MYOGENIC DIFFERENTIATION AND MUSCLE HOMEOSTASIS: NOVEL ROLES OF VASOPRESSIN

The neurohypophyseal nonapeptide arg-vasopressin (AVP) and related peptides constitute a novel family of positive regulators of terminal differentiation of myogenic cell lines and primary satellite cells. By interacting with V1 type receptor, AVP induces activation of phospholipases C and D, regulates cAMP levels, increases cytosolic Ca2+ concentration and up-regulates Myf-5 and myogenin expression, both at the mRNA and at the protein level. In a chemically defined medium, which eliminates the interference of serum components, AVP activates both the calcineurin and the CaMK signaling pathways, whose combined activation leads to the formation of multifactor complexes and is required for the full expression of the differentiated phenotype in vitro. To better clarify the physiological role of AVP in skeletal muscle, we analyzed the AVP effects on muscle regeneration induced by cardiotoxin injection. In particular, to increase skeletal muscle sensibility to circulating AVP, in the absence of systemic effects related to administration of the hormone itself, we over-expressed the V1a AVP receptor in mouse tibialis anterior muscle by electroporation-mediated gene delivery in vivo. The local over-expression of the V1aR in injured muscle results in enhanced regeneration. V1aR over-expressing muscle exhibits: early activation of satellite cells and regeneration markers, accelerated differentiation, increased cell population expressing hematopoietic stem cell markers and its conversion to the myogenic lineage. We demonstrate that V1aR over-expressing muscle increases calcineurin and IL-4 expression levels, and induces the phosphorylation of FOXO trascription factors, inhibiting the expression of atrophic genes. This study highlights a novel in vivo role for the AVP-dependent pathways which may represent a potential gene therapy approach for many diseases affecting muscle homeostasis

Molecular mechanisms regulating skeletal muscle homeostasis: effects of V1a AVP receptor over-expression.

The mantainance a working skeletal musculature is conferred by its remarkable capacity to regenerate after mechanical or pathological injury. Most muscle pathologies are characterized by the progressive loss of muscle tissue due to chronic degeneration combined with the inability of the regeneration machinery to replace damaged myofibers. In particular, cachexia or muscle wasting is characterized by a dramatic loss of adipose and muscle mass associated with a compromised muscle regenerative ability. Arg-vasopressin (AVP) is a potent myogenesis promoting factor and activates both the calcineurin and CaMK pathways, whose combined activation leads to the formation of transcription factor complexes in vitro. The local over-expression of V1a AVP receptor (V1aR) in injured muscle results in enhanced regeneration. V1aR over-expressing muscle exhibits: early activation of satellite cells and regeneration markers, accelerated differentiation, increased cell population expressing hematopoietic stem cell markers and its conversion to the myogenic lineage. Here we investigated the role of V1aR over-expression in animals undergoing cachexia as a result of muscle over-expression of a specific cytokine (TNF-). In these conditions, the local V1aR overexpression counteracts the negative effects of cachexia on muscle, as demonstrated by morphological and biochemical analysis. In particular, the presence of V1aR results in increased Pax-7, myogenin and myosin expression levels both in wild type and in cachectic muscles. The positive effects of V1aR on muscle homeostasis are due to the promotion of the calcinuerin-IL- 4 pathway and to the inhibition of atrophic genes expression mediated by FOXO phosphorilation. This study highlights a novel in vivo role for the AVP-dependent pathways which may represent a potential gene therapy approach for many diseases affecting muscle homeostasis

Cardiac autonomic control in Rett syndrome: Insights from heart rate variability analysis

Rett syndrome (RTT) is a rare and severe neurological disorder mainly affecting females, usually linked to methyl-CpG-binding protein 2 (MECP2) gene mutations. Manifestations of RTT typically include loss of purposeful hand skills, gait and motor abnormalities, loss of spoken language, stereotypic hand movements, epilepsy, and autonomic dysfunction. Patients with RTT have a higher incidence of sudden death than the general population. Literature data indicate an uncoupling between measures of breathing and heart rate control that could offer insight into the mechanisms that lead to greater vulnerability to sudden death. Understanding the neural mechanisms of autonomic dysfunction and its correlation with sudden death is essential for patient care. Experimental evidence for increased sympathetic or reduced vagal modulation to the heart has spurred efforts to develop quantitative markers of cardiac autonomic profile. Heart rate variability (HRV) has emerged as a valuable non-invasive test to estimate the modulation of sympathetic and parasympathetic branches of the autonomic nervous system (ANS) to the heart. This review aims to provide an overview of the current knowledge on autonomic dysfunction and, in particular, to assess whether HRV parameters can help unravel patterns of cardiac autonomic dysregulation in patients with RTT. Literature data show reduced global HRV (total spectral power and R-R mean) and a shifted sympatho-vagal balance toward sympathetic predominance and vagal withdrawal in patients with RTT compared to controls. In addition, correlations between HRV and genotype and phenotype features or neurochemical changes were investigated. The data reported in this review suggest an important impairment in sympatho-vagal balance, supporting possible future research scenarios, targeting ANS

MOLECULAR MECHANISMS REGULATING SKELETAL MUSCLE HOMEOSTASIS: EFFECTS OF V1A AVP RECEPTOR OVER-EXPRESSION

http://www.coram-iim.it/iim/incontri/quarto_meeting.htm

MOLECULAR MECHANISMS REGULATING SKELETAL MUSCLE HOMEOSTASIS: EFFECTS OF V1A VASOPRESSIN RECEPTOR OVEREXPRESSION

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

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-1 beta, 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

Histochemical analysis of damage and regeneration in skeletal muscle wasting

Cachexia is a severe form of muscle wasting, triggered by elevated levels of cytokines in chronic diseases,which interferes with the management of the primary disease and accounts for the death of a significant percentage of patients. In order to identify mechanisms underlying the loss of muscle homeostasis in cachexia, we analyzed the effects of cytokines on skeletal muscle damage and regenerative capacity. We exploited the properties of Evans Blue Dye to detect sarcolemmal damage in a murine model of cachexia. Fiber damage-associated inflammation was also revealed by immuno-histochemistry for activated macrophages invading muscle fibers. Muscles responded to damage and necrosis by activating a satellite cell -mediated response. The cachectic musculature is enriched in satellite cells expressing high levels of Pax7 and MyoD.However, a morphometric evaluation of the muscle regenerative potential indicated that cachectic muscles lack the ability to fully regenerate following satellite cell activation. The uncoupling between increased fiber necrosis and impaired regeneration resulted in a decreased muscle fiber number per cross-section in the cachectic musculature. Following experimentally-induced damage, intramuscular TNF injection significantly decreased the number and size of the regenerating fibers, an effect abolished by treatment with a caspase inhibitor. Active caspases were highlighted in interstitial cells expressing stem cell markers in the absence of apoptotic phenomena. TNF-dependent impairment of regeneration was rescued by muscle gene delivery of the dominant negative form of PW1 (a TNF effector) or the V1a receptor (to sensitize muscle to the myogenic factor Vasopressin). We conclude that both increased damage and decreased regeneration contribute to muscle wasting in cachexia, suggesting to counteract cytokine effects on muscle regeneration by pharmacological and gene therapy approaches

V1a AVP receptor is required for neurohypophyseal hormonedependent differentiation in C2C12 cells

Vasopressin (AVP), oxytocin (OT) and related peptides induce differentiation and hypertrophy in myogenic cells expressing the V1a-vasopressin receptor (V1aR) or the oxytocin receptor (OTR). Either receptor can transduce both ligand signals. Binding of AVP and OT to the V1aR the target cells activates phosphatidylinositol hydrolysis, which in turn releases Ca2+ from internal stores. The AVP-dependent increase in cytosolic Ca2+ induces the activation of calcium/calmodulin-dependent kinase (CaMK) and calcineurin signaling, two pathways required for the full expression of the differentiated phenotype. Here we investigate the role of V1aR in myogenesis and hypertrophy by ectopically restoring V1aR expression and function using the C2C12 cell line, which is an experimental model of satellite cells that do not respond to AVP treatment. Our results show that AVP treatment enhances myogenic differentiation in V1aR-transfected C2C12 cultures alone. Moreover, calcium imaging analyses performed in individual control and V1aR-transfected C2C12 cells demonstrated that the presence of V1aR is sufficient to make C2C12 cells responsive to neurohypophyseal hormones stimulation, as demonstrated by the rapid and sustained release of calcium from internal stores observed in V1aR-transfected cells. These data demonstrate that, despite the high levels of OTR expressed by C2C12 cells, both AVP and OT failed to stimulate the differentiation program, thereby indicating that the presence of V1aR is essential to mediate the effects of neurohypophyseal hormones on myogenic differentiation in C2C12 cells