Myoblast migration is a key step in myogenesis and regeneration. It allows myoblast alignment and their fusion into myotubes. The process has been shown to involve m-calpain or μ-calpain, two Ca²⁺-dependent cysteine proteases. Here we measure calpain activity in cultured cells and show a peak of activity at the beginning of the differentiation process. We also observed a concomitant and transient increase of the influx of Ca²⁺ and expression of TRPC1 protein. Calpains are specifically activated by a store-operated entry of Ca²⁺ in adult skeletal muscle fibres. We therefore repressed the expression of TRPC1 in myoblasts and studied the effects on Ca²⁺ fluxes and on differentiation. TRPC1-depleted myoblasts presented a largely reduced store-operated entry of Ca²⁺ and a significantly diminished transient influx of Ca²⁺ at the beginning of differentiation. The concomitant peak of calpain activity was abolished. TRPC1-knockdown myoblasts also accumulated myristoylated alanine-rich C-kinase substrate (MARCKS), an actin-binding protein and substrate of calpain. Their fusion into myotubes was significantly slowed down as a result of the reduced speed of cell migration. Accordingly, migration of control myoblasts was inhibited by 2-5 μM GsMTx4 toxin, an inhibitor of TRP channels or by 50 μM Z-Leu-Leu, an inhibitor of calpain. By contrast, stimulation of control myoblasts with IGF-1 increased the basal influx of Ca²⁺, activated calpain and accelerated migration. These effects were not observed in TRPC1-knockdown cells. We therefore suggest that entry of Ca²⁺ through TRPC1 channels induces a transient activation of calpain and subsequent proteolysis of MARCKS, which allows in turn, myoblast migration and fusion.

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 Ca²⁺ 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 Ca²⁺ during repeated contractions and help muscles to maintain their force during sustained repeated contractions.

TRP channels have been associated with cell proliferation and aggressiveness in several cancers. In particular, TRPC1 regulates cell proliferation and motility, two processes underlying cancer progression. We and others have described the mechanisms of TRPC1-dependent cell migration. However, the involvement of TRPC1 in cell proliferation remains unexplained. In this study, we show that siRNA-mediated TRPC1 depletion in non small cell lung carcinoma cell lines induced G(0)/G(1) cell cycle arrest resulting in dramatic decrease in cell growth. The expression of cyclins D1 and D3 was reduced after TRPC1 knockdown, pointing out the role of TRPC1 in G(1)/S transition. This was associated with a decreased phosphorylation and activation of EGFR and with a subsequent disruption of PI3K/Akt and MAPK downstream pathways. Stimulation of EGFR by its natural ligand, EGF, induced Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry through TRPC1. Ca(2+) entry through TRPC1 conversely activated EGFR, suggesting that TRPC1 is a component of a Ca(2+)-dependent amplification of EGF-dependent cell proliferation.

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.

Group I metabotropic glutamate receptors, in particular mGluR5, have been implicated in various forms of synaptic plasticity that are believed to underlie declarative memory. We observed that mGluR5 specifically activated a channel containing TRPC1, an isoform of the canonical family of transient receptor potential (TRPC) channels highly expressed in CA1-3 regions of the hippocampus. TRPC1 is able to form tetrameric complexes with TRPC4 and/or TRPC5 isoforms. TRPC1/4/5 complexes have recently been involved in the efficiency of synaptic transmission in the hippocampus. We therefore used a mouse model devoid of TRPC1 expression to investigate the involvement of mGluR5-TRPC1 pathway in synaptic plasticity and memory formation. Trpc1^-/- mice showed alterations in spatial working memory and fear conditioning. Activation of mGluR increased synaptic excitability in neurons from WT but not from Trpc1^-/- mice. LTP triggered by a theta burst could not maintain over time in brain slices from Trpc1^-/- mice. mGluR-induced LTD was also impaired in these mice. Finally, acute inhibition of TRPC1 by Pico145 on isolated neurons or on brain slices mimicked the genetic depletion of Trpc1 and inhibited mGluR-induced entry of cations and subsequent effects on synaptic plasticity, excluding developmental or compensatory mechanisms in Trpc1^-/- mice. In summary, our results indicate that TRPC1 plays a role in synaptic plasticity and spatial working memory processes.