Muscle-specific RING finger-1 (MuRF-1), a ubiquitin ligase and key regulator of proteasome-dependent protein degradation, is highly expressed during skeletal muscle atrophy. The transcription factor forkhead box O3 (FoxO3) induces MuRF-1 expression, but the direct role of other major atrophy-related transcription factors, such as SMAD3, is largely unknown. The goal of this study was to determine whether SMAD3 individually regulates, or with FoxO3 coordinately regulates, MuRF-1 expression. In cultured myotubes or human embryonic kidney cells, MuRF-1 mRNA content and promoter activity were increased by FoxO3 but not by SMAD3 overexpression. However, FoxO3 and SMAD3 coexpression synergistically increased MuRF-1 mRNA and promoter activity. Mutation of the SMAD-binding element (SBE) in the proximal MuRF-1 promoter or overexpression of a SMAD3 DNA-binding mutant attenuated FoxO3-dependent MuRF-1 promoter activation, showing that SMAD binding to DNA is required for optimal activation of FoxO3-induced transcription of MuRF-1. Using chromatin immunoprecipitation, SMAD3 DNA binding increased FoxO3 abundance and SBE mutation reduced FoxO3 abundance on the MuRF-1 promoter. Furthermore, SMAD3 overexpression dose-dependently increased FoxO3 protein content, and coexpression of FoxO3 and SMAD3 synergistically increased FoxO-dependent gene transcription [assessed with a FoxO response element (FRE)-driven reporter]. Collectively, these results show that SMAD3 regulates transcription of MuRF-1 by increasing FoxO3 binding at a conserved FRE-SBE motif within the proximal promoter region, and by increasing FoxO3 protein content and transcriptional activity. These data are the first to indicate that two major transcription factors regulating protein degradation, FoxO3 and SMAD3, converge to coordinately and directly regulate transcription of MuRF-1.

Objective: Whole‐body protein metabolism is dysregulated with obesity. The goal of the study was to determine whether activity and expression of major protein degradation pathways are compromised specifically in human skeletal muscle with obesity.Methods: Primary human skeletal muscle (HSkM) cell cultures were utilized since cellular mechanisms can be studied absent of hormones and contractile activity that could independently influence metabolism. HSkM from 10 lean women (BMI ≤ 26.0 kg/m²) and 8 women with severe obesity (BMI ≥ 39.0) were examined basally and when stimulated to atrophy (serum and amino acid starvation).Results: HSkM from obese donors had a lower proportion of type I myosin heavy chain and slower flux through the autophagic/lysosomal pathway. During starvation, flux through the ubiquitin‐proteasome system diverged according to obesity status, with a decrease in lean subjects and an increase in HSkM from subjects with obesity. HSkM in obesity also displayed elevated proteasome activity despite no difference in proteasome content. Atrophy‐related gene expression and myotube area were similar in myotubes derived from individuals with and without obesity under basal and starved conditions.Conclusions: Our data indicate that muscle cells in lean individuals and in those with severe obesity have innate differences in management of protein degradation, which may explain their metabolic differences.

Bollinger, LM, Brantley, JT, Tarlton, JK, Baker, PA, Seay, RF, and Abel, MG. Construct validity, test-retest reliability, and repeatability of performance variables using a flywheel resistance training device. J Strength Cond Res 34(11): 3149–3156, 2020—Power production is highly associated with physical performance; however, the ability to quantitatively measure power output during resistance exercise is lacking. The purpose of this study was to determine the validity and test-retest reliability of flywheel-based performance testing. Twelve young, resistance trained subjects completed 2 bouts of resistance exercise using a flywheel resistance training device (Exxentric kbox 4 Pro). Each session consisted of 3 sets of 3 exercise (bent-over row, Romanian deadlift, and biceps curl) with varying moments of inertia (0.050, 0.075, and 0.100 kg·m², respectively) in random order. Each set consisted of 5 maximal effort repetitions with 3-minute recovery between sets. Average power, peak concentric and eccentric power, average force, average speed, and total work for each set were recorded. Regression analysis revealed a near-perfect relationship between measured and predicted power, force, and work at given workloads. Pearson's r between trials 1 and 2 revealed good (≥0.70) to excellent (≥0.90) test-retest reliability for all outcomes with the exception of peak eccentric power for biceps curls (r = 0.69), which narrowly missed the cutoff for acceptable reliability. Bland-Altman plots revealed small (approximately 5–15%), but statistically significant bias between the 2 trials for some measures. Coefficient of repeatability for all outcomes was relatively high, indicating poor repeatability. Flywheel-based performance testing provides valid data. However, reliability varies between individual lifts and specific outcomes. Given the poor repeatability between trials, it is likely that subjects who are unaccustomed to this modality may require multiple testing sessions or a thorough familiarization period to ensure accurate measures of power, force, speed, and work during flywheel-based performance testing.

A short-term high-fat diet impairs mitochondrial function and the ability of skeletal muscle to respond to growth stimuli, but it is unknown whether such a diet alters the ability to respond to atrophy signals. The purpose of this study was to determine whether rapid weigh gain induced by a high-fat (HF) diet accelerates denervation-induced muscle atrophy. Adult, male mice (C57BL/6) were fed a control or HF (60 % calories as fat) diet for 3 weeks (3wHF). Sciatic nerve was sectioned unilaterally for the final 5 or 14 days of the diet. Soleus and extensor digitorum longus (EDL) muscles were removed and incubated in vitro to determine rates of protein degradation and subsequently homogenized for determination of protein levels of LC3, ubiquitination, myosin heavy chain (MHC) distribution, and mitochondrial subunits. When mice were fed the 3wHF diet, whole-body fat mass more than doubled, but basal (innervated) muscle weights, rates of protein degradation, LC3 content, mitochondrial protein content, and myosin isoform distribution were not significantly different than with the control diet in either soleus or EDL. However in the 14 day denervated soleus, the 3wHF diet significantly augmented loss of mass, proteolysis rate, amount of the autophagosome marker LC3 II, and the amount of overall ubiquitination as compared to the control fed mice. On the contrary, the 3wHF diet had no significant effect in the EDL on amount of mass loss, proteolysis rate, LC3 levels, or ubiquitination. Fourteen days denervation also induced a loss of mitochondrial proteins in the soleus but not the EDL, regardless of the diet. Taken together, a short-term, high-fat diet augments denervation muscle atrophy by induction of protein degradation in the mitochondria-rich soleus but not in the glycolytic EDL. These findings suggest that the denervation-induced loss of mitochondria and HF diet-induced impairment of mitochondrial function may combine to promote skeletal muscle atrophy.