Integrated Muscle Protein Synthesis During Disuse and Rehabilitation in Late-Midlife Adults

Abstract

Aim:

The purpose of this study was to investigate the sex-specific differences in how late-midlife adults respond to short term disuse and rehabilitation.

Methods:

Sixteen, late-midlife adults, who were free of overt disease (8 males; 58±2 yr; BMI 29.4±0.8 kg·m⁻²; 8 post-menopausal females; 56±2 yr; BMI 29.1±1.1 kg·m⁻²) underwent 7 days of unilateral lower limb suspension (ULLS), followed by 7 days of rehabilitation. Vastus lateralis muscle biopsies were collected prior to and following ULLS (in both control [CON] and immobilized [IMM] legs) and in the IMM leg post-rehabilitation. We applied deuterium oxide to measure muscle protein synthesis (MPS), immunoblotting to assess mTORC1 signaling, and assessed changes in muscle fiber cross sectional area (CSA) and leg strength.

Results:

MPS was 17.8±14.6 and 32.7±10.9 % lower in the IMM compared to the CON leg in males (P=0.32) and females (P<0.05), respectively during immobilization. MPS was 27.5±24.5 and 9.7±38.9 % higher in the IMM leg during the rehab compared to during the IMM phase in the males and females respectively (P>0.05). Leg extension one repetition maximum declined by 24.2±2.4 and 17.1±2.1 % in males and females respectively after IMM (both P<0.01), in the IMM leg with no change in the CON leg (P>0.05).

Conclusion:

Our data show that late-midlife males and females experience similar reductions in MPS and muscle fiber CSA. Seven days of resistance exercise rehabilitation partially reverses the decline in muscle strength, CSA and MPS but longer rehabilitation periods are required for full recovery in late-midlife adults.

New & Noteworthy:

This study provides novel data on the average rate of muscle protein synthesis during 7 days of disuse and 7 days of rehabilitation in late-midlife adults. Both sexes experienced a similar reduction in muscle protein synthesis, strength and fiber cross-sectional area during disuse. Seven days of resistance exercise rehabilitation partially reverses the disuse-induced decline in muscle protein synthesis, strength, and fiber size, however, longer periods of rehabilitation are required for full recovery.

Graphical Abstract

Introduction

Incidences of illness or injury often require a period of short-term muscle disuse to aid recovery. Although these periods of immobilization or bed rest are essential, they result in negative health effects including declines in muscle function [1–6], mass [3–5,7] and insulin sensitivity [8,9]. These negative adaptations can be reversed by exercise rehabilitation [10,11], although older people take longer to recover after a period of disuse [12]. While the physiological responses and cellular mechanisms of muscle disuse atrophy [2,13–15], and subsequent rehabilitation [11,12,16] have been well studied, understanding the sex specific differences requires further investigation.

While the effects of short-term muscle disuse and rehabilitation on skeletal muscle mass, function [3–5,7] and metabolism [10,13,17,18] are relatively well studied. Only a very limited number of studies have directly compared how males and females respond to muscle disuse or rehabilitation. In a study of intensive care unit (ICU) patients, females exhibited a greater increase in muscle weakness compared to males after a period of bed rest [19]. In contrast, in older adults with knee osteoarthritis, only males experienced a significant reduction in myosin heavy chain type 2x (MHC2x) fiber cross sectional area (CSA) [20]. In healthy young subjects after 1 week [21] and 2 weeks [22] of leg immobilization females lost greater knee extension isometric force. Furthermore, females required one extra training session to regain this deficit compared to males, although this was not significant [21]. Understanding any potential differences in how males and females respond to disuse and rehabilitation could lead to better targeted interventions aimed at alleviating the negative effects of muscle disuse as well as optimizing rehabilitation.

Mechanistically, muscle mass is regulated by the balance of muscle protein synthesis and breakdown [23]. It is well established that short periods of muscle disuse reduce muscle protein synthesis rates (MPS) which contribute to muscle atrophy [2,13,17,24,25]. This reduction in MPS can be reversed after disuse with physical rehabilitation [26] or nutritional supplementation [27], particularly in young adults. However, most of these studies have been conducted with young adult male subjects [2,13,24,26,27]. In community dwelling older adults there is some evidence that sex differences in MPS exist in older (70 years old) [28], but not younger (37 years old) populations [29]. However, whether these differences exist during periods of muscle disuse and rehabilitation remains unknown.

The purpose of the present study was to determine the sex specific effect of disuse and rehabilitation on muscle protein synthesis, function and metabolism in late-midlife adults. We focused on molecular parameters including muscle protein synthesis, mTORC1 signaling. As well as functional parameters including leg extension strength and muscle mass. We hypothesized that females would experiences greater deficits in muscle mass and protein synthesis rates during disuse but would recover at a slower rate than males during rehabilitation.

Materials & Methods

Participants

16 late-midlife adults free of overt disease (8 males age; 58±2 y, BMI; 29.4±0.8 kg·m⁻², 8 post-menopausal females, age; 56±1 y, BMI; 29.1±1.1 kg·m⁻²) were included in the present study. Participants were recreationally active (i.e. spent < 8 hr per day with <100 counts per minute based on accelerometry data [30]) without resistance training experience. Participant characteristics are shown in Table 1. Participants attended the clinic for a medical screening visit to assess their eligibility for participation. Inclusion criteria included: males and post-menopausal females aged 50 to 65 y, BMI between 18.5 to 30 kg∙m⁻². Post-menopausal status was confirmed by a combination of self-report (i.e. no menstruation in the previous 12 months) and if estradiol levels were <10pg/ml. Exclusion criteria included: endocrine or metabolic disease (e.g. hypogonadism, type 2 diabetes), cardiovascular disease, acute or chronic infection, compromised musculoskeletal function, recent injury or history of falls (within last 6 months), anabolic steroid use (both sexes) or hormone replacement therapy use (females only) within the last 6 months, use of recreational drugs. During the medical screening subjects’ height, body mass, and blood pressure were measured, a single blood draw was obtained and analyzed for blood count, metabolic/lipid panel, sex hormones, and for infectious diseases (e.g. Hepatitis, HIV). This study was conducted in accordance with the Declaration of Helsinki and approved by the UT Health San Antonio and University of Texas Medical Branch at Galveston institutional review boards (IRB#20230594H and IRB#1900–45). This study was registered as a clinical trial with clinicaltrials.gov (NCT04151901). Participants provided written informed consent before participating in the study.

Table 1.

Subject characteristics

Subject characteristics	Total	Males	Females
Age (y)	57±2	58±2	56±2
Weight (kg)	81.9±4.4	90.1±7.0	73.6±3.3
Height (m)	1.67±0.03	1.75±0.04	1.59±0.02
Body mass index (kg∙m−2)	29.3±0.9	29.4±0.8	29.1±1.1

Values represent means±SEM, n=16, 8 males and 8 females.

Experimental Design

A graphical representation of the experimental study design is shown in Figure 1. Following the medical screening visit all participants attended eight study visits as part of the protocol containing seven days of unilateral lower limb suspension (ULLS) immediately followed by four resistance exercise rehabilitation sessions over a seven-day period. Approximately 10 days before the ULLS period participants received training from a study physical therapist on how to use the assistive devices (e.g. crutches and a walker) to ambulate safely during the disuse period. Participants were also familiarized to the exercise equipment. Participants were instructed to refrain from vigorous exercise and maintain their habitual diet for the remainder of the study. Seven days before the ULLS period participants completed one repetition (1-RM) maximum unilateral leg extension strength testing. All subsequent visits were conducted over the 14-day period of disuse and rehabilitation and participants were asked to fast for 12 h prior to each visit.

Figure 1.

Study schematic. 16 late-midlife adults (8 males and 8 post-menopausal females) underwent 7 days of unilateral leg suspension followed by 7 days of rehabilitation (1 session every other day, 4 sessions total). Blood indicates venous blood sample. Arrows represent bilateral m. vastus lateralis muscle biopsies (i.e., taken from the control and immobilized legs). On day 14 only a single m. vastus lateralis muscle biopsy was taken from the immobilized leg. D2O, deuterated water ingestion. Activity is physical activity measured continuously by GENEactiv accelerometry. Dietary recall is habitual dietary intake recorded by self-reported, written diet diary.

On day 1 of ULLS participants reported to the clinic at 0800 and completed a dual-energy X-ray absorptiometry (DXA) scan (Lunar iDXA; GE Medical Systems, Mississauga, ON). Following this, bilateral skeletal muscle biopsies of the vastus lateralis were obtained under local anesthetic (2% lidocaine) using Bergstrom technique [32] by a licensed medical professional. Immediately following the biopsy, the muscle sample was washed with ice cold saline and quickly assessed, and any non-muscle tissue was discarded. Of the remaining muscle tissue, one section was flash frozen in liquid nitrogen and a second section was oriented and mounted in Tissue Tek media (Sakura Finetek, Torrance CA) on cork and frozen in liquid nitrogen-cooled isopentane. All samples were stored at −80°C until further analysis. A blood sample was obtained via venipuncture of the antecubital vein concomitantly with the muscle biopsies. Post-biopsies subjects ingested a bolus dose of deuterium oxide (D2O, 150ml, 70% Atom excess, Cambridge isotopes, Tewskbury, MA, USA) for measurement of integrated muscle protein synthesis rates (MPS) rates [18,31]. A saliva sample was collected immediately before and once every other day following the consumption of D₂O to measure ²H enrichment in body water. To collect the saliva samples, participants were instructed to lightly chew on a cotton sponge [Celluron, Hartmann, Germany] until saturated. The sponge was then placed into an empty syringe and saliva was ejected into an Eppendorf tube. Participants were fitted with a leg sling and provided crutches and a walker to start the disuse phase and were provided with the supplies to collect their saliva samples at home.

After 4 days of ULLS, participants returned to the clinic for a monitoring visit where they completed an adherence questionnaire, the suspended leg was checked for any discomfort and a blood sample was obtained and tested for markers of thrombosis (e.g. D-dimer). Skin temperature of the dorsal foot was measured on both legs. On Day 7 of ULLS, participants returned to the clinic with their saliva samples and the same measurements that occurred on Day 1 were repeated (DEXA, bilateral vastus lateralis muscle biopsies, a single blood sample and 1RM strength testing). Participants also received a top up dose of D2O (50 mL), a saliva sample was collected before and once every other day throughout the rehabilitation period. Participants were provided with more saliva collection supplies for the rehabilitation phase.

Approximately 1 h following the post-ULLS biopsy, participants began the rehabilitation phase. Participants completed a 5-minute treadmill-based walking warmup and were monitored to maintain a heart rate of 100 beats per minute. Participants then completed a series of resistance exercise rehabilitation sessions which included; 4 × 10 reps of unilateral leg extensions at 70% 1RM, with a 1-minute rest between legs and a 2-minute rest between sets. This was followed by 4 × 10 reps of bilateral leg curls at 70% of 1RM with a 2-minute rest between sets. All exercises were done using standard gym equipment (Precor, Woodinville, MA, USA). Rehabilitation sessions were performed 3x per week on non-consecutive days (Monday [Day 7], Wednesday [Day 9, Friday [Day 11]) and finished on Monday (Day 14). On the final test day participants underwent a DEXA scan, a biopsy of the immobilized leg, 1-RM strength testing and following a 1 hour break the final rehabilitation session.

Immobilization protocol

Unilateral lower limb suspension (ULLS) [33,34] was utilized as the model of leg immobilization. The left leg was suspended via a strap that connected to a belt positioned around the subject’s waist and a cuff around the ankle that held the left leg at 30 degrees of flexion (Figure 1). Participants ambulated with a walker and crutches. The left leg was selected so subjects would still be able to drive an automatic transmission car. Subjects were encouraged to wear the sling for 24 hours per day (any weight bearing on the suspended leg was strictly forbidden). Participants were provided with a shower bench so they could remain seated while showering. To monitor compliance, participants were contacted daily and completed a diary detailing their daily activities. Furthermore, participants continuously wore accelerometers (gt3x, actigraph, USA) around both ankles for the week of ULLS (except for when showering, Supplemental figure 1) and had skin temperature of the dorsal mid-foot of the ULLS and control leg measured before, during and after the week of ULLS (Traceable Infrared thermometer, Cole Palmer, IL, USA). A permanent marker was used to mark the location the temperature reading to ensure the same location was used each time skin temperature was measured.

Leg strength

Unilateral leg extensions exercises were performed for both legs individually using standard gym equipment (Precor, Woodinville, MA, USA). One repetition maximum (1-RM) strength testing was assessed using an incremental repetition procedure that was conducted separately for each leg, with the immobilized leg being tested first. Participants first completed two warm up sets of 4 and 2 repetitions at 25 % and 50 % of 1-RM respectively. Subsequently, single repetitions at 1-RM were undertaken, each separated by a 2-minute rest. Following each successful attempt the weight was increased until no extra weight could be lifted. The final 1-RM was taken as the heaviest single attempt that was successfully completed with full range of motion and correct technique.

Leg lean mass

Leg lean mass (LLM) was determined by dual-energy X-ray absorptiometry (DXA) on days 0, 7, and 14 (Lunar iDXA; GE Medical Systems). To standardize and minimize the effects of fluid shifts, subjects were required to lie supine for 10 min before scanning.

Diet and Physical activity monitoring

For 3 days prior to the Unilateral lower limb suspension (ULLS) period as well as throughout ULLS and rehabilitation participants physical activity was measured using an accelerometer (GENEactiv, USA) that was worn around the waist and each ankle. Participants were asked to maintain their habitual activity level in the habitual period before crutches, as well as during ULLS (to the best of their ability despite using crutches / a walker to avoid whole body sedentariness) and rehabilitation. Participants were asked to wear the accelerometer continuously and data was recorded at 60Hz sampling frequency. Physical activity data from the GENEactiv accelerometers that were worn around the waist were converted into 60 s epochs and used to estimate time spent performing total physical activity (all intensities) using standard cut-off points [30] for the pre-ULLS, ULLS and rehabilitation periods respectively. For the GENEactiv accelerometers that were worn around the ankle of the ULLS and control legs during the ULLS period. Vector magnitude counts were calculated for both legs for each day of the ULLS period to assess total movement in the ULLS leg and control leg respectively as a measure of compliance (Supplemental figure 1). Vector magnitude counts were calculated from the counts in each axis ($\mathrm{x},\mathrm{y}$ and $\mathrm{z}$, which is the raw acceleration data that was converted into counts using Actilife software (Actigraph, Pensacola, FL)) which were combined using the following formula:

$$\mathrm{V}\mathrm{M}=\sqrt{X^2+Y^2+Z^2}$$

where $\mathrm{X},\mathrm{Y}$ and $\mathrm{Z}$ are counts in the $\mathrm{x},\mathrm{y}$ and $\mathrm{z}$ axes.

Participants diet was recorded for three days prior to ULLS, during ULLS and rehabilitation (one weekend day and two weekdays), respectively by a self-reported written diet diary. Subjects were asked to refrain from alcohol intake and maintain a similar diet during and throughout the study. Dietary analysis was completed using ASA24 (National Cancer Institute, Bethesda, MA).

Immunohistochemistry

Muscle samples from both the immobilized (IMM) and control (CON) legs at both pre-ULLS (PRE) and post-ULLS (POST), and the IMM leg post rehab (REHAB) were preserved for immunohistochemistry. Samples were embedded in Tissue Tek compound (Sakura Finetek, Torrance CA) on cork and frozen in liquid nitrogen-cooled isopentane. Subsequently, seven-micron-thick sections were cut in a cryostat (Cryostar NX70, Epredia, Portsmouth, NH) and were air dried for 1 h. For immunofluorescence staining and muscle fiber detection unfixed slides were immersed in acetone for 10 min at −20°C, then rinsed for 1 × 5 min in 1 x phosphate buffered saline (PBS), 7.5 pH. Subsequently, slides were incubated overnight at 4°C in primary antibodies (MyHC-1, BA-D5c, 1:100, MyHC-2a, SC-71c, 1:100, MyHC-2x, 6H1-s 1:10; Developmental Studies Hybridoma Bank, IA, USA, laminin, 1:200, catalog no. L9393, Sigma Aldrich, St. Louis, MO). The next day, after washing the slides in PBS (3 × 5 min) slides were incubated for 1 h at room temperature with secondary antibodies (MyHC-1, Alexa Fluor (AF) 647, 1:250, MyHC-2a, AF488, #A21121, 1:500, MyHC-2x, AF555, #A21426, 1:500, Invitrogen, Carlsbad, CA, USA; laminin 1:500 dilution of AF568, Invitrogen, catalog no. 11011). Subsequently, slides were rinsed three times with PBS and post fixed in methanol before being mounted with fluorescent mounting media (Vectashield,no. H-1000; Vector Laboratories, Burlingame, CA) and a coverslip applied.

Image acquisition and analysis

All muscle sections were imaged at 10X magnification using the tile and stitching functions on a Nikon Eclipse Ti2-U inverted microscope equipped with Nikon Elements Ar software package (Nikon Americas Inc, Melville, NY, USA). Image analysis was completed in a blinded manner and performed using Myovision version 2 software [35] to calculate cross sectional area of type 1, type 2a and type 2x muscle fibers. A pixel conversion ratio value of 0.654μm/pixel was used to account for the size of the images. A detection range from 500 to 12,000μm² was used to ensure artifacts were removed (e.g., structures between laminin stains which were likely small blood vessels or large fibers which may have not been in cross-section).

Myofibrillar bound ²H alanine enrichments and body water ²H enrichments

The enrichments of [2^H]alanine in the myofibrillar fraction of skeletal muscle tissue samples were determined as described previously [36,37]. Briefly, 50 mg of whole frozen muscle was mechanically homogenized in 7.5 volumes of ice-cold homogenization buffer [50 mM Tris-HCL (pH 7.4), 1 mM EDTA, 10 mM b-glycerophosphate, 1 mM EGTA, 50 mM NaF, 0.5 mM activated sodium orthovanadate, and 1 complete mini protease inhibitor cocktail tablet per 50 mL of buffer (Roche Holding AG, Basel, Switzerland)]. Homogenized samples were centrifuged (10 min, 2,200 g at 4°C) and the pellet was washed in 500 μL of ice-cold homogenization buffer, centrifuged (10 min, 700 g at 4C), and solubilized (750 mL of 0.3 M sodium hydroxide at 50°C for 30 min). Following centrifugation (10,000 g at 4°C for 10 min), myofibrillar proteins were precipitated from the supernatant by adding 500 μL of 1 M perchloric acid and vortexing for 40 s and pelleted by centrifugation (10 min, 700 g at 4°C). The pellet was washed twice in 70% ethanol and amino acids were hydrolyzed in 2 mL of 6 M hydrochloric acid at 110°C for 24 h. The samples were subsequently dried under a vacuum (Savant SpeedVac, Thermo Fisher Scientific), reconstituted in 3 mL of acetic acid (25%), passed over cation exchange resin columns (100–200 mesh; H⁺ form; Dowex 50WX8; Sigma Aldrich Company Ltd., St Louis, MO, USA) and eluted with 6M NH₄OH, before being dried again under vacuum. Samples were resuspended in 1mL of 0.1% formic acid in acetonitrile and 1 mL distilled water, centrifuged (3min, 10,000g at 4°C), and the supernatant was aliquoted, dried under a vacuum, and stored at −20°C. Amino acids were derivatized by adding 50 μL N-tert-butyldimethyl-silyl-N-methyltrifluoroacetamide (MTBSTFA) + 1% tert-butyl-dimethylchlorosilane and 50 μL acetonitrile and vortexed and heated for 40 min at 95°C. The samples were then transferred to a gas chromatography vial. Alanine enrichment was analyzed using a Thermo Fisher Delta V Advantage IRMS (Bremen, Germany) fitted with a Trace 1310 gas chromatograph with an online high-temperature thermal conversion oven (HTC) at 1,420°C. The sample (1μL) was injected in splitless mode at an injection port temperature of 250°C. The peaks were resolved on a 30 m 0.25 mm ID × 0.25 μm film Agilent Technologies DB-5 capillary column (temperature program: 110°C for 1 min; 10°C·min⁻¹ ramp to 180°C; 5°C·min⁻¹ ramp to 220°C; 20°C·min⁻¹ ramp to 300°C; hold for 2 min) before pyrolysis. Helium was used as the carrier gas with a constant flow of 1 mL·min⁻¹. Any amino acid eluting from the gas chromatograph was converted to H₂ before entry into the IRMS via pyrolysis reaction performed by the HTC oven. Deuterium enrichment of myofibrillar protein-bound alanine was determined by monitoring ion masses 2 and 3, thereby establishing the 2H/1H ratio of the amino acid. A series of known standards were applied to assess the linearity of the mass spectrometer.

Body water enrichment was measured from collected saliva samples using an inline gas preparation system (GasBench II, Thermo Scientific), connected to the above isotope ratio mass spectrometer, as described previously [38]. In brief, 5 mg of activated charcoal and 200 mg of copper powder was added to an exetainer (Labco Ltd, UK), along with 200 μl of the saliva sample. Afterwards the sample was flushed with 2% H₂ in helium for 7 minutes and allowed to equilibrate at room temperature (4–6 hours) before analysis.

Calculation of fractional synthesis rate

Integrated myofibrillar fractional synthesis rates (FSR) were calculated based on the incorporation of the mean body water ²H enrichment over the ULLS and rehabilitation phases as a precursor pool into myofibrillar-bound proteins. Previous work has shown that the body water ²H pool is a valid precursor pool for the calculation of myofibrillar protein synthesis rates (corrected by a factor of 3·7 based on ²H labelling of alanine during de novo synthesis) which shows excellent agreement with plasma alanine as an alternative precursor pool [24,39]. FSR was calculated as follows:

$$FSR=-Ln\left(\frac{1-\left[\frac{\left({APE}_{Ala}\right)}{{APE}_P}\right]}{t}\right)$$

Where ${\mathrm{A}\mathrm{P}\mathrm{E}}_{\mathrm{A}\mathrm{l}\mathrm{a}}$ is the deuterium enrichment of protein-bound alanine, ${\mathrm{A}\mathrm{P}\mathrm{E}}_{\mathrm{P}}$ is the mean precursor enrichment over the time period, and $\mathrm{t}$ is the time between biopsies [40].

Immunoblotting

20 mg of muscle tissue was homogenized using glass on glass homogenization, in pre-cooled tubes in a buffer containing; 50 mM Tris·HCl, 250 mM mannitol, 50 mM sodium fluoride, 5 mM sodium pyrophophate, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraaceticacid, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 5 g/ml soybean trypsin inhibitor. The homogenates were then centrifuged, and supernatants were reserved, subsequently total protein concentration was determined by bicinchoninic acid (BCA) assay. Protein concentration was normalized, and samples were mixed with Laemmli sample buffer and heated at 70°C for 10 min. Proteins were resolved by Laemmli SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBS-T) plus 5% bovine serum albumin (BSA) and probed with the primary antibody overnight at 4°C. Primary antibodies included phosphorylated rpS6^Ser235/236(Cell Signaling Technology [CST], Cat. #4858) and 4E-BP1^Thr37 (CST, Cat. #2855), with β -tubulin (CST, Cat. #2128) used as a housekeeping protein. Membranes were then washed with TBS-T and probed with fluorescent IRDye 800CW Donkey Anti-Rabbit secondary antibody (LI-COR Biosciences, Lincoln, NE, USA, Cat. #925–32213) for 1 h at room temperature. Proteins were visualized using a LI-COR Odessey CLx Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Subsequently membranes were stripped (Restore Fluorescent western blot striping buffer, Thermo Scientific, Waltham, MA) and re-blocked with TBS-T plus 5% BSA and primary antibodies for total-rs6 (CST, Cat. #9452S) and total-4E-BP1 (CST, Cat. #9452) were applied overnight at 4°C. Membranes were again washed with TBS-T and probed with fluorescent IRDye 800CW Donkey Anti-Rabbit secondary antibody (LI-COR Biosciences, Lincoln, NE, USA, Cat. #925–32213) for 1 h at room temperature. Proteins were again visualized using a LI-COR Odessey CLx Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Mitochondrial respiration

Small bundles of skeletal muscle fibers (2–5 mg) were blotted, weighed, and gently teased apart using fine-tipped forceps to partially separate fibers without removing them from the fiber bundle. Each fiber bundle was permeabilized for 15 min on ice in BIOPS buffer containing 50 μg/mL saponin to selectively permeabilize cell membranes. Fiber bundles were then rinsed for 15 min in ice-cold MiR05 buffer (110 mM sucrose, 60 mM potassium lactobionate, 2 mM MgCl2, 20 mM taurine, 10 mM KH2PO4, 0.5 mM EGTA, 20 mM HEPES, and 1 g/l bovine serum albumin). Following the rinse step, bundles were placed inside the Oroboros Oxygraph-2k (O2k) (Oroboros Instruments, Innsbruck, Austria) with MiR05 buffer, set at 37 °C with the stir bars spinning at 750 revolutions/min. Supplemental oxygen was added to each chamber to maintain O2 concentrations between 400 and 200 μM throughout the experiment. Mitochondrial respiration rate was measured via the sequential titration of substrates (5 mM pyruvate, 2 mM malate, 10 mM glutamate), ADP (5 mM), succinate (10 mM), cytochrome c (10 μM), and finally, the ionphore CCCP (5 μM). Background oxygen consumption was measured following the addition of antimycin A (2.5 μM).

Data analysis

All data are presented as means±SEM, and all statistical analyses were conducted in GraphPad Prism version 10.0 (GraphPad Software, San Diego, CA). A three-way mixed model ANOVA with sex (male vs female, between subject’s), leg (immobilized vs control) and time (pre-ULLS, post-ULLS, post-rehabilitation) as within subject’s factors was used to compare differences in leg extension strength. A two-way mixed model ANOVA was used to assess changes in integrated muscle protein synthesis rates during the disuse period with sex (male vs female, between subjects’), leg (immobilized vs control, within subjects’) as factors. A two-way mixed model ANOVA was used to assess changes in integrated muscle protein synthesis rates in the immobilized leg between the disuse and rehabilitation periods with time (disuse vs rehabilitation, within subjects’) and sex (males vs females, between subject’s) as factors. For muscle fiber CSA for the immobilized leg, a two-way mixed model ANOVA with sex (males vs females, between subject’s) and time (disuse vs rehabilitation, within subject’s) as factors. A one-way repeated measures ANOVA was used to assess for changes in protein content/phosphorylation in the immobilized leg. For all ANOVAs, when a significant interaction was found Bonferroni post hoc tests were applied to locate individual differences. Statistical significance was set at P < 0.05.

Results

Compliance with unilateral lower limb suspension

To check for compliance with the unilateral lower limb suspension (ULLS) protocol, we used both lower limb skin temperature and accelerometry data. Total activity counts in the suspended leg were lower compared to the control leg (mean ULLS leg = 154634±8192 vector magnitude counts/day; mean control leg = 321967±23108 vector magnitude counts/day, P<0.001) throughout every day of the ULLS period (Supplemental Figure 1). Dorsal foot skin temperature in the ULLS leg decreased over time throughout the disuse period (pre-ULLS = 95.0±0.5°F, vs during ULLS (day 4) = 93.5±1.6 °F) and was significantly reduced after 7 days (post- ULLS = 92.9±0.5 °F, P<0.001). The right (weight bearing) leg dorsal foot skin temperature did not change over time (pre- ULLS = 94.6±0.6 °F, during ULLS (day 4) = 96.1±0.4 °F, post- ULLS = 94.9±0.5 °F). Together these data indicate that all subjects were very compliant with the ULLS protocol.

Leg strength

Maximum unilateral leg extension one repetition maximum (1-RM) decreased by in males −24.1±2.1 % (pre disuse 1-RM = 65.1±6.8 kg, post disuse 1-RM = 49.4±4.6 kg) and to the same extent −17.0±2.3 % in females (pre disuse 1-RM = 32.2±1.6 kg, post disuse 1-RM = 26.8±1.0 kg) (leg × time × sex interaction, P>0.05, η²ₚ = 0.08, 95% CI [0.00, 0.102], but time × leg interaction was significant, P<0.05, η²ₚ = 0.30, 95% CI [0.00, 0.34]) (Figure 2). The main effect of sex was significant (P<0.001, η²ₚ = 0.53, 95% CI [0.20, 1.00]) with males showing greater overall strength than females. Males were significantly stronger than females for both IMM and CON leg at pre- ULLS (P<0.05). After the exercise rehabilitation period males leg extension 1-RM was 59.2±6.1 kg (increased 19.8±5.0 % vs post-ULLS) and females was 31.0±2.4 kg (increased 16.0±8.6 % vs post-ULLS). There was no difference in 1-RM between the IMM leg post-ULLS and post-rehab in either the males or females. Furthermore, there was no difference in 1-RM between the IMM leg and CON leg post-rehab in either the males or females. 1-RM did not change in males or females after ULLS or after rehabilitation in the CON leg.

Figure 2.

Leg extension strength in males and females, in both the control and immobilized legs, pre-disuse, post-disuse and post-rehabilitation. A three-way ANOVA was used to see if males and female leg strength was altered by 7 days of unilateral leg suspension and 7 days of resistance exercise rehabilitation (4 sessions, 1 session every other day). The main effect of leg and time were significant (P<0.05), but sex was non-significant (P>0.05). The time × leg and time × sex interaction effects were significant, but the leg × sex and leg × time × sex interaction effects were non-significant P>0.05. The leg × sex and leg × time × sex interaction effects were non-significant P>0.05). * Denotes a significant difference from pre-disuse. N=16, 8 males and 8 females. Data are means±SEM.

Leg lean mass

There was no difference in leg lean mass pre-ULLS in females (IMM leg = 6622.5±16.0 g; CON leg = 6779.8±16.2, P>0.05) or males (IMM leg = 9300.4±35.9 g; CON leg = 9427.5±41.3 g, P>0.05). The main effect of time and leg were non-significant, but the main effect of sex was significant (P<0.001), such that the females had lower leg lean mass than males at all timepoints. The time × leg, time × sex and time × leg × sex interactions were non-significant such that leg lean mass did not change after disuse or after rehabilitation in either the males or females in the IMM and CON legs.

Muscle fiber cross sectional area

Analysis of mean fiber cross sectional area (CSA) showed no change in myosin heavy chain (MyHC) type 1 and type 2x fibers in the immobilized leg after the disuse period or after the rehabilitation period (time × sex interaction and main effect of time both, P>0.05) (Figure 3A and C). Although the main effect of sex was significant with males having greater CSA compared to females at all time points for all fiber types MyHC type 1, type 2a and type 2x (P<0.05). In females for MyHC type 1 fibers approached a trend to decrease from pre- (3501.1±703.2 μm⁻²) to post-disuse (3157.2±647.5 μm⁻²) (P=0.12). MyHC type 2a fibers experienced a reduction in mean CSA from pre- to post-disuse to the same extent in both males (pre-disuse CSA = 4042.2±299.6, post disuse CSA = 3652.9±280.0 μm⁻², −9.6±1.0 %, P<0.05) and females (pre-disuse CSA = 3252.9±722.1 μm⁻², post-disuse CSA = 2919.5±646.4 μm⁻², −10.2±7.0 %, main effect of sex P<0.05, η²ₚ = 0.35, 95% CI [0.006, 0.431]) and time P<0.001, η²ₚ = 0.88, 95% CI [0.758, 0.914]), time × sex interaction P>0.05, η²ₚ = 0.17, 95% CI [0.00, 0.364])) (Figure 3B). This reduction in MyHC type 2a CSA after disuse was partially recovered by rehabilitation in males (post-rehabilitation CSA = 3898.8±244.1 μm⁻², P>0.05 from pre-ULLS) and females (post-rehabilitation CSA = 3040.4±660.0 μm⁻², P>0.05 from pre-ULLS).

Figure 3.

Vastus lateralis muscle fiber cross sectional area in the immobilized leg pre-disuse, post-disuse and post-rehabilitation in males and females, in MyHC type 1 (A), type 2a (B) and type 2x (C) fibers. Data presented are means±SEM. D) representative image. Two-way repeated measures ANOVA were used to compare each fiber type over time. * Denotes a significant difference from pre-disuse P<0.05.

Body water enrichment and daily myofibrillar protein synthesis rates

Saliva ²H enrichment over the week of ULLS and the week of rehabilitation can be observed in Figure 4A and 4B. 24 hours after the D₂O bolus dose body water enrichment increased to 0.54±0.05 % and decayed over the week of ULLS. 24 hours after the top up dose of D₂O on the first day of rehabilitation body water enrichment increased to 0.52±0.04 % and decayed over the week of rehabilitation.

Figure 4.

Myofibrillar protein FSR (%/day) calculated from saliva precursor pools. Previous research (39) has shown that when using deuterium oxide (²H20) as a stable isotope tracer saliva ²H enrichment can be used as a precursor pool to calculate FSR instead of the traditional used plasma precursor pool ([²H]-alanine) as they correlate strongly when they are used to calculate FSR. A and B) Saliva 2H enrichment (%) during the week of ULLS and rehab week respectively. C and D) Myofibrillar FSR (%/day) during the week of ULLS and the week of rehab respectively, males and females combined. E and F) Myofibrillar FSR (%/day) during the week of ULLS and rehab with males and females presented separately. * denotes significant difference from control leg (P<0.05). n=16, 8 males and 8 females. Data presented are means±SEM.

When assessing how integrated muscle protein synthesis (MPS) rates changed during disuse in the IMM and CON leg of males and females, the main effect of leg was significant (P<0.05, η²_p = 0.25, [95% CI: 0.00, 0.36]), but sex was non-significant (P>0.05, η²_p = 0.006 [95% CI: 0.00, 0.12]) and the leg × sex interaction was non-significant (P>0.05, η²_p = 0.02 [95% CI: 0.00, 0.19]) (Figure 4). Seven days of ULLS reduced integrated MPS rates in the disused leg compared to the control leg (P<0.01) and to a similar extent in males (CON leg FSR = 1.43±0.24%·d⁻¹ , IMM leg FSR = 1.17±0.10%·d⁻¹) and females (CON leg FSR = 1.62±0.20%·d⁻¹ , IMM leg FSR = 1.08±0.14%·d⁻¹) (Figure 4). Although not significant the interaction differences ((IMM - CON) males – (IMM - CON) females) was −0.28 which suggests a small non-significant difference toward a greater reduction in MPS in females compared to males.

When assessing how 1 week of physical rehabilitation affected integrated MPS rates in the IMM leg between the disuse and rehabilitation phases. Although numerically higher (IMM leg MPS rates were 22.8±24.7 % and 9.6±39.9 % higher during the rehabilitation compared to the disuse in males and females respectively) the main effect of time (P>0.05, η²_p = 0.04 [95% CI: 0.00, 0.20]) and sex (P>0.05, η²_p = 0.02 [95% CI: 0.00, 0.19]), as well as the time × sex (P>0.05, η²_p = 0.005 [95% CI: 0.00, 0.14]) interaction were all non-significant.

Immunoblotting for mTOR signaling

There was no change in the phosphorylation of rpS6^Ser235/236 or 4E-BP1^Thr37 between pre- and post-ULLS and post-ULLS and post-rehabilitation time points in the IMM leg (both, P>0.05, Figure 5). No change was observed in total-rpS6, total-4E-BP1 or the housekeeping protein β-tubulin at any point during the study in the IMM leg.

Figure 5.

Changes in protein expression of 4E-BP1^Thr37 (A) and rpS6^Ser235/236 (B) pre-disuse, post-disuse and post-rehabilitation in the immobilized leg. Protein phosphorylation for all targets is presented relative to respective total protein expression, with baseline normalized to 1. Values are means±SEM. n=10 males and females combined. Significance was set at P < 0.05. No significant time effect was found for 4E-BP1^Thr37 or rpS6^Ser235/236. Representative western blot lanes appear above each graph for the pre-disuse, post-disuse and post-rehabilitation time points.

Mitochondrial respiration

Leak (state 2) respiration was unaffected by unilateral lower limb suspension in both females and males in both the IMM and CON leg (time × leg × sex, P>0.05). Coupled respiration after the addition of succinate (state 3_+succinate) remained unchanged by unilateral lower limb suspension in males and females and in both legs (time × leg × sex, P>0.05). Lastly electron transfer system capacity (maximal uncoupled respiration) was also unaltered in males and females across the week of ULLS in both the IMM and CON legs (time × leg × sex, P>0.05) (Figure 6).

Figure 6:

Skeletal muscle mitochondrial respiration measurements. A, C and E display mitochondrial respiration in males and females in both the immobilized (IMM) and control (CON) legs pre- and post-disuse. B, D and F, display mitochondrial respiration measurements in the IMM leg in Males and Females at post-ULLS and post-rehab timepoints. Data are means±SEM, n=16 8 males and 8 females.

Skeletal muscle mitochondrial respiratory function was also unaltered during rehabilitation. Leak (state 2) respiration, coupled respiration after the addition of succinate (state 3_+succinate) and electron transfer system capacity (maximal uncoupled respiration) were unaltered in IMM leg after disuse compared to after rehabilitation for both males and females (time × sex, P>0.05) (Figure 6).

Dietary analysis

Table 2 shows participants habitual dietary intake for 3 days preceding unilateral leg suspension, 3 days during unilateral leg suspension and for 3 days during rehabilitation. No differences in macronutrient intake (protein, carbohydrate or fat) or energy intake were observed between males and females over the course of the study (i.e. pre-ULLS, ULLS phase and rehab phase) (sex × time, P>0.05).

Table 2.

Dietary intake and physical activity levels measured during; pre-disuse, disuse and rehabilitation.

	Pre-disuse		Disuse		Rehabilitation
	Males	Females	Males	Females	Males	Females
Energy intake (Kcal·d−1)	2461±354	2031 ± 175	2258±341	1803±140	2264±256	2294±169
Protein intake (g·d−1) Protein intake (g·kg bm·d−1) Fat intake (g·d−1)	114±24 1.2±0.2 107±15	75±7 1.1±0.1 86±7	120±20 1.3±0.2 93±13	77±5 1.1±0.1 77±12	114±20 1.3±0.2 99±9	91±8 1.4±0.2 106±10
CHO intake (g·d−1)	238±46	244±13	224±40	172±15	229±39	236±19
Light physical activity (h·d−1)	1.0±0.1	0.9±0.1	1.2±0.1	1.0±0.1	1.3±0.1	1.1±0.1
Moderate physical activity (h·d−1)	2.1±0.2	1.8±0.2	2.2±0.3	1.7±0.2	3.0±0.5	1.9±0.2
Vigorous physical activity (h·d−1) Total physical activity (h·d−1)	0.3±0.1 3.4±0.3	0.1±0.2 2.7±0.2	0.3±0.2 3.7±0.4	0.1±0.02 2.8±0.2	0.3±0.04 4.6±0.6	0.1±0.1 3.1±0.4

Values represent means±SEM, n=16, 8 males and 8 females.

Discussion

In the current study we applied a deuterated water approach to assess integrated myofibrillar protein synthesis (MPS) rates throughout one week of unilateral lower limb suspension and seven days of physical rehabilitation in healthy late-midlife males and post-menopausal females. We report that late-midlife males and post-menopausal females experienced the same reduction in integrated MPS over seven days of unilateral lower limb suspension. This was accompanied by a reduction in the cross-sectional area (CSA) of type 2a muscle fibers as well as a reduction in leg extension strength both of these reductions were of a similar magnitude in males and females. Although females did approach a trend for a decline in type 1 fiber CSA. Seven days of physical rehabilitation partially recovered MPS, leg extension strength and muscle fiber cross sectional area in males and females.

During the week of unilateral lower limb suspension (ULLS) integrated MPS reduced by ~25.7% (3.7%/day) in all subjects. While there was not a significant time × sex interaction there was a small effect size (η²p = 0.02) as females experienced a 32.7% (4.7%/day) reduction in integrated MPS in the IMM compared to CON leg while males experienced a 17.8% (2.5%/day) reduction. However, the change in integrated MPS during disuse was not accompanied by a change in basal anabolic signaling. Most of the previous work that has measured integrated MPS using deuterated water during short term unilateral leg immobilization has been conducted on younger adults. Previous research in males who underwent short term leg immobilization (i.e. ≤ 1 week) showed that integrated MPS declined by −17% (4%/day) [18] and −36% (5.1%/day) [24]. In contrast in young females the reduction in integrated MPS was slightly lower at −14% (0.67%/day), although this was over 2 weeks [25]. In the present study, it should be noted that although not significant the magnitude of reduction in integrated MPS in females was almost twice that of males (i.e. −32.7% vs −17.8%). This would support previous research that has shown that females experience greater muscle weakness after a period of bed rest during an intensive care unit (ICU) stay [19]. One reason for this could be that short term disuse induces anabolic resistance to protein ingestion [13] and older females have a diminished response to protein ingestion compared to age-matched males in free living conditions [28]. In the present study there was no difference in protein intake between the sexes during the disuse period (Table 2). Thus, it may be the case that the reduced anabolic response to protein ingestion accumulated over the disuse period and resulted in a numerically (but not significantly) lower MPS in females compared to males during disuse. Interestingly, we detected minimal changes in basal mTORC1 signaling following disuse and rehabilitation. This is somewhat to be expected as we didn’t provide a protein anabolic challenge to our participants as we did in our bed rest study in older adults [17].

Seven days (1 session every other day, 4 sessions total) of resistance exercise rehabilitation partially recovered integrated MPS in the immobilized leg by 16.2% (2.3%/day) in all participants. Although there was no significant difference in integrated MPS between males (22.8, 3.3%/day) and females (9.6%, 1.4%/d) during rehab, there was a small interaction effect size (η²_p = 0.005) which may suggest that late-midlife males may recover integrated MPS to a greater extent than females during short-term rehabilitation after short-term muscle disuse. This may be due to older post-menopausal women having a reduced anabolic response to resistance exercise compared to age-matched males [41–43], potentially due to reduced estrogen levels [44]. Alongside integrated MPS not fully recovering during rehabilitation in females, muscle fiber cross sectional area also failed to fully recover compared to pre-ULLS levels in males and to a similar extent in females. This agrees with previous work that states that older individuals require longer periods of rehabilitation to fully recover from a period of disuse [11] and with regards to leg strength, young adult females took one extra session to regain leg strength compared to males after one week of unilateral leg immobilization [21].

In addition to measuring skeletal muscle size and integrated MPS, we examined changes in unilateral leg extension strength. In both males and females leg extension strength was significantly reduced post-ULLS. There was a small interaction effect size (η²_p = 0.008) for males to decline to a greater extent compared to females (−24% vs −17%). Previous research has suggested that young adult females lose a greater amount of leg strength compared to age matched males after 7 [21] and 14 days of unilateral leg immobilization [22]. This difference is unlikely to be due to the reduction in muscle size after disuse as males and females experienced the same reduction in type 2a fiber cross sectional area. Furthermore, in young adults, females also seem to experience a greater loss of neuromuscular function compared to males [45], however further investigation is required to assess if this is the case in older adults.

Most of the previous research that has assessed the effectiveness of interventions aimed at alleviating the negative effects of short-term muscle disuse as well as promoting recovery during rehabilitation have focused on males ([4,6,46–49]). Given that integrated MPS rates declined by a similar amount in late-midlife males and post-menopausal females after short-term ULLS it is likely that interventions aimed at reducing muscle atrophy during disuse i.e. electrical stimulation will work in females, as has already been demonstrated in males [50,51]. For other interventions that have shown mixed results at alleviating reductions in muscle atrophy during short-term disuse, namely protein or essential amino acid supplementation which has sometimes been successful [4,6,52] and other times not [49,53,54]. It is plausible that due to females experiencing a numerically (but not significantly) greater reduction in integrated MPS compared to males, these interventions will have a greater chance at working in late-midlife post-menopausal females. Conversely due to the numerically greater inhibition in integrated MPS in late-midlife females compared to males during disuse and the numerically blunted increase in integrated MPS in females during rehabilitation. Late-midlife females may benefit more from interventions that sensitize the muscle to anabolic stimuli as has been demonstrated by omega 3 supplementation [25]. As basal phosphorylation of mechanistic Target Of Rapamycin Complex 1 (mTORC1) is elevated in skeletal muscle of older adults [55]. Future work should investigate interventions that aim to reduce this hyperphosphorylation of mTORC1, before and during disuse as well as during rehabilitation to assess if this improves the efficacy of interventions that aim to increase integrated MPS during disuse and rehabilitation (i.e. protein supplementation).

The present study has some limitations. Previous research has shown that when using the ULLS model the control (weight bearing) leg experiences overuse [56,57] which must be acknowledged especially when making comparisons to the suspended leg. It should also be noted that participants may vary widely in this compensatory activity. Secondly, to reduce participant burden, participants completed one week of rehabilitation. However, rehabilitation from an injury or surgery (e.g. knee arthroplasty) would require longer periods of rehabilitation [58] which may limit the applicability of this study to a real-world situation. Thirdly, due to the small sample size of the study some of the results presented are exploratory and should be followed up in future research studies.

In conclusion, our data show that late-midlife males and post-menopausal females experience a reduction in integrated MPS and muscle fiber cross sectional area that is similar between males and females. Both males and females experienced reductions in leg strength although males experienced the numerically greater decline. Seven days of resistance exercise rehabilitation partially reverses the decline in muscle strength, cross sectional area and integrated MPS but longer rehabilitation periods are required for full recovery in late-midlife adults.

Supplementary Material

Supplemental figure 1: https://doi.org/10.6084/m9.figshare.29640884.v1