Mario Molinaro

Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle

Aging skeletal muscles suffer a steady decline in mass and functional performance, and compromised muscle integrity as fibrotic invasions replace contractile tissue, accompanied by a characteristic loss in the fastest, most powerful muscle fibers. The same programmed deficits in muscle structure and function are found in numerous neurodegenerative syndromes and disease-related cachexia. We have generated a model of persistent, functional myocyte hypertrophy using a tissue-restricted transgene encoding a locally acting isoform of insulin-like growth factor-1 that is expressed in skeletal muscle (mIgf-1). Transgenic embryos developed normally, and postnatal increases in muscle mass and strength were not accompanied by the additional pathological changes seen in other Igf-1 transgenic models. Expression of GATA-2, a transcription factor normally undetected in skeletal muscle, marked hypertrophic myocytes that escaped age-related muscle atrophy and retained the proliferative response to muscle injury characteristic of younger animals. The preservation of muscle architecture and age-independent regenerative capacity through localized mIgf-1 transgene expression suggests clinical strategies for the treatment of age or disease-related muscle frailty.

Skeletal Muscle Is a Primary Target of SOD1G93A-Mediated Toxicity

The antioxidant enzyme superoxide dismutase 1 (SOD1) is a critical player of the antioxidative defense whose activity is altered in several chronic diseases, including amyotrophic lateral sclerosis. However, how oxidative insult affects muscle homeostasis remains unclear. This study addresses the role of oxidative stress on muscle homeostasis and function by the generation of a transgenic mouse model expressing a mutant SOD1 gene (SOD1(G93A)) selectively in skeletal muscle. Transgenic mice developed progressive muscle atrophy, associated with a significant reduction in muscle strength, alterations in the contractile apparatus, and mitochondrial dysfunction. The analysis of molecular pathways associated with muscle atrophy revealed that accumulation of oxidative stress served as signaling molecules to initiate autophagy, one of the major intracellular degradation mechanisms. These data demonstrate that skeletal muscle is a primary target of SOD1(G93A) -mediated toxicity and disclose the molecular mechanism whereby oxidative stress triggers muscle atrophy.

Local expression of IGF-1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines

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)

Chapter 9 Cell Heterogeneity in The Myogenic Lineage

Publisher Summary This chapter (1) shows that heterogeneity exists among myogenic cells and (2) suggest that this heterogeneity may play an important role in both muscle histogenesis and pathology. It discusses the evidence for the existence of different classes of myogenic cells, emerging at different periods during development and embryologically related to specific aspects of muscle differentiation and further maturation. Additional reasons to examine myogenic cell heterogeneity lies in the well-known histopathological picture of primary myopathies, where certain muscle fibers appear unaffected by the disease while the majority degenerate. Such heterogeneity reflects the existence of different populations of fibers, which might not express, or express to a different level, the mutated gene. The chapter reviews the literature on early and late myoblasts in somites and limbs and on satellite cells, and discusses possible relationships of these cell types with muscle fiber development and with fiber heterogeneity in primary myopathies. Immunocytochemical evidence of heterogeneity among somitic cells with respect to the expression of myosin heavy-chain (MHC) isoforms is obtained. Proteins which cross-react with antibodies directed against slow MHC are expressed by a subpopulation of differentiated muscle cells. This subpopulation apparently migrates to the limb, since it can be observed in cultures of limbs from 10-day and, to a lesser extent, 12-day-old embryos.

High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patients muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA. Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to beta-galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced satellite cells. MyoD-converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo.

In vitro differentiation of satellite cells isolated from normal and dystrophic mammalian muscles. A comparison with embryonic myogenic cells

Satellite cells were isolated from skeletal muscles of adult normal and dystrophic mice (C57/6J/dy strain) by sequential digestion of tissue fragments with collagenase, hyaluronidase and trypsin. These cells exhibit in culture similar behaviour to that of embryonic myoblasts, undergoing an initial duplicative period lasting about 2--3 days, followed by a shorter phase (1--2 days) of rapid cell fusion. During the duplicative phase most of the satellite cells appear round-shaped, whereas embryonic myoblasts appear typically spindle-shaped: both cell types actively incorporate [3H]thymidine. During the subsequent days of culture an increasing number of satellite cells becomes spindle-shaped; afterwards the cells contact each other and fuse into multinucleated myotubes. The majority of spindle-shaped satellite cells is unable to incorporate [3H]thymidine, thus behaving as post-mitotic cells. Concomitantly with satellite cell fusion, an increase of about 80-fold of creatine phosphokinase (CPK) specific activity is observed. Satellite cells are able to recognize co-cultured embryonic myoblasts ([3H]thymidine-labelled): hybrid myotubes containing labelled and unlabelled nuclei are formed in these experimental conditions. Satellite cells from dystrophic animals are able to differentiate in culture and do not show appreciable differences as compared to their normal counterparts. In dystrophic myotubes, however, CPK specific activity is almost twice that observed in normal myotubes. Human dystrophic satellite cells from biopsies of adult muscle cultured in similar conditions grow and fuse into multinucleated myotubes showing a behaviour identical to normal controls.

PKCα-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells

We have previously suggested that PKCα has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated growth arrest and myogenic differentiation in human embryonal rhabdomyosarcoma cells (RD). Here, by monitoring the signalling pathways triggered by TPA, we demonstrate that PKCα mediates these effects by inducing transient activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all referred to as MAPKs). Activation of MAPKs following ectopic expression of constitutively active PKCα, but not its dominant-negative form, is also demonstrated. We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating myogenic-related morphology in differentiated RD cells.

Differential response of satellite cells and embryonic myoblasts to a tumor promoter

Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) reversibly suppressed myotube formation and expression of acetylcholine receptors in cultures of Day 15 mouse embryo presumptive myoblasts, but was totally ineffective in cultures of adult mouse satellite cells. A subpopulation of TPA-resistant myogenic cells became apparent in cultures prepared from older embryos or newborn mice. Thus, limb presumptive myoblasts are a heterogeneous population, and part of the distinct TPA-resistant subpopulation may represent satellite cell precursors.

Tumor Necrosis Factor-α Inhibition of Skeletal Muscle Regeneration Is Mediated by a Caspase-Dependent Stem Cell Response

Skeletal muscle is susceptible to injury following trauma, neurological dysfunction, and genetic diseases. Skeletal muscle homeostasis is maintained by a pronounced regenerative capacity, which includes the recruitment of stem cells. Chronic exposure to tumor necrosis factor‐α (TNF) triggers a muscle wasting reminiscent of cachexia. To better understand the effects of TNF upon muscle homeostasis and stem cells, we exposed injured muscle to TNF at specific time points during regeneration. TNF exposure delayed the appearance of regenerating fibers, without exacerbating fiber death following the initial trauma. We observed modest cellular caspase activation during regeneration, which was markedly increased in response to TNF exposure concomitant with an inhibition in regeneration. Caspase activation did not lead to apoptosis and did not involve caspase‐3. Inhibition of caspase activity improved muscle regeneration in either the absence or the presence of TNF, revealing a nonapoptotic role for this pathway in the myogenic program. Caspase activity was localized to the interstitial cells, which also express Sca‐1, CD34, and PW1. Perturbation of PW1 activity blocked caspase activation and improved regeneration. The restricted localization of Sca‐1+, CD34+, PW1+ cells to a subset of interstitial cells with caspase activity reveals a critical regulatory role for this population during myogenesis, which may directly contribute to resident muscle stem cells or indirectly regulate stem cells through cell‐cell interactions.

Effect of metabolic inhibitors on macromolecular synthesis and early development in the mouse embryo

Abstract Mouse embryos were cultured in vitro for various periods of time, during the interval from the 2-cell stage to the late blastocyst stage, in the continuous presence of actinomycin D or puromycin and labeled precursors. At various time intervals, the incorporation of 3 H-uridine into total RNA and of 3 H-leucine into protein, and the stage of development of the embryos were recorded. Actinomycin D at the concentration of 0.1 μg/ml caused a rapid and almost complete inhibition of the incorporation of 3 H-uridine at all stages of development, and a rapid depression of the incorporation of 3 H-leucine into protein until a level of about 50 % of the control incorporation which was attained after 12–16 h of incubation. Longer incubation with the antibiotic did not further depress the relative incorporation of 3 H-leucine with respect to the control. The development of the embryos in culture was markedly depressed after continuous incubation with 0.01 and 0.1 μg/ml of actinomycin D, but was not completely arrested. Puromycin at the concentration of 50 μg/ml caused an immediate and complete inhibition of 3 H-leucine incorporation and of development in culture. These results were interpreted to indicate that in the mouse embryo protein synthesis and normal development in culture from the 2-cell stage to the blastocyst stage are regulated by a continuous synthesis of RNA. These results correlate with previous biochemical evidence that in the mouse embryo, genes are transcribed very early during development. There is, however, a large fraction of protein synthesis which seems to be dependent on RNA molecules with very long half-life; this fraction of protein synthesis accounts probably for the partial development occurring after actinomycin treatment. The culture methods available at present do not allow to establish whether these stable messengers are synthesized during oogenesis cr after fertilization. The early dependence of embryonic development on gene activity in the mouse contrasts then markedly with the situation observed in sea urchin and amphibians, where embryonic development and protein synthesis until gastrulation are completely independent of simultaneous gene activity, but are probably fully regulated by ribosomes and stable RNA messengers synthesized during oogenesis and stored in the egg cytoplasm to be utilized after fertilization.