Utilizing Stable Isotope Labeling to Measure Changes in Global, Ontology-Grouped, and Individual Protein Flux Rates in Skeletal Muscle

Proteins are the ultimate focal point for organismal composition and the basis for many biological functions that occur within. It is well known that proteins in a biological system are not static; there is flux through synthesis and degradation pathways, resulting in a dynamic steady-state that underlies both stable and adaptive levels of proteins. For many biological functions to occur, the rate of either protein synthesis or degradation deviates from normal to allow for changes to occur .occur. Accordingly, when protein pool size adjusts to new levels a kinetic signature in the form of a fraction of newly synthesized proteins may be present at each stage. In the Hellerstein Lab, we utilize an innovative methodology and approach to measure this signature directly in- vivo with stable isotope labeling.The approach used in this study to measure protein dynamics in- vivo involves administering deuterium (2H) from deuterated “heavy” water (2H2O) to research subjects or experimental animals. During the labeling period, newly synthesized proteins will incorporate the available 2H into amino acids in newly synthesized protein molecules, resulting in “heavy” proteins. These are functionally identical to their non- 2H-incorporated “light” counterparts, and only differ in subtle alterations in the pattern and amount of deuterium atoms in the constituent amino acids of which a peptide chain is composed. Protein mass isotopomers are then quantified through analysis by high performance high-performance liquidliquid chromatography (HPLC) coupled single (MS) and tandem (MS/MS) mass spectrometry and protein flux rates are calculated by use of Mass Isotopomer Distribution Analysis (MIDA). With this approach, we investigated here non-invasive biomarkers of Duchenne muscular dystrophy (DMD) as well as the unique stages of muscle tissue regeneration and effects on tissues distant from acute injury. DMD is the most severe form of muscular dystrophy, with debilitating symptoms that cause patients to succumb to their complications early in life. Currently, there are no existing a few approved treatments for DMD, but these therapies mainly treat the symptoms of the disease and have shown mild improvements in a clinical population so far. and approved therapies have shown little promise in a clinical population. DMD is one of the most studied genetic diseases, as there is a pressing demand for proper treatment or therapy for this indication. Here, we have discovered and validated a clinical biomarker of whole muscle protein synthesis, which is a key marker of skeletal muscle health in DMD, from non-invasive methods. This marker was first discovered in a clinical population, but then reverse translated to an animal model of DMD to validate that it is a faithful marker of whole muscle protein synthesis rates. Our data clearly demonstrate that certain muscle- specific proteins can escape into circulation from the dystrophic muscle and be captured from urinary excretion to understand changes in whole musclewhole-muscle protein flux rates. These findings may provide crucial information to clinicians and researchers alike about a key metric that can determine disease progression or response to treatment/therapy. Both skeletal and smooth muscle tissue have the capacity to regenerate after injury or damage to help maintain optimal functional properties. This regenerative capacity is an important process, and its dysregulation is implicated in many diseases, such as muscular dystrophy and pathological atrophy. Muscle regeneration is a widely studied topic, but no one has utilized the method of in- vivo stable isotope labeling to categorize the global, ontology-groupedgrouped, and individual flux rates of proteins at each sequential unique stage of regeneration. In the work presented here, we examine how protein flux rates change over time as skeletal muscle tissue regenerates from an acute injury. Also, we explore the relationship between protein flux measurements and abundance-based gene expressions to understand their correlation. There is evidence to support the notion that other muscle tissue that is not directly injured will respond to systemic circulating factors from acute tissue injury and enter an altered functional state. Therefore, we also explore the effect that acute local injury has on distant muscle tissue that is undamaged. Our data show clear changes in global, ontology-grouped, and individual protein flux changes at each unique stage of muscle regeneration, and that there is no correlation between measured protein flux changes in regenerating muscle and gene expression of the same proteins. Interestingly, changes are further demonstrated in global, ontology-grouped, and individual protein fluxes in muscle that are distant to local acute injury, which don’t do not reflect the same tissue gene expression rates. This data provides further evidence that other muscle tissue that is not directly injured will respond to systemic circulating factors from acute tissue injury. Therefore, setting up future experiments to deepen our understanding of the systemic communication network that our body utilizes during injury or regenerative events.

Advisor: Hellerstein, Marc K