The decline in skeletal muscle mass and strength occurring in aging, referred as sarcopenia, is the result of many factors including an imbalance between protein synthesis and degradation, changes in ...metabolic/hormonal status, and in circulating levels of inflammatory mediators. Thus, factors that increase muscle mass and promote anabolic pathways might be of therapeutic benefit to counteract sarcopenia. Among these, the insulin‐like growth factor‐1 (IGF‐1) has been implicated in many anabolic pathways in skeletal muscle. IGF‐1 exists in different isoforms that might exert different role in skeletal muscle. Here we study the effects of two full propeptides IGF‐1Ea and IGF‐1Eb in skeletal muscle, with the aim to define whether and through which mechanisms their overexpression impacts muscle aging. We report that only IGF‐1Ea expression promotes a pronounced hypertrophic phenotype in young mice, which is maintained in aged mice. Nevertheless, examination of aged transgenic mice revealed that the local expression of either IGF‐1Ea or IGF‐1Eb transgenes was protective against age‐related loss of muscle mass and force. At molecular level, both isoforms activate the autophagy/lysosome system, normally altered during aging, and increase PGC1‐α expression, modulating mitochondrial function, ROS detoxification, and the basal inflammatory state occurring at old age. Moreover, morphological integrity of neuromuscular junctions was maintained and preserved in both MLC/IGF‐1Ea and MLC/IGF‐1Eb mice during aging. These data suggest that IGF‐1 is a promising therapeutic agent in staving off advancing muscle weakness.
A summary of the molecular pathways responsible for the protective role of IGF‐1 isoforms against sarcopenia.
Angiotensin (1-7) production increases during AT1R (angiotensin type-1 receptor) blockade. The contribution of Ang (1-7) (angiotensin 1-7) and its receptor (MasR) to the favorable effect of ...angiotensin receptor blockers on remodeling and function of resistance arteries remains unclear. We sought to determine whether MasR contributes to the improvement of vascular structure and function during chronic AT1R blockade. Spontaneously hypertensive rats were treated with Ang (1-7) or olmesartan ± MasR antagonist A-779, or vehicle, for 14 days. Blood pressure was measured by tail cuff methodology. Mesenteric arteries were dissected and mounted on a pressurized micromyograph to evaluate media-to-lumen ratio (M/L) and endothelial function. Expression of MasR and eNOS (endothelial nitric oxide synthase) was evaluated by immunoblotting, plasma nitrate by colorimetric assay, and reactive oxygen species production by dihydroethidium staining. Independently of blood pressure, olmesartan significantly reduced M/L and improved NO bioavailability, A-779 prevented these effects. Likewise, Ang (1-7) significantly reduced M/L and NO bioavailability. MasR expression was significantly increased by Ang (1-7) as well as by olmesartan, and it was blunted in the presence of A-779. Both Ang (1-7) and olmesartan increased eNOS expression and plasma nitrite which were reduced by A-779. Superoxide generation was attenuated by olmesartan and Ang (1-7) and was blunted in the presence of A-779. These MasR-mediated actions were independent of AT2R activation since olmesartan and Ang (1-7) increased MasR expression and reduced M/L in Ang II (angiotensin II)-infused AT2R knockout mice, independently of blood pressure control. A-779 prevented these effects. Hence, MasR activation may contribute to the favorable effects of AT1R antagonism on NO bioavailability and microvascular remodeling, independently of AT2R activation and blood pressure control.
In Duchenne muscular dystrophy (DMD) the absence of dystrophin at the sarcolemma delocalizes and downregulates nitric oxide synthase (nNOS); this alters S-nitrosylation of HDAC2 and its chromatin ...association. We show that the differential HDAC2 nitrosylation state in Duchenne versus wild-type conditions deregulates the expression of a specific subset of microRNA genes. Several circuitries controlled by the identified microRNAs, such as the one linking miR-1 to the G6PD enzyme and the redox state of cell, or miR-29 to extracellular proteins and the fibrotic process, explain some of the DMD pathogenetic traits. We also show that, at variance with other myomiRs, miR-206 escapes from the dystrophin-nNOS control being produced in activated satellite cells before dystrophin expression; in these cells, it contributes to muscle regeneration through repression of the satellite specific factor, Pax7. We conclude that the pathway activated by dystrophin/nNOS controls several important circuitries increasing the robustness of the muscle differentiation program.
► Dystrophin controls gene expression by nNOS relocalization and HDAC2 nitrosylation ► Epigenetic control of miRNA expression is involved in DMD pathogenesis ► miR-1 controls G6PD expression and the redox state of the cell ► miR-206 contributes to satellite cell differentiation by repressing Pax7
Duchenne muscular dystrophy (DMD) is characterized by progressive lethal muscle degeneration and chronic inflammatory response. The mdx mouse strain has served as the animal model for human DMD. ...However, while DMD patients undergo extensive necrosis, the affected muscles of adult mdx mice rapidly regenerates and regains structural and functional integrity. The basis for the mild effects observed in mice compared with the lethal consequences in humans remains unknown. In this study, we provide evidence that interleukin-6 (IL-6) is causally linked to the pathogenesis of muscular dystrophy. We report that forced expression of IL-6, in the adult mdx mice, recapitulates the severe phenotypic characteristics of DMD in humans. Increased levels of IL-6 exacerbate the dystrophic muscle phenotype, sustaining inflammatory response and repeated cycles of muscle degeneration and regeneration, leading to exhaustion of satellite cells. The mdx/IL6 mouse closely approximates the human disease and more faithfully recapitulates the disease progression in humans. This study promises to significantly advance our understanding of the pathogenic mechanisms that lead to DMD.
The adult heart displays poor reparative capacities after injury. Cell transplantation and tissue engineering approaches have emerged as possible therapeutic options. Several stem cell populations ...have been largely used to treat the infarcted myocardium. Nevertheless, transplanted cells displayed limited ability to establish functional connections with the host cardiomyocytes. In this study, we provide a new experimental tool, named 3D eX vivo muscle engineered tissue (X-MET), to define the contribution of mechanical stimuli in triggering functional remodeling and to rescue cardiac ischemia. We revealed that mechanical stimuli trigger a functional remodeling of the 3D skeletal muscle system toward a cardiac muscle-like structure. This was supported by molecular and functional analyses, demonstrating that remodeled X-MET expresses relevant markers of functional cardiomyocytes, compared to unstimulated and to 2D- skeletal muscle culture system. Interestingly, transplanted remodeled X-MET preserved heart function in a murine model of chronic myocardial ischemia and increased survival of transplanted injured mice. X-MET implantation resulted in repression of pro-inflammatory cytokines, induction of anti-inflammatory cytokines, and reduction in collagen deposition. Altogether, our findings indicate that biomechanical stimulation induced a cardiac functional remodeling of X-MET, which showed promising seminal results as a therapeutic product for the development of novel strategies for regenerative medicine.
The antidiabetic drug phenformin displays potent anticancer activity in different tumors, but its mechanism of action remains elusive. Using Shh medulloblastoma as model, we show here that at ...clinically relevant concentrations, phenformin elicits a significant therapeutic effect through a redox-dependent but complex I-independent mechanism. Phenformin inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), a component of the glycerophosphate shuttle, and causes elevations of intracellular NADH content. Inhibition of mGPD mimics phenformin action and promotes an association between corepressor CtBP2 and Gli1, thereby inhibiting Hh transcriptional output and tumor growth. Because ablation of CtBP2 abrogates the therapeutic effect of phenformin in mice, these data illustrate a biguanide-mediated redox/corepressor interplay, which may represent a relevant target for tumor therapy.
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•Therapeutic doses of phenformin suppress Hedgehog-dependent tumor growth•Phenformin inhibits mGPD in cancer cells but does not affect complex I activity•Inhibition of tumor mGPD mimics phenformin and increases redox state/NADH content•Elevated NADH promotes Gli1/CtBP2 complex formation and inhibition of tumor growth
Di Magno et al. investigate the therapeutic properties of phenformin in Hedgehog-dependent tumors. At clinically relevant doses, phenformin works independent of respiratory complex I through mGPD-mediated increase of the redox state. This promotes CtBP2/Gli1 complex formation and consequent inhibition of Hedgehog transcriptional output and tumor growth.
The interaction of RNA with specific RNA-binding proteins (RBP) leads to the establishment of complex regulatory networks through which gene expression is controlled. Careful consideration should be ...given to the exact environment where a given RNA/RBP interplay occurs, as the functional responses might depend on the type of organism as well as the specific cellular or subcellular contexts. This requisite becomes particularly crucial for the study of long non-coding RNAs (lncRNA), as a consequence of their peculiar tissue-specificity and timely regulated expression. The functional characterization of lncRNAs has traditionally relied on the use of established cell lines that, although useful, are unable to fully recapitulate the complexity of a tissue or organ. Here, we detail an optimized protocol, with comments and tips, to identify the RNA interactome of given RBPs by performing cross-linking immunoprecipitation (CLIP) from mouse embryonal hearts. We tested the efficiency of this protocol on the murine pCharme, a muscle-specific lncRNA interacting with Matrin3 (MATR3) and forming RNA-enriched condensates of biological significance in the nucleus. Key features • The protocol refines previous methods of cardiac extracts preparation to use for CLIP assays. • The protocol allows the quantitative RNA-seq analysis of transcripts interacting with selected proteins. • Depending on the embryonal stage, a high number of hearts can be required as starting material. • The steps are adaptable to other tissues and biochemical assays.
Long noncoding RNAs (lncRNAs) are emerging as critical regulators of heart physiology and disease, although the studies unveiling their modes of action are still limited to few examples. We recently ...identified pCharme, a chromatin-associated lncRNA whose functional knockout in mice results in defective myogenesis and morphological remodeling of the cardiac muscle. Here, we combined Cap-Analysis of Gene Expression (CAGE), single-cell (sc)RNA sequencing, and whole-mount in situ hybridization analyses to study pCharme cardiac expression. Since the early steps of cardiomyogenesis, we found the lncRNA being specifically restricted to cardiomyocytes, where it assists the formation of specific nuclear condensates containing MATR3, as well as important RNAs for cardiac development. In line with the functional significance of these activities, pCharme ablation in mice results in a delayed maturation of cardiomyocytes, which ultimately leads to morphological alterations of the ventricular myocardium. Since congenital anomalies in myocardium are clinically relevant in humans and predispose patients to major complications, the identification of novel genes controlling cardiac morphology becomes crucial. Our study offers unique insights into a novel lncRNA-mediated regulatory mechanism promoting cardiomyocyte maturation and bears relevance to Charme locus for future theranostic applications.
The transcription factor MEF2C (Myocyte Enhancer Factor 2C) plays an established role in the early steps of myogenic differentiation. However, the involvement of MEF2C in adult myogenesis and in ...muscle regeneration has not yet been systematically investigated. Alternative splicing of mammalian MEF2C transcripts gives rise to two mutually exclusive protein variants: MEF2Cα2 which exerts a positive control of myogenic differentiation, and MEF2Cα1, in which the α1 domain acts as trans‐repressor of the MEF2C pro‐differentiation activity itself. However, MEF2Cα1 variants are persistently expressed in differentiating cultured myocytes, suggesting a role in adult myogenesis. We found that overexpression of both MEF2Cα1/α2 proteins in a mouse model of muscle injury promotes muscle regeneration and hypertrophy, with each isoform promoting different stages of myogenesis. Besides the ability of MEF2Cα2 to increase differentiation, we found that overexpressed MEF2Cα1 enhances both proliferation and differentiation of primary myoblasts, and activates the AKT/mTOR/S6K anabolic signaling pathway in newly formed myofibers. The multiple activities of MEF2Cα1 are modulated by phosphorylation of Ser98 and Ser110, two amino acid residues located in the α1 domain of MEF2Cα1. These specific phosphorylations allow the interaction of MEF2Cα1 with the peptidyl‐prolyl isomerase PIN1, a regulator of MEF2C functions. Overall, in this study we established a novel regulatory mechanism in which the expression and the phosphorylation of MEF2Cα1 are critically required to sustain the adult myogenesis. The described molecular mechanism will represent a new potential target for the development of therapeutical strategies to treat muscle‐wasting diseases. Stem Cells 2017;35:725–738
MEF2C protein splice variants play different functions during adult myogenesis. Phosphorylated MEF2Cα1 promotes the proliferation of SC‐derived myoblasts (red cells) by inducing, directly or not, the immediate early genes Plk2 and JunB. Upon differentiation, MEF2Cα2 is upregulated and it drives cell cycle exit, terminal differentiation and myoblasts fusion to give newly formed myofibers, by stimulating the expression of the CDK inhibitor P21 as well as P38 and MyoD. Dephosphorylated MEF2Cα1 contributes to the activation of muscle genes in nascent myofibers and it promotes myofiber growth by activating the anabolic AKT/mTOR/S6K pathway.
ABSTRACT
Muscle regeneration following injury is characterized by myonecrosis accompanied by local inflammation, activation of satellite cells, and repair of injured fibers. The resolution of the ...inflammatory response is necessary to proceed toward muscle repair, since persistence of inflammation often renders the damaged muscle incapable of sustaining efficient muscle regeneration. Here, we show that local expression of a muscle‐restricted insulin‐like growth factor (IGF)‐1 (mIGF‐1) transgene accelerates the regenerative process of injured skeletal muscle, modulating the inflammatory response, and limiting fibrosis. At the molecular level, mIGF‐1 expression significantly down‐regulated proinflammatory cytokines, such as tumor necrosis factor (TNF)‐alpha and interleukin (IL)‐1beta, and modulated the expression of CC chemokines involved in the recruitment of monocytes/macrophages. Analysis of the underlying molecular mechanisms revealed that mIGF‐1 expression modulated key players of inflammatory response, such as macrophage migration inhibitory factor (MIF), high mobility group protein‐1 (HMGB1), and transcription NF‐KB. The rapid restoration of injured mIGF‐1 transgenic muscle was also associated with connective tissue remodeling and a rapid recovery of functional properties. By modulating the inflammatory response and reducing fibrosis, supplemental mIGF‐1 creates a qualitatively different environment for sustaining more efficient muscle regeneration and repair.—Pelosi, L., Giacinti, C., Nardis, C., Borsellino, G., Rizzuto, E., Nicoletti, C., Wannenes, F., Battistini, L., Rosenthal, N., Molinaro, M., Musaro, A. Local expression of IGF‐1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines. FASEB J. 21, 1393–1402 (2007)
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