Single-arm resistance training study to determine the relationship between training outcomes and muscle growth factor mRNAs in older adults consuming numerous medications and supplements

Abstract

Objectives

Determine if the muscle mRNA levels of three growth factors (insulin-like growth factor-1 [IGF1], ciliary neurotropic factor [CNTF], and vascular endothelial growth factor-D [VEGFD]) are correlated with muscle size and strength gains from resistance exercise while piloting a training program in older adults taking medications and supplements for age-associated problems.

Design

Single-arm prospective study.

Setting

US Veterans Affairs hospital.

Participants

Older (70±6 yrs) male Veterans (N=14) of US military service.

Intervention

Thirty-five sessions of high-intensity (80% one-rep max) resistance training including leg press, knee curl, and knee extension to target the thigh muscles.

Measurements

Vastus lateralis biopsies were collected and body composition (DEXA) was determined pre- and post-training. Simple Pearson correlations were used to compare training outcomes to growth factor mRNA levels and other independent variables such as medication and supplement use.

Results

Average strength increase for the group was ≥ 25% for each exercise. Subjects averaged taking numerous medications (N=5±3) and supplements (N=2±2). Of the growth factors, a significant correlation (R>0.7, P≤0.003) was only found between pre-training VEGFD and gains in lean thigh mass and extension strength. Mass and strength gains were also correlated with use of α-1 antagonists (R=0.55, P=0.04) and pre-training lean mass (R=0.56, P=0.04), respectively.

Conclusions

Muscle VEGFD, muscle mass, and use of α-1 antagonists may be predisposing factors that influence the response to training in this population of older adults but additional investigation is required to determine if these relationships are due to muscle angiogenesis and blood supply.

Introduction

Sarcopenia is a serious health problem for older adults since loss of muscle mass and strength leads to disabilities (1, 2). Resistance exercise is an effective intervention but the magnitude of benefit varies between individuals (3, 4, 5). The causes of this variability are numerous and complex (e.g. lifestyle, genetics, comorbidities) though the sum of their effects are expected to be reflected by the expression of genes that control muscle atrophy and hypertrophy (6). Thus, an increased understanding of the molecular correlates of muscle adaptation in the context of aging, exercise, and individual characteristics may be important for optimizing strategies for muscle maintenance in older adults particularly those prone to lower adaptive responses.

Resistance training studies are having success identifying molecular correlates of muscle adaptation that are influenced by aging (7, 8). Our efforts identified mRNAs encoding three growth factors that were expressed at lower levels in muscle of old (72±5 yrs) compared to young (32±7 yrs) adults (9) The growth factors included insulin-like growth factor-1 (IGF1), ciliary neurotropic factor (CNTF), and vascular endothelial growth factor-D (VEGFD). IGF1 is a commonly studied regulator of muscle mass thought to promote muscle growth by activating satellite cells and by stimulating protein synthesis (10). Less is known about the functions of CNTF and VEGFD though they may serve in maintaining neuromuscular junctions and adequate capillary density, respectively (11, 12). Due to their potential role in muscle adaptation, our prior study also investigated the relationships between the transcripts of these growth factors and training outcomes (13). Healthy older (68±6 yrs) adults participated in a resistance training program for the thigh muscles. The post-training gains in muscle size (7±2%) and strength (30±6%) varied between individuals but were strongly correlated with the pre-training muscle levels of the IGF1, CNTF, and VEGFD mRNAs (13). The results suggested that these growth factors may be predictive biomarkers which also function in adaptation to training. However, the older adults were quite healthy and taking few, if any, medications or supplements for age-associated problems. Thus, it remains to be determined whether the relationships between training response and growth factors also exist in less healthy populations of older adults.

Veterans of United States military service possess a high prevalence for age-associated morbidities that are detrimental to muscle (14, 15). The U.S. Department of Veterans Affairs is committed to rehabilitation research for older Veterans and this population would likely benefit from resistance training programs. The current study sought to confirm the positive relationships, described above, between training outcomes and muscle growth factor levels for IGF1, CNTF, and VEGFD while piloting a high-intensity resistance training program in 60-80 year olds who receive care at a Veterans Affairs hospital. The correlations between training outcomes and individual characteristics that could influence training outcomes such as age, pre-training muscle mass, and use of medications and supplements for age-associated problems were also determined. This information may improve our understanding of the determinants and correlates of training response and could be useful in designing exercise trials for older adults.

Methods

Overview

Conduct of this single-arm resistance training study was approved by the institutional review board of the Central Arkansas Veterans Healthcare System and met the research guidelines of the Veterans Health Administration. Recruitment utilized advertisements posted in the hospital. Recruits provided written consent at their initial visit. Participants completed a high-intensity resistance exercise training program and donated muscle and had body composition measured prior to and after completion of the program. Medication and nutritional supplement use was collected from the hospital record and self-report.

Inclusion and Exclusion Criteria

The inclusion criteria included: 60-80 years old, body mass index (BMI) of 18.5-29.9 kg/m², non-smoker, and no participation in resistance exercise in the past three months. Further screening was performed by a physician based on medical history, physical exam, clinical labs, and electrocardiogram at rest and during strength testing. Individuals were excluded for lidocaine allergy or bleeding disorder, or using prescription anti-coagulants. Subjects taking over the counter products that affect bleeding halted consumption prior to biopsy (e.g. aspirin for ten days or non-steroidal anti-inflammatory drugs for three days). Subjects were also excluded based on the contraindications put forth by the American College of Sports Medicine (16). Subjects had fasting blood sugar <200 mg/dl and blood pressure <160/100 mmHg.

Resistance Training

The goal of the supervised resistance exercise program was to increase muscle mass and strength of the thigh muscles. Exercise sessions included bilateral knee extension and curl, and leg press performed on pneumatic strength training equipment (Keiser, Fresno, CA). Exercise sessions began with a low-intensity 10 minute warm-up on a cycle ergometer and included stretching as needed. Subject strength was tested for one-repetition maximum (1RM) three times, at least two weeks apart, prior to training and the third test was considered pre-training strength. Strength was then tested every sixth session for resistance progression. Subjects completed five sessions at low-intensity (graduated from 30-70% 1RM) followed by 35 high-intensity (80% 1RM) training sessions. Sessions consisted of three sets of eight repetitions for each exercise and a fourth set completed until voluntary failure. Subjects rested two minutes between sets and five minutes between exercises. Subjects alternated weeks of exercising two or three times per week. The program was optimally completed in 14-weeks though scheduling flexibility was allowed due to subject necessity.

Dual Energy X-Ray Absorptiometry

Whole body and thigh lean and fat mass was measured by DEXA using a QDR Series Discovery Wi densitometer (Hologic, Bedford, MA). Measurements were taken prior to the introduction to exercise and three days after the final training bout. Regional measurements for the dominant leg were obtained for a trapezoid area surrounding the thigh and lower gluteal region at the top between the femoral head and greater trochanter, at the bottom just proximal to the lateral condyle of the tibia, and on the sides by the medial and lateral surfaces of the thigh.

Muscle Biopsy

Biopsies were taken from the vastus lateralis under local anesthetic (1% lidocaine HCl) using a 6mm Bergstrom trocar. The pre-training biopsy was taken from the dominant leg (per subject self-report) prior to the introductory exercise sessions and the post-training biopsy was taken from the non-dominant leg three days after the final training bout. The three day post-training time point was based on our previous gene expression and exercise studies which yielded significant results for measuring the molecular response to exercise (9, 13, 17, 18). Flexibility in altering the total number of exercise sessions from 35 was allowed (i.e. 34 or 36 sessions) in order to maintain correct timing of the post-training biopsy since biopsies were not performed on weekends. Subjects were required to refrain from exercise and unaccustomed physical activity for three days prior to the pre-training biopsy.

Tissue Processing

Muscle tissue was stabilized by placing in RNAlater (Life Technologies, Grand Island, NY) overnight at 4ºC and then transferring to -20ºC for storage. Total RNA was isolated from 25mg of each specimen. After RNAlater removal, muscle was powdered using liquid N2 and a stainless steel pulverizer. RNA was then purified according to the manufacturer's instructions using Tri Reagent and DNA-free followed by a second purification using the RNAqueous Micro kit (Life Technologies, Grand Island, NY). RNA quantity and quality were assessed with the 2100 Bioanalyzer and RNA 6000 Nano Labchips (Agilent Technologies, Palo Alto, CA). The average RNA yield was 4.1±1.7 μg and the average Agilent RNA integrity value was 7.7±0.5.

Gene Expression Analysis

Expression of the CNTF, IGF1, VEGFD genes and an endogenous control β2-microglobulin (B2M) was measured by quantitative reverse transcriptase real-time polymerase chain reaction (RT-PCR) using the 7900HT System (Life Technologies, Grand Island, NY). Assays were performed according to the instructions of the iScript cDNA Synthesis Kit and iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA). The primer sequences specific to the mRNAs of interest were reported previously (9). The forward and reverse primer sequences for the B2M control were 5'-CCTAGCTTGATCATGCGATAGATG-3' and 5'-GAAACCGCCCTGCTATCTTCT-3'. Data was collected by absolute quantitation using standard curves generated using serial dilutions of a pooled cDNA that ranged from 50 ng to 0.8 ng RNA-equivalents of cDNA for the genes of interest and 8.0 ng to 0.03 ng for B2M. The standard curves assay efficiencies were greater than 90%, R2=1.0 with y-intercepts of 31.3±0.9 cycles for the genes of interest and 25.1 cycles for B2M. Input per assay for individual specimens was 3.1 ng and 0.2 ng for the genes of interest and B2M respectively. Assay specificity was confirmed by melting curves and controls lacking template. Assays were run at least in duplicate. Standards showed a CV of <1% and individual samples <20%. mRNA levels were normalized to B2M, a control previously used in gene expression studies of the vastus lateralis and resistance exercise (19) and validated again here in that B2M was not correlated with training outcomes (P>0.05) and was unaffected by exercise training (P=0.12).

Statistics

Data are presented as mean and standard deviation. The training outcomes were absolute change in lean mass of the thigh and absolute and percent strength gain for knee extension. Analysis focused on the results of knee extension since it stimulates the vastus lateralis, i.e. site of the biopsy, to a greater extent than knee curl and leg press (20). Training outcomes were continuous variables and compared for change from pre- to post-training using the paired samples t-test. Gene expression data were also continuous variables. Medication and supplement use was a dichotomous variable (0 = not using, 1 = using). Medication use was included rather than comorbidities since it was expected to be a better indicator of disease severity than diagnosis. Pearson's test was used to determine the correlations between the dependent variables (training outcomes), the independent variables (gene expression) and secondary independent variables (age, pre-training thigh lean mass, use of medications and supplements). Relationships were tested for significance (α 0.05) using an approximate t-test. Due to the small sample size, exact P-values are presented rather than making adjustments for multiple comparisons.

Results

Recruitment and Participation

A total of 456 Veterans responded to recruitment though many declined due to travel distance (N=109, 24%) or were excluded for obesity (N=72, 16%) at an initial contact by telephone. Other reasons for decline or exclusion existed but few (N=6, 1%) individuals were excluded for already exercising. The protocol was started by 17 subjects and 14 finished after two withdrew due to time commitment and one due to fainting during biopsy. Other study related adverse events were uncommon. Soreness was reported after 4% (N=27/700) of exercise sessions and two mild reactions to the muscle biopsy (skin irritation from the bandage tape and extended temporary numbness of the site) were reported. The post-training biopsy did not yield tissue for one subject making molecular data available pre-training for N=14 and post-training for N=13. Otherwise, protocol deviations did not affect subject safety, data, or study integrity.

Subject Characteristics

The 14 subjects who completed the study were male and of white (N=10, 71%), black (N=3, 21%), or Native American (N=1, 7%) race. Their characteristics and frequency of medication and nutritional supplement use are shown in Table 1. Subjects were 70±6 yrs old and had a body composition at study entry of 25±5% fat and 72±5% lean mass. Subjects typically possessed multiple (N=5±2) age-associated comorbidities and were taking multiple medications (Mean ± SD = 5 ± 3, range = 2 - 12) and supplements (Mean ± SD = 2 ± 2, range = 0 - 5). The problems possessed and products consumed by at least five of the fourteen subjects are listed in Table 1.

Table 1.

Characteristics, health problems, and medication and nutritional supplement usage for the subjects (N=14) who completed the study protocol

Characteristic	Mean ± SD	Range
Age (yrs)	69.9 ± 6.4	60 – 78
Height (cm)	177.3 ± 6.9	167.6 – 193.0
Weight (kg)	85.0 ± 10.9	63.0 – 107.0
BMI (kg/m2)	27.0 ± 2.5	21.8 – 30.9
Fat (%)1	25.0 ± 5.4	15.3 – 31.7
Lean (%)1	71.7 ± 5.1	65.3 – 80.6
Bone (%)1	3.3 ± 0.4	2.4 – 4.1
Prevalent Age-Associated Health Problems2	N of 14	N/14 (%)
Hyperlipidemia	12	85.7
Benign Prostate Hyperplasia	9	64.3
Hypertension	8	57.1
Depression or Anxiety	6	42.9
Diabetes	6	42.9
Chronic Back Pain	6	42.9
Acid Reflux	5	35.7
Medication or Nutritional Supplement2	N of 14	N/14 (%)
Aspirin	8	57.1
Statins	8	57.1
Fish Oil	7	50.0
B Vitamins	7	50.0
Multivitamin or Vitamin E	7	50.0
Vitamin D	7	50.0
α1 Antagonists	6	42.9
Non-steroidal Anti-inflammatory Drugs	5	35.7
H2 Antagonists and Proton Pump Inhibitors	5	35.7

^1.Body composition was measured by whole body DEXA prior to exercise training;

^2.List only includes problems possessed or medications and supplements used by ≥ 5 of 14 subjects.

Training Outcomes

Of the 14 subjects, 10 completed all 35 training sessions targeting the thigh muscles, three completed 34 exercise sessions, and one completed 36 in order to maintain timing of the post-training biopsy. The average time to completion of the program was 17.4±1.3 weeks (range 16 - 20 weeks). The pre- and post-training measures of thigh composition and strength are shown in Table 2. Regional analysis of the DEXA scan showed that training increased lean mass of the thigh by approximately 3% (0.2±0.29 kg, P=0.02) but bone mineral content and fat did not change. Training also significantly (P<0.0001) increased subject strength by 25% for the knee curl (15.3±6.5 kg), 28% for knee extension (18.8±7.3 kg), and 27% for leg press (54.7±27.8 kg).

Table 2.

Muscle mass and strength for older adults before and after 35 sessions of progressive high-intensity resistance training of the thigh muscles

Outcome	Pre-Training	Post-Training	Absolute Change	Percent Change	P-Value3
Thigh BMC1 (kg)	0.20 ± 0.04	0.20 ± 0.03	0.00 ± 0.02	-0.29 ± 9.7	0.686
Thigh Fat Mass1 (kg)	1.93 ± 0.62	1.98 ± 0.56	0.05 ± 0.18	4.09 ± 10.8	0.291
Thigh Lean Mass1 (kg)	5.99 ± 1.01	6.19 ± 1.19	0.20 ± 0.29	3.09 ± 4.3	0.022
Thigh Total Mass1 (kg)	8.13 ± 1.28	8.38 ± 1.45	0.25 ± 0.32	2.91 ± 3.7	0.012
Curl 1RM2 (kg)	64.2 ± 17.9	79.4 ± 20.8	15.3 ± 6.5	24.7 ± 10.6	<0.0001
Extension 1RM2 (kg)	67.7 ± 17.7	86.5 ± 24.0	18.8 ± 7.3	27.5 ± 6.8	<0.0001
Press 1RM2 (kg)	202.8 ± 55.3	257.4 ± 72.6	54.7 ± 27.8	27.1 ± 14.2	<0.0001

1. Thigh composition including bone mineral content (BMC) was measured by regional analysis of the whole body DEXA scan; 2. Strength capability measured by 1-repetition maximum testing (1RM); 3. Significant changes between pre- and post-training determined by paired samples t-test.

Training Outcomes and Muscle Molecular Analysis

The correlations between training outcomes (changes in thigh lean mass and knee extension strength) and muscle mRNA levels of CNTF, IGF1, and VEGFD or other independent variables were determined. The significant relationships are summarized in Table 3. Pre-training VEGFD levels were significantly correlated with both percent gain in strength (R=0.74, P=0.002, Figure 2A) and absolute change in lean thigh mass (R=0.73, P=0.003, Figure 2B). However, the relationship between pre-training VEGFD levels and change in absolute strength gain was not significant (R=0.45, P=0.107, Table 3). The pre-training levels of IGF1, and CNTF were not correlated (P>0.05) with any training outcome (supplementary Table S1). Post-training, levels for the molecular variables were not correlated (P>0.05) with training outcomes (supplementary Table S1). Other independent variables included in the analyses were age, pre-training thigh lean mass, and use of the medications and nutritional supplements listed in Table 1. Pre-training lean mass was correlated with percent (R=0.56, P=0.037) and absolute (R=0.90, P<0.0001) strength gain but did not reach significance for change in lean mass (R=0.51, P=0.060). However, change in lean mass was correlated with use of α-1 antagonist medications (R=0.55, P=0.041). The outcomes were not correlated with age or use of other medications or supplements.

Figure 1.

Relationship between pre-training VEGFD mRNA levels and training outcomes

Table 3.

Relationships between training outcomes, muscle VEGFD, and covariates

INDEPENDENT VARIABLES
TRAINING OUTCOMES	Pre-Training VEGFD		Pre-Training Lean Mass		α-1 Antagonists		Age
	R	P-Value3	R	P-Value	R	P-Value	R	P-Value
Strength Gain1 (%)	0.74	0.002	0.56	0.037	0.21	0.461	0.06	0.832
Strength Gain1 (kg)	0.45	0.107	0.90	< 0.0001	0.15	0.610	0.46	0.102
Mass Change2 (kg)	0.73	0.003	0.51	0.060	0.55	0.041	0.32	0.268

1. Strength gain for knee extension was analyzed as percent and absolute gain; 2. Change in lean mass of the thigh was measured by regional DEXA scan; 3. Significance of Pearson’s R was determined by approximate t-test.

Supplementary Table 1.

Relationships between training outcomes and pre- and post-training levels of IGF1, CNTF, and VEGFD muscle transcripts

Outcome	Strength Gain1 (%)		Strength Gain1 (kg)		Mass Change2 (kg)
	R	P-Value3	R	P-Value3	R	P-Value3
Pre-Training IGF1	0.27	0.356	-0.01	0.985	0.38	0.184
Post-Training IGF1	-0.19	0.525	-0.09	0.773	0.09	0.778
Pre-Training CNTF	0.00	0.996	-0.02	0.951	0.195	0.503
Post-Training CNTF	-0.01	0.96	0.08	0.800	0.06	0.846
Pre-Training VEGFD	0.74	0.002	0.45	0.107	0.73	0.003
Post-Training VEGFD	-0.08	0.803	0.11	0.727	0.18	0.557

1. Strength gain for knee extension was analyzed as percent and absolute gain; 2. Change in lean mass of the thigh was measured by regional DEXA scan; 3. Significance of Pearson’s R was determined by approximate t-test.

Discussion

Participants (age 60-78 yrs) experienced a wide range of gains in thigh muscle mass (-5% to +9%) and strength (9% to 57%) after 17-weeks of resistance training. This reiterates our previous findings and those of others: some individuals respond well while others have modest gains in strength or even loss of muscle with training (5, 13, 21). The study investigated whether varied training responses were related to mRNA levels of specific muscle growth factors though significant correlations (R > 0.7) with training outcomes were only found for VEGFD. This contrasts with our previous study which found multiple growth factors to be strongly correlated (R≥0.9) with strength gain (13). The basis for the breakdown in relationships is unknown but could be due to the health of the study populations. Participants in the previous study were taking few, if any, medications and supplements for age-associated problems whereas the current participants were taking an average of eight products each. Statins and NSAIDs have been found to influence the response to resistance training and our results suggest that α-1 antagonists may as well (22, 23, 24). These findings illustrate an important issue. Individuals being treated for comorbidities of aging benefit from resistance exercise but are often excluded and more studies are needed to understand the determinants of the training response in such populations.

The correlations between VEGFD, α-1 antagonists, strength gain, and muscle mass could be related to muscle blood supply and angiogenesis. VEGFD is the “D isoform from the VEGF family of growth factors which, at least in animals, regulate muscle angiogenesis (12, 25). In humans, muscle capillary content decreases with aging while angiogenesis and blood supply are considered important determinants of resistance training response (26, 27). Training studies to date have measured the VEGF “A isoform (28, 29, 30) which leaves our studies as the first evidence that VEGFD may be involved in angiogenesis which supports adaptation to training. Similarly, our finding that change in lean mass was positively correlated with use of α-1 antagonists also points to blood supply and angiogenesis as being important. These medications lower blood pressure through vasodilation which increases blood flow to muscle by up to 100% (31). Thus, VEGFD and α-1 antagonists may have overlapping influences on muscle. It would be interesting to assess factors such as VEGFD receptor activation, the angiogenic signaling cascade, capillary density, and peripheral blood flow in the contexts of exercise and/or α-1 antagonist treatment.

The current pilot study has several limitations. The participants were limited to older U.S. Veterans, a group which has a high prevalence of age-associated medical problems (15, 32). However, multimorbidity (hyperlipidemia, prostate hyperplasia, hypertension, depression, etc.) is common in older males and thus, the study results should be generalizable to individuals of similar health. Obesity was a frequent (16%) exclusion criterion despite the benefits of resistance exercise during weight loss (33) with the rationale being that weight loss could negate gains in muscle mass from training. However, obese individuals were referred to an alternate exercise program, our “Geriatric Walking Clinic (34). Another limitation was that functional abilities were not measured which hinders discussion of the clinical significance of the findings. Progressive high-intensity training of the thigh muscles has shown some success for improving performance in tasks such as gait, balance, and mobility from a seated position and the effects appear to be dampened in less healthy older adults (35). This also emphasizes the importance of understanding the molecular and individual determinants of muscle maintenance and rehabilitation in older adults so that improved strategies can be developed. Towards this goal, we are assessing the effects of resistance training and nutritional supplementation on strength and functional performance in a larger sample size of older adults who possess multiple age-associated health problems (36).

In summary, this pilot study conducted in older adults consuming numerous medications and supplements for age-associated problems identified muscle VEGFD, muscle mass, and use of α-1 antagonist medications as pre-training individual characteristics that were correlated with post-training improvements in muscle mass and strength. Further investigation is needed to understand the mechanistic relationship between these variables and their clinical significance.

Conflict of Interest

All authors (RAD, KKG, PMK, CMP, MMB, SL, KPP, PRP, and DHS) declare that no real or perceived conflicts of interest existed during the conduct of the study.

Ethical standard

The study complied with the US Code of Federal Regulations for research, all current U.S. law, and the Veterans Health Administration requirements for the protection of human subjects.