Participants

Data from PAD patients and healthy adults were used from an existing biobank of samples (PK). The protocol for the collection of such specimens was approved by the University of Texas at Austin Dell Medical School (CR-23–058) and the Baylor Scott and White Medical Center (402,270). We specifically selected 56 patients with symptomatic PAD and 30 non-PAD control patients with a known medical history of alcohol consumption. A PAD diagnosis was based on medical history, physical examination, reduced ankle-brachial index (ABI) at rest (< 0.90), and computerized or standard arteriography. Non-PAD control patients presented to the medical clinics for indications other than PAD, had normal lower extremity pulses and blood flow, normal ABIs at rest and after stress, no history of PAD symptoms, and all led a sedentary lifestyle. Individuals with musculoskeletal or neurologic symptoms unrelated to PAD, or acute lower extremity ischemic events secondary to thromboembolic disease or trauma, were excluded from the study.

Alcohol consumption

Habitual alcohol use was determined from medical history information and patient interviews with medical personnel. Using the definition of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) [53], 28 PAD patients were classified as heavy drinkers/with AUD (i.e., consumed > 14 or > 7 drinks/week, for males and females, respectively). An additional 28 PAD patients and 30 non-PAD healthy adults consumed moderate-to-low or no alcohol and served as experimental controls for this study.

Functional assessments

The six-minute walk test was used to assess lower extremity function. The test was performed indoors in a long, flat, straight 20-m hallway, and the total distance walked over a 6-min timeframe (6MWD) was recorded in meters [54]. A Gardner graded exercise treadmill test was also used to determine the absolute claudication distance (ACD); i.e., the distance at which claudication pain became intolerable, forcing the patient to stop walking [55, 56]. In the non-PAD controls, data for the ACD variable represents the distance corresponding to exercise cessation due to volitional fatigue rather than exertional leg pain. Muscle strength was measured using an isometric dynamometer (Biodex System 4.0; Biodex Medical Systems, Shirley, NY). Isometric contraction of the ankle plantar flexor muscle peak force was measured during a maximum isometric contraction held for 10 s, and the data are represented in N*m [25, 26].

Muscle biopsy

For the PAD patients with intermittent claudication (IC) and the non-PAD controls, biopsies were obtained from the anteromedial aspect of the gastrocnemius, 10 cm distal to the tibial tuberosity via fine needle (12G) aspiration. Gastrocnemius samples were acquired during lower leg amputation in patients with critical limb ischemia (CLI) and taken from the same anatomical region described for the IC and non-PAD control patient samples. Approximately 250 mg muscle was obtained from all patients. For histological analyses, a portion of the biopsy was immediately placed in cold methacarn, transferred to cold ethanol:H2O (50:50 v/v) after 48 h, and subsequently embedded in paraffin [19, 25, 26, 57]. For mitochondrial measures, another portion of the sample was immediately placed in fresh ice-cold relaxation solution (10 mM EGTA, 3 mM free Mg2+, 20 mM taurine, 0.5 mM DTT, 20 mM imidazole, 5 mM ATP, 15 mM phosphocreatine, and 160 mM K-MES, pH 7.2) and transferred to the lab for respiration assays [12]. The remaining tissue was snap-frozen in liquid nitrogen for all other analyses.

Histology and quantitative fluorescence microscopy

Muscle biopsy specimens embedded in paraffin were sectioned at 4 microns and labeled with fluorescent reagents to quantify markers of oxidative damage; carbonyl groups and the Michael adduct of hydroxynonenal (4-HNE), as previously described [5, 19, 25]. Briefly, individual myofibers were identified by sarcolemma labeling with 10 µg/mL Alexa Fluor 647 conjugated Wheat Germ Agglutinin (ThermoFisher, Waltham, MA, W32466). For protein carbonyl staining, endogenous biotin groups were first blocked and then carbonyl groups were biotinylated overnight at 4 °C by treating the slide specimens with 5 mM biocytin-hydrazide (Setareh Biotech, Eugene, OR, 6769). The slides were subsequently labeled with Alexa Fluor 488 conjugated streptavidin (10 µg/mL) (ThermoFisher, S32354) for 1 h at room temperature. To assess 4-HNE adducts, a monoclonal mouse antibody (20 µg/mL) (R&D Systems, Minneapolis, MN, MAB3249) was applied to slide specimens and incubated overnight at 4 °C. The slides were then labeled with Alexa Fluor 594 conjugated anti-mouse IgG (10 µg/mL) (ThermoFisher, A11005) for 1 h at room temperature. All fluorescent-labeled specimens were mounted in ProLong Gold anti-fade medium with DAPI nuclear stain (ThermoFisher, P36931) prior to image acquisition.

Image acquisition and analysis

Quantification of inter-myofiber carbonyls and 4-HNE adducts was based on three-channel imaging of microscopic fields, with channels corresponding to 1) the sarcolemma, 2) carbonyl groups, and 3) 4-HNE adducts. Fluorescence imaging was captured with the 10 × objective of an epifluorescence microscope (EVOS M7000, ThermoFisher). Celleste software (ThermoFisher) was used for data acquisition and analysis. Several microscopic fields were captured per specimen, corresponding to 3,500–15,000 myofibers. The fluorescent signal within each myofiber was corrected for background and expressed as the mean pixel intensity in grayscale units (gsu) on a 12-bit gray scale, which corresponded to the concentration of carbonyls or 4-HNE within the myofiber [5, 19, 25]. The mean fluorescence signal from all myofibers present in each specimen was determined.

Morphometric measures

Morphometric parameters of myofiber size and shape were determined from the Wheat Germ Agglutinin-marked myofibers as previously described in detail [10, 26]. Cross-sectional area (in square microns) was assessed as the number of pixels enclosed within a segmented myofiber, diameter (in microns) was defined as the diameter of a circle with the same area as the segmented myofiber region, and roundness was determined as the equivalent diameter x Π, divided by the perimeter of the segmented region [10].

Mitochondrial respirometry

Mitochondrial respiration was evaluated in saponin-permeabilized muscle fibers using an Oroboros O2K high-resolution fluororespirometer (Oroboros Instruments, Austria) [13, 43, 58]. Four separate assays were performed to assess respiration at each complex of the electron transport chain (ETC). Complex I-dependent respiration was measured by the addition of 5 mM glutamate, 5 mM malate, and 1 mM ADP; Complex II assessments used 1 mM ADP, 10 mM succinate, and 3 μM rotenone (to inhibit complex I); Complex III required 3 μM rotenone, 1 mM ADP, and 1 mM duroquinol; and Complex IV respiration was assessed using 3 μM rotenone, 1 mM ADP, 0.2 mM N,N,N,N-tetramethyl-p-phenylenediamine (TMPD), and 10 mM ascorbate to prevent TMPD auto-oxidation [59]. The respiration rates were then normalized to citrate synthase (CS) activity (nanoatomO2/min/unit CS activity) as a proxy for intramuscular mitochondrial content.

CS activity was determined colorimetrically via the reduction of the substrate 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) by coenzyme A (CoA-SH) and recorded as the increase in absorbance at a wavelength of 412 nm (DU Series 640 spectrophotometer, Beckman Instruments, Fullerton, CA). CS activity was then normalized to sample protein concentration (µmol/min/mg protein) determined using a Pierce BCA assay (Thermo Scientific, Rockford, IL, 23,225) as previously described [2, 59].

Real time polymerase chain reaction

Total RNA was isolated from frozen muscle biopsy samples using the Direct-zol™ RNA Microprep Kit (Zymo Research, Irvine, CA, R2063) and RNA concentration and quality was determined using a NanoDrop One spectrophotometer (Thermo Scientific). RNA purity was accepted if the A260/A280 ratio was between 1.8–2.1. The RNA was then reverse transcribed to cDNA using the iScript™ Reverse Transcription Supermix for RT-qPCR Kit (Bio-Rad, Hercules, CA, 1,708,841). PCR reactions used the following target genes of the ETC: NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8, complex I; qHsaCEP0040853), Succinate Dehydrogenase Complex Iron Sulfur Subunit B (SDHB, complex II; qHsaCID0011704), Ubiquinol-Cytochrome C Reductase Core Protein 2 (UQCRC2, complex III; qHsaCIP0033016), Cytochrome c oxidase (COX10, complex IV; qHsaCEP0049189), ATP synthase F1 subunit alpha (ATP5A1, complex V; qHsaCEP0025064), as well as target genes for alcohol-metabolizing enzymes: alcohol dehydrogenase 1B (ADH1B; qHsaCEP0039662) and ALDH2 (qHsaCEP0050628). The housekeeping gene was glyceraldehyde-3-phosphate dehydrogenase (GAPDH; qHsaCED0038674). All primers were obtained from and validated by Bio-Rad Laboratories (Bio-Rad Laboratories, Hercules, CA, 10,025,220).

PCR reactions were run on a CFX Opus Real-Time PCR System (Bio-Rad, Hercules, CA) using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 1,725,271). The quantification cycle (Cq) of all target genes was normalized to the housekeeping gene (GAPDH). The relative normalized expression (the delta delta Ct value) was then calculated using the 2^(-delta delta Cq) method [60]. All samples were run in triplicate, and results were averaged.

Protein expression analysis by western blot

Protein was extracted from frozen muscle samples using an ice-cold lysis buffer containing 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% triton X-100, 0.5% sodium-deoxycholate, 0.1% sodium dodecyl sulfate, and 1 × protease inhibitor cocktail (Sigma Aldrich, Burlington, MA, P8340) and the protein content within the muscle homogenate was determined using a BCA assay (Thermo Scientific, 23,225). Following the addition of 2 × Laemmli sample buffer (Bio-Rad Laboratories, 1,610,747) and 2-mercaptoethanol reducing agent (Bio-Rad Laboratories, 1,610,710), sample protein (20 µg) was separated using electrophoresis in a Criterion Cell Tank (Bio-Rad Laboratories) and 4–20% Criterion TGX Precast Midi Protein Gels (Bio-Rad Laboratories, 5,671,095). Proteins were then transferred to 0.2 µm Amersham Hybond P polyvinylidene difluoride (PVDF) transfer membranes (Cytiva, Malborough, MA, 10,600,021) and total protein per lane was established using Ponceau S stain (Boston BioProducts, Milford, MA, ST180). The membranes were subsequently incubated with the following primary antibodies: 4-HNE (1:1000, Abcam, Waltham, MA, ab46545), OxiSelect Protein Carbonyl anti-DNP antibody (1:1000, Cell Biolabs, Inc, San Diego, CA, STA-308), ALDH2 (1:500, Proteintech, Rosemont, IL, 15,310–1-AP), ADH1B (1:1000, Proteintech, 66,939–1-Ig), and OXPHOS human antibody cocktail (1:200, Thermo Scientific, 45–8199). Secondary antibodies used included Goat anti-Rabbit IgG (1:10,000, Invitrogen, Waltham, MA, 31,462) or Goat anti-Mouse IgG (1:10,000, Invitrogen, 31,430). Band visualization and detection was achieved using Clarity ECL Substrate (Bio-Rad, 1,705,060) and the ChemiDoc MP Imaging System (Bio-Rad Laboratories). The band intensity of each target protein was quantified using Image Lab software (Version 6.1, Bio-Rad Laboratories) and normalized to sample total protein (Ponceau S stain). The protein content of each target is expressed as mean fold change relative to non-PAD control muscle ± SD.

Colorimetric measures of oxidative stress

Acetaldehyde accumulation: An acetaldehyde assay kit (Sigma Aldrich, Burlington, MA, MAK434) was used to quantify acetaldehyde content in patient muscle homogenate (20 μg protein) and serum (undiluted). The assay is based on the aldehyde dehydrogenase-catalyzed oxidation of acetaldehyde and reduction of NAD to NADH. The resulting NADH reduces MTT, producing a colored formazan compound detected at 565 nm, which is directly proportional to the sample acetaldehyde concentration. Measurements were performed in duplicate and acetaldehyde content is expressed as mg/dL (serum) or normalized to sample total protein (mg/dL/mg protein) (muscle homogenate).

Superoxide dismutase (SOD) activity: The enzymatic activity of the antioxidant SOD was assessed in the muscle lysates (20 μg protein) using an SOD activity kit (Sigma Aldrich, CS0009) as described in detail elsewhere [58]. All samples, controls, and standards were assayed in duplicate and SOD activity was expressed as units of activity/mL and normalized to total protein within the muscle homogenate (U/mL/mg).

Statistical analysis

Demographic characteristics of PAD patients with AUD, low/no-alcohol consuming PAD patients and non-PAD control subjects were compared using chi-square or Fisher’s exact tests for categorical variables and one-way analysis of variance (ANOVA) with Tukey post-hoc tests for continuous variables. ANOVA with Tukey post-hoc adjustment was also used to determine group differences in all other biological targets of interest. Prism statistical software (version 10.5.0, GraphPad, Boston, MA) was used to perform the statistical analyses. Data are reported as mean ± SD for continuous variables, and frequencies with proportions for categorical variables, and significance was accepted at P < 0.05.