DOI: 10.5061/dryad.xksn02vtj
title: The ankle–brachial index, gastrocnemius mitochondrial respirometry, and walking performance in people with and without peripheral artery disease

description: Background: Mitochondrial abnormalities exist in lower-extremity
 peripheral artery disease (PAD), yet the association of the ankle–brachial
 index (ABI) with mitochondrial respiration in gastrocnemius muscle is
 unknown. The association of gastrocnemius mitochondrial respiration with
 6-minute walk distance in PAD is unknown. This objective of this study was
 to describe associations of the ABI with mitochondrial respiratory
 function in gastrocnemius muscle biopsies and associations of
 gastrocnemius mitochondrial respirometry with 6-minute walk distance in
 people with and without PAD. Methods: People with (ABI ⩽ 0.90) and without
 (ABI 1.00–1.40) PAD were enrolled. ABI and 6-minute walk distance were
 measured. Mitochondrial function of permeabilized myofibers from
 gastrocnemius biopsies was measured with high-resolution respirometry.
 Results: A total of 30 people with PAD (71.7 years, mean ABI: 0.64) and 68
 without PAD (71.8 years, ABI: 1.17) participated. In non-PAD participants,
 higher ABI values were associated significantly with better mitochondrial
 respiration (Pearson correlation for maximal oxidative phosphorylation
 PCI+II: +0.29, p = 0.016). In PAD, the ABI correlated negatively and not
 significantly with mitochondrial respiration (Pearson correlation for
 PCI+II: –0.17, p = 0.38). In people without PAD, better mitochondrial
 respiration was associated with better 6-minute walk distance (Pearson
 correlation: +0.51, p &lt; 0.001), but this association was not
 present in PAD (Pearson correlation: +0.10, p = 0.59). Conclusions: Major
 differences exist between people with and without PAD in the association
 of gastrocnemius mitochondrial respiration with ABI and 6-minute walk
 distance. Among people without PAD, ABI and walking performance were
 positively associated with mitochondrial respiratory function. These
 associations were not observed in PAD.
description: # **The ankle–brachial index, gastrocnemius mitochondrial respirometry,
 and walking performance in people with and without peripheral artery
 disease** Dataset DOI: [10.5061/dryad.xksn02vtj](10.5061/dryad.xksn02vtj)
 **Description of the data and file structure** Participants with and
 without PAD were enrolled from the Chicago, Illinois area. The study was
 approved by a university Institutional Review Board. All participants
 provided written informed consent. Participants with and without PAD were
 identified using lists of patients and by advertisements placed on Chicago
 Transit Authority buses and trains, and by mailed recruitment postcards,
 using a purchased mailing list. Enrollment occurred between 7/23/2018 and
 8/26/2021. PAD was defined as an ABI &lt; 0.90 in either leg (914).
 Absence of PAD was defined as an ABI of 1.00-1.40. Potential participants
 with ABI of 0.90 to 0.99 were excluded, because they may have mild lower
 extremity atherosclerosis. To attain similar ages between people with vs.
 without PAD, only older people were included among those without PAD.
 Individuals taking part in a randomized clinical trial or who completed a
 randomized clinical trial in the past three months were not eligible.
 Potential participants with diabetes were initially excluded. Due to
 challenges with recruitment, diabetes mellitus was not an exclusion
 criterion beginning 9/20/2019. ABI Measurement: A hand-held Doppler probe
 (Pocket Dop II; Nicolet Biomedical Inc) was used to measure systolic
 pressures twice in the right and left brachial, dorsalis pedis, and
 posterior tibial arteries while the participant was supine. The ABI was
 measured by staff, following study protocol. Staff were certified by
 co-investigator MMM prior to beginning data collection and approximately
 every six months. The ABI was calculated by dividing the mean of the
 dorsalis pedis and posterior tibial pressures in each leg by the mean of
 the four brachial pressures. Six-minute Walk: The 6-minute walk test was
 used to assess functional performance, because the 6-minute walk is a well
 validated measure of walking endurance that reflects walking ability in
 daily life better than treadmill walking performance. For example, the
 6-minute walk was more closely correlated with physical activity in daily
 life than treadmill walking performance. Participants walked up and down a
 100-foot hallway after receiving scripted instructions from a trained and
 certified research coordinator. Participants were advised that the goal of
 the test was to cover as much distance as possible in six minutes. The
 distance completed after six minutes was recorded. Distance to first
 report of leg symptoms during the walk and whether the participant
 reported leg symptoms at the end of the walk was recorded. The intra-class
 correlation coefficient for the test-retest reliability of the 6-minute
 walk among 155 participants who performed the 6-minute walk test twice,
 1-2 weeks apart, was 0.90 (p&lt;0.001). Physical Activity: Physical
 activity was measured over seven days with the ActiGraph accelerometer.
 Low, medium, and high intensity activity were combined into a single
 “activity count” outcome as previously described. Gastrocnemius biopsy: An
 open muscle biopsy was performed in the medial head of the gastrocnemius
 muscle of the leg with the lower ABI. If the left and right legs had
 identical ABI values, the leg for biopsy was selected randomly. All muscle
 biopsies were performed by two co-investigators (RS and KJH). Anesthesia
 was achieved with subcutaneous lidocaine. Subcutaneous tissue was
 dissected until muscle was identified. After muscle tissue was removed,
 10-20 mg was transported in cold biopsy media for immediate respirometry
 testing. Remaining muscle was immediately prepared for freezing at -80
 degree Celsius. Mitochondrial respiratory function: High-resolution
 respirometry was performed using an Oroboros O2k (Oroboros Instruments,
 Innsbruck, Austria) per established protocols. Respirometry was performed
 on muscle immediately after the biopsy was obtained. Muscle fiber bundles
 were separated in ice-cold preservation media (BIOPS) under a dissecting
 microscope to obtain four replicates per participant (approximately 2-3mg
 each), which were permeabilized with saponin for 20 min followed by a 10
 min wash in mitochondrial respiration media (MiR05). All data were
 collected at 37°C in hyperoxygenated (200–450µM O2) conditions in MiR05 to
 avoid limitations to oxygen diffusion. Two substrate-uncoupler-inhibitor
 titrations (SUIT) respiration protocols (carbohydrate and fatty acid)
 assessed maximal phosphorylation (OXPHOS) and electron transport chain
 (ETC) capacity of complex-I and complex-II mediated respiration. Malate,
 pyruvate (carbohydrate) or octanoylcarnitine (fatty acid), ADP, glutamate,
 cytochrome c, and succinate were sequentially added, followed by
 titrations of carbonyl cyanide m-chlorophenyl hydrazine (CCCP), rotenone,
 and antimycin A. The state of respiration in the presence of malate,
 pyruvate, glutamate and ADP was defined as carbohydrate complex-I mediated
 maximal OXPHOS capacity (PCI). Respiration after subsequent succinate
 addition was defined as carbohydrate complex-I+II mediated maximal OXPHOS
 capacity(PCI+II). The state of respiration after adding CCCP (uncoupler)
 was considered maximal ETC capacity (ECI+II). Fatty acid-supported
 respiration in the presence of malate, octanoylcarnitine, and ADP was
 measured using complex-I and electron-transferring flavoprotein complex
 mediated maximal OXPHOS (PCI/FA). Results from replicates that increased
 by more than 15% after adding cytochrome c were considered
 over-permeabilized and not included in analyses. Mitochondrial protein
 measures: Protein expression of mitochondrial ETC complexes I, II, III, IV
 and V (ATP synthase) subunits; voltage-dependent anion channels (VDAC);
 and peroxisome-proliferator activated receptor-γ coactivator (PGC-1α) were
 measured by Western blot. Statistical analyses:  Characteristics of
 participants with and without PAD were summarized as means and standard
 deviations (SD) for continuous variables and as frequencies and
 percentages for categorical variables. Two sample t-tests were used to
 compare continuous characteristics. Chi-squared or Fisher’s exact tests
 were used to compare categorical characteristics of participants with vs.
 without PAD. Pearson correlation coefficients were used to evaluate
 associations of the ABI, six-minute walk distance, and physical activity,
 respectively, with mitochondrial respiratory function and with abundance
 of the mitochondrial proteins. Analyses were performed separately among
 people with and without PAD. We studied whether these associations were
 significantly different between people with and without PAD by testing the
 interaction between PAD status and ABI in a linear regression model with
 ABI as the independent variable of interest and mitochondrial respirometry
 as the dependent variable of interested. We also tested the interaction
 between PAD status with mitochondrial respirometry as the independent
 variable of interest and 6-minute walk and physical activity as separate
 dependent variables of interest. Statistical tests were two-sided and the
 criterion for statistical significance was P-value&lt;0.05. Statistical
 analyses were conducted using SAS 9.4. **Files and variables** **File:
 MDD_dataset.xlsx** **Description:** This file contains all variables from
 this research project, except for those that could be used to identify
 specific individuals.  Missing variables are gender and race.  Each row in
 the dataset is one individual, and the ID_Index variable included in the
 submission has been anonymized **Variables** * PAD or no PAD *
 Ankle-brachial index (ABI) * Body Mass Index (BMI) * Current Smoker - 0=no
 * Diabetes - 0=no * History of Myocardial Infarction - 0=no * History of
 Heart Failure - 0=no * History of stroke - 0=no * History of angina - 0=no
 * History of cancer - 0=no * Baseline 6-minute walk distance in feet *
 Actigraph activity counts per minute * City blocks walked * Flights of
 stairs climbed * Report of regular walking * Baseline CHO Phosphorylation,
 Complex I * Baseline CHO Phosphorylation Complexes I & II * Baseline
 CHO electron transport chain Complexes I& II * Baseline Fatty acid
 phosphorylation, Complexes I * Baseline Fatty acid phosphorylation,
 Complexes I & II * Baseline Fatty acid electron transport chain,
 complexes I/II * PGC1 alpha_Normalized Value (AU - Arbitrary Units) *
 VDAC_Normalized Value (AU - Arbitrary Units) * Complex I \_Normalized
 Value (AU - Arbitrary Units) * Complex II \_Normalized Value (AU -
 Arbitrary Units) * Complex III \_Normalized Value (AU - Arbitrary Units) *
 Complex IV \_Normalized Value (AU - Arbitrary Units) * Complex IV
 \_Normalized Value (AU - Arbitrary Units) **Code/software** None **Access
 information** Other publicly accessible locations of the data: * N/A Data
 was derived from the following sources: * N/A ## Human subjects data This
 file contains de-identified data from this study which looked at
 mitochondrial function in people with and without peripheral artery
 disease. Only de-identified data is available here.