Functional and molecular responses to
concurrent exercise of the arm extensors
Björn Hansson
THE SWEDISH SCHOOL OF SPORT
AND HEALTH SCIENCES
Master Degree Project: 39:2018
Sports Science Program: 2016-2018
Supervisor: Tommy Lundberg
Examiner: Örjan Ekblom
Abstract
Aim
As most concurrent exercise studies to date have focused on lower limb muscles, this study explored the acute response to concurrent exercise of the arm extensors. Specifically, the effects of a preceding bout of aerobic exercise on the subsequent molecular and functional response to resistance exercise was explored.
Method
Eleven men performed unilateral consecutive bouts of arm extensor aerobic exercise (~40 min) and resistance exercise (4 sets of 7 reps) interspersed by 15 min recovery. The contralateral arm performed resistance exercise only. Peak concentric power was assessed during the resistance exercise bout. Muscle biopsies were taken from the m. triceps brachii of each arm immediately before, 15 minutes and 3 h after the resistance exercise bout. Muscle samples were assessed for gene expression of markers involved in regulating protein turnover.
Results
There was no difference in mean concentric peak power in AE + RE vs. RE limb. Gene expression of MuRF-1, atrogin-1, and PGC-1a were significantly greater in AE + RE
compared to RE (arm x time interactions P < 0.05). Myostatin expression generally decreased in both AE + RE and RE (main effect of time P < 0.05).
Conclusions
Inconclusive results suggest that aerobic exercise does not alter power output during
subsequent resistance exercise. Aerobic exercise, performed prior to resistance exercise, alters the expression of markers involved in muscle remodelling processes and anabolic signalling in the arm extensors compared to resistance exercise alone.
Table of content
1 Introduction……….1
1.2 Adaptations to aerobic and resistance training………..1
1.3 Acute molecular responses to aerobic and resistance exercise……….2
1.4 Concurrent training………...4
2 Methods………...7
2.2 General design………...7
2.3 Subjects……….7
2.4 Exercise equipment and description……….8
2.5 Pre-testing and familiarization………..9
2.6 Exercise protocol………...9
2.7 Muscle biopsies………...10
2.8 Gene expression analysis………10
2.8.1 RNA extraction………..10
2.8.2 cDNA reverse transcription………...11
2.8.3 Real-time PCR………...11
2.9 Statistics………..11
2.10 Validity and reliability……….12
3 Results………...12
3.2 Workload test and baseline data………..12
3.3 Exercise characteristics………...12
3.4 Gene expression………..13
4 Discussion……….14 5 References
1. Introduction
Despite there being hundreds of different sports with diverse types of stressors for the human body, the human body adapts very well to the challenges it is exposed to. The human skeletal muscle is a plastic tissue (turnover rate of 1-2% per day) capable of modifying the phenotype depending on which type of stress it is repeatedly exposed to. This is evident when looking at athletes coming from different sports with divergent training backgrounds. Although many team sports require the athlete to possess both strength and endurance simultaneously, it is unlikely that an athlete can concurrently achieve the extreme features displayed by the marathon runner and the powerlifter. Repeatedly performing either resistance or aerobic exercise will alter the phenotype of the skeletal muscle, inducing adaptations reflecting the nature of the exercise stimuli imposed.
1.2 Adaptations to aerobic and resistance training
Common end-point adaptations to chronic resistance exercise include increased force
generating capacity of the skeletal muscle, connective tissue stiffness and cross-sectional area (Knuttgen and Kraemer, 1987). Resistance exercise is typically performed over several repeated cycles (sets) with clusters of coupled concentric-eccentric isotonic repetitions. Most commonly, 3-4 sets of 6-12 RM (repetition maximum) repetitions is recommended to increase size of the skeletal muscle (American College of Sports Medicine, 2009), with the lower range steering adaptations towards increased neural drive and force output and the upper range towards increased size (Fry, 2004). However, this has recently been a matter of debate, as recent research suggest that intensities as low as 30-40 RM may promote skeletal muscle hypertrophy (Grgic and Schoenfeld, 2018) . Resistance is in general applied with free weights or machines with cable stacks, although iso-inertial dependent loading (e.g flywheel) is an proven method to impose resistance exercise adaptations as well (Maroto-Izquierdo et al., 2017).
On the contrary, aerobic exercise increases the ability to transport and utilize oxygen for energy production, thus leading to an improved sustainability to fatigue during prolonged work (Brooks, 2012). Central adaptations to aerobic exercise includes an increase in the maximal oxygen uptake (VO2max), which is mainly achieved through an augmented cardiac output (Ekblom, 1968). The local aerobic capacity of the skeletal muscle is due to the accretion, and greater density, of capillaries, increased activity of oxidative enzymes, and mitochondrial biogenesis (Joyner and Coyle, 2008). Aerobic exercise may be performed in
the form of low-intensity continuous high-volume or high-intensity intermittent low-volume exercise (Gibala et al., 2014).
Although the adaptations to the two exercise modes outlined above represent end-point adaptations, it is important to acknowledge that some of the adaptations show minor
specificity across the training modalities, as resistance exercise may augment capillarization (Nader et al., 2014) and oxidative enzyme activity (Tesch et al., 1989), and aerobic exercise may promote skeletal muscle hypertrophy of the type-I muscle fibres (Harber et al., 2012).
1.3 Acute molecular responses to aerobic and resistance exercise
As previously described, resistance and aerobic exercise induce different adaptations that are specific to the exercise modality performed. Contractions of various intensity and durations results in an immediate cellular response. This initiates transcription of specific DNA sequences and the subsequent translation of the genetic code into series of amino acids to create new proteins (Flück and Hoppeler, 2003). Indeed, readouts of mRNA and proteins may provide a valuable insight on ongoing processes and a theoretical base for chronic
adaptations, however it has been postulated that single molecular markers will likely not conclusively predict long-term adaptations (Fernandez-Gonzalo et al., 2013; Mitchell et al., 2014; Phillips et al., 2013).
Resistance exercise training, characterized by high-force short-duration contractions, stimulates muscle protein synthesis (MPS) positively, causing a positive net protein balance (NPB), which results in protein accretion over time and thus muscle growth (Stokes et al., 2018). The mechanistic target of rapamycin complex 1 (mTORC1) and its downstream regulators eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and P70S6 kinase (P70S6K), are thought to be key mediators to muscle growth through
upregulating muscle protein synthesis (MPS) (Bodine, 2006). Indeed, their role in promoting muscle mass have been highlighted in both animal and human models (Baar and Esser, 1999; Bodine et al., 2001; Terzis et al., 2007). NPB is also influenced by the muscle protein
breakdown (MPB). Atrophy, or muscle catabolism, is caused when rates of MPB are greater than the rates of MPS, causing a negative NPB (Phillips, 2014). The two ubiquitin proteins atrogin-1 and muscle ring finger protein (MuRF) have been shown to be involved in the proteasome dependent degradation progress, and they have consistently been shown to be upregulated in disuse models (Coffey and Hawley, 2007). However, their role in resistance
exercise remains to be elucidated, as atrogin-1 decreases and MuRF-1 increases in response to resistance exercise (Louis et al., 2007; Mascher et al., 2008). Although not classified as a marker for atrophy, myostatin is a well-known negative regulator of hypertrophy through its inhibitory effect on MPS (McNally, 2004). In further highlighting its role in hypertrophy, myostatin-deficient animals and humans display extraordinary muscle mass (McPherron and Lee, 1997; Schuelke et al., 2004). Acute resistance exercise reduces myostatin expression, reverting its inhibitory effects on MPS (Louis et al., 2007). Following resistance exercise there is an concomitant increase in MPB and MPS, resulting in an augmented net protein turnover (Phillips et al., 1997). However, following an acute resistance exercise bout, upregulation of MPS is generally measurable up to 48 hrs, while the increased MPB has returned to baseline after about 24 hrs (Phillips et al., 1997). Since resistance exercise-induced skeletal muscle damage disrupts the compromised integrity of the architectural structure of myofibrils (Gibala et al., 1995), MPB may occur to facilitate muscle remodelling and to restore muscle function (Stokes et al., 2018).
In response to aerobic exercise, characterized by low-force, long-duration contractile activity, the build-up of intracellular stressors due to metabolic perturbations upregulates the amp-activated protein kinase (AMPK) which acts on the coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1a), which is an important transcriptional regulator of mitochondrial biogenesis (Hardie et al., 2012; Norrbom et al., 2004). Aerobic exercise also promotes an increase in the vascular endothelial growth factor (VEGF), which is a key regulator of capillary density (Gustafsson et al., 1999).
It is important to acknowledge that the previous outline of the acute transcriptional and translational response to aerobic and resistance exercise-like stimuli may share similarities in untrained muscle, as research suggests that aerobic exercise may promote increased mTORC1 signalling and MPS (Mascher et al., 2007; Mascher et al., 2011). Also, evidence suggest that resistance exercise may promote AMPK signalling (Dreyer et al., 2006; Vissing et al., 2013). While the molecular response to a novel exercise-stimuli may be generic among untrained (Coffey and Hawley, 2017), trained-accustomed musculature show a more refined response to exercise (Vissing et al., 2013; Wilkinson et al., 2008), hence reflecting the specific muscle phenotype displayed by athletes with divergent training backgrounds.
1.4 Concurrent training
The term concurrent training defines the implementation of both resistance and aerobic exercise concurrently within the same exercise program, on the same workout or separated by hours or days (Leveritt et al. 1999). The combination of resistance and aerobic exercise is essential for sports performance for many athletes. Concurrent exercise training are also important in the prevention of several disorders which might impact physical capacity and health, such as sarcopenia, diabetes type-II, and obesity (Pijnappels et al. 2008; Kelley et al. 2002; Morino et al., 2005).
In 1980, Hickson’s pioneer study suggested that performing aerobic exercise on top of resistance exercise training may lead to attenuated strength adaptations, thus an interference effect was proposed. Research also suggest that concurrent training may interfere with muscle growth and power development (Bell et al., 2000; Chtara et al., 2008; Dudley and Djamil, 1985; Hunter et al., 1987; Jones et al., 2013; Kraemer et al., 1995; Putman et al., 2004; Rønnestad et al., 2012). Although several studies have reported an interference effect, there has been contradictory findings (Hendrickson et al., 2010; McCarthy et al., 1995; Sale et al., 1990; Shaw et al., 2009). Interestingly, some research suggest that concurrent training may augment muscle growth (Kazior et al., 2016; Lundberg et al., 2013), and recent papers have in fact challenged the interference effect regarding muscle hypertrophy (Murach and Bagley, 2016). The implementation of resistance-like exercise training is generally held to augment, rather than attenuate, adaptations to aerobic training (Coffey and Hawley, 2017).
Although the evidence supporting the interference on resistance training-induced adaptations is evident in some cases, little is known about the orchestrating mechanism. It has been suggested that AMPK may inhibit the anabolic signalling response induced by resistance exercise (Atherton et al., 2005; Bolster et al., 2002). However, this work originates from animal models and subsequent work has indeed shown that neither the mTORC1 signalling complex nor the MPS is compromised after acute bouts of concurrent exercise (Carrithers et al., 2007; Donges et al. 2012; Apro et al. 2013; Fernandez-Gonzalo et al. 2013). There are also research indicating that concurrent exercise may promote an even greater anabolic signalling acutely, compared to resistance exercise alone (Lundberg et al. 2012; Pugh et al. 2015; Kazior et al. 2016; Fernandez-Gonzalo et al., 2013a). It is unclear whether the mechanisms by which aerobic exercise compromises acute resistance exercise-induced
signalling in animal models are shared with those in humans. Rather, in humans, the interference effect is likely multifaceted and affected by several different variables, which may be related both to acute signalling and chronic adaptations (Baar, 2014; Fyfe et al., 2014; Hamilton and Philp, 2013; Murach and Bagley, 2016) which will be discussed in the
following section.
It was previously outlined that the training status of participants will likely alter the acute response molecular to training. In symphony with this, the early work of Hickson (1980) suggested that interference in strength adaptations was not evident until the 8th week of concurrent training. An interesting rationale for these results were suggested in recent study by Fyfe and his group (2018), which suggested, in a training-accustomed state, resistance exercise further enhances mTORC1 signalling and ribosomal RNA expression compared to concurrent exercise. This would suggest that long-term concurrent training may interfere with hypertrophic adaptations through a decreased ribosomal biogenesis and thus, decreased translational capacity. However, it is still unclear when participants should be classified as being ‘training-accustomed’ and display a refined response (Coffey and Hawley, 2017).
Further highlighting the complexity of inferring research conducted on concurrent exercise training are the variability in individual training variables (Fyfe et al., 2014; Murach and Bagley, 2016). In a meta-analysis from Wilson and colleagues (2012) it was reported that the total training volume (i.e duration and frequency) of endurance training in a concurrent training protocol negatively correlates with hypertrophic and strength adaptations. Research suggests that, in order to maximize adaptations to resistance exercise training, no more than two aerobic exercise sessions should be conducted each week (Fyfe et al., 2014; Jones et al., 2013). It is unclear whether intensity of aerobic exercise mediates resistance exercise induced adaptations and responses, as little research have directly compared the two within the same training protocol (Fyfe et al., 2016; Silva et al., 2012). However, intensity may mediate acute anabolic signalling (Coffey et al., 2009a, 2009b) and high-intensity interval training may attenuate strength development, but not muscle growth (Sabag et al., 2018). Conducting both resistance and aerobic exercise within the same session may be a time-efficient way to reap benefits from the two different exercise modalities. It is unclear whether the order of the two exercise modalities modulates the response to exercise (Apró et al., 2015; Coffey et al., 2009a, 2009b). It has been postulated, however, that performing resistance and aerobic exercise in close proximity may increase the likelihood of compromised performance
(Bentley et al., 2000; Sporer and Wenger, 2003), strength development (Robineau et al., 2016) and muscle growth (Wilson et al., 2012). Lundberg and co-workers examined the effects of proximity of on resistance exercise induced adaptations, employing either 6 hrs (Lundberg et al., 2013) or 15 mins (Lundberg et al., 2014) between aerobic and resistance exercise. Subjects performed unilateral leg-extensions alone or preceded by ~45 min continuous one-leg aerobic exercise. Results suggested that proximity did not influence adaptations, as limbs subjected to concurrent exercise training similarly increased muscle size twice as much as resistance exercise alone in both protocols (Lundberg et al., 2013; Lundberg et al., 2014). The molecular response similarly suggested augmented anabolic and muscle remodelling signalling (Fernandez-Gonzalo et al., 2013; Lundberg et al., 2014). However, 15 minutes, but not 6h, attenuated power output during resistance exercise. Thus, short recovery between sessions likely leads to attenuated strength/power development, but not muscle growth, in response to concurrent training.
To date, almost all concurrent training studies have investigated the lower-limb muscles. In fact, Abernethy and Quigley (1993) conducted the first and only study investigating whether aerobic exercise compromises adaptations to resistance exercise training in the upper limbs (arm extensors). The results revealed that there was no interference in strength development compared to resistance training alone (Abernethy and Quigley, 1993). Indeed, the acute molecular responses and adaptations to concurrent exercise and/or training of the lower body training are well documented as outlined in the section above. However, findings derived from lower body exercise may not necessarily transfer into upper body exercise modalities, as research suggests that some factors may differ between the upper and lower limbs. For
example, skeletal muscle architecture, structure, and length-tension characteristics varies among the upper and lower limb muscles (Chen et al., 2011). Also, compared to the legs, the arm muscles have a higher percentage of the fast-twitch type-II fibres, rely more heavily on carbohydrate utilization and extract less oxygen during aerobic exercise (Calbet et al., 2005; Helge, 2010; Mittendorfer et al., 2005; Van Hall et al., 2003). Due to the low exposure to eccentric contractions in the upper body in daily activity, the elbow extensors may be more susceptible to muscle damage (Chen et al., 2011). Moreover, hypertrophic response and strength development to resistance training may vary between the upper and lower limbs (Loveless and Ihm, 2015; Paulsen et al., 2003; Rønnestad et al., 2007; Wernbom et al., 2007). It has been postulated that the myofibrillar protein synthesis may vary between the arms and legs, however this variation was considered to be within biological variation (~15 %)
according to the authors (Mittendorfer et al., 2005). While most individuals, through daily activity and motion, engage in movement involving the postural lower limbs, upper body exercise represents a novel exercise modality for most individuals not participating in regular resistance and/or aerobic exercise. Along with the previous outline, this may suggest that the acute response and adaptations to upper body concurrent exercise may be different compared to the legs. Importantly, athletes participating in sports partly involving the upper body (i.e swimming, rowing, cross-country skiing) may, during periods of time, want to focus solely on maximizing adaptations in the upper body musculature. Injured athletes may through periods of time be limited to upper body aerobic and resistance exercise training, further highlighting the need to investigate the response to concurrent exercise in the upper body.
To this background, the objective of this study was to investigate whether the acute molecular and functional response to concurrent exercise is different to that of resistance exercise alone. The research questions were:
• Will the addition of aerobic exercise interfere with the anabolic molecular response to resistance exercise in the arm extensors?
• Will aerobic exercise, performed prior to resistance exercise, compromise
performance during the resistance exercise bout?
2. Method
2.2 General design
Eleven men performed unilateral consecutive bouts of arm extensor aerobic exercise (~40 min) and resistance exercise (4 sets of 7 reps) interspersed by 15 mins recovery. The
contralateral limb performed resistance exercise only. Muscle biopsies were taken from the m. triceps brachii of each arm immediately before, 15 mins and 3 hrs after resistance exercise (Fig. 2). Peak concentric power was measured during the resistance exercise bout, and muscle samples were assessed for gene expression of muscle growth regulators.
2.3 Subjects
The subjects were 11 healthy male volunteers (181 cm ± 6 cm, 81 ± 8 kg, and 28 ± 5 years). They were recreationally active individuals who generally performed either aerobic or resistance exercise 2-3 days/week. All of the subjects had previous experience of resistance training. The study experiments and procedures were explained before subjects gave their
written informed consent to participate. The study was approved by the Regional Ethical Review Board in Stockholm.
2.4 Exercise equipment and description
Aerobic exercise was carried out in an Isokinetic dynamometer (Biodex) with isotonic resistance (Figure 2A). Subjects performed concentric arm extensions in a seated, upright position with their exercising upper arm strapped to a resting pad. The eccentric portion of the movement (elbow flexion) was unresisted. The flexion movement was assisted by the
researcher to minimize fatigue. The resistance exercise equipment consisted of a flywheel ergometer (Exxentric™, Bromma, Sweden) which offers inertial resistance (0.025 kg/m2) during coupled eccentric and concentric actions. The ergometer was placed upside down on a bracket attached to the wall of which the subjects were placed underneath and performed the triceps “pushdown” exercise (Figure 2B). Subjects were instructed to stay tall, not lean forward, and keep their upper arm parallel to their upper body. To ensure eccentric overload, subjects were instructed to break hard after they had passed a 90-degree angle in the elbow joint. Concentric peak power was sampled for each repetition using a sensor attached to the flywheel (kMeter, Exxentric™, Bromma, Sweden).
2.5 Pre-testing and familiarization
Subjects were required to attend to a total of two sessions before the test day. During the first session, subjects filled out a health form, signed the informed consent, described their training background and the study were described verbally by the researcher. They were also
familiarized with the training protocol and equipment. This familiarization consisted of 3 sets of 3-5 submaximal reps in the flywheel ergometer and 2-3 minutes of exercise for each arm in the dynamometer. During the second session, subjects performed a warm-up (1 x 5 at 80-90% effort) and then performed 2 x 7 maximal reps to determine dominant arm and baseline concentric power data was collected. After the dominant arm was determined, the arms were randomised to either RE or AE + RE. Subjects then performed an incremental workload test with the arm chosen for AE + RE to determine maximal workload (MWL). During the
workload test, resistance increased by 2 N each 30 secs (starting at 1 N) until an RPE of 18-20 and/or that the participant could not keep up with the prescribed RPM. Between the
familiarization days and the test day, subjects were instructed to not engage in any upper body resistance exercise. Before the test day, subjects were instructed to refrain from any upper body resistance training 48 h and any strenuous exercise 24 h before the experimental trial. Subjects were recommended to eat their habitual breakfast (but ensuring both carbohydrates and proteins were consumed) about 1 h before arriving to the lab, and they were not allowed to consume any food between biopsies on the test day.
2.6 Exercise protocol
All experimental trials were performed in the morning between 7 and 9 am, with a minimum of 48 h after the last familiarization trial. The AE + RE arm was first subjected to 40 minutes of aerobic exercise in the isokinetic-dynamometer with a workload set to 70% of the MWL using a cadence of 60 RPM. During the bout of aerobic exercise, ratings of perceived exertion (RPE) and heart rate (HR) were collected to ensure that the exercise was sufficiently
strenuous. After 40 minutes, resistance increased by 2 Nm each 30 secs until an RPE of 18-20 and/or that the participant could not keep up with the prescribed RPM. After 15 min rest, subjects performed unilateral resistance exercise (4 x 7 reps) alternating between arms on each set. All the subjects performed resistance exercise with their right arm first. Between each set, there was a 2-minute rest period. A standardized warm-up (1 x 5 at 80-90% effort) preceded the resistance exercise bout for both arms. Subjects were verbally encouraged during exercise bouts to ensure maximal effort during each repetition. Average concentric peak
power was sampled for each repetition using a sensor attached to the flywheel (kMeter, Exxentric™, Bromma, Sweden).
Figure 2. Schematic overview of the study protocol. AE, aerobic exercise; RE, resistance
exercise; B, biopsy.
2.7 Muscle biopsies
Muscle biopsies were obtained from the m. triceps brachii under local anaesthesia immediately before (PRE), 15 mins (POST) and 3 hrs after (POST3) the acute resistance exercise bout of either arm (Fig. 2). The 3-hr time point was chosen to accommodate for changes in both protein phosphorylation and gene expression. Biopsies were always taken from the right arm first. After a small incision on the skin, a conchotome (Patel et al., 2011) was used to obtain ~50 mg of tissue sample. Subsequent biopsies were obtained from separate incisions, moving in direction distal to proximal. Samples were cleansed from excess blood, fat, and connective tissue before being frozen in liquid nitrogen and stored at -80°C.
2.8 Gene expression analysis
RNA extraction. One piece of ~20 mg frozen muscle tissue was put in the Mini-bead beater along with 1ml of TRIzol (Invitrogen Life Technologies, Carlsbad, CA) and zirconia beads. To separate the RNA, 200 µl of chloroform was added and the tube was centrifuged. After the
separation, the RNA was put into a separate tube and 450 µl of Isopropanol was added to precipitate the RNA. After incubation and centrifugation, the supernatant was discarded and 1 ml of ethanol (75 %) was added to the tube to wash the pellet. After the last centrifugation, the ethanol was discarded, and the tube was cleansed with a pipette and pellets were then put to dry before 20 µl of DH2O was added. After RNA had been extracted, the amount of RNA per 1 µl of dilution was determined in the NanoDrop (ThermoFisher).
cDNA reverse transcription. One microgram of total RNA was added to each well of a
PCR-plate. D2HO was added to the remaining volume up to 10 µl. Finally, 10 µl of High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was added. The plate was then short-spinned and put into the PCR machine, which ran at 25°C for 10 mins, 37°C for 120 mins, and lastly 85°C for 5 mins. Before removal, the plate was chilled to 4°C.
Real-time PCR. Real-time PCR was performed on ABI-PRISMA 7700 Sequence Detector
System (Perkin-Elmer Applied Biosystems, Foster City, CA). All reactions were performed using MicroAmp® Fast Optical 96-well reaction plates (Life Technologies). Amplification mixes (10 µL) contained the diluted (1:100) cDNA sample (4.5 µl), TaqMan® Fast Universal PCR Master Mix (5.0 µl) and specific primers (0.5 µl). The cycling procedures were 2 min at 50°C and 10 min at 90°C followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. TaqMan primers for atrogin-1 (Hs01041408_m1), MuRF-1 (Hs00822397_m1), myostatin (Hs00193363_m1), and PGC-1a (Hs01016724_m1) were derived from the TaqMan® Gene Expression Assays (Applied Biosystems). Samples from each individual were assayed on the same plate. Rps18 (HS01375212_g1) was used as the reference gene. For further control, GAPDH (Hs99999905_m1) were analysed as an additional reference gene. Gene expression levels were determined using the 2-ΔCT method, relating mRNA changes as a ratio to the reference gene.
2.9 Statistics
Gene expression were analysed by two-way repeated-measures ANOVA (factors: time and arm). Data skewness was assessed through histograms and the Shapiro-Wilk test. When two-way interaction was found significant, a priori planned simple effect comparison within each level was performed. The false discovery rate (FDR) procedure was employed to adjust for these comparisons (Curran-Everett, 2000). A paired samples t-test was used to detect
differences in concentric power output. Significance was accepted at the 5% level (P < 0.05). Data are presented as means ± SD.
2.91 Validity and reliability
In order to measure power during resistance exercise we used the kMeter (Exxentric™). The kMeter system consists of a module which measures the rotation of the flywheel, and
monitors power, energy and estimates force during flywheel resistance exercise.
Unfortunately, no study has yet been conducted to validate this system. Muscle biopsies were obtained from the m. triceps brachii. Repeated muscle biopsies may be affected by previously taken biopsies (Aronson et al., 1998), however separating subsequent incisions with ~3 cm seem to minimize effects on muscle signalling elicited by muscle injury (Guerra et al., 2011). When quantification of genes are conducted, TaqMan, in comparison to SYBR Green, is generally held to be more specific in quantifying the gene of interest (Tajadini et al., 2014). We compared the relative expression levels of genes to a housekeeping gene by using the
2-ΔCT method. It is generally held that that both relative and absolute quantification of the chosen gene are valid ways to present the expression of selected genes (Livak and Schmittgen, 2001).
3. Results
3.2 Workload test and baseline data
Workload during the incremental test amounted to an average of 26 ± 7 N, which lasted 6:14 min ± 23 sec. During resistance exercise, the RE arm produced an average of 94 ± 42 W and AE + RE 96 ± 39 W. There were no significant differences between limbs in baseline
measurements (P > 0.05).
3.3 Exercise characteristics
Average workload during 40 min aerobic exercise was 12 ± 5 N. Workload increased to 22 ± 7 N during the final increment to exhaustion, which lasted 2:37 min ± 37 sec (Figure 3A). RPE were 15 during the 40 min bout and increased to 19 after the final increment (Figure 3B). Average HR during aerobic exercise was 98 ± 8 beats/min and amounted to 126 ± 15
beats/min after the final exercise stage (Figure 3C). In the subsequent resistance exercise, the AE + RE showed 12% lower (78 ± 37 W; P > 0.05) average peak concentric power than the RE arm (88 ± 40 W) (Figure 4).
12
Figure 3. A-C: Mean workload, RPE and HR during the aerobic exercise bout.
Figure 4. Mean concentric peak power with (AE + RE) or without (RE) preceding aerobic
3.4 Gene expression
There was an arm * time interaction effect on PGC1a expression (F = 21.7, P < 0.0005; Figure 7A), since the expression were greater from POST to POST3 for AE + RE (6.0-fold, P < 0.0005) compared to RE (2.0-fold, P = 0.005). Likewise, there was an interaction effect for MuRF-1 (F = 9.2, P = 0.001), due to greater expression in AE + RE from PRE to POST (2.3-fold, P = 0.006) and PRE to POST3 (4.0-(2.3-fold, P = 0.001), compared to RE (0.8-(2.3-fold, P > 0.05 and 1.71-fold P > 0.05). Atrogin-1 (F = 4.1, P = 0.032) showed an interaction effect due to a general decrease in RE (0.8-fold, P = 0,006) and a tendency for increased expression in AE + RE (1,2-fold, P = 0.083) from PRE to POST. Expression of myostatin did not display an interaction effect, however expression generally decreased in both AE + RE and RE as evident by the main effect of time (F = 7,95, P = 0.003).
Figure 5. A-D: PGC1a, Myostatin, MuRF-1, and Atrogin-1 before (PRE), 15 minutes after
(POST) and 3 h after (POST3) resistance exercise with (AE+RE) or without (RE) preceding aerobic exercise. Significant effects (P < 0.05): a = interaction, b = leg, c = time. Significant
effects (P < 0.05) within timepoint vs. ×opposite arm, *within arm vs. PRE, #within arm vs. POST.
4. Discussion
The current study investigated whether functional and molecular outcomes of an acute bout of resistance exercise were compromised if preceded by a 40 min aerobic exercise bout in the arm extensor musculature. Our novel results suggest that concurrent exercise of the arm extensors induces a molecular response that differs from resistance exercise alone. More specifically, given the generally greater molecular signal induced by AE + RE, it is suggested that concurrent exercise favoured more extensive tissue remodelling than RE. Moreover, aerobic exercise performed prior to resistance exercise did not alter power production during subsequent resistance exercise.
The average concentric peak power output was not statistically different between limbs. However, there was a rather unclear variation between groups and the AE + RE limb did indeed show an average reduction in power of ~12 %. The execution and technique of exercise were closely monitored by experienced research staff at all occasions, nonetheless, the variance may be due to inconsistent execution between the arms. Inevitably, more
participants would have decreased the effect of this variance on the statistical output. As such, it is unclear whether the lack of significance is a ‘real finding’ or simply a lack of power, since this would argue with most research conducting aerobic and resistance exercise in close proximity (Bentley et al., 2000; Sporer and Wenger, 2003). The average reduction seen in the concurrent exercised limb is indeed of a similar magnitude to that seen in leg-extension exercise in previous work (Lundberg et al., 2014) and congruent with adaptations to aerobic and resistance exercise, when performed in close succession, also show compromised force output (Craig et al., 1991; Lundberg et al., 2014; Robineau et al., 2016).
To answer whether specific mRNA markers would be altered with the addition of an aerobic exercise stimuli, we measured gene expression from muscle samples taken from the m. triceps brachii on three different time-points from both limbs. Concurrent exercise prompted a
marked increase of PGC-1a expression, a common response to stimulus induced by aerobic and concurrent exercise, reflecting a molecular response favouring mitochondrial biogenesis (Baar, 2014; Coffey and Hawley, 2007; Fyfe et al., 2014). Recent work suggests that the resistance exercise-induced upregulation of the a4 truncated splice variant of PGC1-a (PGC1-a4), may play a role in the anabolic signalling response to resistance exercise by inducing
insulin growth factor 1 and repressing myostatin (Ruas et al., 2012). However, its role in regulating muscle mass is questionable since some work suggest that mice lacking all isoforms of PGC-1a still undergo hypertrophy (Perez-Schindler et al., 2013) and isoform upregulation of PGC-1a does not seem to be exercise mode-specific (Lundberg et al., 2014; Ydfors et al., 2013).
The limb that performed concurrent exercise showed a more profound downregulation of myostatin. As downregulation of myostatin have been shown to be important in inducing hypertrophic adaptations after resistance exercise (McNally, 2004), this may suggest that a combined bout of resistance and aerobic exercise augments anabolic signalling, at least in the early phase. In comparison to the lower limbs, earlier work have also suggested augmented anabolic molecular responses after concurrent exercise compared to resistance exercise alone (Carrithers et al., 2007; Fernandez-Gonzalo et al., 2013; Lundberg et al., 2014). Compared to earlier work by Lundberg and co-workers (2014), however, the downregulation of myostatin are less profound, as no interaction effect was found. Indeed, it has been suggested by Coffey and Hawley (2017) that the acute response to exercise may be dependent on contraction-volume, rather than being mode-specific per se. In support of this, Donges et al. (2012) studied untrained subjects that either performed a single bout of single-mode resistance or aerobic exercise, or a combined bout equivalent to 50% of that performed during single-mode bouts. They reported comparable increases in myofibrillar protein synthesis between the concurrent bout and resistance exercise alone (Donges et al., 2012), suggesting that facilitated anabolic molecular signaling prompted by the concurrent exercise bout may be caused by the greater volume.
At the transcriptional level, the ubiquitin proteins MuRF-1 and atrogin-1 showed a profound expression in the concurrent, but not resistance, exercised limb. This is line with previous work, suggesting that protein degradation processes are augmented in response concurrent exercise (Fernandez-Gonzalo et al., 2013; Kazior et al., 2016; Lundberg et al., 2014). Work in both animal and human models suggest that adaptations to exercise are reliant on protein degradation to enable muscle remodelling (Cunha et al., 2012; Hwee et al., 2014; Stefanetti et al., 2015). Thus, the augmented activity in the ubiquitin pathway and myostatin suggests a facilitated global muscle remodelling process. Indeed, the earlier work conducted by
Lundberg and co-workers (2014), similarly suggested that concurrent exercise downregulated myostatin and upregulated activity in the ubiquitin pathway in the lower limbs. However,
although the magnitude of suppression was similar in MuRF-1 mRNA expression, the greater upregulation of atrogin-1 suggested greater protein turnover in the lower limbs (Lundberg et al., 2014) compared with the arms in this study.
Thus, the gene expression data suggest a difference in the degree to which concurrent exercise facilitates anabolic molecular and remodelling response in the arm extensors compared to the knee extensors. Congruent with these findings, it has been documented that the vastus
lateralis, as opposed to the triceps brachii, have a higher percentage of the high oxidative slow-twitch type-I fibre (Mittendorfer et al., 2005), which have a higher capacity for protein synthesis and proteasome dependent protein degradation (Mittendorfer et al., 2005; van Wessel et al., 2010). The arm musculature may similarly reach an earlier plateau in muscle growth from resistance exercise (Rønnestad et al., 2007; Wernbom et al., 2007), suggesting that the legs, but not arms, are dependent on higher training volumes to maximize muscle remodelling and training-induced adaptations prompted by exercise stimuli.
The expression of the four previously mentioned genes were used as proxy markers of
mitochondrial biogenesis, protein degradation, and anabolic signalling, we are also measuring specific mRNAs involved in ribosomal biogenesis. As recently suggested by Fyfe and
colleagues (2018), decreased ribosomal content may be a potential candidate for long-term interference of adaptations to resistance exercise training. Moreover, we are also looking into protein phosphorylation of specific proteins related to metabolic perturbations, MPS and mechano-transduction. While these measurements were not completed in time to be included in this thesis, together, they will provide a more complete picture of the molecular response to concurrent exercise of the elbow extensors.
The current study employed an identical aerobic exercise stimulus previously used in the lower limbs (Lundberg et al., 2014). The purpose of this stimuli is to serve as a local aerobic stimulus, as opposed to stress on the cardiovascular system. Previous results suggest that this type of aerobic exercise does indeed reduce glycogen content and increase endurance capacity and citrate synthase activity in the exercised limb (Lundberg et al., 2014). We chose to use a flywheel ergometer to impose the resistance exercise stimuli. The iso-inertial device offers the capability to produce unlimited resistance (via inertia) during the entire range of motion. If the concentric phase is performed with maximal intention, and the trainee uses an appropriate technique, i.e. resisting the inertial force gently during the first third of the eccentric action,
and then applying maximal effort to stop the movement at the end of the range of motion, eccentric overload can be produced in force/power values (Maroto-Izquierdo et al., 2017). More specifically, the current resistance exercise protocol consisted of 4 sets of 7 repetitions, which is known to induce robust muscle growth and power increases (Lundberg et al., 2013; Lundberg et al., 2014).
We chose to subject participants to unilateral exercise to assess the effects of concurrent stimuli (MacInnis et al., 2017). Conducting within-subject comparisons increases statistical power as each individual serve as their own control. Further highlighting the strength of the unilateral design, the variation of resting gene expression may vary between the limbs of two individual participants up to five times more than within one individual (Lindholm et al., 2014). As variability in nutritional and training status of participants may play a key role in deciding concurrent exercise induced outcomes (Coffey and Hawley, 2017; Murach and Bagley, 2016), these variables are less likely to influence in within-comparisons. Exercise may cause a release in hormones that enters the circulation, thus increasing the risk of contamination in with-subject comparisons (MacInnis et al., 2017; West and Phillips, 2012). However, this likely has no effect on acute measures (West and Phillips, 2012, 2010) and biochemical signalling molecules (i.e mRNA, proteins) do not show measurable signs of transfer between limbs (MacInnis et al., 2017).
In conclusion, aerobic and resistance exercise, interspersed by 15 minutes recovery, induces greater expression of markers involved in the muscle remodelling process and anabolic signalling in the arm extensor musculature compared to resistance exercise alone.
Inconclusive results suggested that force output was not altered when resistance exercise was preceded by aerobic exercise. Furthermore, the current results suggest that concurrent exercise of the elbow extensors in recreationally active individuals respond similar to the knee
extensors in the upregulation of specific gene expression markers. However, compared to the legs, myostatin repression and upregulation of atrogin-1 by the concurrent exercise may be less profound in the arms. Collectively, the current study highlights that muscle molecular responses to lower body concurrent exercise protocols might differ from upper body exercise. Thus, if the focus for an individual is on upper-body muscles, one should be cautious when making inferences from concurrent training studies using leg muscles.
18
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Appendix 1 – Literature search
Purpose
Question formulation
1. Will the addition of aerobic exercise interfere with the anabolic molecular response to resistance exercise?
2. Will the short rest period between aerobic and resistance exercise compromise performance during resistance exercise?
Which words have you been using during your literature search?
‘concurrent training’, ‘concurrent exercise’, ‘combined resistance and aerobic training’, ‘combined resistance and aerobic exercise’, ‘upper body concurrent training’, ‘upper body concurrent exercise’
