High-resolution analyses of the secretomes from murine C2C12 cells and primary human skeletal muscle cells reveal distinct differences in contraction-regulated myokine secretion

Background: Skeletal muscle is a recognized endocrine organ secreting muscle-specific proteins, known as myokines. Myokine release during muscle contraction may contribute to the health-promoting effects of exercise. Electrical pulse stimulation (EPS) is a widely used in vitro method to induce muscle contraction and mimic exercise. However, the similarities and differences in the secretory response of different cell models remain unexplored, which limits the interpretation of these results. Murine C2C12 cells and satellite cell-derived primary human skeletal muscle cells (HSkMCs) are among the most commonly used skeletal muscle cell models to investigate contraction-induced myokine secretion. Nevertheless, the extent to which the secretomes of these cell models are similar or distinct remains to be fully elucidated.

Research question: The objective of this study is to conduct a comprehensive analysis and comparison of the contraction-regulated secretomes of two widely utilized skeletal muscle cell models, namely murine C2C12 and primary human myotubes. The analysis aims to identify the commonalities and differences in their secretory responses.

Methodology: Murine C2C12 and HSkMCs were cultivated and differentiated into multinucleated contractile myotubes. The cells were subjected to the most frequently utilized established protocols of low-frequency EPS (11.5 V for C2C12 and 40 V for HSkMCs, 2 ms pulses) for a duration of 6 hours, thereby simulating a single bout of exercise. A non-targeted high-resolution mass spectrometry analysis was performed on the conditioned cell culture media. This was followed by bioinformatic prediction analyses and cross-species comparison, supplemented by a literature search.

Results: A total of 5,710 and 3,285 proteins were identified in the secretomes of C2C12 and human myotubes, respectively. 80% of human myokines were also detected in the murine secretome. Differentially regulated proteins during contraction were identified, with 518 proteins identified in C2C12 and 336 in HSkMCs. Additionally, 1,440 and 385 potential novel myokines were identified in the murine and human secretomes, respectively. Ontology analysis revealed species- and cell type-specific differences in the cellular compartments involved in myokine secretion. While the majority of newly identified myokines in the murine secretome were secreted via unconventional protein secretion pathways (UPS), most novel proteins in the human secretome were secreted through the classical ER-to-Golgi pathway.

Conclusion: This comprehensive secretome analysis provides a detailed comparison of contraction-regulated secretomes of C2C12 and HSkMCs, identifying a substantial number of previously unidentified potential myokines that may contribute to the health-promoting effects of exercise. These findings offer valuable insights into the myokine secretory response of these two cell models and may serve as a basis for future studies investigating myokines.

Publication History

Article published online:
28 May 2025