The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction

Glucose restriction (GR) impairs muscle cell differentiation and evokes myotube atrophy. Resveratrol treatment in skeletal muscle cells improves inflammatory-induced reductions in skeletal muscle cell differentiation. We therefore hypothesised that resveratrol treatment would improve muscle cell differentiation and myotube hypertrophy in differentiating C2C12 myoblasts and mature myotubes during GR. Glucose restriction at 0.6 g/L (3.3 mM) blocked differentiation and myotube hypertrophy versus high-glucose (4.5 g/L or 25 mM) differentiation media (DM) conditions universally used for myoblast culture. Resveratrol (10 μM) treatment increased SIRT1 phosphorylation in DM conditions, yet did not improve differentiation when administered to differentiating myoblasts in GR conditions. Resveratrol did evoke increases in hypertrophy of mature myotubes under DM conditions with corresponding elevated Igf-I and Myhc7 gene expression, coding for the ‘slow’ type I MYHC protein isoform. Inhibition of SIRT1 via EX-527 administration (100 nM) also reduced myotube diameter and area in DM conditions and resulted in lower gene expression of Myhc 1, 2 and 4 coding for ‘intermediate’ and ‘faster’ IIx, IIa and IIb protein isoforms, respectively. Resveratrol treatment did not appear to modulate phosphorylation of energy-sensing protein AMPK or protein translation initiator P70S6K. Importantly, in mature myotubes, resveratrol treatment was able to ameliorate reduced myotube growth in GR conditions over an acute 24-h period, but not over 48–72 h. Overall, resveratrol evoked myotube hypertrophy in DM conditions while favouring ‘slower’ Myhc gene expression and acutely ameliorated impaired myotube growth observed during glucose restriction

Leptin Administration Favors Muscle Mass Accretion by Decreasing FoxO3a and Increasing PGC-1α in ob/ob Mice

Absence of leptin has been associated with reduced skeletal muscle mass in leptin-deficient ob/ob mice. The aim of our study was to examine the effect of leptin on the catabolic and anabolic pathways regulating muscle mass. Gastrocnemius, extensor digitorum longus and soleus muscle mass as well as fiber size were significantly lower in ob/ob mice compared to wild type littermates, being significantly increased by leptin administration (P<0.001). This effect was associated with an inactivation of the muscle atrophy-related transcription factor forkhead box class O3 (FoxO3a) (P<0.05), and with a decrease in the protein expression levels of the E3 ubiquitin-ligases muscle atrophy F-box (MAFbx) (P<0.05) and muscle RING finger 1 (MuRF1) (P<0.05). Moreover, leptin increased (P<0.01) protein expression levels of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a regulator of muscle fiber type, and decreased (P<0.05) myostatin protein, a negative regulator of muscle growth. Leptin administration also activated (P<0.01) the regulators of cell cycle progression proliferating cell nuclear antigen (PCNA) and cyclin D1, and increased (P<0.01) myofibrillar protein troponin T. The present study provides evidence that leptin treatment may increase muscle mass of ob/ob mice by inhibiting myofibrillar protein degradation as well as enhancing muscle cell proliferation

The role of ATP and adenosine in the brain under normoxic and ischemic conditions

By taking advantage of some recently synthesized compounds that are able to block ecto-ATPase activity, we demonstrated that adenosine triphosphate (ATP) in the hippocampus exerts an inhibitory action independent of its degradation to adenosine. In addition, tonic activation of P2 receptors contributes to the normally recorded excitatory neurotransmission. The role of P2 receptors becomes critical during ischemia when extracellular ATP concentrations increase. Under such conditions, P2 antagonism is protective. Although ATP exerts a detrimental role under ischemia, it also exerts a trophic role in terms of cell division and differentiation. We recently reported that ATP is spontaneously released from human mesenchymal stem cells (hMSCs) in culture. Moreover, it decreases hMSC proliferation rate at early stages of culture. Increased hMSC differentiation could account for an ATP-induced decrease in cell proliferation. ATP as a homeostatic regulator might exert a different effect on cell trophism according to the rate of its efflux and receptor expression during the cell life cycle. During ischemia, adenosine formed by intracellular ATP escapes from cells through the equilibrative transporter. The protective role of adenosine A1 receptors during ischemia is well accepted. However, the use of selective A1 agonists is hampered by unwanted peripheral effects, thus attention has been focused on A2A and A3 receptors. The protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells. Reduced activation of p38 mitogen-activated protein kinases that are involved in neuronal death through transcriptional mechanisms may also contribute to protection by A2A antagonism. Evidence that A3 receptor antagonism may be protective after ischemia is also reported

Neuroprotection by adenosine in the brain: From A1 receptor activation to A2A receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A1 receptors (A1Rs) and the less abundant, but widespread, facilitatory A2ARs. It is commonly assumed that A1Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A1R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A1Rs in chronic noxious situations. In contrast, A2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease, ischemic brain damage and epilepsy. The greater interest of A2AR blockade compared to A1R activation does not mean that A1R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A1Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different

Transplantation and perfusion of microvascular fragments in a rodent model of volumetric muscle loss injury

Few clinical options are available for the treatment of volumetric muscle loss (VML). An important consideration that needs to be addressed for the development of treatments for these injuries is the establishment of a vascular supply sufficient to support skeletal muscle regeneration. The objective of the current study was to evaluate the potential for microvascular fragments (MVFs) harvested from adipose tissue to support tissue perfusion for VML. Tibialis anterior muscle defects in rats were replaced with constructs that were created on the day of surgery containing either (1) collagen only (COL), (2) freshly isolated microvascular fragments in collagen (MVF), or (3) adipose tissue derived stem cells (ASCs) in collagen. Muscles were harvested 7 and 14 days after surgery. Defects treated with MVFs had a vessel density higher than the other groups at both 7 and 14 days, and those treated with ASCs had a higher vessel density than COL by day 14 (p < 0.05). Perfused vessels were observed in both the ASC and MVF treated defects at day 14, as well as at day 7 in the MVF. This study supports the use of MVFs as a platform to improve tissue perfusion to treat large VML defects. The use of freshly isolated MVFs on the day of surgery supports their clinical use and application

Enhanced proliferation of human skeletal muscle precursor cells derived from elderly donors cultured in estimated physiological (5%) oxygen

Human skeletal muscle precursor cells (myoblasts) have significant therapeutic potential and are a valuable research tool to study muscle cell biology. Oxygen is a critical factor in the successful culture of myoblasts with low (1–6%) oxygen culture conditions enhancing the proliferation, differentiation, and/or viability of mouse, rat, and bovine myoblasts. The specific effects of low oxygen depend on the myoblast source and oxygen concentration; however, variable oxygen conditions have not been tested in the culture of human myoblasts. In this study, muscle precursor cells were isolated from vastus lateralis muscle biopsies and myoblast cultures were established in 5% oxygen, before being divided into physiological (5%) or standard (20%) oxygen conditions for experimental analysis. Five percent oxygen increased proliferating myoblast numbers, and since low oxygen had no significant effect on myoblast viability, this increase in cell number was attributed to enhanced proliferation. The proportion of cells in the S (DNA synthesis) phase of the cell cycle was increased by 50%, and p21Cip1 gene and protein expression was decreased in 5 versus 20% oxygen. Unlike in rodent and bovine myoblasts, the increase in myoD, myogenin, creatine kinase, and myosin heavy chain IIa gene expression during differentiation was similar in 5 and 20% oxygen; as was myotube hypertrophy. These data indicate for the first time that low oxygen culture conditions stimulate proliferation, whilst maintaining (but not enhancing) the viability and the differentiation potential of human primary myoblasts and should be considered as optimum conditions for ex-vivo expansion of these cells