Bianca Maria Scicchitano

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.

Local overexpression of V1a-vasopressin receptor enhances regeneration in tumor necrosis factor-induced muscle atrophy.

Skeletal muscle atrophy occurs during disuse and aging, or as a consequence of chronic diseases such as cancer and diabetes. It is characterized by progressive loss of muscle tissue due to hypotrophic changes, degeneration, and an inability of the regeneration machinery to replace damaged myofibers. Tumor necrosis factor (TNF) is a proinflammatory cytokine known to mediate muscle atrophy in many chronic diseases and to inhibit skeletal muscle regeneration. In this study, we investigated the role of Arg-vasopressin-(AVP-)dependent pathways in muscles in which atrophy was induced by local overexpression of TNF. AVP is a potent myogenesis-promoting factor and is able to enhance skeletal muscle regeneration by stimulating Ca2+/calmodulin-dependent kinase and calcineurin signaling. We performed morphological and molecular analyses and demonstrated that local over-expression of the AVP receptor V1a enhances regeneration of atrophic muscle. By upregulating the regeneration/differentiation markers, modulating the inflammatory response, and attenuating fibrogenesis, the stimulation of AVP-dependent pathways creates a favourable environment for efficient and sustained muscle regeneration and repair even in the presence of elevated levels of TNF. This study highlights a novel in vivo role for AVP-dependent pathways, which may represent an interesting strategy to counteract muscle decline in aging or in muscular pathologies.

Age-dependent alteration in muscle regeneration: the critical role of tissue niche

Although adult skeletal muscle is composed of fully differentiated fibers, it retains the capacity to regenerate in response to injury and to modify its contractile and metabolic properties in response to changing demands. The major role in the growth, remodeling and regeneration is played by satellite cells, a quiescent population of myogenic precursor cells that reside between the basal lamina and plasmalemma and that are rapidly activated in response to appropriate stimuli. However, in pathologic conditions or during aging, the complete regenerative program can be precluded by fibrotic tissue formation and resulting in functional impairment of the skeletal muscle. Our study, along with other studies, demonstrated that although the regenerative program can also be impaired by the limited proliferative capacity of satellite cells, this limit is not reached during normal aging, and it is more likely that the restricted muscle repair program in aging is presumably due to missing signals that usually render the damaged muscle a permissive environment for regenerative activity.

Molecular and Cellular Mechanisms of Muscle Aging and Sarcopenia and Effects of Electrical Stimulation in Seniors

The prolongation of skeletal muscle strength in aging and neuromuscular disease has been the objective of numerous studies employing a variety of approaches. It is generally accepted that cumulative failure to repair damage related to an overall decrease in anabolic processes is a primary cause of functional impairment in muscle. The functional performance of skeletal muscle tissues declines during post- natal life and it is compromised in different diseases, due to an alteration in muscle fiber composition and an overall decrease in muscle integrity as fibrotic invasions replace functional contractile tissue. Characteristics of skeletal muscle aging and diseases include a conspicuous reduction in myofiber plasticity (due to the progressive loss of muscle mass and in particular of the most powerful fast fibers), alteration in muscle-specific transcriptional mechanisms, and muscle atrophy. An early decrease in protein synthetic rates is followed by a later increase in protein degradation, to affect biochemical, physiological, and morphological parameters of muscle fibers during the aging process. Alterations in regenerative pathways also compromise the functionality of muscle tissues. In this review we will give an overview of the work on molecular and cellular mechanisms of aging and sarcopenia and the effects of electrical stimulation in seniors..

Skeletal muscle is enriched in hematopoietic stem cells and not inflammatory cells in cachectic mice.

Abstract Objective: Cachexia, a debilitating syndrome characterized by skeletal muscle wasting, is associated to many chronic diseases and diminishes the quality of life and survival of patients. Tumor-derived factors and proinflammatory cytokines, including TNF-alpha, IL-6 and IL-1beta, mediate cachexia. In response to elevated cytokine levels, increased proteasome-mediated proteolysis and auto-phagocytosis result in muscle wasting. The histologic features of muscle cachexia are not fully elucidated. Therefore, we analysed alterations of different cell populations in cachectic muscle. Methods: By immunohistochemical and cytological approaches, we characterized changes in the abundance of cellular populations in the musculature of a murine model of cancer cachexia (C26-bearing mice). Results: Cachectic muscle displayed a decreased DNA content proportional to muscle mass wastage. A decrease in the number of nuclei occurred in the muscular but not in the stromal compartment. Cachectic muscle showed: mild modulation of myeloperoxidase activity, a neutrophil marker; reduction of macrophages in the endomysium; decrease in CD3+ lymphocyte number. Conversely, a statistically significant enrichment in Sca-1+ CD45+ hematopoietic stem cells (HSCs) occurred in cachectic muscle. Discussion: The elevated levels of cytokines which characterize cachexia may represent a trigger for inflammatory cell activation. However, we find that in cachexia, inflammatory cells in muscle are not increased while muscle tissue nuclei decline. Our data suggest that the inflammatory cell-mediated stress is not an etiologic component of muscle wasting in cachexia. The relative increase in HSCs in cachectic skeletal muscle suggests an attempt to maintain muscle homeostasis by recruitment and/or activation of stem cells.

Stem cell-mediated muscle regeneration and repair in aging and neuromuscular diseases

One of the most exciting aspirations of current medical science is the regeneration of damaged body parts. The capacity of adult tissues to regenerate in response to injury stimuli represents an important homeostatic process that until recently was thought to be limited in mammals to tissues with high turnover such as blood and skin. However, it is now generally accepted that each tissue type, even those considered post-mitotic, such as nerve or muscle, contains a reserve of undifferentiated progenitor cells, loosely termed stem cells, participating in tissue regeneration and repair. Skeletal muscle regeneration is a coordinate process in which several factors are sequentially activated to maintain and preserve muscle structure and function upon injury stimuli. In this review, we will discuss the role of stem cells in muscle regeneration and repair and the critical role of specific factors, such as IGF-1, vasopressin and TNF-alpha, in the modulation of the myogenic program and in the regulation of muscle regeneration and homeostasis.Branching morphogenesis is a multi-step process that controls the formation of polarised tubules starting from hollow cysts. Its execution entails a series of rate-limiting events which include reversible disruption of cell polarity, dismantling of intercellular contacts, acquisition of a motile phenotype, stimulation of cell proliferation, and final re-establishment of cell polarity for creation of the definitive structures. Branching morphogenesis takes place physiologically during development, accounting for the establishment of organs endowed with a ramified architecture such as glands, the respiratory tract and the vasculartree. In cancer, aberrant implementation of branching morphogenesis leads to deregulated proliferation, protection from apoptosis and enhanced migratory/invasive properties, which together exacerbate the aggressive features of neoplastic cells. Under both physiological and pathological conditions, branching morphogenesis is mainly accomplished by a family of growth factors known as scatter factors. In this review, we will summarise the current knowledge on the biological and functional roles of scatter factors during branching morphogenesis, with a special emphasis on the phenotypic (structural and histological) consequences of scatter factor activity in different tissues.We present here findings obtained on a large number of human tissues over a period of more than ten years, by our modification of the Osmium maceration method for high resolution scanning electron microscopy (HRSEM). Data are documented by original pictures which illustrate both some 3-D intracellular features not previously shown in human tissues, and results obtained in our current studies on mitochondrial morphology and on the secretory process of salivary glands. We have demonstrated that mitochondria of cells of practically all human tissues and organs have usually tubular cristae, and that even the cristae that look lamellar are joined to the inner mitochondrial membrane by tubular connexions similar to the crista junctions later seen by electron tomography. Concerning salivary glands an important result is the development of a morphometric method that allows the quantitative evaluation of the secretory events.

Skeletal Muscle Regeneration in Mice Is Stimulated by Local Overexpression of V1a-Vasopressin Receptor

Skeletal muscle has a remarkable capacity to regenerate after mechanical or pathological injury. We show that the V1a receptor (V1aR) for vasopressin, a potent myogenic-promoting factor that stimulates differentiation and hypertrophy in vitro, is expressed in mouse skeletal muscle and modulated during regeneration after experimental injury. We used gene delivery by electroporation to overexpress the myc-tagged vasopressin V1aR in specific muscles, thus sensitizing them to circulating vasopressin. The correct localization on the surface of the fibers of the recombinant product was demonstrated by confocal immunofluorescence directed against the myc tag. V1aR overexpression dramatically enhanced regeneration. When compared with mock-transfected controls, V1aR overexpressing muscles exhibited significantly accelerated activation of satellite cells and increased expression of differentiation markers. Downstream of V1aR activation, calcineurin was strongly up-regulated and stimulated the expression of IL-4, a potent mediator of myogenic cell fusion. The central role of calcineurin in mediating V1aR-dependent myogenesis was also demonstrated by using its specific inhibitor, cyclosporine A. This study identifies skeletal muscle as a physiological target of hormones of the vasopressin family and reveals a novel in vivo role for vasopressin-dependent pathways. These findings unveil several steps, along a complex signaling pathway, that may be exploited as potential targets for the therapy of diseases characterized by altered muscle homeostasis and regeneration.

Neurohypophyseal hormones: novel actors of striated muscle development and homeostasis

Since the 1980’s, novel functional roles of the neurohypophyseal hormones vasopressin and oxytocin have emerged. Several studies have investigated the effects of these two neurohormones on striated muscle tissues, both in vitro and in vivo. The effects of vasopressin on skeletal myogenic cells, developing muscle and muscle homeostasis have been documented. Oxytocin appears to have a greater influence on cardiomyocite differentiation and heart homeostasis. This review summarizes the studies on these novel roles of the two neurohypophyseal hormones, and open the possibility of new therapeutic approaches for diseases affecting striated muscle.

Stem cells and tissue niche: two faces of the same coin of muscle regeneration

Capacity of adult muscle to regenerate in response to injury stimuli represents an important homeostatic process. Regeneration is a highly coordinated program that partially recapitulates the embryonic developmental program. However, muscle regeneration is severely compromised in several pathological conditions. It is likely that the restricted tissue repair program under pathological conditions is due to either progressive loss of stem cell populations or to missing signals that limit the damaged tissues to efficiently activate a regenerative program. It is therefore plausible that loss of control over these cell fates might lead to a pathological cell transdifferentiation, limiting the ability of a pathological muscle to sustain an efficient regenerative process. The critical role of microenvironment on stem cells activity and muscle regeneration is discussed.

Increased Circulating Levels of Interleukin-6 Induce Perturbation in Redox-Regulated Signaling Cascades in Muscle of Dystrophic Mice

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease in which dystrophin gene is mutated, resulting in dysfunctional or absent dystrophin protein. The pathology of dystrophic muscle includes degeneration, necrosis with inflammatory cell invasion, regeneration, and fibrous and fatty changes. Nevertheless, the mechanisms by which the absence of dystrophin leads to muscle degeneration remain to be fully elucidated. An imbalance between oxidant and antioxidant systems has been proposed as a secondary effect of DMD. However, the significance and precise extent of the perturbation in redox signaling cascades is poorly understood. We report that mdx dystrophic mice are able to activate a compensatory antioxidant response at the presymptomatic stage of the disease. In contrast, increased circulating levels of IL-6 perturb the redox signaling cascade, even prior to the necrotic stage, leading to severe features and progressive nature of muscular dystrophy.