This work is a follow-up to a recently published study (Cheng et al. 2020). The information related to the experimental design, the exercise sessions, CHO intake and nutritional control has already been described in the previous article. In this latter study, we determined whether CHO ingestion could accelerate recovery of prolonged low-frequency force depression induced by a glycogen-depleting exercise session combining moderate-intensity and high-intensity sprint exercises.
Participants
As previously described (Cheng et al. 2020), a randomized crossover design was used, in which eleven healthy male participants initially volunteered to participate. The inclusion criteria used in the current study were: aged between 18 and 40 years, being healthy and physically active with normal blood pressure and without medication. The exclusion criteria were: suffering from any kind of disease, having an injury or any other conditions that would compromise the ability to perform the physical tests. Due to the difficulty of recruiting endurance athletes, we only included recreationally active males who regularly exercised up to 5 h/week. It is noteworthy that one participant withdrew during the study and two subjects needed to be excluded from the muscle biological analysis (due to low amount of biological material available or technical issues during RNA isolation). Therefore, eight participants [age: 32.4 ± 6.4 years; height: 185.8 ± 4.6 cm; body mass: 86.8 ± 9.9 kg; percentage body fat: 16.6 ± 5.0%; body mass index: 25.2 ± 3.2 kg/m2; VO2max: 49.9 ± 8.9 mL/kg/min] were finally included in this study. A similar sample size (i.e., N = 7–9) was used in previous studies that analyzed the effects on CHO restriction following exercise on the mRNA levels of genes involved in endurance-training adaptations in skeletal muscle (Pilegaard et al. 2005; Jensen et al. 2015; Mathai et al. 2008). The percentage body fat was measured using bio-electrical impedance analysis (Tanita TBF-300 UK Ltd, West Drayton, UK) and VO2max was determined as previously described (Cheng et al. 2020). The distribution of myosin heavy chain (MHC) isoforms (as described below) was: MHC1: 36.9 ± 13.6%; MHC2A: 54.2 ± 9.4%; MHC2X: 8.9 ± 8.6%. This study was approved by the Kaunas Regional Research Ethics Committee (no. BE-2-17) and was performed in accordance with the last revision of the Declaration of Helsinki.
Experimental design and exercise session
The experimental design was previously described (Cheng et al. 2020). Briefly, the experiments consisted of three visits to the laboratory. During the first visit, a maximal incremental cycling test was performed to determine VO2max and the intensity used during the subsequent prolonged moderate-intensity cycling exercise executed in visits 2–3. During this first visit, the participants were also familiarized with the equipment and protocols used during the subsequent two visits. The second visit was performed approximately a week after the first visit. The two experimental trials (visits 2–3) were completed in a random order and were separated by four weeks during which the participants maintained their regular physical activity. The participants were asked to refrain from any strenuous exercise during the last 3 days prior to each experimental session.
In visits 2–3, the participants arrived at the laboratory in the morning after a 10–12 h overnight fast and an initial biopsy was collected from the vastus lateralis muscle. An overview of the experimental protocol is presented in Fig. 1. It is noteworthy that neuromuscular testing of knee extensors (i.e. electrically evoked torque and maximal voluntary contraction) was executed before (1 test) and after exercise (7 tests). These tests were only performed on the dominant leg, and thus did not interfere with the muscle biological analyses since muscle biopsies were collected from the vastus lateralis muscle of the non-dominant leg (see below). These tests, performed to answer the aim of the initial study (Cheng et al. 2020), are not included in the current analysis. After muscle biopsy, a warm-up was performed, consisting of 8–10 min of cycling at 1 W/kg body mass, followed by light active stretching of the knee extensor muscles and by 2–3 short isometric knee extensions in the dynamometer chair. This latter exercise was used as a warm-up for the neuromuscular testing. After the completion of the baseline neuromuscular testing (~ 10 min), the participants performed a cycling session involving CCE followed by SIE. For a full description of the exercise session, refer to Cheng et al. (2020). In short, this session consisted of performing a 60-min continuous cycling exercise (i.e. CCE) at a power eliciting 60% VO2max followed by six 30-s all-out cycling sprints (SIE) interspaced with 4 min recovery. The 60-min CCE was performed on an electromechanically braked cycling ergometer (Ergoselect 200P, Ergoline, Medical Measurement Systems, Binz, Germany), and a short break (2–3 min) was allowed after 30 min of CCE during which the participants could drink water (up to 0.5 L). Heart rate (HR) and the rating of perceived exertion (RPE, 6–20 scale) were determined every 10 min during the CCE to evaluate the perceived effort level. The all-out cycling sprints were performed using a mechanically braked cycling ergometer (Monark 824E, Monark, Vansbro, Sweden) with a brake weight corresponding to 7.5% of the participant’s body mass. The peak HR of each cycling sprint and average power were obtained during the SIE. A second muscle biopsy was collected 2–3 min after the last cycling sprint. In a random order, the participants ingested a beverage containing either carbohydrate (CHO) or placebo (PLA) every 15 min during the 150-min period following the completion of the last sprint (post-exercise recovery period). A third biopsy was collected 180 min after the last sprint (i.e. 30 min after the last beverage ingestion). Capillary blood concentration of glucose and lactate was determined from the fingertip before exercise, immediately after CCE, immediately after SIE, and every 30 min during the 180-min recovery period, as previously described (Cheng et al. 2020). Additional neuromuscular testing was executed 15, 30, 60, 90, 120, 150 and 180 min after the completion of the last cycling sprint (not included in the analysis).
Carbohydrate intake during the recovery period and nutritional control
The CHO-enriched beverage contained glucose and fructose at a 2:1 ratio. CHO (final concentration: 100 g/L) was dissolved in a solution containing water and 1:20 lemon juice concentrate (22 g carbohydrate/L of concentrate; Solevita). The volume of CHO beverage was adjusted for each participant to provide 1.5 g/kg body mass/h CHO during the recovery period. An equal volume was ingested every 15 min following the last cycling sprint over a 150-min period to provide the adequate amount of CHO. The total amount of CHO ingested during the CHO trial (i.e. 150-min post-exercise period) was 328.9 ± 37.5 g. The rationale behind this nutritional approach was previously described (Cheng et al. 2020). The PLA beverage was prepared using a solution of water with lemon juice concentrate (1:20 dilution), in which 0.6 g/L of sodium saccharin (Hermesetas, Zurich, Switzerland) and 4 g/L of Suketter (containing sodium cyclamate and sodium saccharin; Cederroth, Upplands Väsby, Sweden) were dissolved. The total amount of CHO ingested during the PLA trial (i.e. 150-min post-exercise period) was negligible (3.6 ± 0.4 g). The volume of beverage consumed was identical for both CHO and PLA trials.
As described above, the participants performed the strenuous exercise sessions in the morning after a 10–12 h overnight fast. They were asked to take a similar meal on the evening before each exercise session and to refrain from any food and caffeine intakes on the morning before each exercise session. Overnight fasting was chosen for two main reasons: (1) to reduce the risks of gastrointestinal issues and vomiting commonly observed during or immediately after all-out cycling sprints; (2) to restrict CHO (that would have been available in the blood stream after breakfast) during exercise, thereby promoting large muscle glycogen depletion. A similar strategy (strenuous exercise after overnight fasting) was accompanied by very low muscle glycogen concentrations after exercise in untrained and trained participants (Hickner et al. 1997; Greiwe et al. 1999). It is also noteworthy that pre-exercise muscle glycogen concentration (see in the result section) was in the same range as that observed in the literature after overnight fast in a similar population (Blom et al. 1987; Maehlum et al. 1977).
Muscle biopsies
Needle biopsies from the non-dominant vastus lateralis muscle were collected by a trained physician, as previously described (Cheng et al. 2020). The non-dominant leg was not subjected to neuromuscular testing. Three biopsy samples (~ 10 mg each) were taken before exercise, 2–3 min after and 180 min after the last cycling sprint. The samples were immediately frozen in liquid nitrogen and stored at − 80 °C until further analysis.
Muscle glycogen measurements
Glycogen concentration was assessed using 5–10 mg of frozen muscle, as previously described (Cheng et al. 2020). Briefly, muscle samples were dissolved in 25 volumes of 2 M NaOH for 50 min at 95 °C, before being neutralized with an equal volume of 2 M HCl. The homogenates (1:50 dilution) were first diluted with distilled water (final dilution: 1:400), and then 5 µL of each diluted homogenate was loaded in duplicates into a 96-well plate. The samples were then analyzed using a fluorometric kit (ab65620, Abcam, Cambridge, UK) following the manufacturer’s instructions. Due to technical reasons, muscle glycogen concentrations could not be expressed in mmol glucosyl units/kg dry weight and were instead expressed as mmol glucosyl units/kg wet weight.
Distribution of myosin heavy chain (MHC) isoforms
The distribution of MHC isoforms was assessed using 5–10 mg of frozen muscle collected before exercise. Muscle samples were homogenized with a bead-homogenizer (TissueLyser LT, Qiagen, Sollentuna, Sweden) in ice-cold lysis buffer (20 µL/mg; pH 7.6) containing 20 mM Hepes, 150 mM NaCl, 5 mM EDTA, 25 mM KF, 1 mM Na3VO4, 5% glycerol, 0.5% Triton X-100, and EDTA-free protease inhibitor cocktail (1 tablet/10 mL buffer; # 04 693 159 001, Sigma-Aldrich, Stockholm, Sweden). Protein concentration of the homogenates was determined using BCA protein assay (#23227, Thermo Fisher Scientific, Stockholm, Sweden), and then adjusted to 2 µg/µL after dilution with the lysis buffer. Samples were then diluted to 10 ng/µL with loading buffer consisting of 125 mM Tris–HCl (pH 6.8), 1 mM EDTA, 20% glycerol, 5% SDS, 5% β-mercaptoethanol and bromophenol blue. Electrophoresis was performed using a Mini Protean III system (Bio-Rad Laboratories, Solna, Sweden). The separating gel consisted of 30% glycerol, 8% acrylamide, 0.16% bisacrylamide, 0.2 M Tris–HCl (pH 8.8), 0.1 M glycine, 0.4% SDS, 0.05% Temed and 1% ammonium persulfate. The stacking gel consisted of 30% glycerol, 4% acrylamide, 0.08% bisacrylamide, 70 mM Tris–HCl (pH 6.8), 4 mM EDTA, 0.4% SDS, 0.1% Temed and 1% ammonium persulfate. Protein lysates were heated at 95 °C for 10 min and 150 ng of protein was loaded into the gel. The gels were run at 4 °C during the whole migration period, including 40 min at 10 mA and 23 h 20 min at 140 V. The gels were silver-stained as described previously (Agbulut et al. 1996). The gels were scanned with an office scanner (CanoScan 9000F, Canon, The Netherlands, Amsterdam) and analysed with UN-SCAN-IT software (version 6.1, Silk Scientific Corporation, Orem, USA). The distribution of MHC isoforms was quantified using positive lane analysis and background correction (single region background value).
RNA isolation, cDNA synthesis and quantitative polymerase chain reaction (qPCR)
Muscle samples (5–10 mg) were disrupted with a bead-homogenizer (TissueLyser LT, Qiagen) in 75 µL RA1 lysis buffer (Macherey-Nage, Dueren, Germany) containing 1% β-mercaptoethanol, and 250 µL TRIzol reagent (Life Technologies, Stockholm, Sweden). Tissue homogenate was then mixed with 100 µL chloroform (Sigma-Aldrich) before centrifugation. The aqueous phase (200 µL) was collected and mixed with an equal volume of chloroform. After centrifugation, 170 µL of the aqueous phase was precipitated with an equal volume of isopropanol at − 20 °C for 10 min. After centrifugation and an extra precipitation step with isopropanol, RNA was washed twice with 70% ethanol to remove excess salt. The pellet was dried at room temperature for 10 min, and dissolved in 12 µL nuclease-free water. The RNA samples were incubated 10 min at 55 °C and then kept on ice. Total RNA concentration and purity were assessed by measuring the optical density (230, 260 and 280 nm) with a spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific). RNA samples were treated with genomic DNA removal kit (Heat and Run DNase, ArcticZymes, Tromsø, Norway) according to the manufacturer’s instructions, and RNA concentration was assessed again.
Total RNA (1 µg) was reversed-transcribed using iScript Select cDNA synthesis kit (Bio-Rad Laboratories). Total RNA was converted into cDNA using a final reaction volume of 20 µL containing: 2 µL of oligodT, 2 µL of random hexamers, 4 µL of 5X-iscript select reaction mix, 1 µL of reverse transcriptase and 9 µL of nuclease-free water. The reaction mix was incubated at 25 °C for 5 min, at 42 °C for 60 min, and at 85 °C for 5 min. After this, the samples were cooled down on ice, aliquoted, and stored at − 80 °C until further analysis. qPCR (final volume: 20 µL) was performed with samples loaded in duplicate using 5 µL of diluted cDNA (1/20 dilution from stock cDNA mixture), 10 µL of Rotor-Gene SYBR Green RT-PCR Master Mix (Qiagen), and 1 µL (Biorad Laboratories) or 0.4 µL (reverse and forward primers at 20 µM) of primers (list of primers presented in Table 1). The sequences of the forward and reverse primers for total PGC1A, PGC1A transcripts from exon 1a (PGC1A-ex1a) and truncated PGC1A were kindly provided by Jorge Ruas’s lab (Karolinska Institutet, Sweden) and were previously used (Ruas et al. 2012). PGC1A-ex1a primers can detect PGC1A1 and NT-PGC1A-a, and truncated PGC1A primers can detect PGC1A4, NT-PGC1A-a and NT-PGC1A-c (not expressed in skeletal muscle) (Martinez-Redondo et al. 2015). PGC1A1 isoform and NT-PGC1A-a are important regulators of mitochondrial biogenesis whereas PGC1A4 isoform is induced after resistance exercise training and could promote muscle hypertrophy (Ruas et al. 2012; Martinez-Redondo et al. 2015). Several transcription factors associated with mitochondrial biogenesis and oxidative metabolism (Scarpulla et al. 2012), including nuclear respiratory factor 1 (NRF1), GA-binding protein transcription factor subunit alpha (GABPA, also called NRF2), mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptors alpha (PPARA) were also studied. The mRNA levels of three genes involved in redox homeostasis, a process closely connected to mitochondrial biogenesis (Ji et al. 2020), were assessed: Sirtuin 1 (SIRT1), nuclear factor erythroid 2-related factor 2 (NFE2L2) and superoxide dismutase 2 (SOD2). Finally, we determined the mRNA levels of two metabolic genes involved in substrate utilization (pyruvate dehydrogenase kinase 4, PDK4) and glucose transport (solute carrier family 2 member 4, SLC2A4, also called GLUT4).
Table 1 List of primers used for qPCR analysis qPCR was performed using a Rotor Gene Q thermocycler (Qiagen) for 40 cycles (95 °C for 5 s and 60 °C for 30 s) followed by melting curve analysis. qPCR efficiency was estimated for each primer pair by performing standard curves obtained from serial dilutions of a pooled sample. We initially tested two reference genes: RPLP0 (ribosomal protein lateral stalk subunit P0) and HPRT1 (hypoxanthine phosphoribosyltransferase 1). HPRT1 was selected and used for normalization because its expression was not affected by the experimental conditions (time and supplementation). The relative mRNA levels were calculated using the ΔΔCT method (Pfaffl 2001). The threshold cycle (CT) was calculated using the Rotor-Gene Q software (Qiagen), based on the qPCR conditions (auto-find threshold with slope correct adjustment) set up from the standard curves obtained for each gene. Since none of the target mRNAs were differently expressed between the two trials at baseline (pre-exercise), the mRNA levels were expressed as fold changes (FC) relative to pre-exercise values, which were set at 100%. This method is commonly used in studies with similar settings (i.e. cross-over design, post-exercise CHO restriction) (Pilegaard et al. 2005; Jensen et al. 2015; Mathai et al. 2008).
Statistical analysis
Data are presented as mean ± standard deviation (SD), and individual values are presented in Fig. 2a, b. All the statistical analyses were performed using GraphPad Prism (Graphpad Prism 8.0.2, San Diego, USA). As explained above, eight participants were included in the analysis of this study. Power output of the SIE was analyzed from only seven subjects because it was not recorded during the SIE of the PLA trial for one subject. In addition, one subject was excluded from the analysis of muscle glycogen (Fig. 2a, b) due to outlier values (i.e. > 2 SD) above the mean at post-exercise for the CHO trial. Similarly, one subject was excluded from the analysis of PDK4 mRNA (Fig. 6a) due to outlier values at the time points post-exercise and 3 h post-exercise for the CHO trial.
Shapiro–Wilk tests were used to check normality before selecting the appropriate parametric or non-parametric statistical tests. Paired t tests were used to analyze the mean power, mean HR, peak HR, mean RPE and the rate of muscle glycogen resynthesis for both conditions. For the analysis of blood glucose concentration, and for the analysis of the mRNA levels of total PGC1A, TFAM, SIRT1 and GABPA, two-way repeated-measures analysis of variance (2-way RM ANOVA) tests were used to assess the effect of time, supplementation (CHO vs PLA), and time × supplementation interaction. When an interaction was observed, a Sidak multiple comparisons test was used to compare the PLA and CHO conditions. For the analysis of blood lactate and muscle glycogen concentration, as well as for the analysis of the mRNA levels of PGC1A-ex1a, truncated PGC1A, NFE2L2, NRF1, PDK4, SOD2, PPARA, SLC2A4, Friedman tests were used to assess the effect of time in both experimental trials. Then, Wilcoxon’s matched-pairs signed-rank tests with Bonferroni corrections were performed to compare the conditions (CHO vs. PLA) at each time point. The level of significance was set at P < 0.05.