Clinical Trial Radar

Distance walked

Total distance walked during the exercise session (standard or M-SET) will be determined using the total time of walking multiplied by the walking speed.

Before and after 12 week exercise program

Energy expenditure

Energy expenditure57 will be assessed via oxygen consumption as measured by a metabolic cart (K5, Cosmed USA, Inc., Chicago, IL). Subjects will stand quietly for five minutes to collect resting energy expenditure. Standing steady state energy expenditure will be calculated for each subject by plotting VO2 and assigning a sliding best fit line over a two-minute window. Steady-state oxygen consumption in ml/kg/min will be calculated during the exercise session. Energy cost of transport will be calculated by subtracting the energy consumption values at standing from walking to achieve net metabolic cost for each bout of exercise.

Before and after 12 weeks after the exercise program

Reactive oxygen species

Quantification of reactive oxygen species (ROS) related markers and free radicals: Electron paramagnetic resonance (EPR), which has been known as a gold standard for free radical assessment, will be used to quantify free radicals such as superoxide as we have previously performed (1-3). Plasma samples will be used for measuring ROS-related markers such as TAC, SOD, GSH:GSSG, ONOO- and MDA with commercially available and previously validated ELISA Assay Kits (CAT#s ab65329, ab65354, ab138881, ab233469, and ab233471 Abcam, UK) according to the manufacturer's instructions60. The average intra-assay variability measured as the coefficient of variation for each sample was 2.9%, 3.8%, and 3.3% for SOD, TAC, and MDA, respectively.

Before exercise, immediately after exercise, 30 minutes after exercise, and 1 hour after exercise

Protein expression of ROS-related enzymes

Protein expression of ROS-related enzymes: Enzymes related to ROS will be quantified with western blots of isolated PBMCs analyzed for catalase, 4 hydroxynonenal (4HNE), and glutathione peroxidase 1 (GPX) (4). All samples will be homogenized in RIPA buffer (Boston Bioproducts) and 100X protease inhibitor (Sigma Aldrich). A 150-volt electrical potential will be applied to the gel for 1 hr to separate proteins based on mass. A 250-volt electrical potential will then be applied to the gel for 7 min to transfer protein bands to transfer paper with TurboTransfer (Biorad). Primary antibodies for catalase, 4HNE, and GPX will be recombinant anti-catalase antibody (ab209211), anti-4 hyroxynonenal antibody (ab243070), and recombinant anti-glutathione peroxidase 1 antibody (ab108429), respectively.

Before exercise, immediately after exercise, 30 minutes after exercise, and 1 hour after exercise

Markers of systemic inflammation

Markers of systemic inflammation: Standard ELISA kits will be used to test blood samples for interleukin 6, tumor necrosis factor-a, vascular cell adhesion protein 1, and vascular endothelial growth factor-a (Cat#s, ab178013, ab285312, ab223591, ab119566). Oxidized low density lipoproteins (LDL) will be measured with an oxidized LDL assay kit (ab242302). All measurements will be made per validated manufacturer specifications.

Before exercise, immediately after exercise, 30 minutes after exercise, and 1 hour after exercise

Initial and absolute walking distances

Initial and absolute walking distances will be determined from the progressive-load treadmill test. Subjects will walk on a treadmill that starts at 0% grade and 2.0 mph. Every two minutes, the grade will be increased by 2% up to a maximum of 15% grade, and the speed held constant throughout the test. Initial claudication distance is the distance walked prior to claudication pain onset. Absolute claudication distance is the total distance walked before subjects had to stop walking due to claudication pain.

Before and after 12 week supervised exercise program

Comprehensive mitochondrial respiratory complex function

Comprehensive mitochondrial respiratory complex function (complex I, II, III, IV, and V) will be completed with previously validated high resolution respirometry with complex agonists/antagonists. Skeletal muscle tissue will be manually teased and permeabilized with 15 l of digitonin for 30 min. Samples will then be rinsed twice in 2 ml of MIRO5 for 10-min. 3-5 mg (dry weight) of tissue will then be isolated and placed in the high-resolution respirometer with 2 ml of MIRO5 solution. The order and quantity of each agonist and antagonist will be applied as follows: 5 l malate and 10 l glutamate (complex I agonist), 20 l adenosine diphosphate (complex V agonist), 20 l succinate (complex II agonist), 1 l rotenone (complex I antagonist), 1 l oligomycin (complex V antagonist), 1 l antimycin A (complex III antagonist), and 5 l ascorbate + 5 l TMPD (complex IV agonist).

Before and after 12 week supervised exercise program

Mitochondrial dynamics

Specific proteins we will assess are Fis-1 and DRP-1 (fission), Mfn-1, Mfn-2 and OPA-1 (fusion), and UPC-1 and UCP-2 (dynamics). We will perform the same Western Blot technique mentioned in Aim #262. Primary antibodies for Fis-1, DRP-1, Mfn-1, Mfn-2, OPA-1, UPC-1, and UCP-2 will be recombinant anti- TTC11/FIS1 (ab156865), recombinant anti-DRP1 (ab184247), anti-mitofusin 1 antibody-N-terminal (ab191853), recombinant anti-mitofusin 2 (ab205236), recombinant anti-OPA1 (ab157457), anti-UCP1 (ab155117), and anti-UCP2 (ab247184), respectively.

Before and after 12 week supervised exercise program

Mitochondrial reactive oxygen species production

Mitochondrial reactive oxygen species production will be assessed with fluorescent imaging of skeletal muscle biopsy samples treated with H2DCFDA (D399, ThermoFisher), which is validated to detect ROS within cells. The validated mitochondrial targeted antioxidant MitoTempo (SML0737, Sigma-Aldrich) will be used to confirm the presence of mitochondrial ROS. EPR will be used to quantify free radicals such as superoxide.

Before and after 12 week supervised exercise program

Metabolic equivalents of task

Metabolic equivalents of task (MET) will be used to quantify exercise dose accumulated over the course of the SET or M-SET session. The MET value (measured by metabolic cart) will be multiplied by the number of minutes exercised during each bout. Session MET/min will be summed to quantify total dose (Exercise intensity).

Immediate

Physical activity enjoyment scale

Physical activity enjoyment scale is an 18-item measure developed to assess the extent to which an individual enjoys a given physical activity. Ratings span from 1(most enjoyment) to 7 (least enjoyment) and the questionnaire has been extensively validated1. The scale will be given at the end of each exercise session.

Immediate

Rating of perceived exertion

Rating of perceived exertion will be recorded using the Borg Scale, which ranges from 6 (no exertion) to 20 (maximal exertion) at the end of exercise session.

Immediate

Physical activity average steps/day

Physical activity will be measured with an accelerometer (Actigraph GT3X, Actigraph, FL, USA) placed on subjects' hip for seven days after the intervention but prior to the assessment visit. Average steps/day will be calculated. Accelerometers will be given to patients at their last exercise session prior to the assessment data collection.

Before and after 12 week supervised exercise program

Muscle strength

Muscle strength will be measured with an isokinetic dynamometer (Biodex 4 Medical Systems, Inc. USA). Peak isokinetic strength (torque) of the ankle and hip of the more symptomatic leg will be measured. Subjects will perform one trial of five repetitions at 60 degrees/second to assess strength.

Before and after 12 week supervised exercise program

Acceptability

In each intervention group, patients will be interviewed after completing the 12 week intervention. Interviews will provide the potential feasibility of exercise by identifying acceptability (satisfaction, intent to continue participation), perceived demand, and practicality (positive/negative effects).

Before and after 12 week supervised exercise program

Maximum cadence/day

Physical activity will be measured with an accelerometer (Actigraph GT3X, Actigraph, FL, USA) placed on subjects' hip for seven days after the intervention but prior to the assessment visit. Accelerometers will be given to patients at their last exercise session prior to the assessment data collection. Cadence is the number of steps per minute and the maximum cadence in a 24 hour period will be recorded as the maximum cadence/day.

Before and after 12 week supervised exercise program

Muscle endurance

Muscle endurance will be measured with an isokinetic dynamometer (Biodex 4 Medical Systems, Inc. USA). Peak isokinetic torque of the ankle and hip of the more symptomatic leg will be measured. Subjects will perform one trial of 15 repetitions at 120 degrees/second to assess endurance.

Before and after 12 weeks supervised exercise program.