Scaffold Protein PDLIM5 Regulates TRPC1 Calcium Channel Mediated Store-Operated Calcium Entry in Mouse Myoblasts

Area of Science:

• Muscle Biology
• Cell Signaling
• Ion Channel Physiology

Background:

• Intracellular calcium (Ca²⁺) signaling is crucial for myogenesis, with Transient Receptor Potential Canonical (TRPC) channels, particularly TRPC1, mediating store-operated Ca²⁺ entry (SOCE) in myoblasts.
• PDLIM5 (ENH1) is a scaffold protein involved in muscle signaling, including ion channel regulation.

Purpose of the Study:

• To investigate the hypothesis that PDLIM5 (ENH1) regulates TRPC1-mediated Ca²⁺ entry in myoblasts and myotubes.
• To elucidate the differential roles of ENH1 and its skeletal muscle-specific variant ENH4 in TRPC1-dependent calcium signaling during myogenic differentiation.

Main Methods:

• Utilized C2C12 myoblasts and differentiated myotubes.
• Employed thapsigargin to induce SOCE, assessed using Ca²⁺ imaging.
• Utilized pharmacological inhibitors (Gd³⁺, 2-APB), small interfering RNA (siRNA) for TRPC1 and ENH1 knockdown, and gene overexpression.
• Performed mRNA expression analysis and co-immunoprecipitation to detect protein interactions.

Main Results:

• TRPC1 is essential for SOCE in myoblasts and myotubes; SOCE inhibition impairs myotube formation and size.
• ENH1 knockdown decreased TRPC1 and STIM1 mRNA, increased basal Ca²⁺, and impaired SOCE and myotube maturation.
• ENH4 overexpression repressed TRPC1-mediated SOCE and myotube formation, while ENH1 overexpression enhanced SOCE in myoblasts but not myotubes.
• Physical interaction was confirmed between ENH1/ENH4 and TRPC1 in myoblasts, with ENH4 interacting with TRPC1 in myotubes, but not ENH1.

Conclusions:

• ENH1 and ENH4 differentially regulate TRPC1-dependent Ca²⁺ entry in C2C12 cells.
• These proteins play distinct roles in modulating myogenic differentiation and skeletal muscle development through calcium signaling pathways.