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Attenuated PGC-1α Isoforms following Endurance Exercise with Blood Flow Restriction

CONCEIÇÃO, MIGUEL SOARES; CHACON-MIKAHIL, MARA PATRICIA TRAINA; TELLES, GUILHERME DEFANTE; LIBARDI, CLEITON AUGUSTO; JÚNIOR, EDSON MANOEL MENDES; VECHIN, FELIPE CASSARO; DE ANDRADE, ANDRÉ LUIS LUGNANI; GÁSPARI, ARTHUR FERNANDES; BRUM, PATRÍCIA CHAKUR; CAVAGLIERI, CLÁUDIA REGINA; SERAG, SARA; SPIEGELMAN, BRUCE M.; HAWLEY, JOHN A.; CAMERA, DONNY M.

Medicine & Science in Sports & Exercise: September 2016 - Volume 48 - Issue 9 - p 1699–1707
doi: 10.1249/MSS.0000000000000970
BASIC SCIENCES
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Introduction Exercise performed with blood flow restriction simultaneously enhances the acute responses to both myogenic and mitochondrial pathways with roles in training adaptation. We investigated isoform-specific gene expression of the peroxisome proliferator-activated receptor gamma coactivator 1 and selected target genes and proteins regulating skeletal muscle training adaptation.

Methods Nine healthy, untrained males participated in a randomized, counterbalanced, crossover design in which each subject completed a bout of low-intensity endurance exercise performed with blood flow restriction (15 min cycling at 40% of V˙O2peak, BFR-EE), endurance exercise (30 min cycling at 70% of V˙O2peak, EE), or resistance exercise (4 × 10 repetitions of leg press at 70% of one-repetition maximum) separated by at least 1 wk of recovery. A single resting muscle biopsy (vastus lateralis) was obtained 2 wk before the first exercise trial (rest) and 3 h after each bout.

Results Total PGC-1α mRNA abundance, along with all four isoforms, increased above rest with EE only (P < 0.05) being higher than BFR-EE (P < 0.05). PGC-1α1, 2, and 4 were higher after EE compared with resistance exercise (P < 0.05). EE also increased vascular endothelial growth factor, Hif-1α, and MuRF-1 mRNA abundance above rest (P < 0.05), whereas COXIV mRNA expression increased with EE compared with BFR-EE (P < 0.05).

Conclusion The attenuated expression of all four PGC-1α isoforms when EE is performed with blood flow restriction suggests this type of exercise provides an insufficient stimulus to activate the signaling pathways governing mitochondrial and angiogenesis responses observed with moderate- to high-intensity EE.

1Faculty of Physical Education, University of Campinas, Campinas, BRAZIL; 2Laboratory of Neuromuscular Adaptations to Resistance Training, Department of Physical Education, Federal University of São Carlos, São Carlos, BRAZIL; 3School of Physical Education and Sport, University of São Paulo, São Paulo, BRAZIL; 4Department of Cell Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; 5Mary MacKillop Institute for Health Research, Centre for Exercise and Nutrition, Australian Catholic University, Melbourne, AUSTRALIA; and 6Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, England, UNITED KINGDOM

Address for correspondence: Miguel Soares Conceição, Ph.D., Faculty of Physical Education, University of Campinas, Av. Érico Veríssimo, 701, Cidade Universitária “Zeferino Vaz” Barão Geraldo, Campinas, São Paulo, CEP: 13083-851, Brazil; E-mail: conceicao.miguel0106@gmail.com.

Submitted for publication February 2016.

Accepted for publication April 2016.

Skeletal muscle is a highly malleable tissue that can alter its phenotype according to the contractile stimulus imposed (39). For instance, moderate-intensity (i.e., <65% of peak oxygen uptake (V˙O2peak)) endurance exercise (EE) training enhances whole-body V˙O2peak (4), increases the maximal activities of oxidative enzymes, and shifts patterns of substrate selection from carbohydrate- to fat-based fuels (18). In contrast, strenuous (80% of one-repetition maximum (1RM)) resistance exercise (RE) has little or no effect on whole-body V˙O2peak and oxidative enzyme profiles (11) but increases myofibrillar protein accretion and muscle cross-sectional area (CSA) (30).

Although RE and EE could be considered at opposite ends of the “adaptation continuum” by virtue of their divergent biochemical and morphological phenotypes, blood flow restriction during low-intensity EE (BFR-EE) improves V˙O2peak, muscle strength, and CSA (1,2). Abe et al. (1) reported increased isometric muscle strength, muscle CSA, and V˙O2peak following 8 wk (24 training sessions) of low-intensity cycle exercise (15 min at 40% V˙O2peak) performed with BFR-EE compared with the same exercise undertaken without BFR. Although these adaptation responses are considerably lower in magnitude relative to conventional endurance and resistance training performed at higher intensities, the local hypoxia induced by BFR appears to induce an additive “metabolic stressor” that perturbs cellular homeostasis (17) and concomitantly enhances both anabolic and oxidative adaptations.

The cellular mechanisms mediating adaptation responses to exercise are complex, involving the crosstalk of several intracellular signaling systems that ultimately form the basis for specific phenotypic responses with divergent contractile modes (17). The transcriptional coactivator peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1α (PGC-1α) is a “master regulator” of many EE-induced adaptations by virtue of its central role in promoting mitochondrial biogenesis, angiogenesis, and inflammatory proteins (17). Transcription of the PGC-1α gene has been shown to be under the control of several promoter regions with activation of the alternative PGC-1α1 promoter resulting in the transcription of three additional isoforms: PGC-1α2, α3, and α4. Ruas et al. (32) recently demonstrated a preferential increase in the PGC-1α4 isoform following RE in human skeletal muscle. However, little is known about the regulation of the α2 and α3 isoforms, and to date, no studies have investigated the expression of all four PGC-1α isoforms to diverse contractile stimuli such as RE and EE, or following BFR, in humans. Accordingly, the aim of the present study was to compare the acute molecular responses mediated by the different PGC-1α isoforms following low-intensity EE (BFR-EE), RE, and moderate EE. Because BFR-EE can promote both endurance capacity and muscle hypertrophy responses, we hypothesized that EE and RE would selectively increase the expression of the PGC-1α1 and α4 isoforms, respectively. In contrast, we hypothesized that BFR-EE would upregulate a molecular signature involving the increase of both isoforms and their respective anabolic and mitochondrial gene targets.

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METHODS

Subjects

Nine untrained, healthy male subjects (age, 22.4 ± 3.0 yr; body mass (BM), 73.5 ± 9.7 kg; height, 1.79 ± 0.05 m; maximal oxygen uptake test (V˙O2peak), 36.8 ± 4.8 mL·kg−1·min−1; leg press 1RM, 266 ± 66 kg; values are presented as mean ± SD) voluntarily participated in this study. The experimental procedures and possible risks associated with the study were explained to all subjects, who provided written informed consent before participation. The study was approved by the local university’s ethics committee and conducted in conformity with the policy statement regarding the use of human subjects according to the latest revision of the Declaration of Helsinki.

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Experimental Design

The study employed a randomized counterbalanced, crossover design in which each subject completed a bout of RE, endurance cycling exercise (EE), or low-intensity cycling exercise combined with blood flow restriction (BFR-EE). Two weeks before the first exercise session, a resting muscle biopsy was obtained before participants underwent V˙O2peak and 1RM testing, and exercise familiarization. Exercise trials were separated by a 1-wk recovery period during which time subjects maintained their habitual diet and physical activity patterns.

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Preliminary Testing

V˙O2peak

Participants performed a maximum graded exercise test on a cycle ergometer with electromagnetic braking (Quinton modelo: Corival 400; Lode BV, Groningen, The Netherlands) based on a protocol used in a previously published article that investigated BFR-EE (1). Briefly, after resting on the bike for 5 min, participants commenced the incremental test protocol. Briefly, subjects commenced cycling at an initial load of 50 W for 1 min, and the workload was increased by 15 W·min−1 until a workload of 200 W was reached, after which further increases were in 10 W·min−1 increments. The test continued until voluntary exhaustion, defined by two of the three following criteria: V˙O2peak plateau (<2.1 mL·kg−1·min−1 of variation), >1.10 RER, and/or HR higher than 90% of the maximum estimated from age (19). Gas exchange data were collected continuously using an automated breath-by-breath metabolic system (CPX; Medical Graphics, St. Paul, MN), and the highest oxygen consumption value was defined as the peak oxygen consumption (V˙O2peak) over any 30-s period. To confirm the appropriateness of this protocol for this study, we performed a pilot study to verify repeatability in V˙O2peak measures and observed a strong repeatability in V˙O2peak (3.0%), power (1.9%), RER (5.6%), and time to exhaustion (1.6%) measures.

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Maximal Strength

The 1RM test was performed on a leg press machine (45° leg press, G3-PL70; Matrix, São Paulo, Brazil) as previously described (8). Briefly, participants performed a 5-min warm-up on a cycle ergometer riding at 25 W. Participants then undertook 1 × 10 repetitions at 50% of their estimated 1RM, followed by 1 × 3 repetitions at 70% of the estimated 1RM with 1-min rest between sets. Participants then performed a series of single repetitions until the maximum load (1RM) lifted was established with complete eccentric–concentric movement with 90° range of motion. Repetitions were separated by a 3-min recovery and were used to establish the maximum load/weight that could be moved through the full range of motion once, but not a second time.

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Diet/Exercise Control

Before each experimental trial (described subsequently), subjects were instructed to refrain from exercise training and vigorous physical activity, and alcohol and caffeine consumption for a minimum of 48 h. Subjects were provided with standardized prepacked meals that consisted of 3 g of carbohydrate per kilogram BM, 0.5 g of protein per kilogram BM, and 0.3 g of fat per kilogram BM consumed as the final caloric intake the evening before reporting for an experimental trial.

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Experimental Testing Sessions

On the morning of an experimental trial, subjects reported to the laboratory after an approximately 10-h overnight fast. After resting in the supine position for approximately 15 min and under local anesthesia (2–3 mL of 1% Xylocaine), a resting biopsy was obtained from the vastus lateralis using a 5-mm Bergstrom needle modified with suction (7). Approximately 100 mg of muscle was removed, dissected free from blood and connective tissue, and snap frozen in liquid nitrogen before being stored at −80°C until subsequent analyses. Because of ethical constraints regarding the total number of muscle biopsies allowed, this single resting biopsy was used as a basal control for all subsequent exercise trials. Two weeks later, participants returned to the laboratory having (after the same pretrial diet and exercise control) to undertake the first of three randomly assigned exercise sessions (described below). Each exercise trial was separated by a 1-wk washout. Following the completion of each exercise session, subjects rested for 180 min after which time a muscle biopsy was obtained. Subsequent incisions were performed 3 cm proximal to each other. Blood samples were collected before each exercise session and immediately, 1, 2, and 3 h postexercise. Blood samples were immediately placed in microtubes containing 1% sodium fluoride and then centrifuged at 3000 rpm for 5 min to separate the plasma before being aliquoted and frozen in liquid nitrogen and stored at −80°C.

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RE

After a standardized warm-up on a cycle ergometer consisting of 5-min light cycling at 25 W, subjects performed 4 sets of 10 repetitions of leg press exercise (45° leg press machine; G3-PL70, Matrix) at 70% of 1RM. Each set was separated by a 1-min recovery period during which time subjects remained seated on the leg press machine. Complete concentric/eccentric movements were performed with 90° range of motion, and strong verbal encouragement was provided during each set. The volume and intensity of this session was based on the recommendations of the American College of Sports Medicine (ACSM) (3). All participants completed every repetition from each respective set.

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EE

Following a standardized warm-up (described previously), subjects performed 30 min of continuous cycling at a power output that elicited at approximately 70% of individual V˙O2peak. Subjects were fan cooled and provided visual feedback for pedal frequency, power output, and elapsed time. The volume and intensity of this session were based on the recommendations of ACSM (4). All participants completed the full 30-min session.

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Low-Intensity Blood Flow Restriction

Subjects performed 15 min of continuous cycling with a cuff strapped over the thigh at a power output that elicited at 40% of V˙O2peak, as previously reported (1). An 18-cm-wide cuff was placed on the proximal portion of the thigh (inguinal fold region) over the tibial artery and, once in position, was inflated until an absence of auditory blood pulse detected through auscultation with a vascular Doppler probe (DV-600; Marted, São Paulo, Brazil). Pressure was then slowly released until the first arterial pulse was detected, which was considered the systolic pressure at the tibial artery. Cuff pressure was set at 80% of the maximum tibial arterial pressure, and the cuff was inflated throughout the entire exercise session (22).

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Analytical Procedures

Blood lactate

Plasma lactate concentration was measured on a spectrophotometer (ELx800; Biotek, Winooski, VT) using a commercial kit (Biotecnica, Varginha, Brazil) according to the manufacturer’s protocol.

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RNA extraction and quantification

Approximately 20 mg of skeletal muscle was homogenized in TRIzol with chloroform added to form an aqueous RNA phase. This RNA phase was then precipitated by mixing with ice-cold isopropanol alcohol, and the resulting pellet was washed and resuspended in 40 μL of RNase-free water. Extracted RNA was quantified using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) by measuring absorbance at 260 and 280 nm.

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Reverse transcription

First-strand complementary DNA (cDNA) synthesis was performed using commercially available TaqMan Reverse Transcription Reagents (Invitrogen, Melbourne, Australia) in a final reaction volume of 20 μL. All RNA and negative control samples were reverse transcribed to cDNA in a single run from the same reverse transcription master mix. Serial dilutions of a template human skeletal muscle RNA (AMBION; Cat. No. AM7982) was included to ensure efficiency of reverse transcription and for calculation of a standard curve for quantitative real-time polymerase chain reaction (PCR).

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Real-time PCR

Quantification (in duplicate) of mRNA was performed using a CFX96 Touch™ real-time PCR Detection System (Bio-Rad, California). TaqMan-FAM–labeled primer/probes for muscle ring finger 1 (MuRF-1) (Cat. No. Hs00822397_m1), COXIV (Cat. No. Hs00971639_m1), IL-6 (Cat. No. Hs00985639_m1), PGC-1α (Cat. No. Hs01016719_m1), HIF-1α (Cat. No. Hs00153153_m1), Myostatin (Hs00976237_m1), insulin-like growth factor 1 (IGF-1) (Hs01547656_m1), and vascular endothelial growth factor (VEGF) (Cat. No. Hs00900055_m1) were used in a final reaction volume of 20 μL. PCR treatments took 2 min at 50°C for uracil N-glycosylase activation, 10 min at 95°C then 40 cycles of 95°C for 15 s and 60°C for 60 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat. No. Hs02758991_g1) was used as a housekeeping gene and was stably expressed between exercise interventions (data not shown). The relative amounts of mRNAs were calculated using the relative quantification (△△CT) method (24). All TaqMan-based PCR experiments were performed in the Centre for Exercise and Nutrition laboratory at the Australian Catholic University.

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Quantification of PGC-1α isoforms

RNA was extracted from a separate piece of snap-frozen muscle (approximately 20 mg) using TRIzol (Invitrogen) and purified using QIAGEN RNeasy minicolumns. Reverse transcription was performed using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Real-Time quantitative PCR was carried out in a SYBR Green ER PCR Master Mix (Invitrogen)/384-well format using an ABI PRISM 7900HT (Applied Biosystems). Relative mRNA levels were calculated using the comparative CT method and normalized to cyclophilin mRNA. Primer sequences are as follows: cyclophilin (forward: GGAGATGGCACAGGAGGAA; reverse: GCCCGTAGTGC TTCAGTTT), PGC-1α1 (forward: ATG GAG TGA CAT CGA GTG TGC T; reverse: GAG TCC ACC CAG AAA GCT GT), PGC-1α2 (forward: AGT CCA CCC AGA AAG CTG TCT; reverse: ATG AAT GAC ACA CAT GTT GGG), PGC-1α3 (forward: CTG CAC CTA GGA GGC TTT ATG C; reverse: CAA TCC ACC CAG AAA GCT GTC T), and PGC-1α4 (forward: TCA CAC CAA ACC CAC AGA GA; reverse: CTG GAA GAT ATG GCA CAT). All SYBR Green-based PCR experiments were performed in the Department of Cell Biology laboratory at the Dana-Farber Cancer Institute, Harvard Medical School (USA).

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Western blots

Approximately 30 mg of muscle was homogenized in a buffer containing 50 mM Tris–HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM DTT, 10 μg·mL−1 trypsin inhibitor, 2 μg·mL−1 aprotinin, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride. After determination of protein concentration (Pierce Biotechnology, Rockford, IL), lysate was resuspended in a Laemmli sample buffer. Lysate was then resuspended in a Laemmli sample buffer with 40 μg of protein loaded onto 4%–20% Mini-PROTEAN TGX Stain-Free™ Gels (Bio-Rad, California). Postelectrophoresis gels were activated according to the manufacturer’s details (Chemidoc; Bio-Rad, Gladesville, Australia) and then transferred to polyvinylidene fluoride membranes. After transfer, a stain-free image of the polyvinylidene fluoride membranes for total protein normalization was obtained before membranes were rinsed briefly in distilled water and blocked with 5% nonfat milk, washed with 10 mM of Tris–HCl, 100 mM of NaCl, and 0.02% Tween 20, and incubated with primary antibody (1/1000) overnight at 4°C. Membranes were incubated with secondary antibody (1/2000), and proteins were detected via chemiluminescence (Amersham Biosciences, Buckinghamshire, UK; Pierce Biotechnology) and quantified by densitometry. All sample time points for each subject were run on the same gel. Polyclonal anti–phospho-mTORSer2448 (no. 2971), p70 S6KThr389 (no. 9206), adenosine monophosphate kinase (AMPK)Thr172 (no. 2531), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) 4E-BP1Thr37/46 (no. 9459), eEF2 eukaryotic translation elongation factor 2 (eEF2) eEF2Thr56 (no. 2331), and p53Ser15 (no. 9284) were purchased from Cell Signaling Technology (Danvers, MA). Volume density of each target protein band was normalized to the total protein loaded into each lane using stain-free technology (15) with data expressed in arbitrary units. Because of low sample availability, phosphorylated proteins were unable to be normalized to their respective total protein content and were therefore also normalized to stain-free protein levels.

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Statistical Analysis

Statistical analysis was performed using SAS version 9.3 for Windows (SAS Institute Inc., Cary, NC). Data normality and variance equality were assessed through the Shapiro–Wilk and Levene tests. One-way ANOVA with repeated measures (factor: condition) was performed for gene and protein expression analyses. A mixed-model ANOVA, assuming group and time as fixed factors and subjects as a random factor, was performed for blood lactate data. Tukey post hoc analysis was used for multiple comparison purposes when significant F-values were found. The significance level was set at P ≤ 0.05. Data are presented as mean ± SD.

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RESULTS

mRNA Expression

Total PGC-1α and isoforms

Total PGC-1α mRNA (Fig. 1A) increased with EE above rest (P < 0.0001), RE (P = 0.0013), and BFR-EE (P > 0.0001). There was a significant increase in PGC-1α1 mRNA with EE above rest (P = 0.0450), RE (P = 0.0069), and BFR-EE (P = 0.0349) (Fig. 1B). There was also a significant increase in PGC-1α2 mRNA (Fig. 1C) with EE above rest (P < 0.0001), RE (P = 0.0003), and BFR-EE (P < 0.0001). PGC-1α3 mRNA (Fig. 1D) increased with EE above rest (P = 0.0389). There was also an increase in PGC-1α4 mRNA (Fig. 1E) with EE above rest (P = 0.0035), RE (P = 0.0469), and BFR-EE (P = 0.0140).

FIGURE 1

FIGURE 1

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VEGF, COXIV, and HIF-1α

There was a significant increase in VEGF mRNA (Fig. 2A) with EE above rest (P = 0.0180) and RE (P = 0.0069). COXIV mRNA expression increased with EE above BFR-EE (P = 0.0550) (Fig. 2B). There was a significant increase in HIF-1α abundance with EE above rest (P = 0.0530) (Fig. 2C).

FIGURE 2

FIGURE 2

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IL-6, IGF-1, myostatin, and MuRF-1

IL-6, IGF-1, and myostatin mRNA expression were unchanged postexercise (Fig. 3A–C). There was a postexercise increase in MuRF-1 mRNA abundance with EE above rest (P = 0.0003), RE (P = 0.0256), and BFR-EE (P = 0.0007) (Fig. 3D).

FIGURE 3

FIGURE 3

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Cell Signaling

mTOR, p70S6K, 4E-BP1, and eEF2

There were no changes in mTORSer2448, p70S6KThr389, 4E-BP1Thr37/46, or eEF2Thr56 phosphorylation postexercise or between exercise groups (Fig. 4).

FIGURE 4

FIGURE 4

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AMPK and p53

AMPKThr156 and p53Ser15 phosphorylation was unchanged postexercise (Fig. 5).

FIGURE 5

FIGURE 5

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Plasma Lactate Concentration

Lactate concentration increased above rest immediately postexercise for all interventions (P < 0.0001 for all comparisons, Table 1). Lactate concentration remained elevated at 1, 2, and 3 h postexercise for EE and RE, and 1 and 2 h for BFR-EE (P < 0.0001 for all comparisons).

TABLE 1

TABLE 1

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DISCUSSION

Low-intensity (<50% of V˙O2peak) endurance training with blood flow restriction has been shown to concomitantly promote isometric muscle strength, muscle CSA, and V˙O2peak (1,2). Although these enhanced adaptation responses are considerably lower in magnitude compared with conventional RE or EE performed without any blood flow restriction, the underlying molecular mechanisms mediating these responses remain largely undefined. For the first time, we report that low-intensity endurance cycling exercise performed with blood flow restriction failed to increase PGC-1α expression to that commonly observed with “conventional” EE. Moreover, we show isoform-specific postexercise increases in the α4 isoform along with Hif-1α and VEGF mRNA expression following higher intensity EE without blood flow restriction. Taken collectively, our novel findings suggest that cycle exercise undertaken with blood flow restriction is unable to provoke the perturbations to cellular homeostasis necessary to induce activation of the cell signaling events regulating mitochondrial biogenesis and angiogenesis that take place with higher intensity EE without blood flow restriction.

A growing body of evidence suggests that exercise undertaken with blood flow restriction can enhance exercise adaptation. A recent meta-analysis reported that both low-load/intensity RE (20%–30% 1RM) and aerobic walking exercise performed with blood flow restriction can induce increases in muscle strength and hypertrophy, although with smaller gains compared with high-intensity RE alone (35). However, little is known about the molecular mechanisms mediating these responses when low-intensity EE is undertaken with blood flow restriction. As such, we compared the expression of key gene and protein targets implicated in a range of exercise adaptation responses such as hypertrophy, mitochondrial biogenesis, muscle proteolysis, substrate metabolism and angiogenesis between BFR-EE, and conventional bouts of RE and EE. We particularly focused on the four different full-length PGC-1α isoforms putatively implicated in anabolic and mitochondrial-related adaptation responses.

In agreement with previous studies (5,23,29), we observed significant increases in total PGC-1α mRNA following continuous EE performed at 70% of V˙O2peak. This increase in PGC-1α mRNA was concomitant with greater abundance of VEGF, a target of PGC-1α (37). However, in contrast to our original hypothesis, this response was absent following a bout of low-intensity EE (40% V˙O2peak) performed with blood flow restriction. In an attempt to identify possible mechanisms responsible for this attenuated PGC-1α response, we investigated IL-6 expression to determine whether an increase in the muscular inflammatory program was implicated in the blunted response. This hypothesis was based on previous data showing an inverse relationship between skeletal muscle PGC-1α and IL-6 expression (16). However, IL-6 mRNA expression postexercise was unchanged in all exercise groups, suggesting any acute increase in muscle inflammation caused by BFR-EE was not responsible for the reduced PGC-1α1 expression observed. We also investigated other cellular markers implicated in exercise adaptation responses that can regulate PGC-1α expression. AMPK is an intracellular “fuel gauge” that can phosphorylate PGC-1α and increase its transcriptional activity (36), whereas the apoptogenic protein p53 has emerged as another signaling regulator of skeletal muscle exercise-induced mitochondrial biogenesis and substrate metabolism that can translocate to the nucleus upon activation and induce PGC-1α expression (17). Phosphorylation of either of these protein targets was unaltered postexercise suggesting other molecular markers and/or physiological mechanisms may be responsible for the upregulation of PGC-1α with high-intensity EE. One plausible explanation for these discrepant findings may be the level of glycogen use between exercise sessions in our untrained subjects. We (10) and others (6,31) have shown greater postexercise PGC-1α expression with low compared with normal or high glycogen concentration, and although we did not measure muscle glycogen use in the current study because of limited muscle tissue availability, the longer duration and higher intensity exercise bout is likely to have induced greater glycogen depletion compared with the EE session performed with blood flow restriction.

Another possible explanation for the discrepancy in PGC-1α1 expression between the two endurance-based exercise bouts is the large differences in estimated energy expenditure. Exercise energy expenditure after BFR-EE was approximately fourfold less compared with the EE protocol with total energy expenditure positively associated with PGC-1α expression (r = 0.73, P = 0.039). Increased PGC-1α mRNA expression has been observed after 30 min of running compared with bouts of 20 and 10 min (37). Thus, total exercise-induced energy expenditure may be an overriding determinant of PGC-1α expression responses postexercise.

Low-intensity EE with BFR was also unable to induce the expression of PGC-1α4 compared with higher intensity EE without blood flow restriction. The PGC-1α4 isoform has been proposed to promote muscle hypertrophy by inducing IGF-1 expression and reducing the expression of myostatin, a negative regulator of muscle growth (32). The increase in PGC-1α4 mRNA expression with EE in the current study was mirrored by a small, nonsignificant, increase and decrease in IGF-1 and myostatin expression, respectively. Ruas et al. (32) were the first to show a selective increase in PGC-1α4 expression (concomitant with decreased myostatin abundance) with RE compared with EE in human skeletal muscle. However, this expression pattern was observed following 8 wk of whole-body resistance training. Thus, a limitation of our study is that we only incorporated a single bout of isolated leg press suggesting that longer training programs/exercise stimulus may be required to induce this selective PGC-1α4 response. Nonetheless, another recent publication reported increased truncated and nontruncated PGC-1α transcripts from both alternative and proximal promoter sites 2 h after an acute bout of RE that incorporated the same volume and intensity as our study (40). This indicates that the RE bout performed in our study was likely sufficient to induce the appropriate signal to increase the expression of this isoform; however, potential differences in postexercise biopsy timing between this study and ours (2 h vs 3 h) may explain why we did not observe this increase with RE.

Increased PGC-1α4 and VEGF expression has also been reported in primary myotubes treated under hypoxic conditions, suggesting low oxygen conditions to be favorable for the activation of this isoform (38). In the current study, the transcription factor Hif-1α, a key regulator of angiogenesis in situations of hypoxia (34), was unchanged after BFR-EE, whereas RE and EE induced twofold higher postexercise changes in lactate compared with BFR-EE. Although it is possible that a greater metabolic and hypoxic stimulus may be required to increase PGC-1α4 signaling, others have reported unchanged blood lactate following aerobic-based exercise with blood flow restriction (26). Moreover, the same occlusion protocol (15-min cycle at 40% V˙O2peak) has been shown to improve muscle volume and V˙O2peak during a chronic training intervention (1). Thus, it is possible that chronic exposure to this occlusion stimulus may be required to elicit increases in PGC-1α4 expression. Because this is the first study to investigate changes in Hif-1α after endurance cycling exercise with BFR, it is difficult to compare our results to those of previous investigations incorporating RE and BFR. However, we speculate that when performed with blood flow restriction, the lower contractile intensity associated with “conventional” EE compared with RE (or sprint) provides adequate blood flow to the exercising musculature and adjoining capillary beds to prevent tissue deoxygenation. Further studies comparing different low-intensity EE protocols with RE that incorporate blood flow restriction are required to corroborate this hypothesis.

Another novel finding from the current study was the postexercise increases in the PGC-1α2 and 3 isoforms. Similar to the α1 and α4 isoforms, both PGC-1α2 and α3 increased above rest with higher intensity EE and were significantly elevated compared with RE. Both isoforms are expressed in skeletal muscle and brown adipose tissue, although little is known about the regulatory targets of these isoforms and their capacity to mediate exercise adaptation responses (27). Based on the elevated response following EE compared with RE, we propose these isoforms to mediate physiological processes related to mitochondrial biogenesis and substrate metabolism.

Considering that low-load EE with BFR can increase muscle strength and hypertrophy (35), we also investigated markers of translation initiation, elongation, and muscle proteolysis. Previous studies have reported increases in mTOR and p70S6K phosphorylation that have formed the basis for enhanced rates of muscle protein synthesis following RE with blood flow restriction (13,14). Nonetheless, the phosphorylation status of these proteins as well as 4E-BP1 and eEF2 were unchanged 3 h postexercise in the current study. This is in agreement with the results of Ozaki et al. (28) who observed no changes in Akt, mTOR, or p70S6K phosphorylation following 20 min of treadmill walking performed with blood flow restriction despite a higher intensity exercise bout (55% V˙O2peak) compared with our protocol. Although our study design was somewhat limited by only having the single postexercise biopsy (9), this sampling time point was specifically chosen based on previous studies showing significant, and in some cases maximal, increases in PGC-1α mRNA expression in response to an exercise challenge (5,23). Future studies investigating EE undertaken with BFR-EE should include a time course of signaling responses to determine the optimal “window” for muscle sampling in subsequent investigations.

Several other factors including the width and pressure of cuff used during BFR must also be considered. Previous studies have reported smaller increases in muscle CSA when lower body resistance training is undertaken with BFR (compared with no BFR) at the site of the cuff (12,20). Although this indicates that a narrow cuff may be advantageous for promoting anabolic adaptation responses due to compressing less muscle tissue, a recent study comparing the effects of a wide versus narrow cuff reported similar increases in maximum strength and muscle cross-sectional area following 12 wk of unilateral elbow flexion performed at 20% of 1RM (21). Also, a recent study showed that there was no difference in either muscle strength or hypertrophy between different occlusion pressures (25). Thus, the use of a wider cuff, as used in our protocol, appears unlikely to attenuate chronic muscle anabolic responses. Regardless, these studies are currently only limited to BFR with RE. Future studies comparing these parameters when EE is performed with BFR are required. Finally, MuRF-1 mRNA expression increased post-EE, which resulted in a higher expression above EE with BFR and RE. MuRF-1 mediates the ubiquitin proteasome system by “labeling” cleaved myofibril segments for degradation (33). It is unclear whether this increase in expression with high-intensity EE represents general tissue remodeling, particularly considering that our participants were untrained and the unaccustomed contractile stimulus, or a greater induction of protein degradation.

In summary, this is the first study to investigate the molecular mechanisms mediating muscle adaptation responses to low-intensity endurance cycling exercise with blood flow restriction. The attenuated expression of all four PGC-1α isoforms when EE is performed with blood flow restriction suggests that this type of exercise is unable to induce the appropriate metabolic perturbation capable of activating the cell signaling machinery responsible for mitochondrial biogenesis and angiogenesis responses with moderate- to high-intensity EE. Longer training programs incorporating EE with BFR that correlate measurements of these molecular markers with functional adaptation responses such as changes in V˙O2peak and cycle time to fatigue will yield important information to the efficacy of this training method to enhance training adaptation and subsequently improve health outcomes in populations that may be unable to perform prolonged exercise.

The authors would like to express gratitude for the FAPESP (2014/00985-0) and FAEPEX for financial support. The writing of this publication was also supported by a Collaborative Research Networks grant awarded to J. A. Hawley (2013000443).

The authors declare no declare no conflicts of interest.

The results of the present study do not constitute endorsement by ACSM.

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Keywords:

MITOCHONDRIAL BIOGENESIS; CELL SIGNALING; SKELETAL MUSCLE; ADAPTATION; ANGIOGENESIS; HIGH-INTENSITY EXERCISE

© 2016 American College of Sports Medicine