We sought to determine the skeletal muscle genome-wide DNA methylation and mRNA responses to one bout of lower load (LL) versus higher load (HL) resistance exercise. Trained college-aged males (n = 11, 23 ± 4 years old, 4 ± 3 years self-reported training) performed LL or HL bouts to failure separated by one week. The HL bout (i.e., 80 Fail) consisted of four sets of back squats and four sets of leg extensions to failure using 80% of participants estimated one-repetition maximum (i.e., est. 1-RM). The LL bout (i.e., 30 Fail) implemented the same paradigm with 30% of est. 1-RM. Vastus lateralis muscle biopsies were collected before, 3 h, and 6 h after each bout. Muscle DNA and RNA were batch-isolated and analyzed using the 850k Illumina MethylationEPIC array and Clariom S mRNA microarray, respectively. Performed repetitions were significantly greater during the 30 Fail versus 80 Fail (p < 0.001), although total training volume (sets × reps × load) was not significantly different between bouts (p = 0.571). Regardless of bout, more CpG site methylation changes were observed at 3 h versus 6 h post exercise (239,951 versus 12,419, respectively; p < 0.01), and nuclear global ten-eleven translocation (TET) activity, but not global DNA methyltransferase activity, increased 3 h and 6 h following exercise regardless of bout. The percentage of genes significantly altered at the mRNA level that demonstrated opposite DNA methylation patterns was greater 3 h versus 6 h following exercise (~75% versus ~15%, respectively). Moreover, high percentages of genes that were up- or downregulated 6 h following exercise also demonstrated significantly inversed DNA methylation patterns across one or more CpG sites 3 h following exercise (65% and 82%, respectively). While 30 Fail decreased DNA methylation across various promoter regions versus 80 Fail, transcriptome-wide mRNA and bioinformatics indicated that gene expression signatures were largely similar between bouts. Bioinformatics overlay of DNA methylation and mRNA expression data indicated that genes related to “Focal adhesion,” “MAPK signaling,” and “PI3K-Akt signaling” were significantly affected at the 3 h and 6 h time points, and again this was regardless of bout. In conclusion, extensive molecular profiling suggests that post-exercise alterations in the skeletal muscle DNA methylome and mRNA transcriptome elicited by LL and HL training bouts to failure are largely similar, and this could be related to equal volumes performed between bouts.

Increasing dietary nitrate (NO3-) through beetroot juice (BRJ) supplementation elicits acute ergogenic benefits. However, it is unknown whether chronic NO3- supplementation can enhance resistance training (RT) adaptations in middle-aged and older individuals. Therefore, we sought to determine whether 12 weeks of combined RT and NO3- supplementation enhanced hypertrophic, vascular, strength, and skeletal muscle angiogenesis adaptations in this population. Twenty-eight apparently healthy, untrained men and women (56±7 years old, 29.1±5.3 kg/m2 body mass index) completed 12 weeks of supervised full-body RT (2x/week) while ingesting either BRJ (140 mL daily, providing 800 mg NO3-; n=14 with 7M/7W) or NO3--depleted BRJ placebo (PLA; n=14 with 7M/7W). Participants underwent a whole-body dual-energy x-ray absorptiometry scan, right mid-thigh ultrasonography for muscle imaging, right leg popliteal artery flow-mediated dilation (FMD) assessments, a biopsy of the right mid-thigh vastus lateralis, and strength testing prior to and following the 12-week intervention. Biopsy analyses included a NO3-/nitrite (NOx) fluorometric assay, immunoblotting for proteins involved in angiogenesis, and immunohistochemistry to quantify fiber type-specific capillaries and cross-sectional areas. Muscle NOx values did not significantly change: +15.4% in BRJ (p=0.073) and +7.8% (p=0.514) in PLA. Both groups significantly improved measures related to muscle hypertrophy, strength, and FMD. However, no significant group × time interactions were observed for whole-body lean mass, mid-thigh muscle cross-sectional area, popliteal artery FMD outcomes, or histological or molecular markers. In conclusion, BRJ supplementation does not enhance RT adaptations in middle-aged and older adults.

Background/Objectives: Creatine monohydrate (CRE) is a popular nutritional supplement that increases lean/muscle mass accretion. Although data regarding CRE and its effects on muscle protein synthesis are mixed, we hypothesized that CRE may potentiate/extend the anabolic response to essential amino acids given that CRE acts as a high-energy phosphate buffer to potentially amplify anabolic signaling. Therefore, we used an in vitro model to determine whether CRE synergistically enhances myotube protein synthesis and the anabolic signaling responses to EAA-rich whey protein (WP). Methods: C₂C₁₂ murine myotubes were treated with control media containing PBS (CTL), WP serum (5 mg/mL), CRE (10 mM), or WP + CRE. Myotubes were collected following 1, 4, and 24 h treatments (n = 6 replicates per treatment and time point) and assayed for relative creatine levels, myotube protein synthesis levels, and phosphorylation markers. Results: Cellular creatine levels were greater in CRE and WP + CRE versus CTL and WP at all treatment time points (p < 0.05). The protein synthesis levels with 4 hr treatments with WP and WP + CRE were greater compared to the CTL (p = 0.036 and p < 0.001, respectively), and 24 h levels were greater with WP versus other treatments (p < 0.05). p-p70S6K (Ser389) and p-rpS6 (Ser235/236) were greater with WP at 1 h compared to all other treatments (p < 0.05). No effects across time points were observed for p-mTOR (Ser2448), p-4E-BP1 (Thr37/46), or p-AMPKα (Thr172). Conclusions: WP increases protein synthesis and anabolic signaling with no additive effect from CRE. However, given that myotubes were not stressed nor stimulated to contract, such models are needed with the current treatment schematic to examine potential interactions.

Recent enthusiasm has surrounded the homeostatic roles that polyamines have in a variety of cell types. Thus, the purpose of this exploratory in vitro study was to determine how spermidine (SPD), a polyamine commonly consumed as a nutritional supplement, affected general markers of cellular health and function in human primary epidermal keratinocytes. Commercial HEKa cells were seeded onto either six-well (transcriptomics and immunoblotting) or 96-well culture plates (viability, ATP, and JC-1 assays) and cultured to ~90+% confluency through complete growth media (CGM) changes every 48 h. Once cells reached this level of growth, treatments included either CGM + phosphate-buffered saline (PBS control, or CTL), CGM + 1 µM SPD, and CGM + 10 µM SPD for either 6 or 24 h depending upon the outcome being assessed. Cellular ATP levels were not significantly affected by 1 µM or 10 µM SPD treatments lasting 24 h. However, cell counts were 9% greater (p = 0.007) when comparing 24 h 10 µM versus CTL treatments indicating increased cell viability. Transcriptomic analyses indicated that 6 h treatments with 10 µM SPD significantly altered 162 transcripts versus non-treated CTL cells (65 up-regulated and 97 down-regulated, p < 0.01). Four pathways were predicted to be enriched based on differential gene expression including protein deubiquitination (GO:0016579), membrane lipid biosynthesis (GO:0046467), DNA metabolic process (GO:0006259), and cell cycle process (GO:0022402). Additionally, the HR gene (essential for keratinocyte hair follicle formation) was significantly up-regulated at the mRNA level with 6 h 10 µM SPD, and immunoblotting confirmed a 96% increase in protein levels with 24 h 10 µM SPD treatments, albeit this did not reach statistical significance (p = 0.102). Pan-keratin protein content was also 60% greater in the 1 µM and 10 µM 24 h treatments than CTL (p ≤ 0.029). Finally, although select markers of mitochondrial content and biogenesis were not significantly altered with 6 h and 24 h treatments, mitochondrial membrane potential (an aspect of mitochondrial function) was 84% greater with 24 h 1 µM versus CTL (p < 0.001). In conclusion, these preliminary screening experiments in unperturbed human keratinocytes suggest that exogenous SPD positively affects various aspects of homeostasis by stimulating transcriptomic and functional alterations (e.g., increased cell viability and enhanced keratinocyte protein levels).