Normalization of gene expression data revisited: the three viewpoints of the transcriptome in human skeletal muscle undergoing load-induced hypertrophy and why they matter

The biological relevance and accuracy of gene expression data depend on the adequacy of data normalization. This is both due to its role in resolving and accounting for technical variation and errors, and its defining role in shaping the view point of biological interpretations. Still, the choice of the normalization method is often not explicitly motivated although this choice may be particularly decisive for conclusions in studies involving pronounced cellular plasticity. In this study, we highlight the consequences of using three fundamentally different modes of normalization for interpreting RNA-seq data from human skeletal muscle undergoing exercise-training induced growth. Briefly, 25 participants conducted 12 weeks of high-load resistance training. Muscle biopsy specimens were sampled from m. vastus lateralis before, after two weeks of training (week 2) and after the intervention (week 12) and were subsequently analysed using RNA-seq. Transcript counts were modelled as (1) per-library-size, (2) per-total-RNA, and (3) per-sample-size (per-mg-tissue). Result: Initially, the three modes of transcript modelling led to the identification of three unique sets of stable genes, which displayed differential expression profiles. Specifically, genes showing stable expression across samples in the per-library-size dataset displayed training-associated increases in per-total-RNA and per-sample-size datasets. These gene sets were then used for normalization of the entire dataset, providing transcript abundance estimates corresponding to each of the three biological viewpoints (i.e., per-library-size, per-total-RNA, and per-sample-size). The different normalization modes led to different conclusions, measured as training-associated changes in transcript expression. Briefly, for 27% and 20% of the transcripts, training was associated with changes in expression in per-total-RNA and per-sample-size scenarios, but not in the per-library-size scenario. At week 2, this led to opposite conclusions for 4% of the transcripts between per-library-size and per-sample-size datasets (↑ vs. ↓, respectively). Conclusion: Scientists should be explicit with their choice of normalization strategies and should interpret the results of gene expression analyses with caution. This is particularly important for data sets involving a limited number of genes or involving growing or differentiating cellular models, where the risk of biased conclusions is pronounced.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.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

Resistance exercise training increases skeletal muscle mitochondrial respiration in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is associated with skeletal muscle mitochondrial dysfunction. Resistance exercise training (RT) is a training modality with a relatively small pulmonary demand that has been suggested to increase skeletal muscle oxidative enzyme activity in COPD. Whether a shift into a more oxidative profile following RT also translates into increased mitochondrial respiratory capacity in COPD is yet to be established. This study investigated the effects of 13 weeks of RT on m. vastus lateralis mitochondrial capacity in 11 per sons with moderate COPD [45% females, age: 69 ± 4 years (mean ± SD), predicted forced expiratory volume in 1 s (FEV1): 56 ± 7%] and 12 healthy controls (75% females, age: 66 ± 5 years, predicted FEV1: 110 ± 16%). RT was supervised and carried out two times per week. Leg exercises included leg press, knee extension, and knee flexion and were performed unilaterally with one leg conducting high-load training (10 repetitions maximum, 10RM) and the other leg conducting low-load training (30 repetitions maximum, 30RM). One-legged muscle mass, maximal muscle strength, and endurance performance were determined prior to and after the RT period, together with mitochondrial respiratory capacity using high-resolution respirometry and citrate synthase (CS) activity (a marker for mitochondrial volume density). Transcriptome analysis of genes associated with mitochondrial function was performed. Resistance exercise training led to similar improvements in one-legged muscle mass, muscle strength, and endurance performance in COPD and healthy individuals. In COPD, mitochondrial fatty acid oxidation capacity and oxidative phosphorylation increased following RT (+13 ± 22%, P = 0.033 and +9 ± 23%, P = 0.035, respectively). Marked increases were also seen in COPD for mitochondrial volume density (CS activity, +39 ± 35%, P = 0.001), which increased more than mitochondrial respiration, leading to lowered intrinsic mitochondrial function (respiration/CS activity) for complex-1- supported respiration ( 12 ± 43%, P = 0.033), oxidative phosphorylation ( 10 ± 42%, P = 0.037), and electron transfer system capacity ( 6 ± 52%, P = 0.027). No differences were observed between 10RM and 30RM RT, nor were there any adaptations in mitochondrial function following RT in healthy controls. RT led to differential expression of numerous genes related to mitochondrial function in both COPD and healthy controls, with no difference being observed between groups. Thirteen weeks of RT resulted in augmented skeletal muscle mitochondrial respiratory capacity in COPD, accompanied by alterations in the transcriptome and driven by an increase in mitochondrial quantity rather than improved mitochondrial quality.publishedVersio

Superior Physiological Adaptations After a Microcycle of Short Intervals Versus Long Intervals in Cyclists

Purpose: To compare the effects of a 1-week high-intensity aerobic-training shock microcycle composed of either 5 short-interval sessions (SI; n = 9, 5 series with 12 × 30-s work intervals interspersed with 15-s recovery and 3-min recovery between series) or 5 long-interval sessions (LI; n = 8, 6 series of 5-min work intervals with 2.5-min recovery between series) on indicators of endurance performance in well-trained cyclists. Methods: Before and following 6 days with standardized training loads after the 1-week high-intensity aerobic-training shock microcycle, both groups were tested in physiological determinants of endurance performance. Results: From pretraining to posttraining, SI achieved a larger improvement than LI in maximal oxygen uptake (5.7%; 95% confidence interval, 1.3–10.3; P = .015) and power output at a blood lactate concentration of 4 mmol·L−1 (3.8%; 95% confidence interval, 0.2–7.4; P = .038). There were no group differences in changes of fractional use of maximal oxygen uptake at a workload corresponding to a blood lactate concentration of 4 mmol·L−1, gross efficiency, or the 1-minute peak power output from the maximal-oxygen-uptake test. Conclusion: The SI protocol may induce superior changes in indicators of endurance performance compared with the LI protocol, indicating that SI can be a good strategy during a 1-week high-intensity aerobic-training shock microcycle in well-trained cyclists

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

The Effect of Different High-Intensity Periodization Models on Endurance Adaptations

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Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2´ site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice expressing human ACE2 against infection when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants

XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes

As revealed using mice heterozygous for the base excision repair (BER) protein XRCC1, BER and mutagenic repair pathways can simultaneously compete for access to single-strand breaks induced by activation-induced deaminase