Cyclin D3 deficiency promotes a slower, more oxidative skeletal muscle phenotype and ameliorates pathophysiology in the mdx mouse model of Duchenne muscular dystrophy.

We previously reported that cyclin D3-null mice display a shift toward the slow, oxidative phenotype in skeletal muscle, improved exercise endurance, and increased energy expenditure. Here, we explored the role of cyclin D3 in the physiologic response of skeletal muscle to external stimuli and in a model of muscle degenerative disease. We show that cyclin D3-null mice exhibit a further transition from glycolytic to oxidative muscle fiber type in response to voluntary exercise and an improved response to fasting. Since fast glycolytic fibers are known to be more susceptible to degeneration in Duchenne muscular dystrophy (DMD), we examined the effects of cyclin D3 inactivation on skeletal muscle phenotype in the mdx mouse model of DMD. Compared with control mdx mice, cyclin D3-deficient mdx mice display a higher proportion of slower and more oxidative myofibers, reduced muscle degenerative/regenerative processes, and reduced myofiber size variability, indicating an attenuation of dystrophic histopathology. Furthermore, mdx muscles lacking cyclin D3 exhibit reduced fatigability during repeated electrical stimulations. Notably, cyclin D3-null mdx mice show enhanced performance during recurrent trials of endurance treadmill exercise, and post-exercise muscle damage results decreased while the regenerative capacity is boosted. In addition, muscles from exercised cyclin D3-deficient mdx mice display increased oxidative capacity and increased mRNA expression of genes involved in the regulation of oxidative metabolism and the response to oxidative stress. Altogether, our findings indicate that depletion of cyclin D3 confers benefits to dystrophic muscle, suggesting that cyclin D3 inhibition may represent a promising therapeutic strategy against DMD.

Brahma is required for cell cycle arrest and late muscle gene expression during skeletal myogenesis.

Although the two catalytic subunits of the SWI/SNF chromatin-remodeling complex--Brahma (Brm) and Brg1--are almost invariably co-expressed, their mutually exclusive incorporation into distinct SWI/SNF complexes predicts that Brg1- and Brm-based SWI/SNF complexes execute specific functions. Here, we show that Brg1 and Brm have distinct functions at discrete stages of muscle differentiation. While Brg1 is required for the activation of muscle gene transcription at early stages of differentiation, Brm is required for Ccnd1 repression and cell cycle arrest prior to the activation of muscle genes. Ccnd1 knockdown rescues the ability to exit the cell cycle in Brm-deficient myoblasts, but does not recover terminal differentiation, revealing a previously unrecognized role of Brm in the activation of late muscle gene expression independent from the control of cell cycle. Consistently, Brm null mice displayed impaired muscle regeneration after injury, with aberrant proliferation of satellite cells and delayed formation of new myofibers. These data reveal stage-specific roles of Brm during skeletal myogenesis, via formation of repressive and activatory SWI/SNF complexes.

Cyclin D3 critically regulates the balance between self-renewal and differentiation in skeletal muscle stem cells.

Satellite cells are mitotically quiescent myogenic stem cells resident beneath the basal lamina surrounding adult muscle myofibers. In response to injury, multiple extrinsic signals drive the entry of satellite cells into the cell cycle and then to proliferation, differentiation, and self-renewal of their downstream progeny. Because satellite cells must endure for a lifetime, their cell cycle activity must be carefully controlled to coordinate proliferative expansion and self-renewal with the onset of the differentiation program. In this study, we find that cyclin D3, a member of the family of mitogen-activated D-type cyclins, is critically required for proper developmental progression of myogenic progenitors. Using a cyclin D3-knockout mouse we determined that cyclin D3 deficiency leads to reduced myofiber size and impaired establishment of the satellite cell population within the adult muscle. Cyclin D3-null myogenic progenitors, studied ex vivo on isolated myofibers and in vitro, displayed impaired cell cycle progression, increased differentiation potential, and reduced self-renewal capability. Similarly, silencing of cyclin D3 in C2 myoblasts caused anticipated exit from the cell cycle and precocious onset of terminal differentiation. After induced muscle damage, cyclin D3-null myogenic progenitors exhibited proliferation deficits, a precocious ability to form newly generated myofibers and a reduced capability to repopulate the satellite cell niche at later stages of the regeneration process. These results indicate that cyclin D3 plays a cell-autonomous and nonredundant function in regulating the dynamic balance between proliferation, differentiation, and self-renewal that normally establishes an appropriate pool size of adult satellite cells.

Role of Hydroxytyrosol and Oleuropein in the Prevention of Aging and Related Disorders: Focus on Neurodegeneration, Skeletal Muscle Dysfunction and Gut Microbiota.

Aging is a multi-faceted process caused by the accumulation of cellular damage over time, associated with a gradual reduction of physiological activities in cells and organs. This degeneration results in a reduced ability to adapt to homeostasis perturbations and an increased incidence of illnesses such as cognitive decline, neurodegenerative and cardiovascular diseases, cancer, diabetes, and skeletal muscle pathologies. Key features of aging include a chronic low-grade inflammation state and a decrease of the autophagic process. The Mediterranean diet has been associated with longevity and ability to counteract the onset of age-related disorders. Extra virgin olive oil, a fundamental component of this diet, contains bioactive polyphenolic compounds as hydroxytyrosol (HTyr) and oleuropein (OLE), known for their antioxidant, anti-inflammatory, and neuroprotective properties. This review is focused on brain, skeletal muscle, and gut microbiota, as these systems are known to interact at several levels. After the description of the chemistry and pharmacokinetics of HTyr and OLE, we summarize studies reporting their effects in in vivo and in vitro models of neurodegenerative diseases of the central/peripheral nervous system, adult neurogenesis and depression, senescence and lifespan, and age-related skeletal muscle disorders, as well as their impact on the composition of the gut microbiota.

PC4/Tis7/IFRD1 stimulates skeletal muscle regeneration and is involved in myoblast differentiation as a regulator of MyoD and NF-kappaB.

In skeletal muscle cells, the PC4 (Tis7/Ifrd1) protein is known to function as a coactivator of MyoD by promoting the transcriptional activity of myocyte enhancer factor 2C (MEF2C). In this study, we show that up-regulation of PC4 in vivo in adult muscle significantly potentiates injury-induced regeneration by enhancing myogenesis. Conversely, we observe that PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that such an effect is MyoD-dependent. We provide evidence revealing a novel mechanism underlying the promyogenic actions of PC4, by which PC4 functions as a negative regulator of NF-κB, known to inhibit MyoD expression post-transcriptionally. In fact, up-regulation of PC4 in primary myoblasts induces the deacetylation, and hence the inactivation and nuclear export of NF-κB p65, in concomitance with induction of MyoD expression. On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels. We also observe that PC4 potentiates the inhibition of NF-κB transcriptional activity mediated by histone deacetylases and that PC4 is able to form trimolecular complexes with p65 and HDAC3. This suggests that PC4 stimulates deacetylation of p65 by favoring the recruitment of HDAC3 to p65. As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis.

Corrigendum: Metabolic Reprogramming Promotes Myogenesis During Aging.

[This corrects the article DOI: 10.3389/fphys.2019.00897.].

pRb-dependent cyclin D3 protein stabilization is required for myogenic differentiation.

The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that cyclin D3 is nearly totally associated with hypophosphorylated pRb in differentiated myotubes, whereas Rb-/- myocytes fail to accumulate the cyclin D3 protein despite normal induction of cyclin D3 mRNA. Here we report that pRb promotes cyclin D3 protein accumulation in differentiating myoblasts by preventing cyclin D3 degradation. We show that cyclin D3 displays rapid turnover in proliferating myoblasts, which is positively regulated through glycogen synthase kinase 3beta (GSK-3beta)-mediated phosphorylation of cyclin D3 on Thr-283. We describe a novel interaction between pRb and cyclin D3 that maps to the C terminus of pRb and to a region of cyclin D3 proximal to the Thr-283 residue and provide evidence that the pRb-cyclin D3 complex formation in terminally differentiated myotubes hinders the access of GSK-3beta to cyclin D3, thus inhibiting Thr-283 phosphorylation. Interestingly, we observed that the ectopic expression of a stabilized cyclin D3 mutant in C2 myoblasts enhances muscle-specific gene expression; conversely, cyclin D3-null embryonic fibroblasts display impaired MyoD-induced myogenic differentiation. These results indicate that the pRb-dependent accumulation of cyclin D3 is functionally relevant to the process of skeletal muscle cell differentiation.

Metabolic Reprogramming Promotes Myogenesis During Aging.

Sarcopenia is the age-related progressive loss of skeletal muscle mass and strength finally leading to poor physical performance. Impaired myogenesis contributes to the pathogenesis of sarcopenia, while mitochondrial dysfunctions are thought to play a primary role in skeletal muscle loss during aging. Here we studied the link between myogenesis and metabolism. In particular, we analyzed the effect of the metabolic modulator trimetazidine (TMZ) on myogenesis in aging. We show that reprogramming the metabolism by TMZ treatment for 12 consecutive days stimulates myogenic gene expression in skeletal muscle of 22-month-old mice. Our data also reveal that TMZ increases the levels of mitochondrial proteins and stimulates the oxidative metabolism in aged muscles, this finding being in line with our previous observations in cachectic mice. Moreover, we show that, besides TMZ also other types of metabolic modulators (i.e., 5-Aminoimidazole-4-Carboxamide Ribofuranoside-AICAR) can stimulate differentiation of skeletal muscle progenitors in vitro. Overall, our results reveal that reprogramming the metabolism stimulates myogenesis while triggering mitochondrial proteins synthesis in vivo during aging. Together with the previously reported ability of TMZ to increase muscle strength in aged mice, these new data suggest an interesting non-invasive therapeutic strategy which could contribute to improving muscle quality and neuromuscular communication in the elderly, and counteracting sarcopenia.

PC4 coactivates MyoD by relieving the histone deacetylase 4-mediated inhibition of myocyte enhancer factor 2C.

Histone deacetylase 4 (HDAC4) negatively regulates skeletal myogenesis by associating with the myocyte enhancer factor 2 (MEF2) transcription factors. Our data indicate that the gene PC4 (interferon-related developmental regulator 1 [IFRD1], Tis7), which we have previously shown to be required for myoblast differentiation, is both induced by MyoD and potentiates the transcriptional activity of MyoD, thus revealing a positive regulatory loop between these molecules. Enhancement by PC4 of MyoD-dependent activation of muscle gene promoters occurs selectively through MEF2 binding sites. Furthermore, PC4 localizes in the nucleus of differentiating myoblasts, associates with MEF2C, and is able to counteract the HDAC4-mediated inhibition of MEF2C. This latter action can be explained by the observed ability of PC4 to dose dependently displace HDAC4 from MEF2C. Consistently, we have observed that (i) the region of PC4 that binds MEF2C is sufficient to counteract the inhibition by HDAC4; (ii) PC4, although able to bind HDAC4, does not inhibit the enzymatic activity of HDAC4; and (iii) PC4 overcomes the inhibition mediated by the amino-terminal domain of HDAC4, which associates with MEF2C but not with PC4. Together, our findings strongly suggest that PC4 acts as a coactivator of MyoD and MEF2C by removing the inhibitory effect of HDAC4, thus exerting a pivotal function during myogenesis.

Lack of cyclin D3 induces skeletal muscle fiber-type shifting, increased endurance performance and hypermetabolism.

The mitogen-induced D-type cyclins (D1, D2 and D3) are regulatory subunits of the cyclin-dependent kinases CDK4 and CDK6 that drive progression through the G1 phase of the cell cycle. In skeletal muscle, cyclin D3 plays a unique function in controlling the proliferation/differentiation balance of myogenic progenitor cells. Here, we show that cyclin D3 also performs a novel function, regulating muscle fiber type-specific gene expression. Mice lacking cyclin D3 display an increased number of myofibers with higher oxidative capacity in fast-twitch muscle groups, primarily composed of myofibers that utilize glycolytic metabolism. The remodeling of myofibers toward a slower, more oxidative phenotype is accompanied by enhanced running endurance and increased energy expenditure and fatty acid oxidation. In addition, gene expression profiling of cyclin D3-/- muscle reveals the upregulation of genes encoding proteins involved in the regulation of contractile function and metabolic markers specifically expressed in slow-twitch and fast-oxidative myofibers, many of which are targets of MEF2 and/or NFAT transcription factors. Furthermore, cyclin D3 can repress the calcineurin- or MEF2-dependent activation of a slow fiber-specific promoter in cultured muscle cells. These data suggest that cyclin D3 regulates muscle fiber type phenotype, and consequently whole body metabolism, by antagonizing the activity of MEF2 and/or NFAT.

MyoD stimulates RB promoter activity via the CREB/p300 nuclear transduction pathway.

The induction of RB gene transcription by MyoD is a key event in the process of skeletal muscle differentiation, because elevated levels of the retinoblastoma protein are essential for myoblast cell cycle arrest as well as for the terminal differentiation and survival of postmitotic myocytes. We previously showed that MyoD stimulates transcription from the RB promoter independently of direct binding to promoter sequences. Here we demonstrate that stimulation by MyoD requires a cyclic AMP-responsive element (CRE) in the RB promoter, bound by the transcription factor CREB in differentiating myoblasts. We also show that both the CREB protein level and the level of phosphorylation of the CREB protein at Ser-133 rapidly increase at the onset of muscle differentiation and that both remain high throughout the myogenic process. Biochemical and functional evidence indicates that in differentiating myoblasts, MyoD becomes associated with CREB and is targeted to the RB promoter CRE in a complex also containing the p300 transcriptional coactivator. The resulting multiprotein complex stimulates transcription from the RB promoter. These and other observations strongly suggest that MyoD functions by promoting the efficient recruitment of p300 by promoter-bound, phosphorylated CREB.