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Neuromuscular Electrical Stimulation and Anabolic Signaling in Patients with Stroke Joni A. Mettler,

PhD,*

Sydney M. Bennett, MS,* Barbara M. Doucet, Dillon M. Magee, MS*

PhD,†

and

Introduction: Stroke results in limited ability to produce voluntary muscle contraction and movement on one side of the body, leading to further muscle wasting and weakness. Neuromuscular electrical stimulation is often used to facilitate involuntary muscle contraction; however, the effect of neuromuscular electrical stimulation on muscle growth and strengthening processes in hemiparetic muscle is not clear. This study examined the skeletal muscle anabolic response of an acute bout of neuromuscular electrical stimulation in individuals with chronic stroke and healthy older adults. Methods: Eleven individuals (59.8 ± 2.7 years old) were divided into a chronic stroke group (n = 5) and a healthy older adult control group (n = 6). Muscle biopsies were obtained before and after stimulation from the vastus lateralis of the hemiparetic leg for the stroke group and the right leg for the control group. The neuromuscular electrical stimulation protocol consisted of a 60-minute, intermittent stimulation train at 60 Hz. Phosphorylation of mammalian target of rapamycin and ribosomal protein S6 kinase beta-1 were analyzed by Western blot. Findings: An acute bout of neuromuscular electrical stimulation increased phosphorylation of mammalian target of rapamycin (stroke: 56.0%; control: 51.4%; P = .002) and ribosomal protein S6 kinase beta-1 (stroke: 131.2%; control: 156.3%; P = .002) from resting levels to postneuromuscular electrical stimulation treatment, respectively. Phosphorylated protein content was similar between stroke and control groups at both time points. Conclusion: Findings suggest that paretic muscles of patients with chronic stroke may maintain ability to stimulate protein synthesis machinery in response to neuromuscular electrical stimulation. Key Words: Neuromuscular—stroke—stimulation—paretic—skeletal muscle—mTORC1. © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.

From the *Department of Health and Human Performance, Texas State University, San Marcos, Texas; and †Department of Occupational Therapy, Louisiana State University Health Sciences Center, New Orleans, Louisiana. Received April 28, 2017; revision received June 17, 2017; accepted July 21, 2017. Work was performed in the Department of Health and Human Performance at Texas State University, San Marcos, Texas. Funding: This study was funded by a Research Enhancement Grant to J.A. Mettler, a Thesis Fellowship Award to S.M. Bennett, and a Graduate Student Research Grant to S.M. Bennett. Address correspondence to Joni A. Mettler, PhD, ATC, CSCS, Texas State University, Department of Health and Human Performance, Division of Exercise and Sport Science, 601 University Drive, San Marcos, TX 78666-4684. E-mail: [email protected]. 1052-3057/$ - see front matter © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2017.07.019

Introduction According to the American Heart Association, stroke is a leading cause of disability and is ranked fifth in causes of death in the United States.1 Individuals suffering from stroke commonly experience hemiparesis, weakness on one side of the body, which often results in significantly compromised muscle function and decreased mobility; these effects of stroke can lead to a vicious cycle of continued muscle atrophy, strength loss, and impaired ability to participate in activities of daily living.2-5 Voluntary resistance training is regularly included in physical rehabilitation programs after a stroke to increase muscle strength and function6-9; however, after a stroke, the individual can be left with little or no ability to perform voluntary muscle contractions. Neuromuscular electrical stimulation (NMES), a therapeutic modality frequently

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used in physical rehabilitation to artificially induce muscle contraction, may therefore be an effective alternative to voluntary resistance training for inducing hypertrophy and strength gains in these individuals. NMES is commonly used as a muscle strengthening treatment for a variety of neuromuscular diseases and disabilities such as stroke,10-12 spinal cord injury,13,14 cerebral palsy,15,16 and orthopedic injury.2 However, equivocal reports regarding the effectiveness of this treatment for muscle strengthening and growth are apparent throughout the literature. In healthy individuals, some studies report that strength increased with NMES training,17-20 whereas others observed no improvements21-23 and that additional strength gains were not associated with combined voluntary resistance training and NMES compared with resistance training alone. 24 Additionally, NMES treatment increased muscle strength in patients with osteoarthritis,25 increased strength after anterior cruciate ligament reconstruction,26 and attenuated muscle loss in individuals during immobilization for limb fracture.27 However, NMES did not prevent postoperative muscle loss28 and results were mixed in critically ill patients.29 Regarding individuals with chronic stroke, our previous work demonstrated improved strength in subjects who received a high-frequency stimulation program for four weeks, but not in those who received the identical low-frequency regimen.10 In other works, NMES training also increased maximal voluntary contraction (MVC) in individuals with stroke30 and improved walking speed.31 As demonstrated, data are inconclusive regarding the effectiveness of NMES for augmenting gains in skeletal muscle mass and strength, particularly in patients with stroke who have impaired neuromuscular function. It is well documented, however, that the primary anabolic signaling pathway responsible for regulation of protein synthesis and skeletal muscle cell growth, the mammalian target of rapamycin complex 1 (mTORC1) pathway, is upregulated in response to voluntary resistance exercise.32-35 Some of the key signaling proteins in the mTORC1 pathway that are upregulated in response to voluntary muscular exercise are mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase beta-1 (S6K1), and eukaryotic translation initiation factor 4Ebinding protein 1 (4E-BP1).32-35 Only a few studies have examined the mTORC1 pathway for the anabolic response to NMES in human skeletal muscle.36,37 Wall et al37 were the first to observe anabolic signaling after a single bout of NMES in the quadriceps muscle of older men with type 2 diabetes. Results showed that anabolic signaling was not significantly different between the control and stimulated legs; however, a trend toward increased phosphorylated protein content of S6K1 and mTOR was observed. In addition, increased skeletal muscle protein synthesis rates, associated with increases in muscle cell size, were obtained after a bout of NMES, demonstrating

that a single NMES treatment is capable of inducing a muscle cell growth response.37 Phosphorylation of mTOR also increased after several days of NMES in bed-ridden comatose patients36 and after high-frequency stimulation was delivered to rodent muscle.38 In another investigation, resting level phosphorylation of mTORC1 signaling proteins was not different between muscle of spinal cord injured and healthy individuals, but total protein levels of S6K1 and 4E-BP1 were lower in the spinal cord injured.39 In summary, very little evidence exists regarding the effect of NMES to induce anabolic changes that might facilitate growth in hemiparetic muscle. Because of motor performance irregularities and movement inconsistencies inherent in persons with stroke,40 it is difficult to determine whether the inconsistent data observed in previous studies are due to the variability of these individuals, variations of the study interventions, or ineffectiveness of the treatment. In addition, because NMES bypasses the motor cortex and the spinal cord and the electrical current directly activates muscle tissue by depolarization of the sarcolemma, the cerebral infarct is bypassed. Accordingly, NMES may be an effective method for determining whether muscle protein synthesis machinery, namely mTORC1 signaling, is intact in hemipartic muscle. Consequently, there is a need to study muscle building processes at the cellular level in human hemiparetic skeletal muscle tissue in response to NMES. Therefore, our primary hypothesis was that a single bout of NMES would increase activation of the mTORC1 signaling pathway similarly in older hemiparetic muscle and older healthy muscle. We also hypothesized that resting level total protein expression of key mTORC1 signaling proteins in hemiparetic muscle would be similar to older healthy muscle. The present study is the first to investigate the anabolic signaling response to NMES in older healthy and hemiparetic human skeletal muscle and to examine how resting level mTORC1 protein expression may be affected by hemiparesis in human muscle of individuals affected by stroke compared with healthy muscle.

Materials and Methods Participants This study employed a two-group, pretest–post-test design. Eleven individuals (5 men, 6 women), consisting of a chronic stroke group (STR: n = 5; age: 61.8 ± 5.4 years; age range: 47-79 years) and a healthy agematched control group (CON: n = 6; age: 58.2 ± 2.3 years; age range: 51-65 years), were studied. The average time since stroke onset was 4.7 ± .6 (range: 3.3-6.4) years before study enrollment. Participants were recruited from Texas State University and surrounding areas through flyers, newspaper advertisements, and stroke support groups. Participants were enrolled in the study if they met the following criteria: (1) age 40 to 85 years; (2) for the stroke group, a stroke onset six months or more before the start

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date of the study; (3) ability to communicate orally and provide informed consent; (4) ability to follow three-step instructions; and (5) ability to comprehend the responsibilities and procedures related to the study. Participants were not enrolled if (1) contraindicating conditions for muscle biopsy were present, eg, currently taking warfarin (Coumadin) or other anticoagulants; (2) contraindicating conditions for electrical stimulation were present, eg, swollen, infected, or painful areas on lower limbs, implanted pacemaker, or surgical hardware implants in the lower limbs; (3) the patient was taking medications that might affect metabolic data (eg, insulin); (4) the patient was participating in physical rehabilitation therapy within two months of the study; or (5) the patient was participating in a regular, rigorous strength training program involving the lower extremity within two months of the study. A health history phone screening was used to determine eligibility of participants based on the inclusion and exclusion criteria. Participants also obtained medical clearance to participate in the study from their personal physician. All subjects provided informed consent and all procedures were approved by the Texas State University Institutional Review Board. Participants who qualified for study participation based on the health history phone screen scheduled three visits to the Neuromuscular Physiology Laboratory at Texas State University.

Study Day 1: Body Mass Index and Physical Function Assessment On Study Day 1, participants provided informed consent. Body mass index (BMI) assessment, physical function assessment, and strength testing familiarization were also performed. Participants’ height and weight were obtained with shoes off (Health-O-Meter Professional 500KL, Alsip, IL) and were used to determine BMI according to American College of Sports Medicine standards.41 Participants then completed functional tests that were used as a comparison of functional mobility between the two study groups (STR and CON). The first functional measure was the Five Repetition Sit-to-Stand test (5XSTS).42,43 For this test, participants were instructed to transition from a seated position in a chair to fully standing with knees in full extension and back to seated, five times as fast and safely as they could.42,43 Grip strength test was performed with each hand, according to the American College of Sports Medicine standard guidelines,41 as a measure of upper body strength. The Timed Up and Go (TUG) test was performed as a measure of overall mobility.44 Participants were seated in a chair and were instructed to rise from the chair, walk three meters, turn around, walk back to the chair, and sit back down on the chair at their self-selected pace.44 Three trials were performed for each functional assessment with a 30-second rest interval between trials, and the fastest time and highest strength measurements were used for analysis.

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After completion of all testing, participants completed a muscle strength testing orientation session on an isokinetic dynamometer (Biodex Systems 4 Pro, Shirley, NY). Submaximal and maximal effort isometric knee extension contractions were practiced with both legs. The participant was seated and stabilized on the isokinetic dynamometer with hip flexion at 85° and knee flexion at 60°. Isokinetic dynamometer settings were consistent for Day 2 and Day 3 testing.

Study Day 2: Strength Testing Study Day 2 was performed at least 48 hours after Day 1 to allow muscle recovery. Knee extension strength tests were performed isometrically on an isokinetic dynamometer consistent with procedures used during the orientation session. Strength testing was performed on both legs and was conducted in random order (Research Randomizer Software, Version 4).45 Three MVCs were performed to evaluate knee extensor strength and to calculate the target torque during NMES. Participants were instructed to kick out as fast and forcefully as possible and to hold for four seconds. If maximal torque continued to increase over the three contractions, a fourth MVC was performed. Verbal encouragement was provided during MVC testing. Torque data were recorded by Biodex software (Version 4.5) on a personal computer and transferred to the PowerLab 16/35 data acquisition system (ADInstruments, Colorado Springs, CO) for later analysis with LabChart software (Version 8.1, ADInstruments). Grip strength and leg MVC data are expressed as a ratio of paretic to nonparetic limb for the STR group and is a common method used to account for bilateral limb discrepancies in strength as would result from hemiplegia.46 For the CON group, limb side was matched to the paretic limb of the stroke group and expressed as a ratio of bilateral limb strength.46

Study Day 3: Muscle Biopsy Study Study Day 3 occurred at least one week after Day 2 to ensure that muscle fatigue, soreness, or residual metabolic effects from Day 2 testing were not present. On Day 3, participants arrived at the laboratory in the morning after an overnight fast. Upon arrival, the participant rested for two hours to control for activity-related metabolic activity. Muscle biopsy #1 was obtained two hours after the participant arrived in the laboratory. The muscle biopsies were obtained from the vastus lateralis muscle from the hemiparetic leg of the STR group and from the right leg for the CON group. Muscle biopsies were obtained following standard techniques.47 Each muscle biopsy (~50 mg) was obtained under local anesthesia (1% lidocaine) from an incision in the vastus lateralis approximately 15-20 centimeters above the mid-patella, using a 5-mm Bergstrom biopsy needle.47 Biopsy #2 was obtained 30 minutes after completion of the NMES protocol from

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the same incision, with the needle positioned at a different angle compared with biopsy #1 (~2 cm between sample sites) to ensure that the muscle sample had not been damaged during the previous biopsy. Immediately after each muscle biopsy was obtained, the muscle sample was removed of adipose tissue, connective tissue, and blood clots, and immediately preserved in liquid nitrogen, and then stored at −80°C for later analysis.

NMES Intervention The participant was seated on the isokinetic dynamometer and setup was consistent with strength testing. The participant was asked to shave the thigh in the region of electrode placement before the visit, and the area was cleaned with alcohol before electrode placement. Four, 3 × 5 inch, stimulating electrodes (ValuTrode Neurostimulation Electrodes, Fallbrook, CA) were placed at the proximal and distal ends of the vastus lateralis and vastus medialis muscles to artificially activate the quadriceps musculature. Stimulating electrodes were placed on the hemiparetic leg of the STR group and on the right leg of the healthy CON group. The NMES intervention consisted of a single bout of electrical stimulation to the quadriceps musculature for 60 minutes using a constant current stimulator (Digitimer DS7AH, MEPs-LLC, Fort Lauderdale, FL). A pulsatile (intermittent), positive, monophasic current with a rectangular waveform was delivered with a duty cycle of 10 seconds on and 15 seconds off at a frequency of 60 Hz with a pulse width of 200 µs. We used a stimulation frequency of 60 Hz to match previous study protocols that have used 60 Hz to investigate the anabolic signaling response to NMES.20,37 The 60-Hz frequency is also within the range applied clinically for muscle rehabilitation.48 To control for the degree of muscle activation, neuromuscular electrical stimulation was delivered at an intensity that consistently produced a target torque equal to 15% MVC as monitored in real time via the Biodex torque output. The muscle quickly accommodates to the current and torque quickly declines during NMES protocols because of muscle fatigue; therefore, torque production was monitored and stimulation intensity was adjusted every five minutes, as needed, within subject tolerance to obtain a torque at 15% MVC. The intended stimulation intensity and the intensity adjustments throughout the treatment were well-tolerated by all participants. Torque output was digitally recorded on an isokinetic dynamometer and the signal was transferred in real time to the PowerLab 16/35 analog-to-digital dataacquisition system (ADInstruments). The NMES stimulation protocol was programmed using LabChart software (Version 8.1, ADInstruments).

Cell Signaling Muscle tissue from both biopsies was used to measure changes in cell signaling from pre-NMES (resting) to

post-NMES intervention, according to procedures previously described32: Samples of frozen muscle tissue were homogenized and the supernatant was removed. Bradford assay analysis was performed in duplicate to quantify the protein concentration of each sample (Bio-Rad SmartSpec plus spectrophotometer, Hercules, CA). The supernatant was diluted in a 1:1 2× sample buffer. Fifty micrograms of total protein was loaded in duplicate into each well of either a 7.5% or a 15% polyacrylamide gel (Bio-Rad), and samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (Criterion Blotter, Bio-Rad) at 150 V for 60 minutes. A known standard and a molecular weight marker were also added to each gel. Proteins were then transferred to a polyvinylidene difluoride membrane (Bio-Rad) at 50 V for 60 minutes. The membrane was then blocked for 60 minutes in either a 5% nonfat dry milk solution or a 5% bovine serum albumin solution and the membrane was then incubated with primary antibody overnight on a rocker at 4°C. The primary antibodies and dilutions used in this study were total mTOR (1:1000; Ser2448), phospho-mTOR (1:500; Ser2448), total p70 S6K1 (1:1000; Thr389), phospho-p70 S6K1 (1:250; Thr389), and total and phospho4E-BP1 (1:1000; Thr37/46) (Cell Signaling Technology, Danvers, MA). Donkey anti-rabbit IgG horseradish peroxidaseconjugated secondary antibody (1:6000) (Santa Cruz Biotechnology, Dallas, TX) was then applied to the membrane and placed on a rocker for 60 minutes at room temperature. Blots were then washed and incubated for five minutes with an enhanced chemiluminescence reagent (ECL Plus Western Blotting Detection System, GE Healthcare Bio-Sciences, Pittsburgh, PA) and were imaged using an optical density imager (FOTO/Analyst Luminary/FX, Fotodyne Inc., Hartland, WI). Band density was analyzed using Quantity One 1-D analysis software (Version 4.5.2, Bio-Rad). Cell signaling data are expressed as a ratio of phosphorylated protein to total protein content.

Statistical Analysis Statistical analysis was performed using SPSS statistical software (Version 24, IBM Corp, Armonk, NY). This study employed a two-way repeated measures analysis of variance (ANOVA) with group (CON and STR) and time (pre-NMES and post-NMES) as the independent factors to compare anabolic signaling activity in response to the NMES intervention. One-way ANOVAs were used to compare baseline (pre-NMES) total protein levels between stroke and control groups. Effect size using Cohen’s d49 was also calculated for baseline total protein content between groups. Repeated measures ANOVA was used to compare NMES intensity needed to attain the target torque at the start and at the end of the protocol. Grip strength, 5XSTS, TUG, MVC, BMI and age were compared by independent t test with group (CON and STR) as the independent factor. Data are reported as mean ± standard error and statistical significance was set a priori at P ≤ .05.

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Results Participant Characteristics, Physical Function, and Strength Participant characteristics comparisons are displayed in Table 1. There were no differences between groups for BMI, age, height, or body weight. For the TUG test, the STR group was significantly slower compared with the CON group (P = .03); 5XSTS time was also significantly slower in the STR compared with the CON group (P = .05) (Table 2). Bilateral hand difference in grip strength was not significantly different between groups (P = .07) when expressed as a ratio between limbs (Table 2). The bilateral strength difference for knee extension strength, as measured by MVC, was significantly greater in the STR group compared with the CON group (P = .05) (Fig 1). One subject from the STR group had severe osteoarthritis in the nonparetic knee; therefore, data from this subject were excluded from this analysis.

MVC Ratios (Nm/Body Weight kg)

1.4 1.2

*

1 0.8 0.6 0.4 0.2 0

Control

Stroke

Figure 1. Knee extensor strength. MVC values are expressed as a ratio of paretic to nonparetic limb for the STR group, and for the CON group, limb side was matched to the paretic limb of the stroke group. Ratios for MVC knee extensor strength were used to account for bilateral limb discrepancies. STR: n = 4; CON: n = 6. * Indicates significantly greater bilateral strength discrepancy as determined by independent t test, P ≤ .05. Data are presented as mean ± SE. Abbreviations: CON, control; MVC, maximal voluntary contraction; STR, stroke; SE, standard error.

Anabolic Signaling Phosphorylated protein content of key anabolic signaling proteins obtained from the vastus lateralis muscle of the CON and STR groups is displayed in Figure 2, and data for each subject are displayed in Table 3. An acute bout of NMES significantly upregulated phosphorylation of mTOR after NMES compared with resting levels by 70.9% in the STR group and by 63.6% in the CON group, compared with pre-NMES (P = .002; power = .96). Additionally, there was a 175.1% increase for phosphorylated S6K1 in the STR group and a 162.2% increase in the CON group (P = .002; power = .96). Phosphorylated 4E-BP1 Table 1. Participant characteristics Age (years)

n

Height (m)

Weight (kg)

BMI

Stroke 5 61.8 ± 5.4 1.66 ± .03 78.6 ± 7.0 28.7 ± 2.8 Control 6 58.2 ± 2.3 1.69 ± .05 68.2 ± 4.1 23.8 ± 1.1 P value .52 .53 .21 .10 Abbreviation: BMI, body mass index.

Table 2. Physical function tests

n Stroke Control P value

5 6

TUG (seconds)

5XSTS (seconds)

Grip strength ratio

9.16 ± 1.14 6.57 ± .19 .03 *

8.52 ± 1.09 5.98 ± .55 .05 *

.93 ± .07 1.25 ± .17 .07

Abbreviations: 5XSTS, Five Repetition Sit-to-Stand test; TUG, Timed Up and Go test. *Indicates stroke group was significantly slower than the control group; P ≤ .05.

Table 3. Changes in phosphorylation status before and after NMES for individual subjects

Subject

% Change pre-post mTOR

% Change pre-post S6K1

% Change pre-post 4E-BP1

STR 1 STR 2 STR 3 STR 4 STR 5 CON 1 CON 2 CON 3 CON 4 CON 5 CON 6

−12.16 134.03 133.77 42.71 56.29 82.36 43.55 50.83 −1.96 103.44 103.57

275.44 237.12 301.76 −42.18 103.49 242.99 129.46 166.72 149.53 183.58 101.20

−32.56 −13.74 −16.17 3.82 −51.38 −17.81 −13.40 −11.14 −25.62 −18.24 11.20

Abbreviations: 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; CON, healthy older control group; NMES, neuromuscular electrical stimulation; STR, stroke group; mTOR, mammalian target of rapamycin; S6K1, ribosomal protein S6 kinase beta-1.

content was downregulated by 22.0% in the STR group and 12.5% in the CON group (P = .005; power = .91) 30 minutes after the NMES treatment. No significant differences for phosphorylated protein content between STR and CON groups were observed for mTOR (P = .64), S6K1 (P = .26) or 4E-BP1 (P = .48) and the interaction (group × time) was not significant for these proteins (P > .05) (Fig 2). Baseline total protein levels (pre-NMES biopsy) were not significantly different between stroke and control groups for mTOR (P = .19), S6K1 (P = .21), or 4E-PB1 (P = .09) (Fig 3). Total 4E-BP1 protein levels, however, were trending toward

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pre

STR STR pre post

STR pre

B

p-mTOR/mTOR

p-S6K1/S6K1

CON

CON

pre

post

CON pre

C Control Stroke

4.5

1.2

CON post

STR pre

STR post

p-4E-BP1/4E-BP1 1.2

4

Arbitrary Units (AU)

1

Arbitrary Units (AU)

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0.8 0.6 0.4 0.2

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Arbitrary Units (AU)

A

post

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0.4

0

0 pre-NMES

0.6

0.2

0.5

0

0.8

pre-NMES

post-NMES

post-NMES

pre-NMES

post-NMES

Figure 2. Phosphorylated protein content for (A) mTOR, (B) S6K1, and (C) 4E-BP1. * Indicates significant main effect for time (before versus after NMES) as determined by two-way repeated measures ANOVA, P ≤ .05. Blots included with each graph are representative band densities of the respective phosphorylated protein. A gap in the blot represents a break in the data. Band images for a specific protein were obtained from the same image. No differences were observed between groups. STR: n = 5; CON: n = 6. Data are presented as mean ± SE. STR and CON are stroke and control groups, respectively. Abbreviations: 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; ANOVA, analysis of variance; mTOR, mammalian target of rapamycin; NMES, neuromuscular electrical stimulation; S6K1, ribosomal protein S6 kinase beta-1; SE, standard error.

in hemiparetic and healthy older adult skeletal muscle. These preliminary data indicate that the anabolic response was similar in hemiparetic and healthy older muscle despite impaired lower extremity physical function in the stroke group compared with the control group for knee extensor strength, TUG test, and 5XSTS test. We also found that resting state total protein expression was not statistically different between stroke and control groups; however, effect sizes were medium to large, indicating that there may be a difference between stroke and control groups, with the stroke group exhibiting lower protein expression. Although previous work has extensively demonstrated the effects of stimulation regimes on muscle performance, the present study is one of the first to validate changes in skeletal muscle anabolic signaling benefits

significance with the stroke group having lower levels. In addition, there was a large effect size, according to Cohen’s d,49 for baseline total protein between STR and CON groups for mTOR (d = .84) and 4E-BP1 (d = 1.18) and a medium effect size for S6K1 (d = .79) with the STR group having lower total protein expression compared with the CON group. The NMES current intensity increased significantly from the start of the protocol to the end of the 60-minute protocol (86.3 ± 4.0 mA and 139.0 ± 7.2 mA, for start and end, respectively; P < .0001).

Discussion This is the first study to demonstrate that a single, 60minute bout of NMES increases anabolic signaling activity

CON pre

CON pre

STR pre

A

STR pre

CON pre

C

B

Total mTOR

Control

Total S6K1

2.5

Stroke

0.4

Total 4E-BP1 1.2

1

0.5

0.3 0.25 0.2 0.15 0.1 0.05

0

0

1

Arbitrary Units (AU)

1.5

Arbitrary Units (AU)

Arbitrary Units (AU)

0.35 2

STR pre

0.8 0.6 0.4 0.2 0

Figure 3. Baseline total protein content for (A) mTOR, (B) S6K1, and (C) 4E-BP1. Total protein content at baseline between stroke (STR) and control (CON) groups. Data were analyzed with a one-way ANOVA. Blots included with each graph are representative band densities of the respective total protein. A gap in the blot represents a break in the data. Band images for a specific protein were obtained from the same image. No differences were observed between groups, P ≤ .05. STR: n = 5; CON: n = 6. Data are presented as mean ± SE. Abbreviations: 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; ANOVA, analysis of variance; mTOR, mammalian target of rapamycin; S6K1, ribosomal protein S6 kinase beta-1; SE, standard error.

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and potential growth mechanisms that are present in hemiparetic muscle. To our knowledge, only two other studies have examined anabolic signaling of the mTORC1 pathway in response to NMES in humans; however, the stimulation protocols and participant populations were different in these studies.36,37

Anabolic Signaling The significant increase in muscle anabolic response of phosphorylated mTOR and S6K1 observed in the present study is consistent with previous research that reported a 60-minute single bout of NMES at 60 Hz trended toward a significant increase in phosphorylation of mTOR and S6K1 in diabetic men.37 The lack of a significant increase after stimulation by Wall et al37 may be due to the small study population or to the lack of a controlled stimulation intensity during the NMES protocol. Participants received stimulation that was set at a level with which the subject was comfortable and one that produced a visible muscle contraction while intensity was increased during only the first 30 minutes of the 60-minute protocol to maintain a visible muscle contraction.37

Consistency in Experimental Procedures Used In the present study, stimulation intensity was adjusted to achieve 15% MVC, within subject tolerance, and torque output was monitored and increased every five minutes when torque produced by the quadriceps was less than 15% MVC. The stimulation intensity necessary to achieve a specific degree of muscle activation may vary between subjects for a number of reasons, including, but not limited to, rate of muscle fatigue, force potentiation, and subcutaneous fat.50,51 To achieve the target torque output throughout the protocol, we found that the current intensity at the beginning of the protocol was on average 86 mA and increased to 139 mA by the end of the 60-minute protocol. In clinical application, the intensity is often adjusted initially and then remains unchanged throughout the remainder of the treatment. Clinicians may consider increasing the stimulation intensity periodically throughout the treatment session to optimize the potential benefits of NMES treatments. Anabolic protein phosphorylation status also changes over the course of time following voluntary resistance training 32,52 and following NMES treatment.37 In the present study, the muscle biopsy was obtained 30 minutes after NMES, whereas Wall et al37 obtained muscle biopsies at 5 minutes and at two and four hours after NMES. Accordingly, the duration of time elapsed between completion of the NMES protocol and the muscle biopsy is important to note. Similar to the present study, a significant increase in phosphorylated mTOR was observed in comatose patients, although no significant change was observed for phosphorylated S6K1 after three to seven days of NMES.36

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During inactivity, merely maintaining S6K1 phosphorylation may be beneficial for muscle anabolism; therefore, a direct comparison between the present study and comatose patients is difficult because in a comatose state catabolic and inflammatory processes are present. We53 and other authors54 have shown that skeletal muscle anabolism is greater in a postprandial state compared with a fasted state in healthy adults. In the present study, subjects were fasted; however, the comatose patients36 were nourished with a nutritional supplement, but whether the subjects were in a postabsorptive or a postprandial state in relation to time of muscle biopsy was not well defined. In addition, the previous study did not specify the time point in relation to the NMES treatment at which the postNMES intervention biopsy was taken,36 and previous work has shown that mTORC1 signaling is upregulated after muscle contraction.32 We also observed a 22% and 13% downregulation in 4E-BP1 phosphorylation 30 minutes after NMES in stroke and control groups, respectively. Similarly, 4E-BP1 phosphorylation decreased immediately after high-intensity cycling exercise55 and after a single bout of high-intensity voluntary resistance training and returned to baseline within two hours after exercise.32 4E-BP1 also trended toward a 40% decrease immediately after NMES in men with diabetes37 and decreased immediately after electrical stimulation in rat muscle.56 Further investigation is needed to determine the time course of the signaling response after NMES. Because 4E-BP1 downregulation was greater after high-intensity than low-intensity voluntary exercise,55 it is possible that blunting of 4E-BP1 phosphorylation with exercise is fiber-type specific as there is evidence that eukaryotic elongation factor 2 signaling is fiber-type specific.55 Blunted protein synthesis has also been observed immediately after muscle contraction in rat muscle, with this effect preferential to fast-twitch fibers.57 If this is the case, it is plausible that the time course of 4E-BP1 recovery is longer after NMES exercise that preferentially stimulates fast-twitch muscle fibers. Because a reverse motor unit recruitment order occurs during NMES,58 fast-twitch muscle fibers are recruited first and would undergo greater metabolic stress. In comparison, slow twitch fibers are activated first during voluntary muscle activation.59 For this reason, fast-twitch muscle accounts for a greater portion of the activated muscle fibers in NMES induced muscle contraction compared with voluntary exercise. It is important to note that although not statistically significant, there was a large effect size for total mTOR and 4E-BP1 expression and a medium effect size of total S6K1expression between stroke and control groups, with the stroke group expressing lower levels of these proteins. It is, therefore, likely that hemiparetic muscle expression of these proteins is lower and in turn the total protein content available for phosphorylation is reduced compared with healthy older muscle. These results are consistent with the lower levels of total protein content

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in paralyzed rat skeletal muscles that were linked to muscle atrophy60 and reduced total protein content in adults with spinal cord injury.39 The degree of phosphorylation of the available proteins in hemiparetic muscle, expressed as a ratio of phosphorylated to total protein, may not be impaired compared with healthy controls, but the absolute number of phosphorylated proteins may be reduced in hemiparetic muscle as the total protein abundance was lower, and this may contribute to the observed reductions in strength and physical function in the stroke group. Léger et al39 also observed no difference in the total to phosphorylated ratio of several anabolic signaling proteins in muscle of individuals with spinal cord injury compared with healthy adult muscle. Future studies with more participants should address these preliminary findings and also whether repeated NMES treatment is able to upregulate total protein expression of mTORC1 signaling proteins.

Physical Function In the present study, the STR group was significantly slower compared with the CON for 5XSTS and TUG test times. Additionally, the paretic side was weaker for maximal knee extensor strength compared with the nonparetic side in the STR group, which was also expected. This finding is in agreement with previous studies in which knee extensor strength and hand grip was also different between affected and nonaffected legs in individuals who had a stroke.46,61 These physical function data demonstrate that individuals in the stroke group had lower extremity physical impairment compared with the healthy agematched controls, yet the ability of the muscle to mount an anabolic response did not appear to be impaired. This finding highlights the critical need for development of appropriate exercise regimens to induce hypertrophy and strength for chronic stroke patients. There is difficulty when studying persons with stroke due to individual differences in residual motor presentation, participants in our study also differed in that the locations of the infarct varied among these individuals. Also, several persons who volunteered did not meet the strict study criteria. It is possible that group differences would be attained with more study participants. These initial discoveries warrant development of larger studies that would potentially expand and further validate these findings.

Conclusions Data from the present study suggest that stimulation of the protein translational machinery, namely the phosphorylation of the mTORC1 pathway, is intact in patients with stroke and may be stimulated with NMES. These preliminary findings can begin to elucidate additional cellular factors that might facilitate the development of optimal NMES programs and rehabilitation strategies to speed motor recovery after stroke. Based on these data, NMES

appears to be an effective physical rehabilitation treatment to initiate skeletal muscle anabolic processes that promote muscle growth and strengthening in hemiparetic and healthy older skeletal muscle; however, larger studies and long-term interventions assessing cellular and functional outcomes are needed to determine whether these processes translate to improvements in muscle mass and voluntary motor function. Acknowledgments: We would like to thank our participants for their time and commitment to this study. We would also like to thank Dr. Michelle Lane for use of equipment and technical advice.

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