DNA damage signaling mediates the functional antagonism between replicative senescence and terminal muscle differentiation.

The molecular determinants of muscle progenitor impairment to regenerate aged muscles are currently unclear. We show that, in a mouse model of replicative senescence, decline in muscle satellite cell-mediated regeneration coincides with activation of DNA damage response (DDR) and impaired ability to differentiate into myotubes. Inhibition of DDR restored satellite cell differentiation ability. Moreover, in replicative human senescent fibroblasts, DDR precluded MYOD-mediated activation of the myogenic program. A DDR-resistant MYOD mutant could overcome this barrier by resuming cell cycle progression. Likewise, DDR inhibition could also restore MYOD's ability to activate the myogenic program in human senescent fibroblasts. Of note, we found that cell cycle progression is necessary for the DDR-resistant MYOD mutant to reverse senescence-mediated inhibition of the myogenic program. These data provide the first evidence of DDR-mediated functional antagonism between senescence and MYOD-activated gene expression and indicate a previously unrecognized requirement of cell cycle progression for the activation of the myogenic program.

Givinostat as metabolic enhancer reverting mitochondrial biogenesis deficit in Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a rare disorder characterized by progressive muscle wasting, weakness, and premature death. Remarkable progress has been made in genetic approaches, restoring dystrophin, or its function. However, the targeting of secondary pathological mechanisms, such as increasing muscle blood flow or stopping fibrosis, remains important to improve the therapeutic benefits, that depend on tackling both the genetic disease and the downstream consequences. Mitochondrial dysfunctions are one of the earliest deficits in DMD, arise from multiple cellular stressors and result in less than 50% of ATP content in dystrophic muscles. Here we establish that there are two temporally distinct phases of mitochondrial damage with depletion of mitochondrial mass at early stages and an accumulation of dysfunctional mitochondria at later stages, leading to a different oxidative fibers pattern, in young and adult mdx mice. We also observe a progressive mitochondrial biogenesis impairment associated with increased deacetylation of peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) promoter. Such histone deacetylation is inhibited by givinostat that positively modifies the epigenetic profile of PGC-1α promoter, sustaining mitochondrial biogenesis and oxidative fiber type switch. We, therefore, demonstrate that givinostat exerts relevant effects at mitochondrial level, acting as a metabolic remodeling agent capable of efficiently promoting mitochondrial biogenesis in dystrophic muscle.

STAT3 inhibition recovers regeneration of aged muscles by restoring autophagy in muscle stem cells.

Age-related reduction in muscle stem cell (MuSC) regenerative capacity is associated with cell-autonomous and non-cell-autonomous changes caused by alterations in systemic and skeletal muscle environments, ultimately leading to a decline in MuSC number and function. Previous studies demonstrated that STAT3 plays a key role in driving MuSC expansion and differentiation after injury-activated regeneration, by regulating autophagy in activated MuSCs. However, autophagy gradually declines in MuSCs during lifespan and contributes to the impairment of MuSC-mediated regeneration of aged muscles. Here, we show that STAT3 inhibition restores the autophagic process in aged MuSCs, thereby recovering MuSC ability to promote muscle regeneration in geriatric mice. We show that STAT3 inhibition could activate autophagy at the nuclear level, by promoting transcription of autophagy-related genes, and at the cytoplasmic level, by targeting STAT3/PKR phosphorylation of eIF2α. These results point to STAT3 inhibition as a potential intervention to reverse the age-related autophagic block that impairs MuSC ability to regenerate aged muscles. They also reveal that STAT3 regulates MuSC function by both transcription-dependent and transcription-independent regulation of autophagy.

An evolutionarily acquired genotoxic response discriminates MyoD from Myf5, and differentially regulates hypaxial and epaxial myogenesis.

Despite having distinct expression patterns and phenotypes in mutant mice, the myogenic regulatory factors Myf5 and MyoD have been considered to be functionally equivalent. Here, we report that these factors have a different response to DNA damage, due to the presence in MyoD and absence in Myf5 of a consensus site for Abl-mediated tyrosine phosphorylation that inhibits MyoD activity in response to DNA damage. Genotoxins failed to repress skeletal myogenesis in MyoD-null embryos; reintroduction of wild-type MyoD, but not mutant Abl phosphorylation-resistant MyoD, restored the DNA-damage-dependent inhibition of muscle differentiation. Conversely, introduction of the Abl-responsive phosphorylation motif converts Myf5 into a DNA-damage-sensitive transcription factor. Gene-dosage-dependent reduction of Abl kinase activity in MyoD-expressing cells attenuated the DNA-damage-dependent inhibition of myogenesis. The presence of a DNA-damage-responsive phosphorylation motif in vertebrate, but not in invertebrate MyoD suggests an evolved response to environmental stress, originated from basic helix-loop-helix gene duplication in vertebrate myogenesis.

p38 pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci.

During skeletal myogenesis, genomic reprogramming toward terminal differentiation is achieved by recruiting chromatin-modifying enzymes to muscle-specific loci. The relative contribution of extracellular signaling cascades in targeting these enzymes to individual genes is unknown. Here we show that the differentiation-activated p38 pathway targets the SWI-SNF chromatin-remodeling complex to myogenic loci. Upon differentiation, p38 kinases were recruited to the chromatin of muscle-regulatory elements. Blockade of p38 alpha/beta repressed the transcription of muscle genes by preventing recruitment of the SWI-SNF complex at these elements without affecting chromatin binding of muscle-regulatory factors and acetyltransferases. The SWI-SNF subunit BAF60 could be phosphorylated by p38 alpha-beta in vitro, and forced activation of p38 alpha/beta in myoblasts by expression of a constitutively active MKK6 (refs. 5,6,7) promoted unscheduled SWI-SNF recruitment to the myogenin promoter. Conversely, inactivation of SWI-SNF enzymatic subunits abrogated MKK6-dependent induction of muscle gene expression. These results identify an unexpected function of differentiation-activated p38 in converting external cues into chromatin modifications at discrete loci, by selectively targeting SWI-SNF to muscle-regulatory elements.

SerpinE1 drives a cell-autonomous pathogenic signaling in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of peripheral heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. Yet, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. A genome-wide analysis of gene expression in cultures of primary HGPS fibroblasts identified SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1), as central gene that propels a cell-autonomous pathogenic signaling from the altered nuclear lamina. Indeed, siRNA-mediated downregulation and pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by the correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector and target for therapeutic interventions in HGPS pathogenesis.

From Development to Aging: The Path to Cellular Senescence.

Significance: Senescence is a cellular state induced by internal or external stimuli, which result in cell cycle arrest, morphological changes, and dysfunctions in mitochondrial and lysosomal functionality as well as the senescence-associated secretory phenotype. Senescent cells accumulate in tissues in physiological and pathological conditions such as development, tissue repair, aging, and cancer. Recent Advances: Growing evidences indicate that senescent cells in vivo are a heterogeneous cell population due to different cell-autonomous activated pathways and distinct microenvironmental contexts. Critical Issues: In this review, we discuss the different contexts where senescence assumes a key role with beneficial or harmful outcomes. The heterogeneous nature of senescence pushes toward resolution of the specific molecular profile and secretome to typify senescent cells in physiological and pathological contexts. Future Directions: Future research will enable exploring the heterogeneity of the senescent population to precisely map the progression of cells through senescent trajectories and study the impact of the therapeutic advantage of senolytic drugs for translational strategies toward supporting the health span. Antioxid. Redox Signal. 34, 294-307.

Reticulon-1C Involvement in Muscle Regeneration.

Skeletal muscle is a very dynamic and plastic tissue, being essential for posture, locomotion and respiratory movement. Muscle atrophy or genetic muscle disorders, such as muscular dystrophies, are characterized by myofiber degeneration and replacement with fibrotic tissue. Recent studies suggest that changes in muscle metabolism such as mitochondrial dysfunction and dysregulation of intracellular Ca2+ homeostasis are implicated in many adverse conditions affecting skeletal muscle. Accumulating evidence also suggests that ER stress may play an important part in the pathogenesis of inflammatory myopathies and genetic muscle disorders. Among the different known proteins regulating ER structure and function, we focused on RTN-1C, a member of the reticulon proteins family localized on the ER membrane. We previously demonstrated that RTN-1C expression modulates cytosolic calcium concentration and ER stress pathway. Moreover, we recently reported a role for the reticulon protein in autophagy regulation. In this study, we found that muscle differentiation process positively correlates with RTN-1C expression and UPR pathway up-regulation during myogenesis. To better characterize the role of the reticulon protein alongside myogenic and muscle regenerative processes, we performed in vivo experiments using either a model of muscle injury or a photogenic model for Duchenne muscular dystrophy. The obtained results revealed RTN-1C up-regulation in mice undergoing active regeneration and localization in the injured myofibers. The presented results strongly suggested that RTN-1C, as a protein involved in key aspects of muscle metabolism, may represent a new target to promote muscle regeneration and repair upon injury.

The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle.

The transcription factor NF-Y promotes cell proliferation and its activity often declines during differentiation through the regulation of NF-YA, the DNA binding subunit of the complex. In stem cell compartments, the shorter NF-YA splice variant is abundantly expressed and sustains their expansion. Here, we report that satellite cells, the stem cell population of adult skeletal muscle necessary for its growth and regeneration, express uniquely the longer NF-YA isoform, majorly associated with cell differentiation. Through the generation of a conditional knock out mouse model that selectively deletes the NF-YA gene in satellite cells, we demonstrate that NF-YA expression is fundamental to preserve the pool of muscle stem cells and ensures robust regenerative response to muscle injury. In vivo and ex vivo, satellite cells that survive to NF-YA loss exit the quiescence and are rapidly committed to early differentiation, despite delayed in the progression towards later states. In vitro results demonstrate that NF-YA-depleted muscle stem cells accumulate DNA damage and cannot properly differentiate. These data highlight a new scenario in stem cell biology for NF-Y activity, which is required for efficient myogenic differentiation.

Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry.

Terminal differentiation exerts a remarkably tight control on cell proliferation. However, the oncogenic products of DNA tumor viruses, such as adenovirus E1A, can force postmitotic cells to proliferate, thus representing a powerful tool to study progression into S phase. In this study, we identified the gene encoding Np95, a murine nuclear phosphoprotein, as an early target of E1A-induced transcriptional events. In terminally differentiated (TD) cells, the activation of Np95 was specifically induced by E1A, but not by overexpression of E2F-1 or of the cyclin E (cycE)-cyclin-dependent kinase 2 (cdk2) complex. In addition, the concomitant expression of Np95 and of cycE-cdk2 was alone sufficient to induce S phase in TD cells. In NIH-3T3 cells, the expression of Np95 was tightly regulated during the cell cycle, and its functional ablation resulted in abrogation of DNA synthesis. Thus, expression of Np95 is essential for S phase entry. Previous evidence suggested that E1A, in addition to its well characterized effects on the pRb/E2F-1 pathway, activates a parallel and complementary pathway that is also required for the reentry in S phase of TD cells (Tiainen, M., D. Spitkousky, P. Jansen-Dürr, A. Sacchi, and M. Crescenzi. 1996. Mol. Cell. Biol. 16:5302-5312). From our results, Np95 appears to possess all the characteristics to represent the first molecular determinant identified in this pathway.

The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration.

Metabolic reprogramming is an active regulator of stem cell fate choices, and successful stem cell differentiation in different compartments requires the induction of oxidative phosphorylation. However, the mechanisms that promote mitochondrial respiration during stem cell differentiation are poorly understood. Here we demonstrate that Stat3 promotes muscle stem cell myogenic lineage progression by stimulating mitochondrial respiration in mice. We identify Fam3a, a cytokine-like protein, as a major Stat3 downstream effector in muscle stem cells. We demonstrate that Fam3a is required for muscle stem cell commitment and skeletal muscle development. We show that myogenic cells secrete Fam3a, and exposure of Stat3-ablated muscle stem cells to recombinant Fam3a in vitro and in vivo rescues their defects in mitochondrial respiration and myogenic commitment. Together, these findings indicate that Fam3a is a Stat3-regulated secreted factor that promotes muscle stem cell oxidative metabolism and differentiation, and suggests that Fam3a is a potential tool to modulate cell fate choices.

DNA damage and cellular differentiation: more questions than responses.

Studies on DNA damage responses in proliferating cells have revealed the relationship between sensing and repair of the DNA lesions and the regulation of the cell cycle, leading to the discovery and molecular characterization of the DNA damage-activated cell cycle checkpoints. Much less is known about the DNA damage response in progenitors of differentiated cells, in which cell cycle arrest is a critical signal to trigger the differentiation program, and in terminally differentiated cells, which are typically post-mitotic. How DNA lesions are detected, processed and repaired in these cells, the functional impact of DNA damage on transcription of differentiation-specific genes, how these events are coordinated at the molecular level, the consequence of defective DNA damage response on tissue-specific functions and its potential relationship with age-related diseases are currently open questions. In particular the biological complexity inherent to the global genome reprogramming of tissue progenitors, such as embryonic or adult stem cells, suggests the importance of an accurate DNA damage response at the transcription level in these cells to ensure the genomic integrity of regenerating tissues.

Differentiation-induced radioresistance in muscle cells.

DNA damage induces cell cycle arrest and DNA repair or apoptosis in proliferating cells. Terminally differentiated cells are permanently withdrawn from the cell cycle and partly resistant to apoptosis. To investigate the effects of genotoxic agents in postmitotic cells, we compared DNA damage-activated responses in mouse and human proliferating myoblasts and their differentiated counterparts, the myotubes. DNA double-strand breaks caused by ionizing radiation (IR) induced rapid activating autophosphorylation of ataxia-teleangiectasia-mutated (ATM), phosphorylation of histone H2AX, recruitment of repair-associated proteins MRE11 and Nbs1, and activation of Chk2 in both myoblasts and myotubes. However, IR-activated, ATM-mediated phosphorylation of p53 at serine 15 (human) or 18 (mouse) [Ser15(h)/18(m)], and apoptosis occurred in myoblasts but was impaired in myotubes. This phosphorylation could be enforced in myotubes by the anthracycline derivative doxorubicin, leading to selective activation of proapoptotic genes. Unexpectedly, the abundance of autophosphorylated ATM was indistinguishable after exposure of myotubes to IR (10 Gy) or doxorubicin (1 microM/24 h) despite efficient phosphorylation of p53 Ser15(h)/18(m), and apoptosis occurred only in response to doxorubicin. These results suggest that radioresistance in myotubes might reflect a differentiation-associated, pathway-selective blockade of DNA damage signaling downstream of ATM. This mechanism appears to preserve IR-induced activation of the ATM-H2AX-MRE11/Rad50/Nbs1 lesion processing and repair pathway yet restrain ATM-p53-mediated apoptosis, thereby contributing to life-long maintenance of differentiated muscle tissues.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells.

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

Muscle-relevant genes marked by stable H3K4me2/3 profiles and enriched MyoD binding during myogenic differentiation.

Post-translational modifications of histones play a key role in the regulation of gene expression during development and differentiation. Numerous studies have shown the dynamics of combinatorial regulation by transcription factors and histone modifications, in the sense that different combinations lead to distinct expression outcomes. Here, we investigated gene regulation by stable enrichment patterns of histone marks H3K4me2 and H3K4me3 in combination with the chromatin binding of the muscle tissue-specific transcription factor MyoD during myogenic differentiation of C2C12 cells. Using k-means clustering, we found that specific combinations of H3K4me2/3 profiles over and towards the gene body impact on gene expression and marks a subset of genes important for muscle development and differentiation. By further analysis, we found that the muscle key regulator MyoD was significantly enriched on this subset of genes and played a repressive role during myogenic differentiation. Among these genes, we identified the pluripotency gene Patz1, which is repressed during myogenic differentiation through direct binding of MyoD to promoter elements. These results point to the importance of integrating histone modifications and MyoD chromatin binding for coordinated gene activation and repression during myogenic differentiation.

Reversal of Defective Mitochondrial Biogenesis in Limb-Girdle Muscular Dystrophy 2D by Independent Modulation of Histone and PGC-1α Acetylation.

Mitochondrial dysfunction occurs in many muscle degenerative disorders. Here, we demonstrate that mitochondrial biogenesis was impaired in limb-girdle muscular dystrophy (LGMD) 2D patients and mice and was associated with impaired OxPhos capacity. Two distinct approaches that modulated histones or peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) acetylation exerted equivalent functional effects by targeting different mitochondrial pathways (mitochondrial biogenesis or fatty acid oxidation[FAO]). The histone deacetylase inhibitor Trichostatin A (TSA) changed chromatin assembly at the PGC-1α promoter, restored mitochondrial biogenesis, and enhanced muscle oxidative capacity. Conversely, nitric oxide (NO) triggered post translation modifications of PGC-1α and induced FAO, recovering the bioenergetics impairment of muscles but shunting the defective mitochondrial biogenesis. In conclusion, a transcriptional blockade of mitochondrial biogenesis occurred in LGMD-2D and could be recovered by TSA changing chromatin conformation, or it could be overcome by NO activating a mitochondrial salvage pathway.

Involvement of cyclin D activity in left ventricle hypertrophy in vivo and in vitro.

OBJECTIVE: Cardiac hypertrophy is induced by a number of stimuli and can lead to cardiomyopathy and heart failure. Present knowledge suggests that cell-cycle regulatory proteins take part in hypertrophy. We have investigated if the D-type cyclins are involved in cardiac hypertrophy. METHODS: The expression and activity of the D-type cyclins and associated kinases in cardiomyocytes were studied during angiotensin II- and pressure overload-induced hypertrophy in rats (Rattus norvegicus) and in isolated, neonatal cardiomyocytes. Expression of the D-type cyclins was manipulated pharmacologically and genetically in neonatal myocytes. RESULTS: In the left ventricle, there was a low, constitutive expression of the D-type cyclins, which may have a biological role in normal, adult myocytes. The protein level and the associated kinase activity of the D-type cyclins were up-regulated during hypertrophic growth. The increase in cyclin D expression could be mimicked in vitro in neonatal cardiac myocytes. Interestingly, the cyclin Ds were up-regulated by hypertrophic elicitors that stimulate different signalling pathways, suggesting that cyclin D expression is an inherent part of cardiac hypertrophy. Treatment of myocytes with the compound differentiation inducing factor 1 inhibited expression of the D-type cyclins and impaired hypertrophic growth induced by angiotensin II, phenylephrine and serum. The response to hypertrophic elicitors could be restored in differentiation inducing factor 1-treated myocytes by expressing cyclin D2 from a heterologous promoter. CONCLUSION: Our results point to the D-type cyclins as important regulators of cardiac hypertrophy. This supports the notion that cell-cycle regulatory proteins regulate hypertrophic growth.