Force dependent effects of chronic overuse on fibrosis-related genes and proteins in skeletal muscles

To examine the chronic effect of force on mRNA and protein expression levels of fibrosis-related genes in flexor digitorum muscles in a rat model of repetitive overuse injury that induces muscle fibrosis at high force levels.

Two groups of rats were trained to perform a voluntary repetitive lever-pulling task at either a high (HFHR) or a low force (LFHR) for 18 weeks, while a control group (FRC) performed no task. RNA and protein were prepared from forelimb flexor digitorum muscles. Fibrosis-related gene RNA transcripts were evaluated using quantitative PCR (qPCR) and analyzed using the geometric mean of three housekeeping genes or the mean of each individually as reference. Protein levels were quantified using ELISA, western blot, or immunohistofluorescence.

Of eight fibrosis-related mRNAs examined, only FGF2 demonstrated a consistent significant increase in the HFHR group, compared to the FRC group. However, protein amounts of collagen type 1, collagen type 3, and TGFβ1 were significantly higher in the HFHR, compared to the FRC and LFHR groups, while CCN2 and FGF2 were higher in both HFHR and LFHR, compared to the FRC group.

Our results suggest that there is steady-state transcription of fibrogenic genes in muscles with established fibrosis, implying that post-transcriptional processes are responsible for the increased protein levels of fibrotic factors during muscle overuse conditions. We hypothesize that targeting such pathways represents a valid approach to treat overuse injury. Alternatively, FGF2 gene expression may represent a valid target for therapy.