Cycling Exercise Training Enhances Mitochondrial Bioenergetics of Platelets in Patients with Peripheral Arterial Disease: A Randomized Controlled Trial

Cycling Exercise Training Enhances Mitochondrial Bioenergetics of Platelets in Patients with Peripheral Arterial Disease: A Randomized Controlled Trial Ming-Lu Lin, PhD; Tieh-Cheng Fu, MD, PhD; Chih-Chin Hsu, MD, PhD; Shu-Chun Huang, MD, PhD; Yu-Ting Lin, PhD; Jong-Shyan Wang, PhD 2, 4 * 1 Healthy Aging Research Center, Graduate Institute of Rehabilitation Science, Medical Collage, Chang Gung University, Tao-Yuan, Taiwan 2 Department of Physical Medicine and Rehabilitation, Chang Gung Memorial Hospital, Keelung, Taiwan 3 Department of Physical Medicine and Rehabilitation, Chang Gung Memorial Hospital, TaoYuan, Taiwan 4 Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, Tao-Yuan, Taiwan + Equal contribution as the first author


Introduction
Peripheral arterial disease (PAD) is a manifestation of atherosclerosis or thrombosis that causes chronic narrowing of peripheral arteries, consequently reducing the capacity of blood flow to contracting muscles in legs [1]. Moreover, reduced exercise capacity negatively affects the ability of PAD patients to perform the activities required for daily life, further decreasing their independence and quality of life [2,3]. However, exercise training may not only improve physical performance but also reduce vascular thrombotic risk in patients with PAD [2,3].
Platelets play a critical role in thrombogenesis of PAD patients [4,5]. Platelet mitochondrial functionality are mainly involved in the cellular redox balance and activation, thereby modulating thrombogenesis [6][7][8]. Cycling exercise training (CET) improves aerobic fitness, concurrence with reducing the risk of major vascular thrombotic events in patients with circulatory disorders [9,10]. According to our early studies, moderateintensity exercise training on a bicycle ergometer depressed platelet adhesion/aggregation at rest and attenuated the enhancement of platelet reactivity caused by acute strenuous exercise [11,12]. Recently, our investigation further demonstrated that CET improved the capacity for platelet mitochondrial bioenergetics [13,14] and reduced platelet-induced thrombin generation [13] in healthy sedentary people [13] or heart failure (HF) patients [14]. However, the effects of CET on platelet mitochondrial bioenergetics in PAD patients have not yet been established.
The electron transport complexes in mitochondrion are interconnected in mitochondrial inner membrane and turn into respiratory supercomplexes [15]. Mitochondrial structure and function are disrupted as isolating independent organelles from whole cells [16]. A highresolution respirometry can measure mitochondrial functionality by the addition of exogenous substrates and inhibitor to the permeablized cells [17]. Recently, we have established two novel reference protocols of the substrate-uncoupler-inhibitor titration (SUIT-RPs), those were employed to determine the capacities of mitochondrial oxidative phosphorylation (OXPHOS) and electron transport system (ETS) in platelets, respectively [18]. Therefore, the present study further evaluated how CET effects systemic aerobic capacity and mitochondrial OXPHOS and ETS activities of platelets in PAD patients.
To answer the abovementioned questions, this study clearly assessed how CET (i.e., cycling exercise at ventilation threshold for 30 min/day, 3 days/week for 12 weeks) affects systemic aerobic capacity and mitochondrial OXPHOS and ETS activities of platelets in patients with PAD. The present study aimed to establish an effective exercise regimen for improving aerobic capacity and to enhance platelet mitochondrial functionality in PAD patients. Fifty-two patients diagnosed with PAD have been surveyed for the interventions from   April 1, 2018 to December 31, 2019 at the Chang Gung Memorial Hospital, Keelung, Taiwan. Inclusion criteria in this investigation were listed as follows: (i) > 20 years old, (ii) PAD for more than 2 weeks; (iii) ankle-brachial index (ABI) < 0.9, and (iii) active voluntary exercise.

Participants
Exclusion criteria were listed as follows: (i) less than 20 years old; (ii) unstable angina; (iiii) systolic blood pressure (SBP) at rest is greater than 200 mm Hg or diastolic blood pressure (DBP) greater than 110 mm Hg; (iv) the orthostatic blood pressure drop is greater than 20 mm Hg with symptoms; (v) severe aortic stenosis with a peak systolic pressure gradient greater than 50 mm Hg, with an aortic valve opening area less than 0.75 cm 2 ; (vi) acute discomfort or fever; (vii) uncontrolled atrial or ventricular dysrhythmias; (viii) uncontrolled sinus rhythm tachycardia (more than 120 per minute); (ix) uncompensated congestive heart failure; (x) third degree atrioventricular block; (xi) acute pericarditis or myocarditis; (xii) recent embolism; (xiii) thrombophlebitis; (xiv) ST segment displacement is more than 2 mm at rest; and (xv) uncontrolled diabetes (glycemic blood glucose greater than 300 mg/dL or greater than 250 mg/dL with ketone bodies). Afterwards, forty eligible PAD patients were randomly divided into general rehabilitation (GR) with CET (GR+CET, n=20) and only received GR (GR, n=20) groups (Fig. S1). All subjects provided informed consent after the experimental procedures were explained. This study was performed in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan (ClinicalTrials.gov Identifier: NCT03965520).

Ankle-brachial index (ABI) measurement
Doppler measurements were performed in accordance with AHA guidelines for ABI measurement [19,20]. A digital vascular doppler HUNTLEIGH Dopplex DMX (Huntleigh Healthcare, United Kingdom) with an 8 MHz probe was used to measure the individual systolic pressures. An appropriately sized pneumatic cuff was applied to the right upper arm, inflated to suprasystolic pressure and deflated slowly until a Doppler flow signal was detected. The process was repeated for right leg and values for both dorsal pedal and anterior tibial arteries were measured, followed by left leg and left arm. ABI was subsequently calculated for each lower limb separately using the value of pressure from the respective arm as a denominator.

Grade exercise test
The participants performed the graded exercise test on a bicycle ergometer (Ergoselect150P, Germany) by one rehabilitation physician who was blinded to the GR+CET or GR subjects to assess their cardiopulmonary parameters 2 days before and 2 days after 12week intervention. Moreover, the data collector was isolated from the data analytic specialist.
Each participant was instructed to fast for at least 8 h and to refrain from exercise for at least 24 h before the test. All participants arrived at the testing center at 9:00 A.M. to eliminate diurnal effects. The subject first collected two minutes of resting parameters on the stationary bicycle. Then, the subjects performed on the unloaded free pedal for two minutes, after which the load was increased by 1 watt every 6 seconds (10 watts per minute) until the subjects were exhausted (i.e., progressive exercise to peak oxygen consumption, VO2peak). During the process, the bicycle speed was required to be maintained at 60 rpm. Minute ventilation (VE), VO2, and carbon dioxide production (VCO2) were measured on a breath-by-breath basis, using a computer-based system (Master Screen CPX, Cardinal-health Germany) [14]. Heart rate (HR) was determined from the R-R interval on a 12 lead electrocardiogram, mean arterial pressure (MAP) was measured using an automatic blood pressure system (Tango, SunTech Medical, UK), and arterial O2 saturation was monitored through finger pulse-oximetry (model 9500, Nonin Onyx, Plymouth, MN, USA) [14]. VO2peak was defined by the following criteria: VO2 increased by <mL/kg/min over at least 2 min, HR ≥85% of its predicted upper threshold, respiratory exchange ratio ≥ 1.10, or some other symptom/sign limitations in accordance with the guidelines of the American College of Sports Medicine for exercise testing [21].
Additionally, the ventilation threshold (VT) was determined when VE/VO2 increased without a corresponding increase in the VE-to-VCO2 ratio, end-tidal PO2 increased without a decrease in end-tidal PCO2, or there was a departure from linearity for VE [21].
Ventilation and VCO2 responses, acquired from the initiation of exercise to the peak values, were used to calculate the VE-VCO2 slope, using least-squares linear regression (y = m · x + b, m = slope) [22]. Additionally, the 6-minute walk test (6 MWT) was used to assess functional capacity and exercise endurance in patients with PAD [23]. The distance covered over a time of 6 minutes was used as the outcome by which to compare changes in performance capacity [23].

Hemodynamic measurements
Noninvasive continuous cardiac output monitoring system (NICOM, Cheetah Medical, Wilmington, Delaware) was used to evaluate cardiac hemodynamic response to exercise, which analyzes the phase shift (ΔΦ) created by alternating electrical current across the chest of the subject as described in our previous study [24]. Four dual surface electrodes were placed on the back of each subject to establish electrical contact with the body and avoid interference of upper body motion with the electrical cables during exercise testing. Stroke volume (SV) was estimated using the following equation: SV = C·VET·dΦ/dtmax, where C is a constant of proportionality, and VET denotes the ventricular ejection time, as determined using the NICOM and electrocardiogram signals. The cardiac output (CO) was then calculated using the following equation: CO = SV x HR [24].

Cycling exercise training (CET) protocol
In addition to the daily PAD rehabilitation course, the GR+CET subjects also performed supervised hospital-based training on a bicycle ergometer (Ergoselect 150P, Germany), completing 3 weekly sessions for 12 weeks. The CET protocol comprised a warm-up at 30% of VO2peak for 3 min, followed by continuous work-rate at VT for 30 min, then a cool-down at 30% of VO2peak for 3 min. All subjects used an HR monitor (Tango, SunTech Medical, UK) to obtain the assigned intensity of exercise. Borg 6-to-20 scale was used to assess the rate of perceived exertion during and after each exercise session. The work-rate of bicycle ergometer was adjusted continuously to ensure that the intensity of exercise matched the target HR throughout the training period. Patients were instructed to immediately stop exercise training if they had leg pain or other signs/symptoms of circulatory disorders. The GR subjects only engaged in general PAD rehabilitation course, as instructed by their rehabilitation physicians.
The rates of compliance with the GR+CET and GR subjects were 100% and 100%, respectively.

Platelet isolation
Before the graded exercise test at the beginning of the present study and 12 weeks later in various groups, 20 ml of blood was sampled from each subject's antecubital vein within 1 min by venipuncture (20-gauge needle). Blood samples (20 mL) were collected in polypropylene tubes containing sodium citrate (3.8 g/dL, 1-9 vol. blood). Platelet rich plasma (PRP) was prepared through centrifugation at 300 g for 10 min at approximately 20°C. Platelets were sedimented through centrifugation of the PRP at 1500 g for 10 min at approximately 20 °C and then washed once with phosphate buffered saline (PBS) containing ethylenediaminetetraacetic acid (EDTA, final concentration, 4 mM) (Sigma) to inhibit platelet activation [13,14]. The number of platelets was adjusted to 2 × 10 8 cells/mL with mitochondrial respiration medium (MiR05, containing sucrose 110 mM, HEPES 20 mM, taurine 20 mM, K-lactobionate 60 mM, MgCl2 3 mM, KH2PO4 10 mM, EGTA 0.5 mM, BSA 1 g/L, pH 7.1). Blood analysis was repeated twice to ensure reproducibility of the results. All platelet fractions were analyzed within 2 h after cell purification. Blood cells were enumerated using a Sysmax SF-3000 cell counter (GMI, Inc., Ramsey, MN, USA).

High-resolution respirometry
Platelet mitochondrial respiration was measured in 2 mL glass chambers of a highresolution respirometry (Oxygraph-2K, Oroboros Instrument, Austria) with a stirrer speed of 750 rpm at a constant temperature of 37 °C. Data was acquired and recorded every 2 s by DatLab software version 6 (Oroboros Instrument, Austria). The O2 sensors and instrument background O2 consumption had been already calibrated before experiments following the manufacturer's instruction [13,14]. Fixed number of platelets (2 x 10 8 cells/mL) were added in the glass chamber filled with 2 mL mitochondrial respiration medium (MiR05) for measurement [13,14]. Air calibration at saturation point was performed each time before the experiment for quality control. O2 concentration was automatically calculated from barometric pressure and solubility factor was 0.92 for MiR05.

Health-related quality of life
Health-related quality of life (HRQoL) in the PAD population were measured using the Short Form-36 Health Survey questionnaire (SF-36). SF-36 is a generic measure and can help differentiate QoL issues related to co-morbidities from those related to PAD [25].

Statistical analysis
Data were expressed as mean±SEM and were analyzed using the statistical software package StatView. Experimental results were analyzed by 2 (groups) × 2 (time sample points; i.e., pre-and post-interventions) repeated measures ANOVA with Bonferroni's post hoc test to compare cardiopulmonary fitness and capacities of mitochondrial OXPHOS and ETS in platelets at the beginning of this study and after 12 weeks in the GR+CET and GR groups.
Pearson correlation analysis was used to determine the association between aerobic capacity (i.e., VO2peak) and platelet mitochondrial OXPHOS and ETS in patients with PAD. The criterion for significance was P<0.05.

Cardiopulmonary fitness and HRQoL
Fifty-two patients diagnosed with PAD have been surveyed for the interventions. A total of 40 eligible subjects completed the study in the GR+CET (n=20) and GR (n=20) groups. No adverse vascular or thrombotic event occurred in the two groups throughout the periods of investigation (Fig. 1S). Moreover, both GR+CET and GR groups did not differ significantly in anthropometric and clinical parameters (  (Table 2). However, the GR alone remained unchanged the scores of SF-36 physical and mental components ( Table 2).

Blood cell count
There were no significant changes in erythrocyte, hemoglobin, hematocrit, lymphocyte and platelet following 12 weeks of the GR with or without CET (Table 3). However, the GR+CET considerably decreased neutrophil count (counts from 5.30 to 3.78 x10 3 /μl, P<0.05) and the ratio of neutrophils to lymphocytes (NLR, ratio from 3.43 to 2.22, P<0.05). Moreover, these blood cell counts were unchanged following the GR alone (Table 3).

Discussion
This investigation clearly exhibits that 12-week GR+CET improves systemic aerobic capacity and ventilatory/hemodynamic efficiencies, which is accompanied by improving health-related quality of life in patients with PAD. Notably, this study is the first to demonstrate that the CET regimen effectively enhances capacities of mitochondrial OXPHOS and ETS in platelet through increasing Complex II activity in PAD patients.

Ventilation/hemodynamic efficiencies and HRQoL
Optimal exercise programs increase the ability of PAD patients to independently perform activities of daily living, thereby further improving their quality of life [2,3]. In this study, 12-week GR+CET increased VO2peak and decreased VE-VCO2 slope, as well as, enhanced hemodynamic responses (such as CO and SV) to exercise in the PAD patients. The ventilatory parameters obtained from the graded exercise test may convey information regarding prognosis of circulatory disorders [22]. The VO2peak is an indicator of aerobic capacity, respectively [22], whereas the VE-VCO2 slope is a powerful predictor of survival in patients with circulatory disorders [22]. Our previous study using patients with HF reported that these indices of ventilatory efficiency modulated by exercise training were correlated with exercise-induced central and peripheral hemodynamic changes [24]. Accordingly, the GR+CET may effectively improve ventilatory and hemodynamic efficiencies, thereby enhancing systemic aerobic capacity in patients with PAD.
Beside an improvement in systemic aerobic capacity, CET for 12 weeks also increased the distance in 6-minute walk test and consequently heightened the Short Form-36 physical/mental component scores. These findings imply that the exercise regimen effectively enhances the ability of patients to cope with the physical demands of daily activity and subsequently improving psychosocial status in PAD patients. Furthermore, the better HRQoL might exhibit less potential for mortality in PAD patients and simultaneously reduce the financial burden in their health care system [2,3].

Mitochondrial functionalities in platelets
Progression to PAD is probably associated with a gradual decline in bioenergetic reserve capacity owing to the inability of endogenous homeostatic mechanisms to compensate for the insufficient energy supply [26]. Moreover, mitochondrial dysfunction in PAD patients may result in systemic inflammation that facilitates susceptibility to energy-based pathologies associated with oxidative stress [27]. Additionally, atherosclerosis and thrombosis are reported to be associated with deterioration of platelet mitochondrial function [6,7]. Hence, it is plausible that platelet mitochondrial bioenergetics is a marker for metabolic stress in PAD progression.
NLR has been associated with systemic inflammation and atherosclerotic burden, leading to inferior outcomes after lower extremity interventions [28]. Mitochondria are highly sensitive to inflammatory stress and respond dynamically to changes in their microenvironment [29].
Decreased oxidative capacity due to mitochondrial respiratory chain impairment may be associated with increased release of reactive oxygen species (ROS) and reduction of calcium retention capacity, leading to enhanced apoptosis of skeletal muscles [30]. The present investigation further demonstrated that CET significantly depressed circulatory inflammatory status, indicated by decreased neutrophil count and NLR in patients with PAD. Hence, we posit that the CET-induced physiological adaptations protect against inflammation associated with mitochondrial dysfunction in PAD patients [31].
A previous study reported that ischemia results in accumulation of intracellular succinate, thus leading to elevated mitochondrial ROS production [32]. Elevation of oxidative stress may also induce succinate accumulation by decreasing SDH activity [33]. Recently, our study including sedentary healthy men reported that CET significantly enhanced platelet SDH activity and Complex II-related respiration following hypoxic stress [13]. In clinical investigation, we further demonstrated that CET elevated platelet mitochondrial O2 consumption rate via increasing Complex II activity, which was accompanied by depressed oxidative stress in patients with HF [14]. In the present study, 12-week CET effectively also enhanced platelet mitochondrial OXPHOS and ETS capacities by increasing Complex II activity in the PAD patients. Therefore, CET-induced elevation in Complex II activity in platelets may rapidly eliminate succinate, further reducing ROS production from platelet mitochondria, eventually depressing circulatory oxidative stress and pro-inflammatory status in patients with PAD. Moreover, we further demonstrate that change of VO2peak was positively correlated with changes of maximal OXPHOS and ETS capacities in platelets. These experimental results suggest that CET-induced platelet metabolic adaptation may be associated with improved systemic aerobic capacity in patients with PAD.

Limitations of this study
Our small sample size in each group is a major limitation. However, results regarding platelet mitochondrial bioenergetics have high values of statistical power ranging from 0.864 to 1.000. Additionally, this study mainly focused on the effects of CET on platelet mitochondrial bioenergetics rather than platelet reactivity (adhesion and aggregation) or platelet-mediated coagulation or thrombin generation. Our previous studies have investigated the effect of CET on platelet adhesiveness and aggregation and their underling mechanisms in healthy people and patients with cardiovascular disorders [9][10][11][12]. Recent study further reported that CET markedly suppressed hypoxia-induced oxidative damage of platelet mitochondria and consequent attenuation of platelet-mediated thrombin generation caused by hypoxia in healthy sedentary men [13]. However, the role of platelet mitochondrial function on CET-mediated platelet reactivity and coagulation in PAD patients warrants further investigation.

Conclusions
In the present study, 12-week CET enhances systemic aerobic capacity through enhancing ventilatory/hemodynamic efficiencies, as well as improved HRQoL in patients with PAD.
Simultaneously, the exercise regimen also increased the capacities of OXPHOS and ETS by enhancing mitochondrial Complex II activity in platelets. These experimental findings may facilitate the identification of effective exercise regimen to increase physical performance and improve the efficacy for platelet mitochondrial bioenergetics in PAD patients.