77. Mechanical Stimulation Alters Gene Expression After Muscular Injury in Rats

PURPOSE: Volumetric muscle loss (VML) injury is associated with a majority of the 150,000 open fractures in the U.S annually. Current therapies -including physical therapy- lead to limited recovery with 50% of patients with severe injuries developing chronic disability. Previously, we had reported that mechanical stimulation improves functional recovery after skeletal muscle injury in rats, with higher tetanic torque, higher resistance to fatigue and endurance on a treadmill, lower tissue fibrosis, and higher tissue angiogenesis compared to untreated controls. Here, we performed RNA sequencing analysis of rats receiving mechanical stimulation after VML, to elucidate underlying genetic expression associated with the postulated mechanisms of action of the treatment. METHODS: An excisional muscle injury (8mm) was created in the left Tibialis Anterior (TA) muscle of female adult (200-250 grams) Sprague Dawley rats (n= 10/group). Post-injury, animals were either followed up with no treatment (control group) or subjected to controlled mechanical stimulation of injured muscles (experimental group). At 28 days post injury muscle tissue was collected in RNALaterTM and stored in -80 °C prior to RNA extraction. A differential gene expression analysis tool (DESeq2) was used to calculate the log2 fold change (log₂FC) and the adjusted p-values between the two groups. Differentially expressed genes with p-value less than 0.05 and log₂FC greater than 0.2 were then individually analyzed to determine function. They were then classified into ‘Pro-regenerative’ (PR) or ‘Anti-regenerative’(AR) genes. Volcano plots were created for illustrative purposes. RESULTS: Out of 10,462 differentially expressed sequences detected,165 were significant (p<0.05) and 92 had a log₂FC greater than 0.2(56 downregulated and 36 upregulated). 13 PR genes and 1 AR gene were upregulated whereas 12 PR and 16 AR genes were downregulated. We could not assign either function to 50 genes. Upregulated genes included those related to positive regulation of angiogenesis (Cdh13, Col22a1), neuronal tissue development (Cobl, Map3k7cl, Chodl, Npy), skeletal muscle development (Synpo2l, Hoxc11), positive regulation of cell cycle progression (Cdc25b, Pbx1), increasing skeletal muscle fatigue resistance (Car3) and negative regulation of leukocyte activation (Lgals5). Downregulated genes included those involved in positive regulation of cytokine production (Cybb, Cyba, LOC498276), leukocyte activation/proliferation (Fcer1g, Csf1r, Csf2ra, Crlf2, RT1-DMa, RT1-DMb, Ccr2, Slfn2), negative regulation of angiogenesis (Klf4), cell death/killing (Ctsc, Ctsl, Fcgr1a) and negative regulation of cell cycle progression (Fam107a). CONCLUSION: In rats, mechanical stimulation improves functional recovery after skeletal muscle injury by altering expression of genes involved in angiogenesis, cell cycle progression, inflammation, muscle development and cell killing. Genetic analysis in large animal and human studies is needed to further explore these findings.