Ablation of Sam50 is associated with fragmentation and alterations in metabolism in murine and human myotubes.

ABSTRACT

The Sorting and Assembly Machinery (SAM) Complex is responsible for assembling ß-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system (MICOS) complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopy (SBF-SEM) and computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.