The global mortality rate resulting from cardiovascular disease (CVD) (including coronary artery disease [CAD] and chronic heart failure [CHF]) is over 17 million persons annually (1). Exercise-based cardiac rehabilitation is recommended to improve cardiovascular function and quality of life and to reduce the risk of secondary CVD events (2). To date, most cardiac rehabilitation exercise programs are designed for the improvement of cardiovascular function; however, structural and functional abnormalities of skeletal muscle are also frequently observed in CHF (3), and lower skeletal muscle mass has been associated with both low aerobic capacity and increased mortality in CAD (4).
Microvascular circulation is essential for the maintenance of function in skeletal muscle (5). Increases in blood flow and thereby shear stress promote the expression of proangiogenic signals, especially endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) protein (6). However, in CVD, peripheral blood flow and oxygen perfusion are often reduced because of cardiac dysfunction and poor vasoactive control (7,8). Previous studies have shown that patients with CAD and CHF have compromised skeletal muscle blood flow and diffusion of oxygen (9,10), lower mitochondrial density with reduced peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) protein expression (11), and unfavorable muscle fiber type distribution (12). Conversely, other studies have shown no difference in measures of capillary density, mitochondrial volume, and enzymes related to oxidative capacity with CHF (13,14). Thus, there is conflicting evidence as to whether differences in skeletal muscle phenotype exist between those with CVD and their healthy counterparts. Critically, beyond CHF patients, far less is known about the skeletal muscle characteristics in individuals with CAD.
Recent studies have reported that enhanced satellite cell activation and expansion are related to greater skeletal muscle fiber capillarization in healthy young individuals (15) and that capillarization is closely associated with skeletal muscle mass in older adults (16). To our knowledge, no study has examined these skeletal muscle characteristics in individuals with CAD compared with healthy controls. Further, little is known about how differing modes of cardiac rehabilitation exercise affect skeletal muscle characteristics in individuals with CAD.
Traditional exercise-based cardiac rehabilitation typically consists of moderate-intensity aerobic exercise for at least 30 min·d−1, anywhere from 3 to 7 d·wk−1 (17). Improvements in peak aerobic capacity as well as increased capillary density and succinate dehydrogenase activity in skeletal muscle in CAD patients have been reported (10). High-intensity interval training (HIIT) has been shown to be a feasible and effective alternative to traditional moderate-intensity continuous exercise program (TRAD). Currie et al. (18,19) showed that 3 and 6 months of stationary bicycle-based HIIT resulted in equivalent improvements in cardiovascular function (V˙O2peak and flow-mediated dilation [FMD]) in CAD patients compared with TRAD. In addition, even one bout of HIIT demonstrated marked improvements in the cardiovascular function, highlighting the potency of HIIT as an exercise stimulus to induce favorable physiological adaptations (20). However, less is known about skeletal muscle adaptation in individuals with CAD after exercise-based cardiac rehabilitation.
The purpose of this study was twofold. First, we characterized differences in skeletal muscle between individuals with CAD and age, body mass index (BMI), and sex-matched healthy participants. We hypothesized that, based on previous work in CHF patients, CAD participants would have deteriorated skeletal muscle characteristics (number of satellite cells and capillary-related factors) and a relative fiber atrophy compared with healthy controls participants. We further aimed to compare the effects of 12 wk of TRAD and stair climbing–based HIIT program (STAIR) on skeletal muscle phenotype in individuals with CAD. We hypothesized that both 12 wk of TRAD and 12 wk of STAIR would improve skeletal muscle characteristics in individuals with CAD and that improvements would be similar between exercise modalities.
METHODS
Ethics approval
All participants were informed of the purpose, experimental procedures, and the possible risks of the study before providing written informed consent. This study was approved by the Hamilton Integrated Research Ethics Board (HIREB#3301) and conformed to the Declaration of Helsinki. This study was registered as a clinical trial at clinicaltrials.gov (NCT03235674). Participants reported on as part of this study were part of the larger research project (data reported separately) examining the effectiveness of STAIR on improving endothelial function measured by brachial artery FMD in CAD patients completing outpatient cardiac rehabilitation.
Participants
Twenty participants (18 M/2F, 61 ± 7 yr) with CAD and who have had a history of previous myocardial infarction, coronary artery bypass graft, and/or percutaneous coronary intervention were recruited for this study. A sample size calculation based on the primary outcome variable of FMD showed that 13 participants per group would be adequate to detect meaningful differences in that outcome. There was no specific sample size calculation performed for the skeletal muscle characteristics.
All participants were nonsmokers, had stable medical therapy, and had registered to participate in exercise-based cardiac rehabilitation at the Cardiac Health and Rehabilitation Centre (CHRC) at the Hamilton General Hospital. Exclusion criteria included any noncardiac surgical procedure within 2 months, symptomatic peripheral arterial disease that limits exercise capacity, coronary heart failure (NYHA class II–IV confirmed via echocardiography), surgically inserted pacemakers, atrial fibrillation, documented peak orifice area valve stenosis, any musculoskeletal abnormality that would limit exercise participation (regular use of a mobility device, neuromuscular or neurometabolic disease), unstable angina, uncontrolled hypertension (>180/100 mm Hg), and documented chronic obstructive pulmonary disease (FEV1 < 60% and/or FVC <60%). Two participants withdrew from the study because of time constraints. We were unable to collect adequate muscle biopsy samples from two participants after the baseline biopsy; hence, a total of 16 participants (16 M/1F, 61 ± 7 yr) were included in the analysis. In addition, we studied muscle samples from nine healthy participants from previous trials, the muscle from whom served as controls, and they were individually matched (according to age, sex, and BMI) with nine randomly selected participants with CAD.
Study design
This study was a nonblinded, parallel group design. To examine differences in resting skeletal muscle characteristics between CAD and healthy controls, muscle biopsies were taken at baseline from the vastus lateralis for the determination of muscle fiber cross-sectional area, satellite cell content, myonuclear content, capillary-related factors, and expression of proteins related to vascular and mitochondrial function. After screening assessments and baseline measures, CAD participants were randomly assigned via a computer-generated random number sequence to participate in 12 wk (six supervised exercised sessions over 4 wk, followed by 8 wk of unsupervised exercise [~24 exercise sessions]) of either TRAD or STAIR. Skeletal muscle biopsies were taken at 4 wk (after six supervised sessions) and after 8 additional weeks (unsupervised session) of the exercise interventions in CAD participants only.
Rehabilitation exercise training
All participants performed a medically supervised cardiopulmonary exercise test on either a treadmill or a cycle ergometer for the measurement of peak cardiorespiratory fitness (V˙O2peak) and peak heart rate (HRpeak) using metabolic cart (Vmax 229; Sensor Medics, Yorba Linda, CA) ahead of enrollment at the CHRC. For the cycle ergometer, the workload was increased by 100 kpm per minute. For the treadmill, the test was conducted in accordance with the Bruce protocol. The treadmill workload started at 2.0 mph with 0% grade, and after 1 min, only the speed increased to 3.0 mph. After each minute, the incline increased by 2.5% grade. Once the grade reached 20%, the speed was increased by 0.5 mph·min−1.
The TRAD training consisted of 30 min of moderate-intensity continuous exercise on combination of stationary cycling, treadmill, and/or self-paced walking The exercise intensity for the TRAD group was determined using a target training heart rate from individual cardiopulmonary exercise test results through the heart rate reserve method. Exercise was completed at 60%–80% heart rate reserve, with an intensity goal of 11–13 on Borg’s RPE 6–20 scale. STAIR exercise sessions consisted of three bouts of ascending and descending of single flight of stairs (12 steps) six times at a self-selected vigorous intensity. Individuals in STAIR were asked to climb up and down the stairs one step at a time and ascend at a pace that challenged them (RPE 14–15/20) and to descend at a pace that was comfortable. Between each bout of stair climbing, participants performed 90 s of active recovery consisting of light walking on flat ground. Continuous heart rate was monitored by a chest-worn heart rate monitor (Polar A300, Polar H9 heart rate sensor; Polar Electro Oy, Kempele, Finland) to assess exercise intensity. Both TRAD and STAIR sessions began with a 10-min warm-up and finished with a 5 min cooldown consisting of light walking. To ensure the use of proper technique, all participants performed six exercise sessions of their assigned exercise modality at CHRC under the supervision of a certified health care professional for the first 4 wk. For the subsequent 8 wk, both groups were asked to continue to perform their exercise program, unsupervised 3 d·wk−1 at home or in a community-based exercise facility. Participants continued to use heart rate monitoring during exercise sessions at home, and these data were available to study investigators after upload by the participants.
Muscle biopsy procedure
All muscle biopsies were collected after an overnight fast (>10 h) and were taken at baseline, after 4 wk, and after 12 wk of exercise training in CAD participants and at baseline only in healthy controls. Participants were advised to refrain from exercise and alcohol consumption for 24 h and caffeine consumption for 10 h before muscle biopsy sampling. All prescribed medications and vitamins were taken as usual, except for any vasoactive medications (i.e., nitroglycerin). All muscle biopsies were obtained using a 5-mm Bergström needle that was adapted for manual suction under 1% xylocaine local anesthesia. Muscle tissue was then carefully freed from visible connective tissue, fat, and blood through manual dissection. A piece of the muscle tissue was embedded in optical cutting temperature compound (OCT; Tissue-Tek, The Netherlands) for histochemical analysis, and OCT embedded tissue and whole muscle were both rapidly frozen in liquid nitrogen and stored at −80°C for further analysis.
Immunohistochemistry
Muscle tissue embedded in OCT was cut on a cryostat (5 μm) maintained at a temperature of −20°C and transferred to a positively charged glass slide. After fixation in a 4% paraformaldehyde solution for 5 min, the slides were submerged in methanol and stored at −20°C for 10 min to remove fat. After placement in a blocking solution (goat serum and 0.1% triton/phosphate-buffered saline at a 9:1 ratio) for 20 min, the slides were incubated with primary antibodies against Pax7 (MAB 1675, 1:100; R&D Systems, Minneapolis, MN), myosin heavy chain type I (MHC I, A4.951, 1:1; Developmental Studies Hybridoma Bank, Iowa City, IA), MHC II (ab91506, 1:1000; Abcam, Cambridge, MA), laminin (ab11575, 1:500, Abcam), and CD31 (ab28364, 1:20, Abcam). Secondary antibodies used for Pax7 (Alexa Fluor 594, 1:500; Thermo Fisher Scientific, Waltham, MA), MHC I (Alexa Fluor 488 or 594, 1:500, Thermo Fisher Scientific), MHC II (Alexa Fluor 647 or 350, 1:500, Thermo Fisher Scientific), laminin (Alexa Flour 488 or 350, 1:500, Thermo Fisher Scientific), and CD31 (Alexa Flour 488, 1:500, Thermo Fisher Scientific). Nuclei were stained with 4,6-diamidino-2-phenylindole (1:20000; Sigma-Aldrich, Oakville, ON, Canada). Lastly, the slides were mounted with Prolong Diamond Antifade Reagent (Life Technologies, Burlington, ON, Canada). All analysis was completed with the investigator blinded to group and time point.
Samples were imaged by Nikon Eclipse 90i microscope equipped with high-resolution Photometrics CoolSNAP HQ2 fluorescence camera (Nikon Instruments, Melville, NY). All images were obtained with ×20 objective and analyzed using Nikon NIS element AR software (Nikon Instruments). To ensure the reliability of cross-sectional analysis (CSA), muscle fiber type, satellite cell content, and myonuclear content, ~200 muscle fibers per sample were analyzed (21). In addition, 50 muscle fibers per sample were analyzed for quantification of the capillary-to-fiber ratio (C/Fi) on an individual fiber basis and the capillary-to-fiber perimeter exchange index (CFPE) for the measurement of capillary-to-fiber surface area (22).
Western blotting
To analyze protein expression and phosphorylation, snap-frozen muscle tissue was homogenized with ice-cold lysis buffer (10 μL·mg−1; 25 mM Tris 0.5% vol/vol Triton X-100 and protease/phosphatase inhibitor cocktail tablets; Complete Protease inhibitor Mini-Tabs [Roche] and aPhosSTOP [Roche Applied Science]) and centrifuged at 1500g for 10 min at 4°C. The protein concentration of the supernatant (sarcoplasmic fraction) was determined via bicinchoninic acid assay (Thermo Scientific, Waltham, MA). A 4× Laemmli buffer (0.25 M Tris, 4% SDS, 20% glycerol, 0.015% bromophenol blue, and 10% 2-mercaptoethanol) was added to working samples, and equal amounts of protein (10 μg) from each sample were loaded into wells on 4%–15% TGX Stain-Free Precast Gels (Bio-Rad, Hercules, CA). A protein ladder (Precision Plus Protein Standard, Bio-Rad) and four internal standard calibration curves were loaded on every gel. Gel electrophoresis was run at 200 V for 45 min. The protein transfer from the gel to a nitrocellulose membrane was carried out by turbo transfer (#1704150, Bio-Rad). To ensure a complete protein transfer, membrane pre- and posttransfer images were checked using the ChemiDoc MP Imaging System (#12003154, Bio-Rad). Membranes were then blocked with 5% bovine serum albumin for 90 min at room temperature. Primary antibodies, total endothelial nitric oxide synthase (eNOS, 9572, 1:1000; Cell Signaling, Danvers, MA), phospho-eNOSSer1177 (9571S, 1:1000, Cell Signaling), VEGF (ab46154, 1:1000, Abcam), PGC-1α (Ab3242, 1:1000; Merck Millipore, Billerica, MA), and cytochrome c oxidase subunit IV (COX IV, Ab110261, 1:1000, Abcam) were incubated in a blocking solution for 12 h at 4°C. The membranes were then washed 3 × 5 min with Tris-buffered saline and Tween 20 (TBS-T; Millipore Sigma, Oakville, ON, Canada) and incubated with the appropriate host species secondary antibody for 90 min at room temperature. The secondary antibodies were removed by 3 × 5 min washing with TBS-T. Membrane bands were detected by chemiluminescence solution (Clarity Western ECL substrate, Bio-Rad), and images were scanned using the ChemiDoc MP Imaging system and analyzed by Image Lab Software for PC version 6.0.1.
Statistical analysis
The distribution of data was assessed using the Shapiro–Wilk test. All nonnormally distributed data are specified in the figure legend. Characteristics of CAD and healthy control participants, and TRAD versus STAIR participants at baseline were analyzed with an independent-sample t-test for normally distributed data or a Mann–Whitney test for nonnormally distributed data. A two-way repeated-measures ANOVA was used to compare TRAD and STAIR with exercise intervention as the between-subjects variable and time (baseline, 4 wk, and 12 wk) as the within-subjects variable. All significant interactions from the ANOVA analysis were further examined via Tukey’s post hoc test. Differences in nonnormally distributed data in this comparison were analyzed with robust a two-way between-within ANOVA in R. Trimmed means were used as a measure of location rather than the mean, which is subject to outlier effects, with the trimming parameter set to 0.2. Hochberg-adjusted multiple comparisons for interaction and main effects were conducted using the “bwmcppb.adj” function to control for family-wise error, as described in Wilcox (23). Statistical significance was set at P < 0.05. Data are presented as mean ± SD or graphed as means with individual data and showing the 95% confidence interval (CI). Statistical analyses were completed using the SPSS Statistics software package (SPSS Statistics, Version 26.0 for Windows; IBM Corp., Armonk, NY) or R (version 4.0.2).
RESULTS
Participant characteristics
Baseline characteristics of the participants are presented in Table 1. There were no differences in anthropometric measures between CAD and healthy controls (P > 0.05) or between TRAD and STAIR (P > 0.05). There were no differences in clinical outcomes, aerobic fitness (V˙O2peak), CVD risk factors (type 2 diabetes, hypertension, and dyslipidemia), blood variables (fasted glucose, fasted insulin, HDL, LDL, triglycerides, and cholesterol), or medication between TRAD and STAIR (P > 0.05).
TABLE 1 -
Baseline characteristics of the participants.
| Variables |
Healthy Controls (n = 9) |
CAD (n = 9) |
CAD (n = 16) |
| TRAD (n = 7) |
STAIR (n = 9) |
| Sex (M/F) |
(9/0) |
(9/0) |
(7/0) |
(8/1) |
| Age (yr) |
66 ± 4 |
64 ± 5 |
61 ± 10 |
62 ± 6 |
| Height (cm) |
175 ± 6 |
173 ± 5 |
174 ± 3 |
175 ± 6 |
| Body mass (kg) |
91 ± 14 |
90 ± 12 |
97 ± 20 |
90 ± 11 |
| BMI (kg·m−2) |
30 ± 4 |
30 ± 4 |
30.2 ± 3.7 |
29.8 ± 3.3 |
| V˙O2peak (L·kg−1⋅min−1) |
|
21.7 ± 3.9 |
23.2 ± 2.5 |
21.4 ± 4.5 |
| Clinical |
|
|
|
|
| STEMI, n (%) |
– |
3 (33.3) |
1 (14.3) |
2 (22.2) |
| NSTEMI, n (%) |
– |
4 (44.4) |
4 (57.1) |
5 (55.6) |
| Angina, n (%) |
– |
1 (11.1) |
1 (14.3) |
2 (22.2) |
| PCI, n (%) |
– |
6 (66.7) |
4 (57.1) |
7 (77.8) |
| CABG, n (%) |
– |
3 (33.3) |
3 (42.9) |
2 (22.2) |
| Time since event (wk) |
– |
9 ± 6 |
8 ± 4 |
9 ± 5 |
| Medications |
|
|
|
|
| Beta-blockers, n (%) |
– |
8 |
6 (85.7) |
9 (100) |
| ACE inhibitors, n (%) |
– |
6 |
6 (85.7) |
6 (66.6) |
| ASA, n (%) |
– |
9 |
7 (100) |
9 (100) |
| Lipid lowering, n (%) |
– |
9 |
7 (100) |
9 (100) |
| Metformin, n (%) |
– |
2 |
1 (14.3) |
1 (11.1) |
| CVD risk factors |
|
|
|
|
| T2DM, n (%) |
– |
3 (33.3) |
2 (28.6) |
1 (11.1) |
| Hypertension, n (%) |
– |
6 (66.6) |
6 (85.7) |
6 (66.7) |
| Dyslipidemia, n (%) |
– |
6 (66.6) |
6 (85.7) |
6 (66.7) |
| Blood markers |
|
|
|
|
| Fasted glucose (mM·L−1) |
|
5.6 ± 1.0 |
5.4 ± 1.0 |
5.8 ± 1.00 |
| Fasted insulin (mIU·L−1) |
|
10.1 ± 6.7 |
7.3 ± 2.3 |
12.8 ± 6.8 |
| HDL (mM·L−1) |
|
1.0 ± 0.3 |
1.1 ± 0.4 |
1.0 ± 0.4 |
| LDL (mM·L−1) |
|
1.3 ± 0.4 |
1.3 ± 0.4 |
1.4 ± 0.4 |
| Triglycerides (mM·L−1) |
|
1.0 ± 0.4 |
0.8 ± 0.2 |
1.1 ± 0.4 |
| Cholesterol (mM·L−1) |
|
2.7 ± 0.6 |
2.7 ± 0.8 |
2.9 ± 0.7 |
Data are expressed as mean ± SD.
STEMI, ST elevation myocardial infarction; NSTEMI, non-ST elevation myocardial infarction; PCI, percutaneous intervention; CABG, coronary artery bypass graft; ACE, angiotensin-converting enzyme; ASA, acetylsalicylic acid; T2DM, type 2 diabetes mellitus.
Characteristics of cardiac rehabilitation exercise
Participants in both TRAD (3.0 ± 2.2 d·wk−1) and STAIR (3.0 ± 3.2 d·wk−1) groups adhered to their respective exercise programs throughout the 12-wk intervention. During exercise, STAIR elicited a greater HRpeak compared with TRAD (112 ± 14 vs 129 ± 11 bpm, P = 0.008). In addition, the average %HRpeak was 12% higher in STAIR compared with TRAD (P = 0.028). The average total exercise time per session at the prescribed intensity of STAIR was significantly shorter compared with TRAD (STAIR, 5 ± 2 min, vs TRAD, 33 ± 8 min; P < 0.001). Exercise protocol values after 12 wk of exercise training are presented in Supplemental Digital Content 1, https://links.lww.com/MSS/C212.
Skeletal muscle phenotype in CAD and healthy controls
Participants with CAD had a 52% ± 88% lower prevalence of Type I fibers (P = 0.034) and a 14% ± 21% higher percentage of Type II fibers (P = 0.034) (Fig. 1A). There were no differences in fiber CSA for Type I or II fibers between CAD and healthy controls (Type I fibers, P = 0.141; Type II fibers, P = 0.084; Fig. 2B). There was no difference in satellite cell (P = 0.282) or myonuclei content per fiber in Type II fibers between CAD and healthy controls (P = 0.094); however, Type I muscle fiber-associated satellite cell and myonuclei content per fiber were 181% ± 354% (P = 0.019) and 24% ± 22% (P = 0.017) lower in CAD compared with healthy controls, respectively (Fig. 1C and D). There were no differences in C/Fi number (P = 0.063) or CFPE index (P = 0.123) in Type I fibers between CAD and healthy controls. However, Type II fiber C/Fi number and CFPE index were 35% ± 25% (P = 0.06) and 23% ± 29% (P = 0.034) lower in CAD compared with healthy controls, respectively (Fig. 1E and F).
;)
FIGURE 1: Skeletal muscle characteristics in CAD patients and healthy controls. The ratio of the fiber type composition to Type I and Type II (A) and the cross-sectional area (B). The number of Pax7+ satellite cells (C) and myonuclei (D) per fiber. Individual muscle fiber C/Fi (E) and CFPE per 1000 μm of cross-sectional area (F). White bars represent healthy controls; gray bars represent CAD. Data are expressed as mean ± SD and 95% CI. * P < 0.05, significantly different from healthy controls within each fiber type.
FIGURE 2: Expression of capillary and mitochondria-related proteins. The ratio of phosphorylation to total endothelial nitric oxide synthase (eNOS)Ser1177 (A), VEGF (B), PGC-1α (C; Mann–Whitney test), and cytochrome c oxidase subunit IV (COX IV) (D; Mann–Whitney test); representative Western blotting bands (E). AU, arbitrary unit; HC, healthy controls. White bars represent TRAD; gray bars represent STAIR. Data are expressed as mean and 95% CI. *P < 0.05, significantly different from healthy controls.
Capillary and mitochondria-related protein expression between CAD and healthy controls
There was no difference in the ratio of phosphorylation to total eNOSSer1177 (P = 0.24) or COX IV (P = 0.44) protein expression between CAD and healthy controls (Fig. 2A and D). PGC-1α and VEGF protein expression values in CAD were 128% ± 149% (P = 0.024) and 120% ± 173% (P = 0.044), respectively, lower compared with healthy controls (Fig. 2B and C).
Changes in skeletal muscle phenotype with exercise training in CAD patients
There were no changes in fiber CSA during the exercise training interventions (Type I fibers: P = 0.39; Type II fibers: P = 0.911) and no differences between CAD groups (Type I fibers: P = 0.67; Type II fibers: P = 0.638) at any time point (Figs. 3A and B). Satellite cell content per fiber in Type I fibers increased at 4 wk (22% ± 56%, P = 0.011) with no difference between training groups (P = 0.076). Satellite cell content per fiber in Type I fibers returned to those similar to baseline at 12 wk (P = 0.284; Fig. 3C). Satellite cell content per fiber in Type II fibers was greater at 12 wk of training compared with baseline (35% ± 35%, P = 0.046) and at 4 wk (25% ± 33%, P = 0.003) in STAIR only (Fig. 3D). There was no difference in the number of myonuclei per fiber at 4 wk (Type I fibers: P = 0.406; Type II fibers: P = 0.05); however, myonuclei number per fiber increased by 8% ± 15% in Type I fibers (P = 0.012) and 12% ± 19% in Type II fibers (P = 0.006) after 12 wk of training with no differences between groups (Type I fibers: P = 0.359; Type II fibers: P = 0.952) (Fig. 3E and F).
FIGURE 3: Changes in skeletal muscle characteristics at 4 and 12 wk after TRAD and STAIR training in CAD patients. Skeletal muscle CSA for Type I (A; robust ANOVA) and Type II (B). The number of Pax7+ satellite cells per fiber for Type I (C) and Type II (D). The number of myonuclei per fiber for Type I (E) and Type II (F). BL, baseline; 4w, 4 wk; 12w, 12 wk. White bars represent TRAD; gray bars represent STAIR. Data are expressed as means and 95% CI. *P < 0.05, significantly different from BL within group. #P < 0.05, significantly different from 4w within group. †P < 0.05 significantly different from BL.
Capillarization with exercise training in CAD patients
There was no difference in C/Fi number in Type I fibers after TRAD or STAIR training at any time point (P > 0.05; Fig. 4A). C/Fi in Type II fibers increased by 17 ± 12 at 4 wk (P < 0.001) and by 23 ± 14 at 12 wk (P < 0.001) with no difference between groups (P = 0.30) (Fig. 4B). CFPE index in Type I fibers increased by 11% ± 14% at 4 wk (P < 0.01) and by 10% ± 23% at 12 wk (P < 0.01) with no difference between groups (P = 0.066) (Fig. 4C). CFPE index increased by 18% ± 14% at 4 wk (P < 0.001) and by 18% ± 22% at 12 wk (P = 0.002) in Type II fibers with no difference between groups (P = 0.17) (Fig. 4D).
FIGURE 4: Changes in capillarization at 4 and 12 wk after TRAD and STAIR training in CAD patients. Individual muscle fiber C/Fi for Type I (A) and Type II (B; Robust ANOVA). Capillary-to-fiber perimeter exchange (CFPE) per 1000 μm for Type I (C; Robust ANOVA) and Type II (D). BL, baseline, 4w, 4 wk; 12w, 12 wk. White bars represent TRAD; gray bars represent STAIR. Data are expressed as means and 95% CI. †P < 0.05, significantly different from BL.
Capillary and mitochondria-related protein expression with exercise training in CAD patients
There was a significant increase in the ratio of phosphorylation to total eNOSSer1177 at 4 wk of training by 19% ± 33% (P < 0.01) with no difference between groups (P = 0.127). This ratio was not changed from baseline at 12 wk (P = 0.073; Fig. 5A). There was no difference in VEGF protein expression at 4 wk or 12 wk (P > 0.05; Fig. 5B). Protein expression of PGC-1α increased by 19% ± 52% at 4 wk (P = 0.046) and 23% ± 41% at 12 wk (P = 0.014), with no differences between groups (P = 0.11) (Fig. 5C). There was a main effect for time in protein expression of COX IV (P = 0.048). However, there was no significant post hoc effect when P values are adjusted for multiple comparisons (Fig. 5D).
FIGURE 5: Changes in expression of capillary and mitochondria-related proteins at 4 and 12 wk after TRAD and STAIR training in CAD patients. The ratio of phosphorylation to total endothelial nitric oxide synthase (eNOS)Ser1177 (A; Robust ANOVA), VEGF (B), PGC-1α (C), and cytochrome c oxidase subunit IV (COX IV) (D; Robust ANOVA), representative, Western blotting bands (E). AU, arbitrary unit; BL, baseline; 4w, 4 wk; 12w, 12 wk. White bars represent TRAD; gray bars represent STAIR. Data are expressed as means and 95% CI. †P < 0.05, significantly different from BL.
DISCUSSION
We discovered marked differences in skeletal muscle characteristics in CAD patients compared with age, sex, and BMI-matched healthy older adults as evidenced by a lower percentage of Type I fibers, fewer satellite cells, and decreased number of myonuclei per fiber in Type I fibers. In addition, skeletal muscle from CAD patients exhibited significantly reduced capillary-related indices C/Fi and CFPE in Type II fibers, as well as lower VEGF and PGC1α protein expression compared with controls. We are uncertain whether these findings are a determinant or consequence of CAD, but they highlight that CAD patients have a lower skeletal muscle metabolic quality and display a presarcopenic skeletal muscle phenotype (24). Critically, we show for the first time, to our knowledge, that despite reduced satellite cell and myonuclear content, and lower capillary-related factors compared with healthy controls, individuals with CAD are able to ameliorate these decrements with 12 wk of either TRAD or STAIR exercise training. In addition, despite a sixfold shorter average exercise time required by STAIR (~5 ± 2 min) versus TRAD (~33 ± 8 min), we observed comparable changes in skeletal muscle phenotype with training. Interestingly, we did observe increased satellite cell proliferation in Type II fibers to a greater extent after the STAIR program, which is deserving of follow-up.
SKELETAL MUSCLE CHARACTERISTICS IN INDIVIDUALS WITH CAD
In general, aging results in the atrophy of Type II fibers along with a reduction in the number of satellite cells and myonuclei per fiber (25). We could not detect differences in the number of Type II fiber satellite cells and myonuclei per fiber between CAD and healthy participants; however, CAD participants exhibited a lower number of satellite cells and myonuclei in Type I fibers compared with healthy participants. Satellite cells play a vital role in skeletal muscle repair, remodeling, and growth (26). In aging muscle, declines in satellite cell content are associated with Type II fiber atrophy, which includes loss of muscle mass as well as a loss of the total number of muscle fibers due to denervation (27). We note that CHF patients have compromised mitochondrial and capillary-related phenotypes, but we could not find an investigation examining satellite cell characteristics in CHF or CAD patients (28); hence, our data in CAD patients are a first and without an easy CVD-related comparison. Therefore, although the profile of Type II fibers in CAD was in line with what would be expected with a natural sarcopenic decline (Fig. 1), the presence of CAD is associated with a Type I fiber-specific deterioration in aging skeletal muscle (Fig. 1).
Muscle perfusion can have substantial impacts on substrate delivery and many cellular processes such as protein turnover (5) and oxygen delivery (29). We observed that C/Fi and CFPE in Type II fibers in CAD were lower compared with healthy controls. In addition, VEGF, an angiogenic growth factor, showed significantly lower protein expression in CAD participants compared with healthy controls, findings that are line with the observed reduction in C/Fi in CAD. However, there was no difference in eNOS protein expression between CAD patients and healthy controls (Fig. 2A and B). We also found that Type II fibers in the skeletal muscle of CAD participants had a lower CFPE index compared with healthy controls. Hepple et al. (30) reported a positive correlation between V˙O2peak and CFPE index, which supports the notion that impaired oxygen delivery to skeletal muscle in CAD patients may be due to reduced capillarization in their skeletal muscle. Previous studies have reported increases in the ubiquitin–proteasome system through increases in ubiquitin ligases, MuRF1 and MAFbx in CHF patients (31), and decreased mitochondria content by reduced PGC-1α protein expression in CVD (11). In the present study, besides PGC-1α protein expression, which was lower in CAD, there were no differences in the CSA of Type I or II fibers or differences in the protein expression of COX IV in CAD participants compared with healthy controls. Further studies are required to fully elucidate the mechanisms resulting in altered skeletal muscle phenotypes in CAD patients.
CARDIAC REHABILITATION AND SKELETAL MUSCLE ADAPTATION IN CAD PATIENTS
To our knowledge, there are very few studies examining the impact of cardiac rehabilitation on skeletal muscle metabolism in individuals with CAD (4,10), with no study that has investigated the effects on skeletal muscle fiber type adaptations. After 12 wk of training, there were no differences in the CSA of Type I or II fibers in either group; however, 4 wk of both TRAD and STAIR was associated with an increase in the number of satellite cells in Type I fibers. Satellite cells are robustly activated in earlier phases of exercise training, an effect that may not be due to muscle damage but may be a normal part of phenotypic adaptation (32). Although there were no differences in the change in satellite cell number in Type I fibers between groups, 12 wk of STAIR increased satellite cell content in Type II fibers to a greater extent compared with TRAD. Previous studies indicated that HIIT may induce a greater increase in satellite cell content, particularly in Type II fibers versus moderate-intensity endurance exercise (33,34). In line with these observations, the higher-intensity nature of STAIR may have increased the recruitment of Type II fibers in comparison with TRAD, resulting in the greater satellite cell content in Type II fibers at the 12-wk time point (35). Satellite cell-mediated myonuclear accretion is a hallmark of largely with skeletal muscle hypertrophy when the upper limits of the myonuclear domain are reached (36). However, previous studies have shown that satellite cell-mediated myonuclear accretion can occur with nonhypertrophic stimuli, including endurance exercise, and may contribute to remodeling of muscle fibers (32).
Both endurance exercise (37) and resistance exercise (38) can promote the expression of angiogenesis-related signals by including an increase in blood flow and thereby shear stress (6). Indeed, Tan et al. (39) reported that 6 wk of HIIT using a cycle ergometer increased capillary contacts (CC) in Type I and Type II fibers in overweight women, and Cocks et al. (40) also reported increased muscle microvascular eNOS content and capillarization after 6 wk sprint interval training and traditional endurance training. We report that 4 wk of both TRAD and STAIR increased C/Fi in Type II fibers in CAD participants and that increase was maintained at 12 wk of training in both groups. This finding is paralleled by the observed increase in maximal V˙O2peak after training (Dunford and MacDonald, personal communication). As increased capillary density is not only closely connected to the delivery of oxygen and nutrients but also enhances activation and expansion of satellite cell content for skeletal muscle repair (15), the increased capillary density after TRAD and STAIR may allow for the regeneration and repair of comprised skeletal muscle in CAD. Indeed, individuals who have a lower capillarization in skeletal muscle at baseline showed the lower extent of skeletal muscle mass increase after resistance exercise in older men (16), and 12 months of longer exercise-based traditional cardiac rehabilitation increased individual fiber area accompanied with increased capillary density in CAD patients (10). Although Tan et al. (39) reported the increase in CC for both Type I and Type II fibers after HIIT, we observed the increase in C/Fi in Type II fibers only. The disparate findings may be due to the characteristics of different participants performing the exercise training.
The secretion of VEGF protein during exercise plays critical roles in promoting angiogenesis by stimulating endothelial cells to proliferate, migrate, and differentiate (41). In addition, VEGF upregulates the expression of eNOS, which synthesizes endothelial nitric oxide (NO) (42). The release of endothelial NO induces vasodilation and thereby improves blood flow and perfusion (43). We observed no further increase in C/Fi at 12 wk of both TRAD and STAIR from 4 wk (Fig. 4). In line with our observations, 4 wk of both TRAD and STAIR increased phosphorylation of eNOS, but these values returned to those similar to baseline at 12 wk. However, there were no changes in VEGF protein expression in either group after exercise training despite an increased C/Fi. An increase in VEGF protein expression after exercise is transient, with levels returning to baseline within 2 h after exercise cessation (44). Given that muscle biopsies were collected at rest and participants were required to refrain from physical activity 24 h before muscle biopsy sampling, the lack of augmented VEGF is perhaps not surprising.
Individuals with CHF had reduced oxidative capacity of skeletal muscle indicative of lower mitochondria density (9). Here we show that both TRAD and STAIR increased expression of the PGC-1α after 4 and 12 wk, whereas there was no difference in protein expression of COX IV after either exercise training program (45). Six sessions of HIIT over 2 wk led to the increase in mitochondria content measured by citrate synthase activity and respiration in young men (46). In addition, 6 wk of HIIT increased mitochondrial content in older men and women (47). Given the former study, although the 2 wk of HIIT training duration was possible to induce mitochondrial biogenesis in young adults, CAD patients who have relatively compromised skeletal muscle characteristics may need a longer period of training to lead to mitochondrial biogenesis although the expression of PGC-1α was increased at 4 and 12 wk of training. Mitochondrial volume and content have been correlated with V˙O2peak (48), which is a strong predictor of mortality (49), and CAD patients generally have a lower oxygen consumption capacity. Thus, exercise training after a cardiac event should be considered as a means to improve muscle oxidative capacity in CAD individuals.
Our novel findings highlight that although CAD patients have compromised skeletal muscle function compared with healthy controls, both TRAD and STAIR, a practical and time-efficient alternative, are equally effective in improving skeletal muscle metabolic characteristics after a cardiac event. However, we recognize that our small sample size is a limitation as this would inflate the risk of a type II statistical error. Also, 8 wk of the training was unsupervised, so we were not able to control the exact exercise intensity performed by participants and had to rely on self-report and heart rate monitoring results for adherence estimates and confirmation.
In conclusion, we show that CAD is associated with smaller muscle fibers, reductions in satellite cell and myonuclei number, and capillary-related perfusion capacity of skeletal muscle in comparison with healthy controls. Critically, we found that 4 and 12 wk of cardiac rehabilitation in the form of either TRAD or STAIR improved these compromised skeletal muscle characteristics by increasing the number of satellite cells, myonuclei, and capillary-related factors induced by CAD. We conclude that skeletal muscle metabolism in individuals with CAD can be improved with exercise-based cardiac rehabilitation. Also, HIIT-based stair climbing, which is easily accessible, could be a feasible and practical alternative to traditional cardiac rehabilitation exercise, despite shorter average exercise time.
C. L. is supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1A6A3A03033939). S. M. P. thanks the Canada Research Chairs program. E. C. D. was supported by the Canadian Institutes of Health Research-Institute of Gender and Health grant. M. J. M. and S. M. P. are supported by the Natural Sciences and Engineering Research Council Discovery grant.
The authors have no professional relationships with companies or manufacturers who will benefit from the results of the present study. The results of the present study do not constitute endorsement by the American College of Sports Medicine. The authors declare that the results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation.
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