The NAD(+)-dependent SIRT1 deacetylase translates a metabolic switch into regulatory epigenetics in skeletal muscle stem cells.

Stem cells undergo a shift in metabolic substrate utilization during specification and/or differentiation, a process that has been termed metabolic reprogramming. Here, we report that during the transition from quiescence to proliferation, skeletal muscle stem cells experience a metabolic switch from fatty acid oxidation to glycolysis. This reprogramming of cellular metabolism decreases intracellular NAD(+) levels and the activity of the histone deacetylase SIRT1, leading to elevated H4K16 acetylation and activation of muscle gene transcription. Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in SCs. Moreover, mice with muscle-specific inactivation of the SIRT1 deacetylase domain display reduced myofiber size, impaired muscle regeneration, and derepression of muscle developmental genes. Overall, these findings reveal how metabolic cues can be mechanistically translated into epigenetic modifications that regulate skeletal muscle stem cell biology.

SirT1 in muscle physiology and disease: lessons from mouse models.

Sirtuin 1 (SirT1) is the largest of the seven members of the sirtuin family of class III nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylases, whose activation is beneficial for metabolic, neurodegenerative, inflammatory and neoplastic diseases, and augments life span in model organisms (Finkel et al., 2009; Lavu et al., 2008). In vitro studies show that SirT1 protects genome integrity and is involved in circadian physiological rhythms (Asher et al., 2008; Nakahata et al., 2008; Oberdoerffer et al., 2008). In the last few years, a fundamental role for SirT1 in the metabolism and differentiation of skeletal muscle cells has been uncovered (Fulco et al., 2003), and the use of specific transgenic or knockout SirT1 mouse models implicates it in the protection of heart muscle from oxidative and hypertrophic stresses (Alcendor et al., 2007). In this Perspective, we review the recent exciting findings that have established a key role for the 'longevity' protein SirT1 in skeletal and heart muscle physiology and disease. Furthermore, given the multiple biological functions of SirT1, we discuss the unique opportunities that SirT1 mouse models can offer to improve our integrated understanding of the metabolism, as well as the regeneration and aging-associated changes in the circadian function, of skeletal and heart muscle.

Comparing and contrasting the roles of AMPK and SIRT1 in metabolic tissues.

The ability to adapt and respond to nutrients is an ancient cellular function, conserved from unicellular to the most complex multicellular organisms, including mammals. Mammals adapt to changes in nutritional status through the modulation of tissue-specific metabolic pathways so as to maintain energy homeostasis. At least two proteins are activated in response to reduced nutrient availability: AMP-activated protein kinase (AMPK) and NAD(+)-dependent deacetylase SIRT1. AMPK functions as a sensor of cellular energy status and as a master regulator of metabolism. When ATP levels decrease, AMPK is activated to boost ATP production and to inhibit ATP usage, thus restoring energy balance. Similarly, SIRT1 is activated in response to changes in the energy status to promote transcription of genes that mediate the metabolic response to stress, starvation or calorie restriction. Several observations support a model where, in response to stress and reduced nutrients, a metabolic pathway is activated within which AMPK and SIRT1 concordantly function to ensure an appropriate cellular response and adaptation to environmental modifications. In this perspective, we compare and contrast the roles of SIRT1 and AMPK in several metabolic tissues and propose a working model of how the AMPK-SIRT1 axis may be regulated to control functions relevant to organismal physiology and pathophysiology.

Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of Nampt.

It is intuitive to speculate that nutrient availability may influence differentiation of mammalian cells. Nonetheless, a comprehensive complement of the molecular determinants involved in this process has not been elucidated yet. Here, we have investigated how nutrients (glucose) affect skeletal myogenesis. Glucose restriction (GR) impaired differentiation of skeletal myoblasts and was associated with activation of the AMP-activated protein kinase (AMPK). Activated AMPK was required to promote GR-induced transcription of the NAD+ biosynthetic enzyme Nampt. Indeed, GR augmented the Nampt activity, which consequently modified the intracellular [NAD+]:[NADH] ratio and nicotinamide levels, and mediated inhibition of skeletal myogenesis. Skeletal myoblasts derived from SIRT1+/- heterozygous mice were resistant to the effects of either GR or AMPK activation. These experiments reveal that AMPK, Nampt, and SIRT1 are the molecular components of a functional signaling pathway that allows skeletal muscle cells to sense and react to nutrient availability.

A role of p73 in mitotic exit.

The p53-related p73 proteins regulate developmental processes, cell growth, and DNA damage response. p73 function is regulated by post-translational modifications and protein-protein interactions. At the G2/M transition, p73 is phosphorylated at Thr-86 by the p34cdc2/cyclin B complex; this is associated with its exclusion from condensed chromosomes and loss of DNA binding and transcriptional activation ability. Here we showed that p73 hypo-phosphorylated species reappear during mitotic exit, concomitant with p73 relocalization to telophase nuclei and recovered ability to activate transcription. Functional knock-out of p73 gene expression by small interfering RNAs (siRNAs) alters mitotic progression, yielding an increase of ana-telophase cells, the accumulation of aberrant late mitotic figures, and the appearance of abnormalities in the subsequent interphase. This p73 activity at the M-to-G1 transition is mediated by its transactivating function because expression of the transcription dominant negative mutant p73DD induces the same mitotic exit phenotype. We also found that the cyclin-dependent kinase inhibitor Kip2/p57 gene is a specific target of p73 regulation during mitotic exit and re-entry into G1. Both knock-out of p73 gene expression by siRNAs and abrogation of p73-dependent transcription by the p73DD mutant abrogate Kip2/p57 increase at the M-to-G1 transition. Moreover, similar abnormalities (e.g. delay in late mitotic stages with the accumulation of aberrant ana-telophase figures, and abnormalities in the following interphase) are observed in cultures in which the expression of Kip2/p57 is abrogated by siRNAs. These results identify a novel p73-Kip2/p57 pathway that coordinates mitotic exit and transition to G1.

Molecular and cellular determinants of skeletal muscle atrophy and hypertrophy.

The maintenance of adult skeletal muscle mass is ensured by physical exercise. Accordingly, physiological and pathological situations characterized by either impaired motor neuron activity, reduced gravity (microgravity during space flights), or reduced physical activity result in loss of muscle mass. Furthermore, a plethora of clinical conditions, including cancer, sepsis, diabetes, and AIDS, are associated with varying degrees of muscle atrophy. The cellular and molecular pathways responsible for maintaining the skeletal muscle mass are not well defined. Nonetheless, studies aimed at the understanding of the mechanisms underlying either muscular atrophy or hypertrophy have begun to identify the physiological determinants and clarify the molecular pathways responsible for the maintenance of muscle mass.

Regulation of the p300 HAT domain via a novel activation loop.

The transcriptional coactivator p300 is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells. However, the molecular events that regulate p300 HAT activity are poorly understood. We evaluated autoacetylation of the p300 HAT protein domain to determine its function. Using expressed protein ligation, the p300 HAT protein domain was generated in hypoacetylated form and found to have reduced catalytic activity. This basal catalytic rate was stimulated by autoacetylation of several key lysine sites within an apparent activation loop motif. This post-translational modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases by autophosphorylation. We therefore propose that this autoregulatory loop could influence the impact of p300 on a wide variety of signaling and transcriptional events.

p73 is regulated by phosphorylation at the G2/M transition.

p73 is a p53 paralog that encodes proapoptotic (transactivation-competent (TA)) and antiapoptotic (dominant negative) isoforms. TAp73 transcription factors mediate cell cycle arrest and/or apoptosis in response to DNA damage and are involved in developmental processes in the central nervous system and the immune system. p73 proteins may also play a role in the regulation of cell growth. Indeed, p73 expression is itself modulated during the cell cycle and TAp73 proteins accumulate in S phase cells. In addition, the function of p73 proteins is also regulated by post-translational modifications and protein-protein interactions in different cellular and pathophysiological contexts. Here we show that p73 is a physiological target of the p34cdc2-cyclin B mitotic kinase complex in vivo. Both p73beta and p73alpha isoforms are hyperphosphorylated in normal mitotic cells and during mitotic arrest induced by microtubule-targeting drugs. p34cdc2-cyclin B phosphorylates and associates with p73 in vivo, which results in a decreased ability of p73 to both bind DNA and activate transcription in mitotic cells. Indeed, p73 is excluded from condensed chromosomes in meta- and anaphase, redistributes throughout the mitotic cytoplasm, and unlike p53, shows no association with centrosomes. Together these results indicate that M phase-specific phosphorylation of p73 by p34cdc2-cyclin B is associated with negative regulation of its transcriptional activating function.

Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state.

Sir2 is a NAD(+)-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span. Prompted by the observation that the [NAD(+)]/[NADH] ratio is subjected to dynamic fluctuations in skeletal muscle, we have tested whether Sir2 regulates muscle gene expression and differentiation. Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation. Conversely, cells with decreased Sir2 differentiate prematurely. To inhibit myogenesis, Sir2 requires its NAD(+)-dependent deacetylase activity. The [NAD(+)]/[NADH] ratio decreases as muscle cells differentiate, while an increased [NAD(+)]/[NADH] ratio inhibits muscle gene expression. Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated [NAD(+)]/[NADH] ratio. These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor. In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression.

DNA damage-dependent acetylation of p73 dictates the selective activation of apoptotic target genes.

The tumor suppressor p53 and its close relative p73 are activated in response to DNA damage resulting in either cell cycle arrest or apoptosis. Here, we show that DNA damage induces the acetylation of p73 by the acetyltransferase p300. Inhibiting the enzymatic activity of p300 hampers apoptosis in a p53(-/-) background. Furthermore, a nonacetylatable p73 is defective in activating transcription of the proapoptotic p53AIP1 gene but retains an intact ability to regulate other targets such as p21. Finally, p300-mediated acetylation of p73 requires the protooncogene c-abl. Our results suggest that DNA damage-induced acetylation potentiates the apoptotic function of p73 by enhancing the ability of p73 to selectively activate the transcription of proapoptotic target genes.