Increased biological relevance of transcriptome analyses in human skeletal muscle using a model-specific pipeline

Abstract Background: Human skeletal muscle responds to weight-bearing exercise with signifcant inter-individual diferences. Investigation of transcriptome responses could improve our understanding of this variation. However, this requires bioinformatic pipelines to be established and evaluated in study-specifc contexts. Skeletal muscle subjected to mechanical stress, such as through resistance training (RT), accumulates RNA due to increased ribosomal biogenesis. When a fxed amount of total-RNA is used for RNA-seq library preparations, mRNA counts are thus assessed in diferent amounts of tissue, potentially invalidating subsequent conclusions. The purpose of this study was to establish a bioinformatic pipeline specifc for analysis of RNA-seq data from skeletal muscles, to explore the efects of diferent normalization strategies and to identify genes responding to RT in a volume-dependent manner (moderate vs. low volume). To this end, we analyzed RNA-seq data derived from a twelve-week RT intervention, wherein 25 participants performed both low- and moderate-volume leg RT, allocated to the two legs in a randomized manner. Bilateral muscle biopsies were sampled from m. vastus lateralis before and after the intervention, as well as before and after the ffth training session (Week 2). Result: Bioinformatic tools were selected based on read quality, observed gene counts, methodological variation between paired observations, and correlations between mRNA abundance and protein expression of myosin heavy chain family proteins. Diferent normalization strategies were compared to account for global changes in RNA to tissue ratio. After accounting for the amounts of muscle tissue used in library preparation, global mRNA expression increased by 43–53%. At Week 2, this was accompanied by dose-dependent increases for 21 genes in rested-state muscle, most of which were related to the extracellular matrix. In contrast, at Week 12, no readily explainable dose-dependencies were observed. Instead, traditional normalization and non-normalized models resulted in counterintuitive reverse dose-dependency for many genes. Overall, training led to robust transcriptome changes, with the number of diferentially expressed genes ranging from 603 to 5110, varying with time point and normalization strategy. Conclusion: Optimized selection of bioinformatic tools increases the biological relevance of transcriptome analyses from resistance-trained skeletal muscle. Moreover,normalization procedures need to account for global changes in rRNA and mRNA abundance.publishedVersio

Systemic and muscular responses to effort-matched short intervals and long intervals in elite cyclists

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Scandinavian Journal of Medicine & Science In Sports published by John Wiley & Sons LtdThe purpose of this study was to compare the acute effects of time- and effort-matched high-intensity intervals on physiological, endocrine, and skeletal muscle molecular variables in elite cyclists. Eight elite cyclists performed short intervals (SI: 30-seconds) and long intervals (LI: 5-minutes) with work:recovery ratio 2:1, using a randomized crossover design. SI was associated with 14% ± 3% higher mean power output (SI; 421 ± 27 vs LI; 371 ± 22 W), and longer working time above 90% of maximal oxygen uptake (VO2max, 54% ± 76%) and 90% peak heart rate (HRpeak, 153% ± 148%) than LI (all P < .05), despite similar degrees of perceived exertion, blood lactate levels and muscle activation measured using EMG root mean square (EMG rms). In blood, SI was associated with more pronounced increases in testosterone and testosterone-tosex hormone-binding globulin (SHBG) ratios, as well as prolonged cortisol responses (P < .05). In skeletal muscle (m. Vastus lateralis), SI and LI led to similar changes in mRNA abundance for a range of transcripts, with the exception of NHE1 mRNA, which decreased after SI (P < .05). Overall, SI was associated with more pronounced physiological and endocrine responses than LI in elite cyclists, suggesting that such training might lead to superior adaptations in elite cyclists.publishedVersio

Chronic obstructive pulmonary disease does not impair responses to resistance training

publishedVersio

Vitamin D3 supplementation does not enhance the effects of resistance training in older adults

Background: Lifestyle therapy with resistance training is a potent measure to counteract age-related loss in muscle strength and mass. Unfortunately, many individuals fail to respond in the expected manner. This phenomenon is particularly common among older adults and those with chronic diseases (e.g. chronic obstructive pulmonary disease, COPD) and may involve endocrine variables such as vitamin D. At present, the effects of vitamin D supplementation on responses to resistance training remain largely unexplored. Methods: Ninety-five male and female participants (healthy, n = 71; COPD, n = 24; age 68 ± 5 years) were randomly assigned to receive either vitamin D3 or placebo supplementation for 28 weeks in a double-blinded manner (latitude 61°N, September-May). Seventy-eight participants completed the RCT, which was initiated by 12 weeks of supplementation-only (two weeks with 10 000 IU/day, followed by 2000 IU/day), followed by 13 weeks of combined supplementation (2000 IU/day) and supervised whole-body resistance training (twice weekly), interspersed with testing and measurements. Outcome measures included multiple assessments of muscle strength (nvariables = 7), endurance performance (n = 6), and muscle mass (n = 3, legs, primary), as well as muscle quality (legs), muscle biology (m. vastus lateralis; muscle fibre characteristics, transcriptome), and health-related variables (e.g. visceral fat mass and blood lipid profile). For main outcome domains such as muscle strength and muscle mass, weighted combined factors were calculated from the range of singular assessments. Results: Overall, 13 weeks of resistance training increased muscle strength (13% ± 8%), muscle mass (9% ± 8%), and endurance performance (one-legged, 23% ± 15%; whole-body, 8% ± 7%), assessed as weighted combined factors, and were associated with changes in health variables (e.g. visceral fat, -6% ± 21%; [LDL]serum , -4% ± 14%) and muscle tissue characteristics such as fibre type proportions (e.g. IIX, -3% points), myonuclei per fibre (30% ± 65%), total RNA/rRNA abundances (15%/6-19%), and transcriptome profiles (e.g. 312 differentially expressed genes). Vitamin D3 supplementation did not affect training-associated changes for any of the main outcome domains, despite robust increases in [25(OH)D]serum (∆49% vs. placebo). No conditional effects were observed for COPD vs. healthy or pre-RCT [25(OH)D]serum . In secondary analyses, vitamin D3 affected expression of gene sets involved in vascular functions in muscle tissue and strength gains in participants with high fat mass, which advocates further study. Conclusions: Vitamin D3 supplementation did not affect muscular responses to resistance training in older adults with or without COPD. Keywords: Cholecalciferol; Muscle plasticity; Strength training. © 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.publishedVersio

Increased biological relevance of transcriptome analyses in human skeletal muscle using a model-specific pipeline

Abstract Background: Human skeletal muscle responds to weight-bearing exercise with signifcant inter-individual diferences. Investigation of transcriptome responses could improve our understanding of this variation. However, this requires bioinformatic pipelines to be established and evaluated in study-specifc contexts. Skeletal muscle subjected to mechanical stress, such as through resistance training (RT), accumulates RNA due to increased ribosomal biogenesis. When a fxed amount of total-RNA is used for RNA-seq library preparations, mRNA counts are thus assessed in diferent amounts of tissue, potentially invalidating subsequent conclusions. The purpose of this study was to establish a bioinformatic pipeline specifc for analysis of RNA-seq data from skeletal muscles, to explore the efects of diferent normalization strategies and to identify genes responding to RT in a volume-dependent manner (moderate vs. low volume). To this end, we analyzed RNA-seq data derived from a twelve-week RT intervention, wherein 25 participants performed both low- and moderate-volume leg RT, allocated to the two legs in a randomized manner. Bilateral muscle biopsies were sampled from m. vastus lateralis before and after the intervention, as well as before and after the ffth training session (Week 2). Result: Bioinformatic tools were selected based on read quality, observed gene counts, methodological variation between paired observations, and correlations between mRNA abundance and protein expression of myosin heavy chain family proteins. Diferent normalization strategies were compared to account for global changes in RNA to tissue ratio. After accounting for the amounts of muscle tissue used in library preparation, global mRNA expression increased by 43–53%. At Week 2, this was accompanied by dose-dependent increases for 21 genes in rested-state muscle, most of which were related to the extracellular matrix. In contrast, at Week 12, no readily explainable dose-dependencies were observed. Instead, traditional normalization and non-normalized models resulted in counterintuitive reverse dose-dependency for many genes. Overall, training led to robust transcriptome changes, with the number of diferentially expressed genes ranging from 603 to 5110, varying with time point and normalization strategy. Conclusion: Optimized selection of bioinformatic tools increases the biological relevance of transcriptome analyses from resistance-trained skeletal muscle. Moreover,normalization procedures need to account for global changes in rRNA and mRNA abundance

Systemic and muscular responses to effort-matched short intervals and long intervals in elite cyclists

The purpose of this study was to compare the acute effects of time- and effort-matched high-intensity intervals on physiological, endocrine, and skeletal muscle molecular variables in elite cyclists. Eight elite cyclists performed short intervals (SI: 30-seconds) and long intervals (LI: 5-minutes) with work:recovery ratio 2:1, using a randomized crossover design. SI was associated with 14% ± 3% higher mean power output (SI; 421 ± 27 vs LI; 371 ± 22 W), and longer working time above 90% of maximal oxygen uptake (VO2max, 54% ± 76%) and 90% peak heart rate (HRpeak, 153% ± 148%) than LI (all P < .05), despite similar degrees of perceived exertion, blood lactate levels and muscle activation measured using EMG root mean square (EMG rms). In blood, SI was associated with more pronounced increases in testosterone and testosterone-tosex hormone-binding globulin (SHBG) ratios, as well as prolonged cortisol responses (P < .05). In skeletal muscle (m. Vastus lateralis), SI and LI led to similar changes in mRNA abundance for a range of transcripts, with the exception of NHE1 mRNA, which decreased after SI (P < .05). Overall, SI was associated with more pronounced physiological and endocrine responses than LI in elite cyclists, suggesting that such training might lead to superior adaptations in elite cyclists

Chronic obstructive pulmonary disease does not impair responses to resistance training

Background: Subjects with chronic obstructive pulmonary disease (COPD) are prone to accelerated decay of muscle strength and mass with advancing age. This is believed to be driven by disease-inherent systemic pathophysiologies, which are also assumed to drive muscle cells into a state of anabolic resistance, leading to impaired abilities to adapt to resistance exercise training. Currently, this phenomenon remains largely unstudied. In this study, we aimed to investigate the assumed negative effects of COPD for health- and muscle-related responsiveness to resistance training using a healthy control-based translational approach. Methods: Subjects with COPD (n = 20, GOLD II-III, FEV1predicted 57 ± 11%, age 69 ± 5) and healthy controls (Healthy, n = 58, FEV1predicted 112 ± 16%, age 67 ± 4) conducted identical whole-body resistance training interventions for 13 weeks, consisting of two weekly supervised training sessions. Leg exercises were performed unilaterally, with one leg conducting high-load training (10RM) and the contralateral leg conducting low-load training (30RM). Measurements included muscle strength (nvariables = 7), endurance performance (nvariables = 6), muscle mass (nvariables = 3), muscle quality, muscle biology (m. vastus lateralis; muscle fiber characteristics, RNA content including transcriptome) and health variables (body composition, blood). For core outcome domains, weighted combined factors were calculated from the range of singular assessments. Results: COPD displayed well-known pathophysiologies at baseline, including elevated levels of systemic low-grade inflammation ([c-reactive protein]serum), reduced muscle mass and functionality, and muscle biological aberrancies. Despite this, resistance training led to improved lower-limb muscle strength (15 ± 8%), muscle mass (7 ± 5%), muscle quality (8 ± 8%) and lower-limb/whole-body endurance performance (26 ± 12%/8 ± 9%) in COPD, resembling or exceeding responses in Healthy, measured in both relative and numeric change terms. Within the COPD cluster, lower FEV1predicted was associated with larger numeric and relative increases in muscle mass and superior relative improvements in maximal muscle strength. This was accompanied by similar changes in hallmarks of muscle biology such as rRNA-content↑, muscle fiber cross-sectional area↑, type IIX proportions↓, and changes in mRNA transcriptomics. Neither of the core outcome domains were differentially affected by resistance training load. Conclusions: COPD showed hitherto largely unrecognized responsiveness to resistance training, rejecting the notion of disease-related impairments and rather advocating such training as a potent measure to relieve pathophysiologies. Trial registration: ClinicalTrials.gov ID: NCT02598830. Registered November 6th 2015, https://clinicaltrials.gov/ct2/show/NCT02598830. Keywords: Anabolic resistance; COPD; Pathophysiology; Skeletal muscle; Strength training; Training load

Chronic obstructive pulmonary disease does not impair responses to resistance training

Background: Subjects with chronic obstructive pulmonary disease (COPD) are prone to accelerated decay of muscle strength and mass with advancing age. This is believed to be driven by disease-inherent systemic pathophysiologies, which are also assumed to drive muscle cells into a state of anabolic resistance, leading to impaired abilities to adapt to resistance exercise training. Currently, this phenomenon remains largely unstudied. In this study, we aimed to investigate the assumed negative effects of COPD for health- and muscle-related responsiveness to resistance training using a healthy control-based translational approach. Methods: Subjects with COPD (n = 20, GOLD II-III, FEV1predicted 57 ± 11%, age 69 ± 5) and healthy controls (Healthy, n = 58, FEV1predicted 112 ± 16%, age 67 ± 4) conducted identical whole-body resistance training interventions for 13 weeks, consisting of two weekly supervised training sessions. Leg exercises were performed unilaterally, with one leg conducting high-load training (10RM) and the contralateral leg conducting low-load training (30RM). Measurements included muscle strength (nvariables = 7), endurance performance (nvariables = 6), muscle mass (nvariables = 3), muscle quality, muscle biology (m. vastus lateralis; muscle fiber characteristics, RNA content including transcriptome) and health variables (body composition, blood). For core outcome domains, weighted combined factors were calculated from the range of singular assessments. Results: COPD displayed well-known pathophysiologies at baseline, including elevated levels of systemic low-grade inflammation ([c-reactive protein]serum), reduced muscle mass and functionality, and muscle biological aberrancies. Despite this, resistance training led to improved lower-limb muscle strength (15 ± 8%), muscle mass (7 ± 5%), muscle quality (8 ± 8%) and lower-limb/whole-body endurance performance (26 ± 12%/8 ± 9%) in COPD, resembling or exceeding responses in Healthy, measured in both relative and numeric change terms. Within the COPD cluster, lower FEV1predicted was associated with larger numeric and relative increases in muscle mass and superior relative improvements in maximal muscle strength. This was accompanied by similar changes in hallmarks of muscle biology such as rRNA-content↑, muscle fiber cross-sectional area↑, type IIX proportions↓, and changes in mRNA transcriptomics. Neither of the core outcome domains were differentially affected by resistance training load. Conclusions: COPD showed hitherto largely unrecognized responsiveness to resistance training, rejecting the notion of disease-related impairments and rather advocating such training as a potent measure to relieve pathophysiologies

Vitamin D3 supplementation does not enhance the effects of resistance training in older adults

Background: Lifestyle therapy with resistance training is a potent measure to counteract age-related loss in muscle strength and mass. Unfortunately, many individuals fail to respond in the expected manner. This phenomenon is particularly common among older adults and those with chronic diseases (e.g. chronic obstructive pulmonary disease, COPD) and may involve endocrine variables such as vitamin D. At present, the effects of vitamin D supplementation on responses to resistance training remain largely unexplored. Methods: Ninety-five male and female participants (healthy, n = 71; COPD, n = 24; age 68 ± 5 years) were randomly assigned to receive either vitamin D3 or placebo supplementation for 28 weeks in a double-blinded manner (latitude 61°N, September-May). Seventy-eight participants completed the RCT, which was initiated by 12 weeks of supplementation-only (two weeks with 10 000 IU/day, followed by 2000 IU/day), followed by 13 weeks of combined supplementation (2000 IU/day) and supervised whole-body resistance training (twice weekly), interspersed with testing and measurements. Outcome measures included multiple assessments of muscle strength (nvariables = 7), endurance performance (n = 6), and muscle mass (n = 3, legs, primary), as well as muscle quality (legs), muscle biology (m. vastus lateralis; muscle fibre characteristics, transcriptome), and health-related variables (e.g. visceral fat mass and blood lipid profile). For main outcome domains such as muscle strength and muscle mass, weighted combined factors were calculated from the range of singular assessments. Results: Overall, 13 weeks of resistance training increased muscle strength (13% ± 8%), muscle mass (9% ± 8%), and endurance performance (one-legged, 23% ± 15%; whole-body, 8% ± 7%), assessed as weighted combined factors, and were associated with changes in health variables (e.g. visceral fat, -6% ± 21%; [LDL]serum , -4% ± 14%) and muscle tissue characteristics such as fibre type proportions (e.g. IIX, -3% points), myonuclei per fibre (30% ± 65%), total RNA/rRNA abundances (15%/6-19%), and transcriptome profiles (e.g. 312 differentially expressed genes). Vitamin D3 supplementation did not affect training-associated changes for any of the main outcome domains, despite robust increases in [25(OH)D]serum (∆49% vs. placebo). No conditional effects were observed for COPD vs. healthy or pre-RCT [25(OH)D]serum . In secondary analyses, vitamin D3 affected expression of gene sets involved in vascular functions in muscle tissue and strength gains in participants with high fat mass, which advocates further study. Conclusions: Vitamin D3 supplementation did not affect muscular responses to resistance training in older adults with or without COPD. Keywords: Cholecalciferol; Muscle plasticity; Strength training. © 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders