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.

Although transcriptome profiling has been used in several resistance training studies, the associated analytical approaches seldom provide in-depth information on individual genes linked to skeletal muscle hypertrophy. Therefore, a secondary analysis was performed herein on a muscle transcriptomic dataset we previously published involving trained college-aged men (n = 11) performing two resistance exercise bouts in a randomized and crossover fashion. The lower-load bout (30 Fail) consisted of 8 sets of lower body exercises to volitional fatigue using 30% one-repetition maximum (1 RM) loads, whereas the higher-load bout (80 Fail) consisted of the same exercises using 80% 1 RM loads. Vastus lateralis muscle biopsies were collected prior to (PRE), 3 h, and 6 h after each exercise bout, and 58 genes associated with skeletal muscle hypertrophy were manually interrogated from our prior microarray data. Select targets were further interrogated for associated protein expression and phosphorylation induced-signaling events. Although none of the 58 gene targets demonstrated significant bout x time interactions, ~57% (32 genes) showed a significant main effect of time from PRE to 3 h (15↑ and 17↓, p < 0.01), and ~26% (17 genes) showed a significant main effect of time from PRE to 6 h (8↑ and 9↓, p < 0.01). Notably, genes associated with the myostatin (9 genes) and mammalian target of rapamycin complex 1 (mTORC1) (9 genes) signaling pathways were most represented. Compared to mTORC1 signaling mRNAs, more MSTN signaling-related mRNAs (7 of 9) were altered post-exercise, regardless of the bout, and RHEB was the only mTORC1-associated mRNA that was upregulated following exercise. Phosphorylated (phospho-) p70S6K (Thr389) (p = 0.001; PRE to 3 h) and follistatin protein levels (p = 0.021; PRE to 6 h) increased post-exercise, regardless of the bout, whereas phospho-AKT (Thr389), phospho-mTOR (Ser2448), and myostatin protein levels remained unaltered. These data continue to suggest that performing resistance exercise to volitional fatigue, regardless of load selection, elicits similar transient mRNA and signaling responses in skeletal muscle. Moreover, these data provide further evidence that the transcriptional regulation of myostatin signaling is an involved mechanism in response to resistance exercise.

We examined how set-volume equated resistance training using either the back squat (SQ) or hip thrust (HT) affected hypertrophy and various strength outcomes. Untrained college-aged participants were randomized into HT (n = 18) or SQ (n = 16) groups. Surface electromyograms (sEMG) from the right gluteus maximus and medius muscles were obtained during the first training session. Participants completed 9 weeks of supervised training (15–17 sessions), before and after which gluteus and leg muscle cross-sectional area (mCSA) was assessed via magnetic resonance imaging. Strength was also assessed prior to and after the training intervention via three-repetition maximum (3RM) testing and an isometric wall push test. Gluteus mCSA increases were similar across both groups. Specifically, estimates [(−) favors HT (+) favors SQ] modestly favored the HT versus SQ for lower [effect ±SE, −1.6 ± 2.1 cm²; CI_95% (−6.1, 2.0)], mid [−0.5 ± 1.7 cm²; CI_95% (−4.0, 2.6)], and upper [−0.5 ± 2.6 cm²; CI_95% (−5.8, 4.1)] gluteal mCSAs but with appreciable variance. Gluteus medius + minimus [−1.8 ± 1.5 cm²; CI_95% (−4.6, 1.4)] and hamstrings [0.1 ± 0.6 cm²; CI_95% (−0.9, 1.4)] mCSA demonstrated little to no growth with small differences between groups. mCSA changes were greater in SQ for the quadriceps [3.6 ± 1.5 cm²; CI_95% (0.7, 6.4)] and adductors [2.5 ± 0.7 cm²; CI_95% (1.2, 3.9)]. Squat 3RM increases favored SQ [14 ± 2 kg; CI_95% (9, 18),] and hip thrust 3RM favored HT [−26 ± 5 kg; CI_95% (−34, −16)]. 3RM deadlift [0 ± 2 kg; CI_95% (−4, 3)] and wall push strength [−7 ± 12N; CI_95% (−32, 17)] similarly improved. All measured gluteal sites showed greater mean sEMG amplitudes during the first bout hip thrust versus squat set, but this did not consistently predict gluteal hypertrophy outcomes. Squat and hip thrust training elicited similar gluteal hypertrophy, greater thigh hypertrophy in SQ, strength increases that favored exercise allocation, and similar deadlift and wall push strength increases.

As a follow-up to our original investigation by Michel JM et al. J Appl Physiol (1985) 138: 450–467, 2025, we sought to examine how limb immobilization and recovery resistance training affect molecular outcomes related to angiogenesis in younger adults with or without a prior training history. Regardless of resistance training history, the molecular responses are largely similar between participant cohorts and are suggestive of a reduced (PRE-MID) and increased (MID-POST) angiogenic response, with disuse and subsequent recovery resistance training.

Results from this study suggest that 12 wk of dietary nitrate supplementation does not significantly alter skeletal muscle hypertrophic, strength, or vascular outcomes in previously untrained middle-aged and older adults who performed resistance training.

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.