Myoglobinemia, Peripheral Arterial Disease, and Patient Mortality

BACKGROUND:

Peripheral arterial disease (PAD) causes leg muscle damage due to inadequate perfusion and increases cardiovascular events and mortality 2- to 3-fold. It is unclear if PAD is a biomarker for high-risk cardiovascular disease or if skeletal muscle injury harms arterial health. The objective of this work is to test if serum myoglobin levels (myoglobinemia) are a marker of PAD, and if so, whether myoglobin impairs vascular health.

STUDY DESIGN:

Patient blood samples were collected from PAD and control (no PAD) patients and interrogated for myoglobin concentrations and nitric oxide bioavailability. Patient mortality over time was captured from the medical record. Myoglobin activity was tested on endothelial cells and arterial function.

RESULTS:

Myoglobin is a biomarker for symptomatic PAD and was inversely related to nitric oxide bioavailability; 200 ng/mL myoglobin in vitro increased endothelial cell permeability in vitro and decreased nitrate bioavailability. Ex vivo, 100 ng/mL myoglobin increased vascular tone in naive murine aortas approximately 1.5 times, impairing absolute vessel relaxation. In vivo, we demonstrated that myoglobinemia caused impaired flow-mediated dilation in a porcine model. Patients presenting with myoglobin levels of 100 ng/mL or greater had significantly more deaths than those with myoglobin levels of less than 100 ng/mL.

CONCLUSIONS:

Using a combination of patient data, in vitro, ex vivo, and in vivo testing, we found that myoglobin is a biomarker for symptomatic PAD and a potent regulator of arterial health that can increase vascular tone, increase vascular permeability, and cause endothelial dysfunction, all of which may contribute to the vulnerability of PAD patients to cardiovascular events and death.

Peripheral arterial disease (PAD) causes muscle damage and is associated with a 2× to 3× increase in cardiovascular events and mortality. Is PAD a biomarker or does it have a biologic component of PAD contributing to these events? This work presents myoglobin as a biomarker for symptomatic PAD with biologic impact on arterial health.

Peripheral arterial disease (PAD) is a significant age-related medical condition that is increasing in incidence¹ and affects 12% to 20% of the population older than 65.^2-4 PAD is the third leading cause of atherosclerotic disease morbidity,⁵ and PAD increases endothelial dysfunction and patient risk of cardiovascular events and mortality 3 times more than that of non-PAD cohorts.^5-12 The incidence of PAD increases with age, but PAD in younger people can have a more pernicious course,^13,14 making PAD important to all adult age groups. PAD affects approximately 8.5 million Americans more than 40 years of age.¹⁵ The vast majority of these PAD patients (99%) are not at imminent risk of major amputation, but they can be heavily burdened by pain, walking dysfunction, and depression.^16,17

Ischemic myopathy is the pathologic hallmark of PAD from arterial obstruction, which is typically from bulky atherosclerotic plaque and/or thrombotic disease.^18,19 While treatment paradigms for PAD continue to evolve, optimal medical therapy, risk factor modification, and supervised or structured exercise therapy are all part of the first-line therapies for PAD patients.²⁰

Because PAD affects arterial and skeletal muscle health, we examined the literature for possible biologic links that may provide insight to the increased cardiovascular events in PAD. We found that the globin domain in hemoglobin shared homology with the globin domain in myoglobin, which is an important component of skeletal muscle health that is released into circulation when skeletal muscle is injured. Gladwin and colleagues^21-23 have linked hemoglobinemia in sickle cell disease with decreased nitric oxide (NO) bioavailability and impaired vascular health and cardiovascular events.

The objective of this body of work was to examine whether myoglobinemia is a biomarker for PAD, and if so, does myoglobinemia impact arterial health? We hypothesized that myoglobinemia would be a marker of severe PAD that impaired arterial health. To test this hypothesis, we layered clinical data with in vitro, ex vivo, and in vivo measurements of endothelial cell (EC) dysfunction and arterial health.

METHODS

Study design

This study was approved separately by the IRBs of Baylor University and Emory University and was conducted in accordance with relevant guidelines and regulations governing human research. The study complies with the Declaration of Helsinki, and informed consent was obtained from all participants.

The initial cohort used for testing myoglobinemia as a biomarker and correlating myoglobin concentrations with NO bioavailability comprised patients recruited from Baylor Scott and White Hospital and the University of Texas Health Science Center at San Antonio in a cross-sectional study design. Thirty-three healthy age-matched non-PAD control patients without PAD undergoing an alternative procedure, 40 patients with symptomatic intermittent claudication due to infrainguinal PAD undergoing a revascularization procedure, and 36 critical limb ischemia (CLI) patients with arterial insufficiency with gangrene, nonhealing ischemic ulcers, or consistent rest pain, undergoing a major amputation procedure were recruited and consented to participate. Myoglobin was collected in all patients. NO bioavailability was collected in 13 control, 13 claudicant, and 12 CLI patients. All diagnoses were made after physical and medical history examination, ankle brachial index measurement, and arteriography. Patients with any underlying nonischemic musculoskeletal or neurologic conditions, or acute lower extremity ischemic events secondary to thromboembolic disease or trauma, were excluded from the study.

Additional 11 control and 10 PAD patients were included from a separate IRB-approved study at Emory. All PAD patients had revascularization procedures. Control patients were age-matched and had either fibular free flaps, carotid endarterectomy, or abdominal aortic aneurysm operations. There were 4 claudication and 6 CLI patients. These persons had blood drawn just before their vascular operation and were included with this cohort in the myoglobin concentration and mortality association.

Patient myoglobin ELISA

Patient and pig serum myoglobin was measured with a commercial myoglobin assay (Abnova, Taipei, Taiwan) by using a sandwich enzyme immunoassay. A standard curve and serum samples were prepared per the manufacturer instructions. All samples, controls, and standards were assayed in duplicate. The optical density of the wells was determined using a Varioskan LUX multimode microplate reader set to 450 nm (ThermoFisher, Waltham, MA).

Nitric oxide determination

A commercial Nitric Oxide Assay Kit (Invitrogen, Carlsbad, CA) to determine serum NO by the measurement of nitrate and nitrite, as previously published.²⁴ Levels of endogenous nitrite were detected as products of the Griess reaction, with a colored azo dye that absorbs at 540 nm. Next, all the nitrate in the samples was converted into nitrite using the enzyme nitrate reductase, and the total nitrite was measured. To obtain the nitrate concentration in the samples, the endogenous nitrite was subtracted from the total nitrite value. Serum was diluted 1:2 for the first assay and 1:20 for the second assay. All samples, controls, and standards were assayed in duplicate. The optical density of the wells was determined using a Varioskan LUX multimode microplate reader set to 540 nm (ThermoFisher, Waltham, MA).

Cell culture

Commercially available human umbilical vein ECs (Cell Applications 200-05n, donor sex: mixed donors) cultured with complete Endothelial Cell Media MV2 with MV2 (PromoCell) and 1× penicillin streptomycin (Pen-strep; Sigma-Aldrich, St. Louis, MO) in T25 or T75 polystyrene culture flasks (Corning, Corning, NY) depending on experimental design. Cell culture medium was replaced every 2 days, and cells were subcultured before 100% confluency was attained. Cell cultures were passaged (less than 6 passages) using trypsin and trypsin-neutralizing solution (Lonza, Basel, Switzerland) and were maintained in a cell culture incubator (37° C, 5% CO₂; Sanyo, Osaka, Osaka, Japan).

Permeability assay

A 10 mg/mL gelatin solution was prepared by dissolving 1 g of gelatin (Sigma-Aldrich, St. Louis, MO) in 100 mL of sodium bicarbonate (pH 8.3; Sigma-Aldrich, St Louis, MO) heated to 70° C with constant stirring and centrifuged for 5 minutes at 10,000g. Next, a 5.7 mg/mL solution of EZ-Link NHS-LC-LC-biotin (ThermoFisher, Waltham, MA) was prepared from a 50 mg/mL dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) stock solution and diluted 1:10 into the gelatin solution. Twenty-four–well plates with inserted glass coverslips (ThermoFisher, Waltham, MA) were coated with 1 mL of this biotinylated gelatin solution at room temperature for approximately 30 minutes. A total of 250,000 ECs were then seeded into each well of the 24-well plates and placed in a cell culture incubator overnight to achieve confluency. Cells were then treated with vascular endothelial growth factor 165 (ThermoFisher, Waltham, MA), albumin (ThermoFisher, Waltham, MA), human myoglobin (Sigma-Aldrich, St. Louis, MO), and thrombin (Sigma-Aldrich, St. Louis, MO) solutions prepared from stock solutions diluted 1:100 in EC media. After 1 hour of drugging, media were aspirated, cells were washed with 1× Dulbecco’s dPBS, and a 2 mg/mL solution of avidin-conjugated fluorescein isothiocyanate (ThermoFisher, Waltham, MA) was diluted to 25 µg/mL in EC media and incubated with the cells for 5 minutes in a cell culture incubator at 37° C. After 5 minutes, the cells were washed 3 times for 5 minutes with 1× dPBS to remove excess avidin-conjugated fluorescein isothiocyanate fluorophore and fixed with 4% formaldehyde before being mounted with 4′,6-diamidino-2-phenylindole and added to microscope slides. Experiments were imaged with a Keyence BZ-X810 (Keyence, Osaka, Osaka, Japan) automated inverted microscope and subsequent images were analyzed using FIJI to quantify total fluorescent signal per image.

Griess assay procedure

Laminar or oscillatory wall shear stress vinyl stickers were placed in 6-well plates and sterilized using 70% ethanol; 400,000 ECs were seeded into each well of the 6-well plate and were placed in a cell culture incubator overnight to achieve confluency. Upon confluency, cells were placed on an orbital shaker at 100 rpm for 24 hours to induce shear stress. After 24 hours, cells were then treated with human myoglobin at various concentrations or no treatment for 1 hour before two 85 µL aliquots of supernatant were removed from each well and quantified for either nitrite or nitrate using the microplate colorimetric assay protocol described by the manufacturer (ab65328).

Arterial ring testing

Six male and 6 female C57B6 mice were euthanized under Institutional Animal Care and Use Committee approval, and their aortas were freshly harvested and placed in physiologic buffer. All arteries were tested twice, first in the absence and second in the presence of increasing myoglobin (100ng/mL; 300ng/mL).

Vessels were cut into 5-mm endothelium-intact aortic ring segments and tested immediately. Isometric force testing was performed as previously described.²⁵ In brief, vessels were taken from experimental conditions and immediately mounted between stainless steel wires in an organ chamber containing Krebs-Henseleit buffer (118 mmol/L sodium chloride [NaCl], 4.73 mmol/L potassium chloride [KCl], 1.2 mmol/L magnesium sulfate [MgSO₄], 0.025 mmol/L ethylenediaminetetraacetic acid [EDTA], 1.2 mmol/L potassium dihydrogen phosphate [KH₂PO₄], 2.5 mmol/L calcium chloride [CaCl₂], 11 mmol/L glucose, and 25 mmol/L sodium bicarbonate [NaH₂CO₃], pH 7.4, in 95% O₂-5% CO₂ at 37° C) that was connected to a Harvard apparatus differential capacitor force for the remainder of the experiment. Resting tension was adjusted to 20 milliNewtons (mN) for a 30-minute period, which was maintained for the duration of the study. Vessels were subjected to relaxation testing and contraction testing in a sequential manner with a 30-minute washout period. Data were obtained using PowerLab hardware and analyzed with Labchart software (ADInstruments, Colorado Springs, CO).

Contraction testing

Concentration-isometric force curves were generated in response to receptor-mediated agonist phenylephrine (0.1 nmol/L to 10 mmol/L). Developed forces were expressed both as a percentage of the maximal force generated in response to KCl or phenylephrine and force/cross-sectional area. Cross-sectional area estimation is based on vessel geometry and wet weight [cross-sectional area = 2 × wet weight/ (circumference)].

Relaxation testing

Relaxation responses were examined by precontracting the vessel with 300 nmol/L phenylephrine, a concentration that yields 80% maximum contraction, and relaxation was examined after addition of the endothelium-dependent vasorelaxant methacholine (1 nmol/L to 10 μmol/L). Thirty percent or more relaxation was considered effective for endothelium-dependent function.²⁶

Data analysis

Data were tested and found to be normally distributed. Arterial function was compared between buffer condition and fresh samples using unpaired, two-tailed t test. P<.05 was considered statistically significant. Data analysis was performed using GraphPad Prism version 9.2.0.332 (GraphPad Software, San Diego, CA).

Exertional myoglobinemia model in swine

A single skeletally mature female Yorkshire pig was used for model development. The first stage of the operation excluded the animal’s right external iliac artery, as published.²⁷ To make the animal myoglobinemic, the next week we excluded the animal’s left external iliac artery in the same manner and deployed a vascular plug in the right internal iliac artery trunk. The animal underwent pre/postoperative (and then weekly) forelimb to hindlimb measurements that are analogous to an ankle brachial index, and 60-second flow-mediated dilation of her femoral artery was performed after 5 minutes of cuff occlusion of the animal’s bilateral calves.

Swine supervised exercise therapy

Swine were accommodated to treadmill exercise for 1 week before any operations. They were subsequently exercised to fatigue twice per week starting 1 week after the second operation. Blood was drawn after exercise for myoglobin levels, flow-mediated dilation was performed bilaterally in the hindlimbs, and hoof pressures were measured.

Moxy Protocol

Hemoglobin oxygen saturation was measured using 2 Moxy Monitors (Fortiori Design LLC, Hutchinson, MN), as published.²⁸ The monitors were placed on the belly of the medial gastrocnemius and secured with cover-roll tape strips. Each monitor was connected to a Samsung Galaxy Tablet (Samsung, Seoul, South Korea) for live data tracking throughout the study.²⁹

RESULTS

Myoglobin and nitric oxide bioavailability in patient serum

Myoglobin is a biomarker for symptomatic PAD with claudication patient having significantly higher myoglobin concentrations compared with control patients (86 ± 23 ng/mL vs 60 ± 83 ng/mL), and CLI patients (192 ± 91 ng/mL) were further increased compared with claudication patients (Fig. 1A). NO bioavailability was significantly decreased in PAD patients compared with controls (Fig. 1B). Finally, when we plotted myoglobin vs NO levels, myoglobin was inversely related to NO bioavailability (Fig. 1C). Figures 1A, B data were analyzed using 1-way ANOVA with Bonferroni; *p < 0.01. For Figure 1C, nonlinear fit regression was used to determine significance of inverse relationship between NO and myoglobin in Figure 1C with R² = 0.65, p < 0.001.

Figure 1.

Peripheral arterial disease status for control patients, claudicant patients, or CLI patients compared with serum concentrations of myoglobin (A) and NO (B). C, Inverse relationship between NO and myoglobin in CLI patients. Data analyzed using 1-way ANOVA with post hoc Tukey HSD test. *p < 0.5. CLI, critical limb ischemia; NO, nitric oxide.

Myoglobin and nitric oxide in vitro

To test the effect of myoglobin on NO bioavailability in a clean system, we added increasing myoglobin concentrations to ECs under laminar or oscillatory wall shear stress conditions. We found that myoglobin significantly decreased nitrate (NO₃) beginning at 200 ng/mL in ECs under laminar shear (Figs. 2, A/C). Under oscillatory wall shear stress conditions, which are known to limit NO bioavailability, myoglobin significantly decreased nitrate only at 500 ng/mL. Data analyzed using a 1-way ANOVA with post hoc Tukey HSD Test. *p < 0.05.

Figure 2.

Extracellular concentrations of nitric oxide (NO) products measured by Griess reagent analysis for human umbilical vein endothelial cells pooled from 3 separate donors (P3 to P5) exposed to 24 hours of laminar shear stress (A) NO₂^– (nitrite) and (B) NO₃^– (nitrate) and oscillatory shear stress (C) NO₂^– and (D) NO₃^– followed by 1 hour of treatment with human myoglobin at decreasing concentrations compared with no treatment. Four replicate experiments were conducted in duplicate. Data analyzed using a 1-way ANOVA with post hoc Tukey HSD Test. *p < 0.05. NT, no treatment.

Myoglobin induces endothelial cell permeability

Compared with 100 ng/mL, no treatment, or similar concentration of 200 ng/mL of albumin, 200 ng/mL human myoglobin significantly increased endothelial permeability, but myoglobin induced significantly less permeability than the positive controls (thrombin and vascular endothelial growth factor-165; Fig. 3) Data were analyzed using a 1-way ANOVA with post hoc Tukey HSD Test. *p < 0.05, **p < 0.01.

Figure 3.

Representative images (A) and quantification (B) of endothelial permeability for confluent human umbilical vein endothelial cells pooled from 3 separate donors (P3 to P5) after 1 hour of treatment with human myoglobin, VEGF-165, thrombin, or no treatment compared with 1 hour of treatment with albumin. Four replicate experiments were conducted in duplicate. Images were analyzed using FIJI, and subsequent data were analyzed using a 1-way ANOVA with post hoc Tukey HSD Test. *p < 0.05; **p < 0.01. FITC, fluorescein isothiocyanate; NT, no treatment; VEGF-165, vascular endothelial growth factor 165.

Arterial response to myoglobin

Naïve male and female murine aortas exposed to myoglobin (100 and 300 ng/mL) have similar EC-dependent relaxation profiles but increased contraction (tone; Figs. 4, A/B). Increasing myoglobin concentrations were directly related to increasing aorta contraction (Fig. 4C). Data were analyzed using repeated-measures ANOVA; *p < 0.05.

Figure 4.

Effects of myoglobin on endothelial-dependent relaxation (A) and arterial contraction (B). All aortas were harvested from healthy C57Bl6 mice and tested fresh in the absence of myoglobin and then again after treatment with 100 ng/mL and 300 ng/mL myoglobin. (C) Dose-response effect of increasing concentrations of myoglobin on aortic contraction. Data analyzed using 1-way ANOVA with repeated measures. *p < 0.05. n = 6, female; n = 6, male. MGB, myoglobin.

Large animal model of myoglobinemia

Successful exclusion of bilateral external iliac arteries and vascular plugging of the right internal iliac artery was able to impair collateral inflow to the right hindlimb (compared with left; Figs. 5, A/B). We did this to limit the initial arteriogenic response of the internal iliac artery in our model (Figs. 5, C/D).

Figure 5.

(A) Aortogram demonstrating the pilot animal’s aortic trifurcation after bilateral external iliac artery stenting and vascular plugging of the stent (yellow arrows). (B) Aortogram at terminal procedure (2 weeks after bilateral ischemia) demonstrates the additional vascular plug (yellow arrow) in the right internal iliac artery trunk, which results in decreased pelvic blood flow on the right (treated side) compared with left side (no additional vascular plugs). (C) Standard aortogram demonstrating the trifurcation with the central common iliac artery giving rise to bilateral internal iliac arteries and middle sacral artery (top left). Arteriogenic growth of the internal iliacs is demonstrated in top right aortogram (animal has external iliac arteries occluded). (D) Growth of the internal iliac artery as % diameter change in animals undergoing standard unilateral (right side) external iliac artery occlusion. Data demonstrate need for the internal iliac artery plugging in the myoglobinemic animal. EIA, external iliac artery; IIA, internal iliac artery.

The animal was successfully exercised on a treadmill at week 1 and 2 after the second operation (Fig. 6A). The animal had sustained depressed hindlimb/forelimb indices and sustained muscle hypoxia in the right hindlimb. This led to increased myoglobinemia postexercise (Figs. 6, B–D). To test endothelial dysfunction in vivo, animals had flow-mediated dilation testing. Preoperatively, the flow-mediated dilation value was normal at –3% ± 5% (<7.5%), but at week 2, the animal possessed endothelial dysfunction with a value of –12% ± 2.5%.

Figure 6.

Porcine myoglobinemia model. (A) Image of pig reward after completing structured exercise therapy session. (B) We use the hindlimb/forelimb index as a surrogate of a patient ankle/brachial index to quantify hoof perfusion pressures. Postoperatively in this model the right hindlimb indices are immeasurable immediately postoperatively and at 1 week. Index recovers but stays under 0.4 at termination. (C) Oxygen saturation (Smo₂) in muscle measured using a microoxygen sensor, which demonstrates increased ischemia in the right hindlimb compared with the left during structured exercise session. (D) This results in a serial increase in serum myoglobin at 1 and 2 weeks postischemic insult. (E) Resultant myoglobinemia is associated with increased deficit in flow-mediated dilation consistent with endothelial dysfunction. Normal cutoff for endothelial dysfunction marked by red dotted line. Preoperative flow-mediated dilation was within normal limits.

Association of myoglobinemia and patient mortality

Our experimental data suggest that myoglobinemia levels of 100 ng/mL are sufficient for impairing arterial health. Setting this as our cutoff level, we then grouped patients above and below this level. We found that the age match was equivalent (61.0 ± 9.7 years of age at less than 100 ng/mL and 61.2 ± 6.5 years of age at more than 100 ng/mL). After querying the medical record, we found significantly more deaths in the patient group with 100 ng/mL myoglobin or higher. The time to death was significantly longer in the lower myoglobin group, suggesting that these patients had greater longevity (877.3 ± 384 days for patients with less than 100 ng/ml myoglobin compared with 636.3 ± 418.2 days for patients with more than 100 ng/mL myoglobin; Fig. 7). Mortality differences were statistically determined by Fisher exact test with **p = 0.026. Average time differences calculated with an unpaired t test with Welch’s correction with **p = 0.01.

Figure 7.

(A) Mortality differences between patients with serum myoglobin concentrations greater than and less than 100 ng/mL. (B) Average time from blood draw to mortality in patients who died. (C) Distribution of all patients in the study with myoglobin measurements (x axis) and corresponding serum myoglobin concentrations (y axis). Red arrows = deceased patients. Mortality differences were statistically determined by Fisher exact test with **p = 0.026. Average time differences calculated with an unpaired t test with Welch’s correction with **p = 0.01.

DISCUSSION

A number of PAD-specific biomarkers have been identified, and some inflammatory biomarkers such as, C-reactive protein, interleukin-6, and tumor necrosis factor α have been found to correlate with PAD severity, these also correlate with PAD comorbidities, such as diabetes, which may limit their usefulness to acknowledging resolution with current therapies.^30,31 Additionally, microRNAs may provide diagnostic markers for PAD screening and may have a biologic effect as well.³² Still, we have identified that not only is myoglobin a biomarker for symptomatic PAD, but we have also found that myoglobin increases arterial tone, EC permeability, decreases NO bioavailability, and causes endothelial dysfunction in vivo (Fig. 8).

Figure 8.

Summary of data supporting a biologic role for myoglobin in peripheral arterial disease. Myoglobin impairs arterial health through various mechanisms. EC, endothelial cell; NO, nitric oxide.

To the best our knowledge, myoglobin is the first biomarker of PAD to demonstrate a potential biologic effect on arterial health and mortality. We demonstrate a clear association between myoglobin levels and decreased NO bioavailability. This is interesting for many reasons. First, in sickle cell disease, the spilled globin domain from hemoglobin during crises is responsible for decreased NO bioavailability. Because globin units are similar between hemoglobin and myoglobin, myoglobinemia may cause a NO effect similar to that of sickle cell crisis. This could explain both a propensity to cardiovascular events (platelet aggregation, exuberant vascular tone) and possibly contribute to the increased mortality seen in this study. As such, patients with elevated myoglobin levels theoretically would have decreased arterial health and possibly decreased NO bioavailability that could affect their physiologic reserve to vascular insults like a heart attack or stroke.

Additionally, recent evidence has shown that the α-monomer of hemoglobin is similar in homology to myoglobin and can act as a nitrite reductase in the endothelium of mice under hypoxic conditions leading to a reduction in nitrite and increase in nitric oxide.^33,34 In this study we quantified NO bioavailability using both nitrate and nitrite (referred to as nitric oxides or, simply, NOx), which is a standard and accurate method to determine total NO production.³⁵

There are a number of limitations in this article that warrant further study. We are excited to pursue these in future work. First, our linkage of myoglobinemia with mortality suffers from not knowing definitively how many of these deaths were from cardiovascular events. There is also a likelihood that major amputation played a role in the increased mortality rates given that many CLI patients in this study had an amputation as their index operation. However, recent publications suggest that mortality after major amputation is currently much lower than that published in older literature.³⁶ Existing literature supports that the majority of these deaths would likely be cardiovascular in nature,³⁷ but this needs to be better demonstrated. Nor do we have robust follow-up data from patients undergoing revascularization to see whether decreases in myoglobinemia are associated with “successful” revascularization or protection from mortality. This deserves future evaluation and may be pursued in shorter-term studies using flow-mediated dilation as a surrogate of endothelial dysfunction and as an independent risk factor for cardiovascular death. It is also interesting that revascularization in relatively young patients, like those included here, may not only improve quality of life but also mortality.³⁸ Certainly, more work is needed to link myoglobin (or other biomarkers) to these findings, but we hope this work spurs the next stage of studies that will help answer these important questions. Certainly, the association of increased myoglobin and decreased NO was supported by our in vitro testing. However, the exact interactions will likely be more complex than myoglobin simply being a sink for NO₃. There will likely be more than one mechanism by which myoglobinemia impacts arterial health, and we aim to better define these mechanisms in the future. We believe our pilot myoglobinemic model may be useful in this pursuit.

CONCLUSIONS

In summary, our multi-institutional research partnership has identified myoglobinemia as a biologically active biomarker of PAD. Future work will define the tolerable myoglobin levels and pathophysiology of arteriopathic injury by myoglobin. The long-term goal of this work will be to decrease the cardiovascular event and mortality rate from these events in PAD patients.

Author Contributions

Data curation: Scrivner, Fletcher, Hoffmann, Li, Wilkinson, Sutliff, Koutakis, Brewster

Formal analysis: Scrivner, Koutakis, Brewster

Investigation: Scrivner, Fletcher, Hoffmann, Wilkinson, Miserlis, Smith, Bohannon, Sutliff, Jordan, Koutakis, Brewster

Methodology: Scrivner, Fletcher, Hoffmann, Li, Wilkinson, Miserlis, Smith, Bohannon, Sutliff, Jordan, Koutakis, Brewster

Writing – original draft: Scrivner, Koutakis, Brewster

Writing – review & editing: Scrivner, Koutakis, Sutliff, Jordan, Brewster

Conceptualization: Sutliff, Koutakis, Brewster

Funding acquisition: Koutakis, Brewster

Project administration: Brewster

Resources: Brewster

Supervision: Brewster

Validation: Brewster

Visualization: Brewster