The primary purpose of the present study was to evaluate the effect of manipulating load and repetition duration (i.e. time under tension) during RE performed to task failure on muscle fibre activation, which we quantified via fibre type‐specific glycogen depletion (Bell & Jacobs, 1989 ; Robergs et al . 1991 ; Koopman et al . 2006 ). In addition, we measured surface EMG to determine how well EMG amplitude aligned with muscle fibre type‐specific glycogen depletion. Additionally, to obtain mechanistic insight into how muscle fibre activation would be translated, we examined the phosphorylation of select signalling proteins prominent in contraction‐related anabolism. We hypothesized that performing RE to task failure, independent of any specific RE variable, would result in the activation of type I and type II muscle fibres to an equivalent extent and show comparable increases in anabolic signalling. In addition, we hypothesized that surface EMG would be a poor indicator of muscle fibre type‐specific glycogen depletion (i.e. fibre activation) and that muscle fibre glycogen depletion and anabolic signalling would be related.

According to the size principle of motor unit recruitment, performing submaximal contractions results predominantly in the recruitment of smaller (i.e. lower threshold) motor units that innervate type I fibres, although increasing fatigue necessitates the recruitment of larger (i.e. higher threshold) motor units that innervate type II muscle fibres (Mendell, 2005 ). Accordingly, several acute aerobic (Gollnick et al. 1973 , 1974b ; Vollestad et al . 1984 ; Vollestad & Blom, 1985 ; Prats et al . 2013 ; Kristensen et al . 2015 ) and resistance (Bell & Jacobs, 1989 ; Robergs et al . 1991 ; Koopman et al . 2006 ) exercise studies have shown that sustained submaximal contractions result in the substrate depletion (which is indicative of preceding depolarization or ‘activation’) of type II muscle fibres as fatigue ensues. Nonetheless, despite considerable debate on the ability of surface EMG to provide insight into motor unit recruitment during fatiguing contractions (Dideriksen et al. 2010 , 2011 ; Enoka & Duchateau, 2015 ; Vigotsky et al . 2016 ), the thesis that type II fibre activation is confined to or superior with the lifting of heavier loads has been asserted.

It has been proposed that performing resistance exercise (RE) with heavier loads [greater than 60% one repetition maximum (1RM) strength] is required to elicit muscle hypertrophy and to recruit and result in hypertrophy of type II muscle fibres (Ratamess et al . 2009 ; Grgic & Schoenfeld, 2018 ). By contrast, studies show that performing RE training with relatively lighter loads to task failure (i.e. volitional fatigue) results in hypertrophy of both type I and type II muscle fibres (Mitchell et al . 2012 ; Morton et al . 2016 ; Schoenfeld et al . 2017 ). Indeed, type II muscle fibre hypertrophy, even when lighter loads are lifted to task failure, is indicative of recurrent type II fibre activation (Mitchell et al . 2012 ; Morton et al. 2015 , 2016 ). However, on the basis of greater surface electromyography (EMG) amplitude (Jenkins et al . 2015 ; Looney et al . 2016 ; Haun et al . 2017 ) or decomposition of the EMG signal (Muddle et al . 2018 ), other studies have reported that heavier loads are superior to lighter loads in terms of recruiting higher threshold motor units and thus the eventual hypertrophy of type II fibres (Grgic & Schoenfeld, 2018 ).

Methods

Ethical approval All participants were informed of the purpose, methodology, and potential risks of the study before giving verbal and written informed consent. The study conformed to the standards set by the latest revision of the Declaration of Helsinki and to the most recent Canadian Tri‐Council policy statement on the use of human participants in research (http://www.pre.ethics.gc.ca/eng/policy-politique/initiatives/tcps2-eptc2/Default). The study was approved by the Hamilton Integrated Research Ethics Board (Project Number 0802) and was registered at clinicaltrials.gov (NCT03991117).

Study participants Ten recreationally‐trained young men (mean ± SD: 22 ± 3 years, 81.6 ± 8.9 kg, 178 ± 6 cm) volunteered to participate in the present study. We defined ‘recreationally‐trained’ as engaging in at least one to three RE sessions per week for at least 2 years.

Resistance exercise training conditions Participants’ legs were assigned in randomized cross‐over fashion to perform one of four unilateral RE protocols. The four RE conditions varied in the repetition duration and load (percentage of single maximal voluntary isotonic strength: %1RM). The conditions were: 80 %1RM Regular [80R; 1s:1s:1s (eccentric:pause:concentric)], 80 %1RM Slow (80S; 3:1:3), 30 %1RM Regular (30R; 1:1:1) and 30 %1RM Slow (30S; 3:1:3). Three sets were performed for each condition and each set was separated by 180 s rest. Repetition cadence was maintained by an in‐ear metronome at 60 beats min–1; however, for greater accuracy, repetition duration was quantified with the rise and fall of vastus lateralis (VL) EMG activity. RE volume (kg) was calculated by multiplying the number of repetitions in all three sets by the load lifted per repetition. Total time under load (TUL; s) was calculated by multiplying repetition duration by the number of repetitions in all three sets determined from signal from the VL EMG. Finally, impulse (kg·s) was calculated by multiplying the load lifted per repetition by the repetition duration and by the number of repetitions in all three sets.

Study design Each participant came in for a familiarization session before the RE trials began, which was used to obtain an independent assessment of 1RM for each leg during knee extension (Atlantis, Laval, QC, Canada) and to familiarize them with performing isometric maximum voluntary contractions (MVC; leg curl and knee extension; Biodex dynamometer, System 3; Biodex Medical Systems Inc., Shirley, NY, USA). Using a unilateral within‐subject cross‐over design, participants came in on two separate occasions (separated by at least 72 h) to perform two of the four RE conditions each day (one on each leg) in a randomized order (Fig. 1). Briefly, on each of the two trial days, participants arrived following an overnight fast and a muscle biopsy was taken from their VL under local anaesthesia (2% xylocaine) to serve as the baseline for both conditions performed that day. After the muscle biopsy, dry reusable electrodes (Biometrics SX230; Biometrics Ltd., Newport, UK) were placed on each participant's VL, vastus medialis (VM) and semi‐tendinosus (ST; in line with the direction of muscle action) along with a reference electrode and electronic joint goniometer (SG 150, Biometrics Ltd.) on the head of the fibula and about their knee joint, respectively. When the electrodes were in place, each participant performed three isometric knee extensions with their leg positioned at 60° and isometric leg curls at 45° to record peak torque and maximum voluntary excitation (MVE; i.e. the highest EMG signal: knee extension and leg curls, respectively) (Mathiassen et al. 1995) of the quadriceps and hamstrings, respectively. Afterwards, each participant performed two of the four conditions consecutively (one on each leg), which involved three sets to task failure (i.e. the participant was unable to complete another concentric muscle action) with three isometric knee extension MVCs between each condition's set (∼15 s delay between the knee extension machine and their first MVC). One h following the last MVC in each condition, a muscle biopsy was taken from the VL (one each leg). Figure 1. Study schematic representing one of the two trial days Open in figure viewer PowerPoint The two arrows represent each of the participant's legs.

Reduction in peak torque and EMG analyses Muscle fatigue was quantified in the knee extensors as the reduction in isometric peak torque relative to the pre‐testing peak toque. Surface EMG was recorded on a Biometrics data logger (DataLOG MWX8, Biometrics Ltd.; band‐pass 20–450 Hz, input impedance ∼1015 Ω, common mode rejection ratio >96 dB) and analysed with LabVIEW, version 8.2 (National Instruments, Austin, TX, USA). The raw EMG signals were sampled at 2048 Hz, full‐wave rectified and smoothed with a 6 Hz low pass filter. The skin was shaved and marked (with a dot from a permanent marker) prior to bipolar integral dry reusable electrode (Biometrics SX230; Biometrics Ltd.) placement with a fixed inter‐electrode distance of 2 cm. Care was taken not to place electrodes directly over a biopsy site in the case that the biopsy‐induced oedema impaired motor unit recruitment or EMG signal. The average for each phase of each repetition was modelled with a second order polynomial regression equation, and a fast Fourier transformation was performed on each 250 ms window to calculate mean power frequency (MnPF). The peak EMG amplitude (EMG amp ) of the second repetition of each set is referred to as the ‘initial EMG amp ’. Similarly, the peak EMG amp of the last repetition of each set is referred to as ‘final EMG amp ’. The integrated (or total) EMG is the area under the curve throughout each set. The initial EMG amp , final EMG amp and integrated EMG were calculated as %MVE for each muscle (VL, VM and ST). MVE was measured each trial day during the initial isometric knee extension (VL and VM) and leg curl (ST) MVCs. MnPF and average EMG of the second repetition of each set (initial MnPF and initial average EMG) and the last repetition of each set (final MnPF and final average EMG) were also calculated.

Muscle glycogen and fibre‐type histochemistry Muscle tissue from each biopsy was mounted in OCT media, frozen in liquid nitrogen‐cooled isopentane and stored in a −80 °C freezer until analysis. Cross‐sections were cut 5 µm thick using a Microm HM550 Cyrostat (Thermo Fisher Scientific, Waltham, MA, USA) with particular care taken not to expose samples to any freeze‐thaw cycles (Fairchild & Fournier, 2004). Fibre type‐specific glycogen depletion was quantified by combining a brightfield periodic acid‐Schiff stain (PAS), as described previously (McManus, 1948; Gollnick et al. 1973; Gollnick et al. 1974a, 1974b; Vollestad et al. 1984; Vollestad & Blom, 1985; Robergs et al. 1991; Koopman et al. 2006; Cumming et al. 2014), with a immunofluorescent myosin heavy chain (MHC) stain (Bloemberg & Quadrilatero, 2012; Morton et al. 2016; Jakubowski et al. 2019) on single cross‐sections similar to the methodology described elsewhere (Schaart et al. 2004). Briefly, cross‐sections were fixed using 3.7% formaldehyde in PBS for 60 min, treated with 1% periodic acid in distilled water for 5 min (#3951; Sigma‐Aldrich, Toronto, ON, Canada), rinsed in tap water, stained with Schiff's reagent for 15 min (#3952016; Sigma‐Aldrich), rinsed with distilled water and then rinsed in PBS prior to fluorescence staining. For fluorescence immunohistochemistry, antibodies raised against dystrophin [MANDYS1 (3B7)], MHC I (BA‐F8), MHC IIA/X (SC‐71) and MHC IIX (6H1) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) were combined with secondary isotype‐specific antibodies [488 (A‐21131), 594 (A‐21125) and 647 (A‐21238)] (Alexa Fluor, Thermo Fisher Scientific) before they were mounted with Prolong Diamond Antifade Reagant (Life Technologies, Toronto, ON, Canada) (Bloemberg & Quadrilatero, 2012). Each slide included muscle sections from a single participant within a single day (e.g. slide 1: pre, 80R and 30R; slide 2: pre, 30S and 80S) and all staining was performed within a period of 2 weeks in batches of three to five slides per day. One day after each stain cross‐sections were imaged (brightfield before fluorescence, similar to a previous study; Schaart et al. 2004) with a CoolSNAP HQ2 fluorescence camera (Nikon Instruments, Melville, NY, USA) at 20× magnification with the exposure times: 400 ms (FITC), 100 ms (TRITC) and 200 ms (Cy5).

Muscle glycogen and fibre type analyses Fibre type, cross‐sectional area and glycogen content were determined by tracing the fibre dystrophin border in ImageJ, version 2 (NIH, Bethesda, MD, USA). Each trace was converted to a region of interest (ROI) and saved before being superimposed to another image of interest (i.e. brightfield or another fluorescence channel). Quantification of PAS intensity was determined by first converting the image to a greyscale image and then calibrating the stain to 0.68 µm pixel–1. In addition, by setting thresholds for background vs. stain intensity, we excluded the quantification of freezing‐induced artefact from each ROI on every channel. To quantify fibre type, the intensity of each colour within each ROI was exported alongside the brightfield data for objective quantification of type I and type II fibres. Only fibres with a circularity >0.85 were used for analyses and care was taken not to circle any fibres along the outside of the cross‐section. An average of 275 ± 167 and 191 ± 126 fibres per section (1322 ± 400 and 896 ± 350 fibres per participant) were used for the fibre type/PAS and cross‐sectional area analysis, respectively. The tracer was blinded to both the participant and conditions during the image analysis.

Western blot analysis Muscle samples were homogenized using RIPA buffer (#R0278; Sigma‐Aldrich) and a bead homogenizer with protease and phosphatase inhibitors (#05892970001 and 04906837001; Sigma‐Aldrich). A bicinchoninic acid assay (#23227; Thermo Fisher Scientific) was performed on the whole muscle homogenate to quantify the protein content of each sample. Samples were prepared in Laemmli buffer (#1610747; Bio‐Rad, Hercules, CA, USA) with beta‐mercaptoethanol (M6250; Sigma‐Aldrich) and brought to equal concentrations of 20 µg µL–1. SDS‐PAGE was performed on 7.5 µL per sample along with two 7.5 µL prestained protein standards (#1610375; Bio‐Rad) and a calibration curve (2.5, 5, 7.5 and 10 µL of all post‐training samples pooled) on 26‐well gels (4‐15% Criterion TGX Stain‐Free, #5678085; Bio‐Rad). As a quality check for protein separation along the gel, the gel was imaged by ultraviolet activation with the Chemidoc MP StainFree Imager (Bio‐Rad) before it was transferred to a nitrocellulose membrane via a Trans‐Blot Turbo Transfer System (Bio‐Rad) at 100 V for 30 min in 4°C transfer buffer (25 mm Tris, 192 mm glycine, 0.1% SDS and 20% methanol, pH 8.3). Transfer success was visualized with ultraviolet activation of both the gel and membrane via a Chemidoc MP StainFree Imager (Bio‐Rad). Nitrocellulose membranes were blocked in BSA for 2 h, washed three times for five minutes with Tris‐buffered saline‐Tween 20 (TBST), cut into specific sections according to the molecular weights of our protein targets, and incubated in primary antibodies at 4°C with the 5% BSA block at concentrations between 1:500 and 1:1500 (depending on the affinity of the primary antibody). The primary antibodies we used were total mTOR (#2972), phosphorylated mTOR (Ser2448; #5536), total p70 S6k (#9202), phosphorylated p70 S6k (Thr389; #9205), total 4E‐BP1 (#9452), phosphorylated 4E‐BP1 (Thr37 and Thr46; #2855), total S6 ribosomal protein (#2217), phosphorylated S6 ribosomal protein (Ser240 and Ser244; #2211), total AkT (#4691), phosphorylated AkT (Ser473; #9271), total FAK (#13009), phosphorylated FAK (Tyr397; #8556), total p44/42 MAPK ERK1/2 (#9102) and phosphorylated p44/42 MAPK ERK1/2 (Thr202 and Tyr204; #9101), which were all obtained from Cell Signaling Technologies (Danvers, MA, USA). After an overnight incubation, membranes were washed again three times for 5 min in TBST, incubated in secondary antibody (dilution 1:20,000; anti‐rabbit, HRP‐linked; #7074; Cell Signaling Technologies) for 1 h at room temperature, washed another three times in TBST, rocked for 5 min in ECL substrate (Clarity Max; #1705062; Bio‐Rad) and then imaged on the ChemiDoc MP (Bio‐Rad). The ladder was imaged in colourmetric mode and the proteins of interest were measured in chemilumescence mode. All image analysis was performed in ImageLab, version 5.2.1 (Bio‐Rad). Each gel lane was calibrated to the gel lanes of our calibration curve and each protein band was calibrated to the protein bands of our calibration curve as described elsewhere (Murphy & Lamb, 2013; MacInnis et al. 2017). Afterwards, the calibrated protein band was divided by the calibrated gel lane to quantify absolute protein band intensity.