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Bioenergetic homeostasis is altered in heart failure and may play an important role in pathogenesis, p53 has been implicated in heart failure, and although its role in regulating tumorigenesis is well characterized, its activities on cellular metabolism are just beginning to be understood. We investigated the role of p53 and its transcriptional target gene TP53-induced glycolysis and apoptosis regulator (TIGAR) in myocardial energy metabolism under conditions simulating ischemia that can lead to heart failure. Expression of p53 and TIGAR was markedly upregulated after myocardial infarction, and apoptotic myocytes were decreased by 42% in p53-deficient mouse hearts compared with those in wild-type mice. To examine the effect of p53 on energy metabolism, cardiac myocytes were exposed to hypoxia. Hypoxia induced p53 and TIGAR expression in a p53-dependent manner. Knockdown of p53 or TIGAR increased glycolysis with elevated fructose-2,6-bisphosphate levels and reduced myocyte apoptosis. Hypoxic stress decreased phosphocreatine content and the mitochondrial membrane potential of myocytes without changes in ATP content, the effects of which were prevented by the knockdown of TIGAR. Inhibition of glycolysis by 2-deoxyglucose blocked these bioenergetic effects and TIGAR siRNA-mediated prevention of apoptosis, and, in contrast, overexpression of TIGAR reduced glucose utilization and increased apoptosis. Our data demonstrate that p53 and TIGAR inhibit glycolysis in hypoxic myocytes and that inhibition of glycolysis is closely involved in apoptosis, suggesting that p53 and TIGAR are significant mediators of cellular energy homeostasis and cell death under ischemic stress. TP53-induced glycolysis and apoptosis regulator; hypoxia; metabolism; mitochondria; glycolysis doi: 10.1152/ajpheart.00250.2010.

Medial artery calcification, which does not accompany lipid or cholesterol deposit, preferentially occurs in elderly population, but its underlying mechanisms remain unclear. In the present study, we investigated the potential role of senescent vascular smooth muscle cells (VSMCs) in the formation of senescence-associated medial calcification. Replicative senescence was induced by the extended passages (until passages 11-13) in human primary VSMCs, and cells in early passage (passage 6) were used as control young cells. VSMC calcification was markedly enhanced in the senescent cells compared with that in the control young cells. We identified that genes highly expressed in osteoblasts, such as alkaline phosphatase (ALP) and type I collagen, were significantly upregulated in the senescent VSMCs, suggesting their osteoblastic transition during the senescence. Knock-down of either ALP or type I collagen significantly reduced the calcification in the senescent VSMCs. Of note, runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates the osteoblastic differentiation, was also upregulated in the senescent VSMCs. Knockdown of RUNX-2 significantly reduced the ALP expression and calcification in the senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of aorta from the [klotho.sup.-/-] aging mouse model demonstrated in vivo emergence of osteoblast-like cells expressing RUNX-2 exclusively in the calcified media. We also found that statin and Rho-kinase inhibitor effectively reduced the VSMC calcification by inhibiting [P.sub.i]-induced apoptosis and potentially enhancing matrix Gla protein expression in the senescent VSMCs. These findings strongly suggest an important role of senescent VSMCs in the pathophysiology of senescence-associated medial calcification, and the inhibition of osteoblastic transition could be a new therapeutic approach for the prevention of senescence-associated medial calcification. runt-related transcription factor-2; statin doi: 10.1152/ajpheart.00455.2009.

Although granulocyte colony-stimulating factor (G-CSF) reportedly plays a cardioprotective role in several models of cardiac injury, clinical use of this drug in cardiac patients has been controversial. Here, we tested, in vivo and in vitro, the effect of G-CSF on cardiac mitochondria, which play a key role in determining cardiac cellular fate and function. Mild stimulation of C57/BL6 mice with doxorubicin (Dox) did not induce cardiac apoptosis or fibrosis but did induce damage to mitochondrial organization of the myocardium as observed through an electron microscope. Cardiac catheterization and echocardiography revealed that Dox did not alter cardiac systolic function or left ventricular size but did reduce diastolic function, an early sign of cardiac damage. Treatment with G-CSF attenuated significantly the damage to mitochondrial organization and rescued diastolic function. In an in vitro model for rat neonatal cardiomyocytes, a subapoptotic dose of Dox induced severe mitochondrial damage, including marked swelling of the cardiac mitochondria and/or decreased mitochondrial membrane potential. These mitochondrial changes were completely blocked by pretreatment with G-CSF. In addition, G-CSF dramatically improved ATP generation, which rescued Dox-impaired mitochondrial electron transport and oxygen consumption mainly through complex IV. These findings clearly indicate that G-CSF protects cardiac mitochondria, which are key organelles in the determination of cardiac cellular fate, in the early phase of cardiac injury. doxorubicin; mitochondria; cardiac diastolic function; oxygen consumption

Although the modulated expression of Dicer is documented upon neoplastic transformation, little is known of the regulation of Dicer expression by environmental stimuli and its roles in the regulation of cellular functions in primary cells. In this study, we found that Dicer expression was downregulated upon serum withdrawal in human umbilical vein endothelial cells (HUVECs). Serum withdrawal induced a time-dependent repression of Dicer expression, which was specifically rescued by vascular endothelial cell growth factor or sphingosine-1-phosphate. When Dicer expression was silenced by short-hairpin RNA against Dicer, the cells were more prone to apoptosis under serum withdrawal, whereas the rate of apoptosis was comparable with control cells in the serum-containing condition. Real-time PCR-based gene expression profiling identified several genes, the expression of which was modulated by Dicer silencing, including adhesion and matrix-related molecules, caspase-3, and nitric oxide synthase 3 (NOS3). Dicer silencing markedly impaired migratory functions without affecting cell adhesion and repressed phosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 in adherent HUVECs. Dicer knockdown upregulated caspase-3 and downregulated NOS3 expression, and serum withdrawal indeed increased caspase-3 and decreased NOS3 expression. Furthermore, the overexpression of Dicer in HUVECs resulted in a marked reduction in apoptosis upon serum withdrawal and a decreased caspase-3 and increased NOS3 expression. The inhibition of NOS activity by [N.sup.[omega]]-nitro-L-arginine methyl ester abrogated the effect of Dicer overexpression to rescue the cells from serum withdrawal-induced apoptosis. These results indicated that serum withdrawal decreases Dicer expression, leading to an increased susceptibility to apoptosis through the regulation of caspase-3 and NOS3 expression. caspase 3; nitric oxide synthase 3

Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation. myoblast; skeletal muscle; differentiation; extracellular signal-regulated kinase; myogenin

Angiotensin II type 2 receptor ([AT.sub.2]R) overexpression ([AT.sub.2]TG) attenuates left ventricular remodeling in a mouse model of anterior myocardial infarction (MI). We hypothesized that the beneficial effects of cardiac [AT.sub.2]TG are mediated via the bradykinin subtype 2 receptor ([B.sub.2]R). Fourteen transgenic mice overexpressing the [AT.sub.2]R ([AT.sub.2]TG mice), 10 mice with a [B.sub.2]R deletion ([B.sub.2]KO mice), 13 [AT.sub.2]TG mice with [B.sub.2]R deletion ([AT.sub.2]TG/[B.sub.2]KO mice), and 11 wild-type (WT) mice were studied. All mice were on a C57BL/6 background. Mice were studied by cardiac magnetic resonance imaging at baseline and days 1, 7, and 28 after MI induced by 1 h of occlusion of the left anterior descending artery followed by reperfusion. Short-axis images from apex to base were used to compare ventricular volumes and ejection fraction (EF). At baseline, end-diastolic volume index (EDVI) and end-systolic volume index (ESVI) were lower and EF higher in [AT.sub.2]TG mice compared with the other three strains. Infarct size was similar between groups. No differences were observed in global remodeling parameters at day 28 between [AT.sub.2]TG and [AT.sub.2]TG/[B.sub.2]KO mice; however, EDVI and ESVI were lower and EF higher in both transgenic groups than in WT or [B.sub.2]KO mice. Both strains lacking [B.sub.2]R demonstrated increased collagen content and less hypertrophy in adjacent noninfarcted regions at day 28. Attenuation of postinfarct remodeling by overexpression of [AT.sub.2]R is not directly mediated via a [B.sub.2]R pathway. However, [B.sub.2]R does appear to have a role in the smaller cavity size and hyperdynamic function observed at baseline in [AT.sub.2]TG mice and in limiting collagen deposition during postinfarct remodeling. magnetic resonance imaging; remodeling

The [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter (NBC) plays a key role in intracellular pH (p[H.sub.i] regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. p[H.sub.i] was measured in single cells using the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)5-(6)carboxy-fluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCel) and SLC4A7 (electroneutral NBC, NBCnl). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCel (SLC4A4) and electroneutral NBCnl (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC ([J.sub.NBC]) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via [Na.sup.+]/[H.sup.+] exchange was also increased in hypertrophied myocytes, the relative enhancement of [J.sub.NBC] was larger, 6) membrane depolarization markedly increased [J.sub.NBC] in hypertrophied myocytes, and 7) losartan, an ANG II A[T.sub.1] receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular [Na.sup.+] overload, which may render the ventricles more vulnerable to [Ca.sup.2+] overload during ischemia-reperfusion. cardiac myocytes; intracellular pH regulation; messenger ribonucleic acid; polymerase chain reaction