Myogenesis is a crucial biological process for the development and maintenance of muscle mass, and the loss of regenerative potential leads to debilitating musculoskeletal disorders. Differentiation of progenitor cells (myoblasts) into mature myotubes is a dynamic process during which cell identity and function shift over time. Traditional, bulk biochemical analyses lack the resolution to tease apart the highly heterogenous myoblast response to differentiation conditions, where subpopulations of cells undergo progressive phenotypic and functional changes. Autophagy, a system of intracellular recycling, has been implicated as essential for myoblast differentiation. Via autophagy, long-lived proteins or damaged organelles are degraded to their amino-acid constituents, providing building blocks for synthesis of new or different subcellular components. A high-resolution study of progressively differentiating myoblasts will illuminate which phenotypically unique subpopulations increase autophagic flux. We use mass cytometry, a single-cell inductively-coupled plasma mass spectrometry technique, to quantify changes in phenotypic and autophagy markers during myoblast differentiation <i>in vitro</i>. The technique can measure upwards of 50 parameters per particle through the detection of isotopically pure lanthanide ions chelated by antibody-based probes specific towards protein targets of interest.<br/><br/>In mass cytometry analysis two major issues which can bias detected signal are, i) antibody binding specificity, and ii) variation in cell size or cell permeabilization. To address these issues, we make use of two control antibodies: an isotype to assess non-specific binding, and a “loading control” to normalize specific antibody signal. Individual cell data is mathematically transformed post-acquisition to yield corrected and normalized signals for biological analysis. We demonstrate the use of this method to reveal accurate levels of autophagy-related proteins in genetically or pharmacologically manipulated myoblast models of autophagy dysregulation. Unaltered data show linear titration curves while transformed data can be accurately fit to Langmuir isotherms. With these data, we have accurately quantified the fold-change of autophagy-related proteins in progressively differentiating myoblasts in during normal myoblast differentiation. Analyses suggests an increase in mitochondrial-specific degradation via autophagy, which may indicate a response to changing metabolic needs.

Quantitative multi-dimensional single-cell analysis of autophagy during myogenesis

255th National Meeting (2018)

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