Changes in protein fluxes and gene expression in non-injured muscle tissue distant from an acute myotoxic injury in male mice.

The response to acute myotoxic injury requires stimulation of local repair mechanisms in the damaged tissue. However, satellite cells in muscle distant from acute injury have been reported to enter a functional state between quiescence and active proliferation. Here, we asked whether protein flux rates are altered in muscle distant from acute local myotoxic injury and how they compare to changes in gene expression from the same tissue. Broad and significant alterations in protein turnover were observed across the proteome in the limb contralateral to injury during the first 10 days after. Interestingly, mRNA changes had almost no correlation with directly measured protein turnover rates. In summary, we show consistent and striking changes in protein flux rates in muscle tissue contralateral to myotoxic injury, with no correlation between changes in mRNA levels and protein synthesis rates. This work motivates further investigation of the mechanisms, including potential neurological factors, responsible for this distant effect. KEY POINTS: Previous literature demonstrates that stem cells of uninjured muscle respond to local necrotic muscle tissue damage and regeneration. We show that muscle tissue that was distant from a model of local necrotic damage had functional changes at both the gene expression and the protein turnover level. However, these changes in distant tissue were more pronounced during the earlier stages of tissue regeneration and did not correlate well with each other. The results suggest communication between directly injured tissue and non-affected tissues that are distant from injury, which warrants further investigation into the potential of this mechanism as a proactive measure for tissue regeneration from damage.

Changes in protein fluxes in skeletal muscle during sequential stages of muscle regeneration after acute injury in male mice.

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 the manifestation of clinical symptoms in various musculoskeletal diseases, including muscular dystrophies and pathological atrophy. Here, we employed a workflow that couples deuterated water (2H2O) administration with 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. 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. 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.

Effects of Testosterone on Mixed-Muscle Protein Synthesis and Proteome Dynamics During Energy Deficit.

CONTEXT: Effects of testosterone on integrated muscle protein metabolism and muscle mass during energy deficit are undetermined. OBJECTIVE: The objective was to determine the effects of testosterone on mixed-muscle protein synthesis (MPS), proteome-wide fractional synthesis rates (FSR), and skeletal muscle mass during energy deficit. DESIGN: This was a randomized, double-blind, placebo-controlled trial. SETTING: The study was conducted at Pennington Biomedical Research Center. PARTICIPANTS: Fifty healthy men. INTERVENTION: The study consisted of 14 days of weight maintenance, followed by a 28-day 55% energy deficit with 200 mg testosterone enanthate (TEST, n = 24) or placebo (PLA, n = 26) weekly, and up to 42 days of ad libitum recovery feeding. MAIN OUTCOME MEASURES: Mixed-MPS and proteome-wide FSR before (Pre), during (Mid), and after (Post) the energy deficit were determined using heavy water (days 1-42) and muscle biopsies. Muscle mass was determined using the D3-creatine dilution method. RESULTS: Mixed-MPS was lower than Pre at Mid and Post (P < 0.0005), with no difference between TEST and PLA. The proportion of individual proteins with numerically higher FSR in TEST than PLA was significant by 2-tailed binomial test at Post (52/67; P < 0.05), but not Mid (32/67; P > 0.05). Muscle mass was unchanged during energy deficit but was greater in TEST than PLA during recovery (P < 0.05). CONCLUSIONS: The high proportion of individual proteins with greater FSR in TEST than PLA at Post suggests exogenous testosterone exerted a delayed but broad stimulatory effect on synthesis rates across the muscle proteome during energy deficit, resulting in muscle mass accretion during subsequent recovery.

Profoundly lower muscle mass and rate of contractile protein synthesis in boys with Duchenne muscular dystrophy.

Boys with Duchenne muscular dystrophy (DMD) experience a progressive loss of functional muscle mass, with fibrosis and lipid accumulation. Accurate evaluation of whole-body functional muscle mass (MM) in DMD patients has not previously been possible and the rate of synthesis of muscle proteins remains unexplored. We used non-invasive, stable isotope-based methods from plasma and urine to measure the fractional rate of muscle protein synthesis (FSR) functional muscle mass (MM), and fat free mass (FFM) in 10 DMD (6-17 years) and 9 age-matched healthy subjects. An oral dose of D3 creatine in 70% 2 H2 O was administered to determine MM and FFM followed by daily 70% 2 H2 O to measure protein FSR. Functional MM was profoundly reduced in DMD subjects compared to controls (17% vs. 41% of body weight, P < 0.0001), particularly in older, non-ambulant patients in whom functional MM was extraordinarily low (<13% body weight). We explored the urine proteome to measure FSR of skeletal muscle-derived proteins. Titin, myosin light chain and gelsolin FSRs were substantially lower in DMD subjects compared to controls (27%, 11% and 40% of control, respectively, P < 0.0001) and were strongly correlated. There were no differences in muscle-derived sarcoplasmic proteins FSRs (creatine kinase M-type and carbonic anhydrase-3) measured in plasma. These data demonstrate that both functional MM, body composition and muscle protein synthesis rates can be quantified non-invasively and are markedly different between DMD and control subjects and suggest that the rate of contractile but not sarcoplasmic protein synthesis is affected by a lack of dystrophin. KEY POINTS: Duchenne muscular dystrophy (DMD) results in a progressive loss of functional skeletal muscle but total body functional muscle mass or rates of muscle protein synthesis have not previously been assessed in these patients. D3 -creatine dilution was used to measure total functional muscle mass and oral 2 H2 O was used to examine the rates of muscle protein synthesis non-invasively in boys with DMD and healthy controls using urine samples. Muscle mass was profoundly lower in DMD compared to control subjects, particularly in older, non-ambulant patients. The rates of contractile protein synthesis but not sarcoplasmic proteins were substantially lower in DMD. These results may provide non-invasive biomarkers for disease progression and therapeutic efficacy in DMD and other neuromuscular diseases.