Nadège Zanou

Skeletal muscle hypertrophy and regeneration: interplay between the myogenic regulatory factors (MRFs) and insulin-like growth factors (IGFs) pathways.

Adult skeletal muscle can regenerate in response to muscle damage. This ability is conferred by the presence of myogenic stem cells called satellite cells. In response to stimuli such as injury or exercise, these cells become activated and express myogenic regulatory factors (MRFs), i.e., transcription factors of the myogenic lineage including Myf5, MyoD, myogenin, and Mrf4 to proliferate and differentiate into myofibers. The MRF family of proteins controls the transcription of important muscle-specific proteins such as myosin heavy chain and muscle creatine kinase. Different growth factors are secreted during muscle repair among which insulin-like growth factors (IGFs) are the only ones that promote both muscle cell proliferation and differentiation and that play a key role in muscle regeneration and hypertrophy. Different isoforms of IGFs are expressed during muscle repair: IGF-IEa, IGF-IEb, or IGF-IEc (also known as mechano growth factor, MGF) and IGF-II. MGF is expressed first and is observed in satellite cells and in proliferating myoblasts whereas IGF-Ia and IGF-II expression occurs at the state of muscle fiber formation. Interestingly, several studies report the induction of MRFs in response to IGFs stimulation. Inversely, IGFs expression may also be regulated by MRFs. Various mechanisms are proposed to support these interactions. In this review, we describe the general process of muscle hypertrophy and regeneration and decipher the interactions between the two groups of factors involved in the process.

Role of TRPC1 channel in skeletal muscle function

Skeletal muscle contraction is reputed not to depend on extracellular Ca2+. Indeed, stricto sensu, excitation-contraction coupling does not necessitate entry of Ca2+. However, we previously observed that, during sustained activity (repeated contractions), entry of Ca2+ is needed to maintain force production. In the present study, we evaluated the possible involvement of the canonical transient receptor potential (TRPC)1 ion channel in this entry of Ca2+ and investigated its possible role in muscle function. Patch-clamp experiments reveal the presence of a small-conductance channel (13 pS) that is completely lost in adult fibers from TRPC1(-/-) mice. The influx of Ca2+ through TRPC1 channels represents a minor part of the entry of Ca(2+) into muscle fibers at rest, and the activity of the channel is not store dependent. The lack of TRPC1 does not affect intracellular Ca2+ concentration ([Ca2+](i)) transients reached during a single isometric contraction. However, the involvement of TRPC1-related Ca2+ entry is clearly emphasized in muscle fatigue. Indeed, muscles from TRPC1(-/-) mice stimulated repeatedly progressively display lower [Ca2+](i) transients than those observed in TRPC1(+/+) fibers, and they also present an accentuated progressive loss of force. Interestingly, muscles from TRPC1(-/-) mice display a smaller fiber cross-sectional area, generate less force per cross-sectional area, and contain less myofibrillar proteins than their controls. They do not present other signs of myopathy. In agreement with in vitro experiments, TRPC1(-/-) mice present an important decrease of endurance of physical activity. We conclude that TRPC1 ion channels modulate the entry of Ca(2+) during repeated contractions and help muscles to maintain their force during sustained repeated contractions.

Essential role of TRPV2 ion channel in the sensitivity of dystrophic muscle to eccentric contractions.

Duchenne myopathy is a lethal disease due to the absence of dystrophin, a cytoskeletal protein. Muscles from dystrophin‐deficient mice (mdx) typically present an exaggerated susceptibility to eccentric work characterized by an important force drop and an increased membrane permeability consecutive to repeated lengthening contractions. The present study shows that mdx muscles are largely protected from eccentric work‐induced damage by overexpressing a dominant negative mutant of TRPV2 ion channel. This observation points out the role of TRPV2 channel in the physiopathology of Duchenne muscular dystrophy.

Trpc1 Ion Channel Modulates Phosphatidylinositol 3-Kinase/Akt Pathway during Myoblast Differentiation and Muscle Regeneration

Background: The PI3K/Akt pathway is involved in muscle development and regeneration. Results: Knocking out Trpc1 channels or inhibiting Ca2+ fluxes decreases PI3K/Akt activation, slows down myoblasts migration and impairs muscle regeneration. Conclusion: Trpc1-mediated Ca2+ influx enhances PI3K/Akt pathway during muscle regeneration. Significance: The activity of PI3K/Akt pathway is modulated by intracellular Ca2+. We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1−/− and Trpc1+/+ murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1−/− muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1−/− mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1−/− muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1−/− primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca2+ or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca2+ through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca2+-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.

Androgen deprivation and androgen receptor competition by bicalutamide induce autophagy of hormone‐resistant prostate cancer cells and confer resistance to apoptosis

Treatment of advanced prostate cancer (PCa) relies on pharmacological or surgical androgen deprivation. However, it is only temporarily efficient. After a few months or years, the tumor relapses despite the absence of androgenic stimulation: a state referred to as hormone‐refractory prostate cancer (HRPCa). Although autophagy confers chemoresistance in some cancers, its role in the development of HRPCa remains unknown.

Effects of Pharmacological AMP Deaminase Inhibition and Ampd1 Deletion on Nucleotide Levels and AMPK Activation in Contracting Skeletal Muscle

AMP-activated protein kinase (AMPK) plays a central role in regulating metabolism and energy homeostasis. It achieves its function by sensing fluctuations in the AMP:ATP ratio. AMP deaminase (AMPD) converts AMP into IMP, and the AMPD1 isoenzyme is expressed in skeletal muscles. Here, effects of pharmacological inhibition and genetic deletion of AMPD were examined in contracting skeletal muscles. Pharmacological AMPD inhibition potentiated rises in AMP, AMP:ATP ratio, AMPK Thr172, and acetyl-CoA carboxylase (ACC) Ser218 phosphorylation induced by electrical stimulation, without affecting glucose transport. In incubated extensor digitorum longus and soleus muscles from Ampd1 knockout mice, increases in AMP levels and AMP:ATP ratio by electrical stimulation were potentiated considerably compared with muscles from wild-type mice, whereas enhanced AMPK activation was moderate and only observed in soleus, suggesting control by factors other than changes in adenine nucleotides. AMPD inhibitors could be useful tools for enhancing AMPK activation in cells and tissues during ATP-depletion.

Osmosensation in TRPV2 dominant negative expressing skeletal muscle fibres

Increased plasma osmolarity induces intracellular water depletion and cell shrinkage (CS) followed by activation of a regulatory volume increase (RVI). In skeletal muscle, the hyperosmotic shock‐induced CS is accompanied by a small membrane depolarization responsible for a release of Ca2+ from intracellular pools. Hyperosmotic shock also induces phosphorylation of STE20/SPS1‐related proline/alanine‐rich kinase (SPAK). TRPV2 dominant negative expressing fibres challenged with hyperosmotic shock present a slower membrane depolarization, a diminished Ca2+ response, a smaller RVI response, a decrease in SPAK phosphorylation and defective muscle function. We suggest that hyperosmotic shock induces TRPV2 activation, which accelerates muscle cell depolarization and allows the subsequent Ca2+ release from the sarcoplasmic reticulum, activation of the Na+–K+–Cl− cotransporter by SPAK, and the RVI response.

TRPC1: Subcellular Localization?

Berbey et al. (1) recently reported that overexpressed TRPC1-YFP fusion protein is targeted to the sarcoplasmic reticulum of adult skeletal muscle fibers and operates as a calcium leak channel. This observation is in accordance with previous reports showing that the movement of TRPC1 to the plasma membrane is dependent on co-expression with TRPC4 or TRPC5 ((2); for review see Refs. 3 and 4) and that, when expressed alone, TRPC1 forms functional endoplasmic reticulum (ER) homotetrameric channels (5). In this paper, the authors also show by immunocytochemistry with a Chemicon antibody that endogenous TRPC1 is localized exclusively in the sarcoplasmic reticulum. This observation contradicts previous data reporting its electrophysiological detection in the plasma membrane (6) and its co-localization and co-immunoprecipitation with dystrophin, α1-syntrophin, and caveolin (7,–9). To clarify this apparent discrepancy, we performed immunocytochemistry with the same anti-TRPC1 antibody as Berbey et al. (1) on FDB muscle fibers isolated from wild-type and TRPC1-deficient mice (10). As shown in Fig. 1, immunolabeling with this antibody gave similar results in WT and TRPC1-deficient mice (upper and lower panel, respectively). We observed a striated pattern in both groups (large staining in A bands and faint staining in M bands, as shown on the profile curves along the red line). Similar results were obtained on longitudinal sections of paraffin-embedded EDL muscles (not shown). This staining seems very similar to that presented by Berbey et al. (1). Our data strongly suggest that anti-TRPC1 Chemicon antibody is not specific of TRPC1 when used in these conditions. Fig. 1. In conclusion, we believe that the results presented by Berbey and colleagues (1) do not exclude the possible localization of endogenous TRPC1 in the plasma membrane and that the possible localization of endogenous TRPC1 in the sarcoplasmic reticulum (SR) needs further investigation.

APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation

Besides its crucial role in the pathogenesis of Alzheimers disease, the knowledge of amyloid precursor protein (APP) physiologic functions remains surprisingly scarce. Here, we show that APP regulates the transcription of the glial cell line‐derived neurotrophic factor (GDNF). APP‐dependent regulation of GDNF expression affects muscle strength, muscular trophy, and both neuronal and muscular differentiation fundamental for neuromuscular junction (NMJ) maturation in vivo. In a nerve‐muscle coculture model set up to modelize NMJ formation in vitro, silencing of muscular APP induces a 30% decrease in secreted GDNF levels and a 40% decrease in the total number of NMJs together with a significant reduction in the density of acetylcholine vesicles at the presynaptic site and in neuronal maturation. These defects are rescued by GDNF expression in muscle cells in the conditions where muscular APP has been previously silenced. Expression of GDNF in muscles of amyloid precursor protein null mice corrected the aberrant synaptic morphology of NMJs. Our findings highlight for the first time that APP‐dependent GDNF expression drives the process of NMJ formation, providing new insights into the link between APP gene regulatory network and physiologic functions.—Stanga, S., Zanou, N., Audouard, E., Tasiaux, B., Contino, S., Vandermeulen, G., René, F., Loeffler, J.‐P., Clotman, F., Gailly, P., Dewachter, I., Octave, J.‐N., Kienlen‐Campard, P. APP‐dependent glial cell line‐derived neurotrophic factor gene expression drives neuromuscular junction formation. FASEB J. 30, 1696–1711 (2016). www.fasebj.org

Sphingosine-1-phosphate-activated TRPC1 channel controls chemotaxis of glioblastoma cells

TRP channels are involved in the control of a broad range of cellular functions such as cell proliferation and motility. We investigated the gating mechanism of TRPC1 channel and its role in U251 glioblastoma cells migration in response to chemotaxis by platelet-derived growth factor (PDGF). PDGF induced an influx of Ca2+ that was partially inhibited after pretreatment of the cells with SKI-II, a specific inhibitor of sphingosine kinase producing sphingosine-1-P (S1P). S1P by itself also induced an entry of Ca2+. Interestingly, PDGF- and S1P-induced entries of Ca2+ were lost in siRNA-TRPC1 treated cells. PDGF-induced chemotaxis of U251 cells was dramatically inhibited in cells treated with SKI-II. This effect was almost completely rescued by addition of synthetic S1P. Chemotaxis was also completely lost in siRNA-TRPC1 treated cells and interestingly, the rescue of migration of cells treated with SKI-II by S1P was dependent on the expression of TRPC1. Immunocytochemistry revealed that, in response to PDGF, TRPC1 translocated from inside of the cell to the front of migration (lamellipodes). This effect seemed PI3K dependent as it was inhibited by cell pre-treatment with LY294002, a PI3-kinase inhibitor. Our results thus identify S1P as a potential activator of TRPC1, a channel involved in cell orientation during chemotaxis by PDGF.