Background-Cardiac hypertrophy involves growth responses to a variety of stimuli triggered by increased workload. It is an independent risk factor for heart failure and sudden death. Mammalian target of rapamycin (mTOR) plays a key role in cellular growth responses by integrating growth factor and energy status signals. It is found in 2 structurally and functionally distinct multiprotein complexes called mTOR complex (mTORC) 1 and mTORC2. The role of each of these branches of mTOR signaling in the adult heart is currently unknown. Methods and Results-We generated mice with deficient myocardial mTORC1 activity by targeted ablation of raptor, which encodes an essential component of mTORC1, during adulthood. At 3 weeks after the deletion, atrial and brain natriuretic peptides and ␤-myosin heavy chain were strongly induced, multiple genes involved in the regulation of energy metabolism were altered, but cardiac function was normal. Function deteriorated rapidly afterward, resulting in dilated cardiomyopathy and high mortality within 6 weeks. Aortic banding-induced pathological overload resulted in severe dilated cardiomyopathy already at 1 week without a prior phase of adaptive hypertrophy. The mechanism involved a lack of adaptive cardiomyocyte growth via blunted protein synthesis capacity, as supported by reduced phosphorylation of ribosomal S6 kinase 1 and 4E-binding protein 1. In addition, reduced mitochondrial content, a shift in metabolic substrate use, and increased apoptosis and autophagy were observed. Conclusions-Our results demonstrate an essential function for mTORC1 in the heart under physiological and pathological conditions and are relevant for the understanding of disease states in which the insulin/insulin-like growth factor signaling axis is affected such as diabetes mellitus and heart failure or after cancer therapy. (Circulation. 2011;123:1073-1082.)Key Words: heart failure Ⅲ hypertrophy Ⅲ myocardial metabolism Ⅲ signal transduction A lthough cardiac hypertrophy is a growth response that initially normalizes wall tension, it is associated with an unfavorable outcome: Affected patients are threatened with sudden death or progression to heart failure. 1 Much research is therefore aimed at understanding myocardial growth regulation, and in this setting, the insulin-like growth factor/PI3-kinase/Akt signaling cascade has been studied extensively. 2,3 Experiments with cultured cardiomyocytes have suggested that downstream of Akt, mammalian target of rapamycin (mTOR) mediates responses to pathological stimuli. 4,5 mTOR is an evolutionary conserved Ser/Thr kinase known to control cell growth. 6 Nutrient, energy, and growth factor shortage will impair mTOR activity, resulting in diverse effects, including the slowdown of macromolecule synthesis, enhanced autophagy, and activation of nutrient-or stressresponsive transcription factors. mTOR is found in 2 structurally and functionally distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2. The 2 best-characterized substrates of mTORC1 ...

Recent evidence supports that TNF-alpha, long considered a catabolic factor, may also have a physiological function in skeletal muscle. The catabolic view, mainly based on correlative studies in human and in vivo animal models, was challenged by experiments with myoblasts, in which TNF-alpha induced differentiation. The biological effects of TNF-alpha in differentiated muscle, however, remain poorly understood. In the present study, we tested whether TNF-alpha has growth-promoting effects in myotubes, and we characterized the mechanisms leading to these effects. Treatment of C(2)C(12) myotubes with TNF-alpha for 24 h increased protein synthesis (PS) and enhanced cellular dehydrogenase activity by 22 and 26%, respectively, without changing cell numbers. These effects were confirmed in myotubes differentiated from primary rat myoblasts. TNF-alpha activated two signaling cascades: 1) ERK1/2 and its target eIF4E and 2) Akt and its downstream effectors GSK-3, p70(S6K), and 4E-BP1. TNF-alpha-induced phosphorylation of Akt, and ERK1/2 was inhibited by an antibody against TNF-alpha receptor 1 (TNF-R1). PD-98059 pretreatment abolished TNF-alpha-induced phosphorylation of ERK1/2 and eIF4E, whereas PS was only partially inhibited. LY-294002 completely abolished TNF-alpha-induced stimulation of PS as well as phosphorylation of Akt and its downstream targets GSK-3, p70(S6K), and 4E-BP1. Rapamycin inhibited TNF-alpha-induced phosphorylation of the mTOR C1 target p70(S6K) without altering TNF-alpha-induced PS and 4E-BP1 phosphorylation. In conclusion, our results provide evidence that TNF-alpha enhances PS in myotubes and that this is based on enhanced protein translation mediated by the TNF-R1 and PI3K-Akt and MEK-ERK signaling cascades.

Our study demonstrates that mTORC2 is implicated in maintaining contractile function of the pressure-overloaded male mouse heart.

Nrg1β is critically involved in cardiac development and also maintains function of the adult heart. Studies conducted in animal models showed that it improves cardiac performance under a range of pathological conditions, which led to its introduction in clinical trials to treat heart failure. Recent work also implicated Nrg1β in the regenerative potential of neonatal and adult hearts. The molecular mechanisms whereby Nrg1β acts in cardiac cells are still poorly understood. In the present study, we analyzed the effects of Nrg1β on glucose uptake in neonatal rat ventricular myocytes and investigated to what extent mTOR/Akt signaling pathways are implicated. We show that Nrg1β enhances glucose uptake in cardiomyocytes as efficiently as IGF-I and insulin. Nrg1β causes phosphorylation of ErbB2 and ErbB4 and rapidly induces the phosphorylation of FAK (Tyr861), Akt (Thr308 and Ser473), and its effector AS160 (Thr642). Knockdown of ErbB2 or ErbB4 reduces Akt phosphorylation and blocks the glucose uptake. The Akt inhibitor VIII and the PI3K inhibitors LY-294002 and Byl-719 abolish Nrg1β-induced phosphorylation and glucose uptake. Finally, specific mTORC2 inactivation after knockdown of rictor blocks the Nrg1β-induced increases in Akt-p-Ser473 but does not modify AS160-p-Thr642 or the glucose uptake responses to Nrg1β. In conclusion, our study demonstrates that Nrg1β enhances glucose uptake in cardiomyocytes via ErbB2/ErbB4 heterodimers, PI3Kα, and Akt. Furthermore, although Nrg1β activates mTORC2, the resulting Akt-Ser473 phosphorylation is not essential for glucose uptake induction. These new insights into pathways whereby Nrg1β regulates glucose uptake in cardiomyocytes may contribute to the understanding of its regenerative capacity and protective function in heart failure.

Abstract-Recently, novel corticotropin-releasing factor-related peptides, named urocortin 1, 2, and 3, and a distinct cardiac and peripheral vascular receptor (corticotropin-releasing factor receptor 2) were described being part of a peripheral corticotropin-releasing factor system modulating cardiovascular function in response to stress. Vasorelaxation and blood pressure lowering have been reported after acute administration of these peptides. No data are available on the acute and chronic effects of urocortin 2 on blood pressure in models of arterial hypertension. To test these effects, hypertensive salt-sensitive and normotensive salt-resistant Dahl rats were randomly assigned to twice-daily applications of urocortin 2 or vehicle for 5 weeks. Blood pressure, heart rate, and left ventricular dimension and function were recorded at baseline, after initial application, and, together with cardiac and aortic expression of urocortin 2 and its receptor, after 5 weeks of treatment. Urocortin 2 significantly reduced blood pressure in hypertensive rats without affecting heart rate. Long-term urocortin 2 treatment in hypertensive rats induced sustained blood pressure reduction and diminished the development of hypertension-induced left ventricular hypertrophy and the deterioration of left ventricular contractile function. Corticotropin-releasing factor receptor 2 expression was preserved despite chronic stimulation by urocortin 2. In conclusion, our study shows that, in an animal model of arterial hypertension, urocortin 2 has immediate and sustained blood pressure-lowering effects. Beneficial effects on blood pressure, left ventricular dimension, and function, together with preserved receptor expression, suggest that corticotropin-releasing factor receptor 2 stimulation by urocortin 2 may represent a novel approach to the treatment of arterial hypertension. A rterial hypertension remains the major risk factor for cardiovascular and related diseases. In its most recent report, the World Health Organization lists high blood pressure (BP) as the leading cause of death worldwide.

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Background: Neuregulin (Nrg)1b is a growth factor that activates PI3K/Akt and Src/FAK via the ErbB2/ErbB4 receptors. Although it is currently in clinical trial to treat haert failure, it remains unclear which cellular mechanisms are responsible for its cardioprotective actions. Here we tested if Nrg1b regulates glucose uptake in cardiomyocytes and analyzed the underlying signaling mechanisms. Methods: Neonatal rat ventricular myocytes were treated with Nrg1b (10ng/ml) in combination with the mTOR inhibitors PP242 (2mM) and rapamycin (20ng/ml), the ErbB2 inhibitor lapatinib (1mM), the Src inhibitor PP2 (5mM), the Akt inhibitor VIII (20mM), or vehicle. Cells were pre-incubated for 30 min with the inhibitors and proteins extracted 30 min after the addition of Nrg1b for analysis by Western blot. Glucose uptake was assessed by measuring the incorporation of 3H-D-glucose for 30 min. ErbB2 or ErbB4 receptors were knocked down with siRNA for 48h before Nrg1b treatment. Results: Similar to IGF-I and Insulin, Nrg1b caused a 1.9 fold increase in 3H-D-glucose incorporation (P, 0.01). Nrg1b induced phosphorylation of mTOR (S2448), Akt (S308) and FAK (Y861), as well as of the mTORC1 targets 4E-BP1, p70-S6K1 and ULK and the mTORC2 target Akt (S473). Lapatinib, PP242 and Akt inhibitor VIII blocked the Nrg1b-induced Akt-, mTOR-, p70-S6K1-, ULK-, and 4E-BP1-phosphorylation, indicating that these effects require ErbB2 and are mediated by Akt and mTOR. However, only lapatinib and Akt inhibitor VIII fully blocked the Nrg1b-induced glucose uptake; PP242 partially blocked it and rapamycin did not block it at all. These results suggest that Akt is required for Nrg1b-induced glucose uptake, and that mTORC2-dependent Akt phosphorylation mediates, at least in part, this response. PP2 blocked phosphorylation of FAK as expected, and it also partially blocked phosphorylation of Akt (S473) and p70-S6K1. PP2 also decreased general glucose uptake (0.6-fold of Ctl, p,0.05) and Nrg1b-induced glucose uptake (1.06-fold of Ctl, p=ns). Knockdown of ErbB4 receptor alone was sufficient to decrease both mTORC1 and mTORC2 signaling, whereas knock-down of ErbB2 affected only the mTORC2 targets. Conclusions: Our results show that Nrg1b increases glucose uptake in cardiomyocytes via Akt. We also show that Nrg1b activates mTORC1 via ErbB4 and mTORC2 via the ErbB2/ErbB4 heterodimer. Our data also support the hypothesis that Src/FAK is upstream of mTORC2 and mediates the Nrg1b-induced phosphorylation of Akt and glucose uptake.

The concomitant investigation of apoptosis (a regulated cell death) and autophagy (a conserved cell survival mechanism) in immune cells is rare. More detailed knowledge of these two types of self-consumption in circulating lymphocytes and monocytes would be important, since conditions such as fasting and acute exercise could promote health by a coordinated/linked modulation of autophagy and apoptosis in these mononuclear cells. In this study we performed flow cytometry to quantify numbers of apoptotic and autophagic mononuclear cells, lymphocytes and monocytes in fasting, standardized fed, and exercise conditions, using Annexin V, LC3B, and p62, respectively. We show that within total mononuclear cells lymphocytes are less apoptotic and autophagic than monocytes during fasting (p < 0.001, p < 0.05, respectively) and after acute exercise (p < 0.01, p < 0.05, respectively). Fasting increased circulating autophagic monocyte concentrations, but not lymphocytes compared to the fed control condition. Acute exercise elevated circulating autophagic lymphocyte concentrations, but not monocytes. Interestingly, Western blotting analysis of the fasting samples showed that higher LC3BII/I ratios were correlated with lower numbers of autophagic mononuclear cells (r = − 0.74, p = 0.02, n = 8), which could be attributed to the monocyte subgroup, but not lymphocytes. These results extend the current knowledge of the two types of self-consumption in circulating immune cells and underline their possible importance in pro-inflammatory monocytes during fasting and exercise as health promoting interventions.