TRPC1 is increased in mdx muscle, binds to caveolin-3 and is regulated by Src kinase: implications for Duchenne muscular dystrophy

O.L. Gervasio, N.P. Whitehead and D.G. Allen, Bosch Institute, School of Medical Sciences, Anderson Stuart Building (F13), The University of Sydney, NSW 2006, Australia.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease caused by the absence of dystrophin, a membrane anchoring protein. We have shown that calcium entry through stretch activated channels (SACs) contributes to muscle damage in the mdx mouse, an animal model of DMD (Yeung et al., 2005). Transient receptor potential canonical 1 (TRPC1) forms SACs in mammalian cells (Maroto et al., 2005) and interacts with caveolin-1 in smooth muscle cells (Lockwich et al., 2000). Caveolin-3 (Cav-3), which is structurally homologous to caveolin-1, is increased in mdx muscle (Vaghy et al., 1998). The aim of this study is to investigate the expression levels and interaction of Cav-3 and TRPC1 in mdx muscle. TRPC1 and Cav-3 co-localized, co-immunoprecipitated and had increased expression levels in mdx muscle (immunohistochemistry, Western blot). Fluorescence Energy Resonance Transfer (FRET) was used to confirm the interaction of the two proteins, in C2C12 myblasts co-transfected with TRPC1-CFP and Cav-3-YFP plasmids. Fluorescence Lifetime Imaging Microscopy (FLIM) showed a shortening of the donor lifetime (TRPC1-CFP) when cells were co-transfected with both plasmids (from 2.7ns to 2.1ns; p < 0.001), confirming the interaction between TRPC1 and Cav-3. As Src kinase can activate channels from the TRPC family (Kawasaki et al., 2005), we investigated the role of this kinase on TRPC1 activity and binding properties with Cav-3. Incubation of C2C12 cells with hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS), increased the levels of both the active form of Src kinase (Western blot, p < 0.01) and the total tyrosine phosphorylation in those cells (immunohistochemistry, p < 0.01). Src activation and tyrosine phosphorylation were partially prevented when cells were incubated with PP2, a Src kinase inhibitor, prior to H₂O₂ treatment. Ratiometric calcium imaging (Fura Red) of C2C12 cells transfected with TRPC1-CFP and Cav-3-YFP revealed that calcium influx was increased upon H₂O₂ treatment only in cells expressing both TRPC1 and Cav-3 plasmids. Furthermore, the calcium influx was prevented when those cells were pre-incubated with PP2. These results suggest that Cav-3 is necessary for TRPC1 activity, which is triggered by activation of Src kinase by ROS. As ROS are known to be increased in mdx/DMD, we suggest that targeting the ROS-Src-TRPC1 pathway could lead to the development of new therapeutic approaches for the treatment of DMD.

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