The role of the volume-regulated anion channel VRAC/LRRC8 in myoblast differentiation

Abstract

Myoblast differentiation and fusion are essential for the formation of multinucleated myofibers during skeletal muscle development and regeneration. An elaborate signaling network, including the action of various ion channels and transporters, is involved in myogenesis. Multiple studies have demonstrated the roles of K+ and Ca2+ channels in skeletal myogenesis, but the involvement of Cl− channels is poorly understood. In this thesis, I determine that the volume-regulated anion channel (VRAC)/leucine-rich repeat containing family 8 (LRRC8) promotes mouse myoblast differentiation. Immunoblotting showed the expression of all five LRRC8 subunits of heteromeric VRAC during myotube formation of murine C2C12 myoblasts. siRNA-mediated knockdown of the essential VRAC subunit LRRC8A did not affect myoblast proliferation but significantly reduced the expression of the myogenic transcription factor myogenin and inhibited myoblast fusion. Suppression of VRAC activity by either pharmacological VRAC inhibitors or overexpression of LRRC8A also effectively reduced myoblast differentiation and fusion. VRAC inhibition blocked plasma membrane hyperpolarization of myoblasts early during differentiation and prevented the steady-state increase of intracellular Ca2+ levels that normally occurs during myogenesis. Consistently, I could show temporary activation of VRAC within the first 2 h of myoblast differentiation by a non-invasive FRET-based sensor, demonstrating that VRAC acts upstream of K+ channel activation. Myoblast differentiation resulted in a significant decrease in intracellular Cl− that was abolished by the VRAC inhibitor carbenoxolone. However, as judged by the expression of myogenin in C2C12 cells, lowering the cytosolic Cl− level by extracellular Cl− depletion did not enhance differentiation. Instead, it suppressed myosin expression and myotube formation. This work provides a possible mechanism for the thinning of skeletal muscle bundles observed in LRRC8A-deficient mice and emphasizes the importance of volume-regulated LRRC8 anion channels in cell differentiation.