Transcriptomic analysis reveals extensive modulation of mitochondrial gene expressions after volumetric muscle loss injury
Authors: Miller David , Greising Sarah , Douglass Eugene , Call Jarrod 
Published: May 2024

Volumetric muscle loss (VML) is characterized by the unrecoverable loss of muscle tissue and function. Therapeutic modalities remain elusive in part because the etiology of the pathology remains unclear. Past RNA-sequencing analyses shed light on transcriptional regulation, but differential gene expression and gene set enrichment analysis (GSEA) results are often dominated by the robust inflammatory and fibrotic response to the injury. The primary objective of this study was to leverage past datasets and interrogate transcriptional regulation of cellular metabolism because our group has previously reported that VML injury disrupts mitochondrial function in the remaining muscle after VML injury. We hypothesized that a metabolic GSEA analysis will identify several differentially expressed mitochondrial gene sets. Utilizing a publicly-available rat species RNAseq dataset, we transformed that data from transcripts per million (TPM) to a Z-score with respect to the uninjured group. Since TPM is not compatible with cross-sample comparisons, we opted for a single-sample GSEA that permits phenotype comparisons. Visualizing the normalized enrichment scores (NES) into heatmaps allowed us to select the top-25 enriched gene sets. The majority of the top-25 could be split into three groups: electron transport chain (ETC, n=8), substrate metabolism (SM, n=5), and mitochondrial maintenance (MM, n=2). The NES at each day post-VML injury (dpi) was compared to its respective uninjured group using a two-tailed t-test. We found that all ETC-related pathways were significantly enriched and downregulated at 7-, 14-, and 28-dpi (p ≤ 0.039). For example, the percent change in NES of ‘complex 1’ was- 436%, -419%, and -816%, respectively (p≤0.002). SM-related pathways followed a similar downward trend (p≤0.044). For example, ‘TCA’ -149%, -303%, and -697%, respectively (p≤0.028). Interestingly, within the MM-related gene sets, mitochondrial ‘fusion’ was significant down at 7- and 28-dpi (-319%, -414%, p≤0.009) while ‘fission’ was significantly down at 3-, 7-, and 14-dpi (-157%, -319%, and -797%, p≤0.030). We next analyzed a publicly-available mouse species scRNAseq data set. To examine the muscle-specific transcriptional response we combined the counts for myonuclei and satellite cells. GSEA revealed six significantly enriched gene sets, notably ‘all metabolism genes’ (NES = 1.33, FDR q = 0.050), and the remaining four were related to the ETC (NES ≥ 1.81, FDR q ≤ 0.050). This retrospective, comprehensive analysis confirms dynamic changes in the expression of mitochondrial-related genes in response to VML injury in both rats and mice. The modulation of genes related to the electron transport chain, mitochondrial maintenance, and substrate metabolism agree with physiological outcomes of impaired mitochondrial carbohydrate metabolism, hyperpolarized mitochondria, and slowed electron conductance from previous mouse studies. R01AR078903 to SG and JC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.