Regulated necrosis (necroptosis) and apoptosis are crucially involved in severe cardiac pathological conditions, including myocardial infarction, ischemia-reperfusion injury and heart failure. Whereas apoptotic signaling is well defined, the mechanisms that underlie cardiomyocyte necroptosis remain elusive. Here we show that receptor-interacting protein 3 (RIP3) triggers myocardial necroptosis, in addition to apoptosis and inflammation, through activation of Ca(2+)-calmodulin-dependent protein kinase (CaMKII) rather than through the well-established RIP3 partners RIP1 and MLKL. In mice, RIP3 deficiency or CaMKII inhibition ameliorates myocardial necroptosis and heart failure induced by ischemia-reperfusion or by doxorubicin treatment. RIP3-induced activation of CaMKII, via phosphorylation or oxidation or both, triggers opening of the mitochondrial permeability transition pore and myocardial necroptosis. These findings identify CaMKII as a new RIP3 substrate and delineate a RIP3-CaMKII-mPTP myocardial necroptosis pathway, a promising target for the treatment of ischemia- and oxidative stress-induced myocardial damage and heart failure.

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...

Abstract-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. (Circ Res. 2007;100:1089-1098.)Key Words: superoxide anion Ⅲ peroxynitrite Ⅲ iNOS monomer Ⅲ mTOR S everal investigators have demonstrated that inducible nitric oxide synthase (iNOS) protein is expressed in cardiac myocytes and endocardial endothelium of patients and animals with ventricular hypertrophy or congestive heart failure (CHF) regardless of cause. 1-4 Thus, iNOS was coexpressed with tumor necrosis factor-␣ in cardiac myocytes from patients with dilated cardiomyopathy 2 and increased in several animal models of ventricular hypertrophy or CHF. 5 Although unregulated NO production by iNOS has been proposed to exert negative effects on cardiomyocyte function, the effect of iNOS expression on ventricular hypertrophy and CHF in the in vivo heart is controversial. Thus, Heger et al 6 reported that overexpression of iNOS in cardiac myocytes increased myocardial NOS activity and NO production but had no effect on cardiac morphology or function. In contrast, Mungrue et al 7 reported that cardiac-specific overexpression of iNOS resulted in inflammatory cell infiltrate, left ventricular (LV) hypertrophy, dilation, fibrosis, and contractile dysfunction. The level of iNOS expression in these transgenic mice would depend on the promoter activity, and the iNOSrelated phenotypes might vary depending on the level of myocardial iNOS expression. Furthermore, the effects of stress-induced iNOS expression in...

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...

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, and type-2 pulmonary hypertension in mice. Thus, aortic constriction-induced left ventricular dysfunction and increased left ventricular end-diastolic pressure is associated with up to 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.

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.

Receptor-interacting protein 140 (RIP140) encodes a histone deacetylase (HDAC) inhibitor-sensitive repressive activity. Direct interaction of RIP140 with HDAC1 and HDAC3 occurs in vitro and in vivo as demonstrated in co-immunoprecipitation and glutathione S-transferase pull-down experiments. The HDAC-interacting domain of RIP140 is mapped to its N-terminal domain, between amino acids 78 and 303 based upon glutathione S-transferase pull-down experiments. In chromatin immunoprecipitation assays, it is demonstrated that histone deacetylation occurs at the chromatin region of the Gal4 binding sites as a result of Gal4 DNA binding domain-tethered RIP expression. The immunocomplexes of RIP140 from cells transfected with RIP140 and HDAC are able to deacetylate histone proteins in vitro. This study presents the first evidence for RIP140 as a negative coregulator for nuclear receptor actions by directly recruiting histone deacetylases and categorizes RIP140 as a novel negative coregulator that is able to directly interact with HDACs.Nuclear receptors play important roles in gene regulation. In most cases, they regulate target gene expression by binding to their cognate DNA response elements and recruiting associate proteins to the transcription machinery (1, 2). A large number of associate proteins of nuclear receptors have been found and demonstrated as co-activators or co-repressors (3). One mechanism of co-repressor and co-activator action has been shown to involve chromatin modification such as acetylation of chromosomal histone proteins (4 -6). For instance, corepressors such as N-CoR/SMRT interact with apo-receptors in their hinge region and recruit histone deacetylases (HDACs) 1 indirectly through the Sin3 complexes (4, 7). However, recent studies have shown that co-repressors bind to the same cavity formed by helices 3-5 and 12 in the ligand binding domain of nuclear receptors as do co-activators (8 -10). Upon ligand binding, the ligand binding domain undergoes a conformational change, releases corepressors, and recruits coactivators by re-positioning helix 12 (AF-2 domain). Several coactivators, mainly the p160 family, have been identified, including SRC-1/NCoA-1, TIF2/GRIP1/NCoA-2, and p/CIP/RAC3/ACTR/AIB1 (11-16). The SRC-1, ACTR, and CBP/p300 have been shown to encode intrinsic histone acetyl transferase activities (5, 6, 17).Two major classes of HDACs have been cloned in higher eukaryotes. Class 1 HDACs (HDAC 1-3) are homologous to the yeast , and class 2 HDACs (HDAC 4 -7) are homologues of the yeast Hda1 (21). It has been demonstrated that disruption of either Rpd3 or Hda1 in yeast resulted in global hyperacetylation of histones (19). The acetylation of chromosomal histones is correlated strongly with active gene transcription; conversely, hypoacetylation of a gene regulatory region is usually indicative of gene silencing (22-25). Whereas acetylation of histones is not sufficient to dictate the efficiency of transcription, an overwhelmingly large volume of convincing evidence has supported the notion that t...