On the Rate of Synthesis of Individual Proteins within and between Different Striated Muscles of the Rat

Abstract

The turnover of muscle protein is responsive to different (patho)-physiological conditions but little is known about the rate of synthesis at the level of individual proteins or whether this varies between different muscles. We investigated the synthesis rate of eight proteins (actin, albumin, ATP synthase alpha, beta enolase, creatine kinase, myosin essential light chain, myosin regulatory light chain and tropomyosin) in the extensor digitorum longus, diaphragm, heart and soleus of male Wistar rats (352 ± 30 g body weight). Animals were assigned to four groups (n = 3, in each), including a control and groups that received deuterium oxide (2H2O) for 4 days, 7 days or 14 days. Deuterium labelling was initiated by an intraperitoneal injection of 10 μL/g body weight of 99.9% 2H2O-saline, and was maintained by administration of 5% (v/v) 2H2O in drinking water provided ad libitum. Homogenates of the isolated muscles were analysed by 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionisation time of flight mass spectrometry. Proteins were identified against the SwissProt database using peptide mass fingerprinting. For each of the eight proteins investigated, the molar percent enrichment (MPE) of 2H and rate constant (k) of protein synthesis was calculated from the mass isotopomer distribution of peptides based on the amino acid sequence and predicted number of exchangeable C–H bonds. The average MPE (2.14% ± 0.2%) was as expected and was consistent across muscles harvested at different times (i.e., steady state enrichment was achieved). The synthesis rate of individual proteins differed markedly within each muscle and the rank-order of synthesis rates differed among the muscles studied. After 14 days the fraction of albumin synthesised (23% ± 5%) was significantly (p < 0.05) greater than for other muscle proteins. These data represent the first attempt to study the synthesis rates of individual proteins across a number of different striated muscles.

Endurance-Type Exercise Increases Bulk and Individual Mitochondrial Protein Synthesis Rates in Rats

“…Furthermore, several proteins within the TCA cycle and electron transport system demonstrated 35-140% higher protein synthesis rates in the exercise group when compared with the sedentary group ( Figure 4A). Increased synthesis rates of these proteins, and ATP synthase in particular, likely contribute to the increase in muscle oxidative capacity observed over more prolonged endurance-type exercise training (Burniston & Hoffman, 2011;Hesketh et al, 2016a;Holloszy et al, 1970). However, we did not detect significantly greater abundances of these proteins in the present study, which is likely due to low subject number or the mere 2-week exercise intervention.…”

“…Even more detailed insight into individual protein translational responses has been obtained by performing 2-dimensional gel electrophoresis before measuring labeled amino acid incorporation (Balagopal et al, 1997a;Balagopal et al, 1994). Using this approach, synthesis rates of key myofibrillar (Balagopal et al, 1997b;Balagopal et al, 1997a;Hasten et al, 1998) and several mitochondrial proteins (Hesketh et al, 2016b;Jaleel et al, 2008) have been determined. However, the separation technique is labor intensive (13-15 h for one sample), technically challenging, and label quantification can require relatively large tissue samples.…”

“…The endogenous labeling of nearly all (non-essential) amino acids increases total label incorporation into newly synthesized proteins, which improves analytical detectability. Recently, investigators have combined deuterated water administration with high-throughput analytical techniques of liquid chromatography-mass spectrometry to determine deuterium enrichment in hundreds of tissue-derived peptides (Hesketh et al, 2016b;Kasumov et al, 2011;Price et al, 2012;Shankaran et al, 2016b;Wang et al, 2014;Zangarelli et al, 2006). Based upon shifts in mass spectra over time, the synthesis rates of several proteins may be assessed simultaneously (Price et al, 2012;Shankaran et al, 2016a).…”

Dynamic proteome profiling of individual proteins in human skeletal muscle after a high‐fat diet and resistance exercise

“…9) has not previously been captured and is a unique aspect of the current investigation. New techniques have recently been developed that incorporate D 2 O labeling with proteomic analyses, including peptide mass spectrometry, which measures the rate constant (k) for synthesis on a protein-by-protein basis in the muscle of animals (33)(34)(35)(36) and humans (20). Collectively, these works and our current data (Fig.…”

Fractional Synthesis Rates of Individual Proteins in Rat Soleus and Plantaris Muscles

“…Infusion of stable isotope-labelled amino acids constrains studies to a relatively short duration (e.g., min-h timescales), whereas the turnover of abundant tissue proteins in vivo occurs over a period of days. Longer-term labelling studies have been conducted in animals fed a diet enriched with a stable-isotope-labelled amino acid [13,17], whereas we have focused on the application of deuterium oxide (D 2 O or 2 H 2 O) or "heavy water" [14,[18][19][20][21]. D 2 O can be administered via the drinking water in free-living animals and the majority of amino acids are labelled intracellularly via transamination reactions [22].…”