Lightning Talk & Poster 28th Annual Lorne Proteomics Symposium 2023

A novel platform integrating single cell muscle physiology with single cell proteomics (#20)

Ronnie Blazev 1 , Yaan-Kit Ng 1 , Jeffrey Molendijk 1 , Benjamin L Parker 1
  1. University of Melbourne, Parkville, VICTORIA, Australia

It is well known that exercise is key to reducing risk factors associated with disease and conferring positive health benefits in addition to promoting optimal skeletal muscle function. Understanding how exercise-regulated signalling mediates the beneficial effects of exercise may lead to improved therapeutic strategies and in this context, we recently identified the uncharacterised protein C18ORF25 as a novel regulator of skeletal muscle function and validated it as a bona-fide AMP-activated protein kinase [1]. We further showed that loss of C18ORF25 results in reduced muscle contractile function at the whole muscle and single muscle cell level.

Here, we present a newly developed proteomics platform that allows functional assessment of excitation-contraction (E-C) coupling pathways in an individual muscle cell to be integrated with single cell proteomics in the exact same cell. This platform has been used to identify specific defects in sarcoplasmic reticulum calcium loading following loss of C18ORF25, with paired single-cell proteomics identifying Calcium/Calmodulin Dependent Protein Kinase II Gamma (CAMK2G) as down-regulated and thus a likely key driving mechanism of the reduced muscle function observed in the absence of C18ORF25. Furthermore, our platform has associated seven discrete phenotypes of the E-C pathway to >1,600 proteins using correlation analysis resulting in the identification of 851 unique phenotype:protein associations. Our data analysis workflow shows hundreds of novel associations with an example of two positive controls being the correlation between (i) Junctional Sarcoplasmic Reticulum Protein 1 (JSRP1) and (ii) Sarcoplasmic Reticulum Histidine-Rich Calcium-Binding Protein (HRC) and calcium handling phenotypes.  

We believe our unique proteomics platform, integrating single-cell physiology with single-cell proteomics in the same cell, will provide deep mechanistic insights into skeletal muscle dysfunction in diseased states like insulin resistance and type-2 diabetes not possible with other research strategies. In addition, the ability to pinpoint defective proteins in diseased conditions will allow for novel therapeutic interventions to improve skeletal muscle contractile and metabolic function.

  1. Blazev, R. et al. Cell Metabolism (2022) Oct 4;34(10):1561-1577.