vary widely in different cell types, suggesting that they have distinct functions in specific tissues. Although cyclins D1 and D2 clearly regulate cell proliferation, the role of cyclin D3 has not been well characterized. Cyclin D3 is implicated in cell cycle control in some types of cells, but it is also widely expressed in nonreplicating mammalian tissues, indicating that it plays a role in quiescent cells. 9,10 Indeed, cyclin D3 is up-regulated during te...

The normal expression of myocardial mitochondrial enzymes is essential to maintain the cardiac energy reserve and facilitate responses to stress, but the molecular mechanisms to maintain myocardial mitochondrial enzyme expression have been elusive. Here we report that congestive heart failure is associated with a significant decrease of myocardial Estrogen-Related Receptor alpha (ERRα), but not PPAR gamma coactivator-1 alpha (PGC1α), in human heart failure samples. In addition, chronic pressure overload in mice caused a decrease of ERRα expression that was significantly correlated to the degree of LV dysfunction, pulmonary congestion and decreases of a group of myocardial energy metabolism related genes. We found that the metabolic sensor AMP activated protein kinase (AMPK) regulates ERRα expression in vivo and in vitro. AMPKα2 KO decreased myocardial ERRα (both mRNA and protein) and its downstream targets under basal conditions, with no change in myocardial PGC1α expression. Using cultured rat neonatal cardiac myocytes, we found that overexpression of constitutively active AMPKα significantly induced ERRα mRNA, protein and promoter activity. Conversely, selective gene silencing of AMPKα2 repressed ERRα and its target gene levels, indicating that AMPKα2 is involved in the regulation of ERRα expression. In addition, over-expression of ERRα in AMPKα2 KO neonatal cardiac myocytes partially rescued the repressed expression of some energy metabolism related genes. These data support an important role for AMPKα2 in regulating the expression of myocardial ERRα and its downstream mitochondrial enzymes.

Studies have demonstrated that increased oxidative stress contributes to the pathogenesis and the development of pulmonary artery hypertension (PAH). Extracellular superoxide dismutase (SOD3) is essential for removing extracellular superoxide anions and it is highly expressed in lung tissue. However, it is not clear whether endogenous SOD3 can influence the development of PAH. Here we examined the effect of SOD3 knockout on hypoxia-induced PAH in mice and a loss-of-function SOD3 gene mutation (SOD3E124D) on monocrotaline (40 mg/kg)-induced PAH in rats. SOD3 knockout significantly exacerbated 2 weeks hypoxia-induced right ventricular (RV) pressure and RV hypertrophy, while RV pressure in SOD3 KO mice under normoxic conditions is similar to wild type controls. In untreated control rats at age of 8 weeks, there was no significant difference between wild type and SOD3E124D rats in RV pressure and the ratio of RV weight to left ventricular weight (0.25±0.02 in wild type rats vs. 0.25±0.01 in SOD3E124D rats). However, monocrotaline caused significantly greater increases of RV pressure in SOD3E124D rats (48.6±1.8 mmHg in wild type vs. 57.5±3.1 mmHg in SOD3E124D rats), of the ratio of RV weight to left ventricular weight (0.41±0.01 vs. 0.50±0.09, p<0.05), and of the percentage of fully muscularized small arterioles in SOD3E124D rats (55.2±2.3 % vs. 69.9 ±2.6 %, p<0.05). Together, these findings indicate that the endogenous SOD3 has no role in the development of PAH under control conditions, but plays an important role in protecting the lung from the development of PAH under stress conditions.