Sam50 is Associated with Fragmentation and Alterations in Metabolism in Human and Murine Myotubes

The sorting and assembly machinery (SAM), comprised of Sam50, Sam35 and Sam37, is important for membrane integration and β-barrel protein assembly [1]. Recently, Sam50 has been shown to interact with the mitochondrial contact site and cristae organizing system (MICOS) complex, which connects the inner and outer mitochondrial membranes, and forms the mitochondrial intermembrane space bridging (MIB) complex [2–3]. Sam50 stabilizes the MIB complex for protein transport, respiratory chain complex assembly, and cristae integrity regulation. To structurally form and sustain the cristae, the MICOS complex assembles at the cristae junction and binds directly to Sam50 [2–3]. To determine the role of Sam50 in mitochondrial morphology in vitro, we knocked out Sam50 in murine myotubes. By using serial block facing-scanning electron microscopy (SBF-SEM) for 3D, we saw increased mitochondrial fragmentation, with some mitochondria exhibiting large, sphere-like protrusions (Figure 1). We also noted abnormal mitochondrial cristae formations and increased presence of autophagosomes and lysosomes in the Sam50 knockouts compared to the controls (Figure 2). To garner a better understanding of the role of Sam50 in metabolism, we used targeted gas chromatography mass spectrometry-based metabolomics to identify differential metabolites in human Sam50 knockout myotubes compared to controls. Our enrichment analysis shows an increase in amino acid metabolism and fatty acid metabolism. We concluded that Sam50 is critical for the establishment and maintenance of mitochondria, mitochondrial cristae structure, and mitochondrial metabolism. Mitochondrial fragmentation associated with loss of Sam50 in murine myotubes. Representative orthoslices from wildtype and Sam50 knockout murine myotubes (top). Approximately 50 of these orthoslices are segmented to perform 3D reconstruction. Control and Sam50-deficient myotubes 3D renderings displayed overlaid on SBF-SEM orthoslice (top-middle). Mitochondrial 3D reconstructions displayed along the transvers (lower-middle) and longitudinal (bottom) orientations. 3D reconstruction of autophagosomes from wildtype and Sam50 knockout murine myotubes.