Changes in Protein Fluxes in Skeletal Muscle During Sequential Stages of Muscle Regeneration After Acute Injury in Male Mice

Background Changes in protein turnover play an important role in dynamic physiological processes, including skeletal muscle regeneration, which occurs as an essential part of tissue repair after injury. The inability of muscle tissue to recapitulate this regenerative process can lead to pathology and clinical symptoms in various musculoskeletal diseases, including muscular dystrophies and pathological atrophy. Methods Here, we employed a workflow that couples deuterated water (2H2O) administration with tandem mass spectrometry (MS) to systematically measure in-vivo protein turnover rates across the muscle proteome in 8-week-old male C57BL6/J mice. We compared the turnover kinetics of over 100 proteins in response to cardiotoxin (CTX) induced muscle damage and regeneration at unique sequential stages along the regeneration timeline. This analysis is compared to gene expression data from mRNA-sequencing (mRNA-seq) from the same tissue. Results The data reveals quantitative protein flux signatures in response to necrotic damage, in addition to sequential differences in cell proliferation, energy metabolism, and contractile gene expression. Interestingly, the mRNA changes correlated poorly with changes in protein synthesis rates, consistent with post-transcriptional control mechanisms. Conclusions In summary, the experiments described here reveal the signatures and timing of protein flux changes during skeletal muscle regeneration, as well as the inability of mRNA expression measurements to reveal changes in directly measured protein turnover rates. The results of this work described here provide a better understanding of the muscle regeneration process and could help to identify potential biomarkers or therapeutic targets. Overall design: Mice were randomly assigned to a group (n=5 per group) that reflected the stage of muscle regeneration being measured (Proliferation Stage, Differentiation Stage, Maturation Stage, or Uninjured Control). Cardiotoxin (CTX)-treated groups received a dose of 50 µL 0.1mg/mL CTX in sterile PBS/0.2% Meloxicam in the form of an intramuscular injection in the Tibialis Anterior (TA) muscle to one limb while the TA from the other limb was not injected. The day of CTX injection is called Day 0, and subsequent days allow for the toxin-induced muscle regeneration to occur. One group of mice did not receive CTX injection to serve as uninjured controls alongside experimental groups.