Silencing MYOT Expression May Inhibit Autophagy in Human Skeletal Muscle Cells

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Muscle diseases are frequently associated with disruptions in cellular autophagy.
• Autophagy plays a critical role in muscle regeneration and function, with its inhibition impacting muscle health.
• Mutations in the MYOT gene are known to cause hereditary myopathies, but its specific role in autophagy regulation within these conditions remains unclear.

Purpose of the Study:

• To investigate the role of MYOT gene expression in regulating autophagy in human skeletal muscle cells.
• To determine if MYOT knockdown affects autophagy pathways relevant to hereditary myopathies.

Main Methods:

• Established MYOT knockdown human skeletal muscle cell models (HSkMCs) using small interfering RNA.
• Utilized Real-time PCR and Western blot to assess the expression of autophagy markers (p62, LC3B-II).
• Employed immunofluorescence (Ad-mCherry-GFP-LC3B adenovirus transfection, monodansylcadaverine staining) and pharmacological treatments (EBSS, BAF A1) to evaluate autophagy flux and activation.

Main Results:

• MYOT knockdown significantly decreased the expression of p62 and LC3B-II, indicating altered autophagy.
• Immunofluorescence and staining revealed that reduced MYOT expression inhibits autophagy.
• Silencing MYOT impaired autophagy activation by EBSS and exacerbated inhibition by BAF A1, also downregulating ATG7 and ATG5 expression.

Conclusions:

• This study provides the first evidence that reduced MYOT expression inhibits autophagy in human skeletal muscle cells.
• The findings suggest that impaired autophagy due to MYOT knockdown may be a contributing factor to hereditary myopathies associated with MYOT mutations.