After damage, skeletal muscle (SkM) regeneration is reliant on SkM-specific stem cells (muscle progenitor cells [MPCs]), which are activated from quiescence and rapidly proliferate until an adequate number of cells to support regeneration are available. The regenerative process is impaired with advancing age. An emerging requirement for rapidly proliferating cells (e.g., cancer cells, activated T cells) are the non-essential amino acids serine (Ser) and glycine (Gly). We have shown that Ser and Gly within the SkM decline with aging (r2 = −0.376, P < 0.004, n = 62). Objective: To determine the requirement of Ser and Gly for human MPC (hMPC) proliferation.
From vastus lateralis biopsy tissue of female donors (n = 5) hMPCs (CD29+/CD56+) were isolated via FACS. hMPCs were cultured in a formulated growth media with 10% dialyzed and charcoal treated fetal bovine serum to control Ser and Gly concentrations.
hMPCs cultured without Ser/Gly did not increase cell number. Addition of Ser/Gly rescued cell number in a dose-dependent manner (P < 0.01). RNA-sequencing (RNAseq) identified up-regulation of genes related to NRF2 signaling (oxidative stress response), Ser/Gly biosynthesis, amino acid transport, and gluconeogenic genes and down-regulation of glutathione metabolism and collagen synthesis genes with Ser/Gly restriction. Supporting our RNAseq data, Ser/Gly restriction increased reactive oxygen species, as measured by flow cytometry (>70%, P = 0.1), reduced total glutathione levels (P < 0.01) as well as the reduced: oxidized glutathione ratio (P < 0.01). However, exogenous glutathione provided a modest rescue in cell number (P < 0.05) while pyruvate, a gluconeogenic precursor with antioxidant properties, fully rescued cell number (P < 0.01). With Ser/Gly depletion, we also observed an increase in ATF4 (P < 0.01), a stress-responsive transcription factor downstream of NRF2 and an upregulation of ATF4 target genes.
Ser and Gly restriction in hMPCs leads to an increased oxidative state, which likely underlies increased NRF2/ATF4 activity and loss of proliferation. This research underscores the essentiality of Ser and Gly for human MPC proliferation and therefore, the SkM regenerative process.
Canadian Institutes of Health Research Doctoral Foreign Study Award to BG.