Abstract
Mitochondrial dysfunction plays a critical role in the pathogenesis of cardiovascular and metabolic diseases. However, direct assessment of mitochondrial respiration in human vascular tissue remains technically challenging. In this study, we present an ex vivo approach for real-time analysis of mitochondrial respiration in human left internal mammary artery (LIMA) grafts obtained during coronary artery bypass grafting (CABG). LIMA segment was collected intraoperatively and processed for bioenergetic assessment using the Seahorse XF Flex 3D Analyzer. Mitochondrial respiration was evaluated using the Mito Stress Test, enabling real-time measurement of oxygen consumption rate (OCR). In parallel, tissue nucleotide levels were quantified using high-performance liquid chromatography (HPLC), allowing complementary assessment of cellular energy status and redox balance. We demonstrate the feasibility of measuring mitochondrial respiration in intact human arterial tissue ex vivo. To our knowledge, this is the first application of the Seahorse XF Flex 3D platform for real-time bioenergetic analysis in intact human vascular tissue. Combined analysis of OCR and nucleotide levels enabled integrated assessment of vascular bioenergetic status. The applied protocol enabled reliable assessment of key bioenergetic parameters, including basal and maximal respiration. This study establishes a novel proof-of-concept workflow for ex vivo bioenergetic profiling of human vascular grafts, providing a platform for future investigations of vascular metabolism in cardiovascular and metabolic disorders.
Introduction
Mitochondria play a central role in vascular homeostasis, extending beyond ATP production to regulate redox signalling, calcium handling and cellular stress responses (). In endothelial cells, mitochondria function primarily as signalling organelles rather than major energy producers, critically modulating vascular tone, inflammation and adaptation to metabolic stress (; ).
Accumulating evidence indicates that mitochondrial dysfunction contributes to the pathogenesis of cardiovascular and metabolic disorders. Impaired mitochondrial function leads to excessive reactive oxygen species (ROS) production, endothelial activation and reduced nitric oxide bioavailability, promoting vascular stiffness, inflammation and atherosclerosis (). These processes are particularly relevant in patients undergoing coronary artery bypass grafting (CABG), where the functional integrity of vascular grafts is essential for long-term outcomes ().
The left internal mammary artery (LIMA) is considered the gold standard conduit in CABG due to its superior long-term patency and resistance to atherosclerosis (). Despite its clinical importance, the bioenergetic profile of LIMA remains poorly characterized, largely due to technical limitations in assessing mitochondrial function in intact human vascular tissue.
Recent advances in extracellular flux analysis, such as the Seahorse XF platform, have enabled real-time assessment of mitochondrial respiration in intact cells and isolated mitochondria (; ). However, their application to intact human vascular tissues remains limited.
In this study, we aimed to establish a novel ex vivo approach for real-time bioenergetic analysis of intact human vascular tissue using the Seahorse XF Flex 3D Analyzer. Using LIMA grafts obtained during CABG, we demonstrate the feasibility of measuring mitochondrial respiration in structurally preserved human arterial segments. This proof-of-concept study introduces, to our knowledge, the first application of Seahorse XF Flex 3D technology to intact human vascular tissue and provides a new platform for investigating vascular mitochondrial function in clinically relevant settings.
Materials and methods
Patient characteristics
A 67-year-old male patient undergoing elective CABG was included in this study. The patient presented with stable two-vessel coronary disease, arterial hypertension and orally treated diabetes mellitus. LIMA was harvested during a standard surgical technique with the sternotomy access and used as a graft to the left anterior descending artery. A surplus/residual segment of LIMA was collected for ex vivo analysis. All procedures were conducted in accordance with the Declaration of Helsinki and approved by the local bioethics committee at the Medical University of Gdańsk, Poland (No. KB/259/2025). Written informed consent was obtained from the patient prior to inclusion in the study.
Tissue processing
LIMA segment was obtained intraoperatively during CABG procedure. Immediately after harvesting, the vessel was placed in Agilent Seahorse XF DMEM Medium (pH 7.4) and transported to the laboratory. The vessel was cleaned of surrounding tissue and sectioned into small fragments suitable for analysis. The vascular rings were then cut longitudinally and gently flattened to obtain planar tissue segments. The samples with the luminal surface facing upwards were immobilized and placed onto Seahorse XF capture microplates equipped with islet capture screens, ensuring that the endothelial layer was exposed to the measurement chamber (Figure 1).
FIGURE 1
Histological staining
LIMA sections were fixed in 4% buffered formaldehyde and embedded in paraffin. The embedded artery fragments were cut into 6 µm-thick cross-sections and placed on microscope slides. LIMA sections were deparaffinized with xylene and rehydrated in a descending alcohol series to water. Slides were stained with hematoxylin and eosin (HE) to assess vascular structure and confirm the presence of an endothelial lining.
Seahorse XF flex mito stress assay
The mitochondrial function of vascular tissue was assessed using the Agilent Seahorse XF Flex analyzer with the Seahorse XF Flex 3D Capture Microplate-L. LIMA segments were cut into 2–3 mm long fragments (n = 3) to fit the microplate wells. One day prior to the assay, the sensor cartridge was hydrated with 1 mL of Seahorse XF calibrant solution per well and incubated at 37 °C in a non-CO2 incubator overnight. On the day of the experiment, vessel segments were loaded into wells containing assay medium, and the capture screen was positioned to secure the tissue. The mitochondrial stress test was conducted by sequential injection of compounds in the following order: oligomycin A at a final well concentration of 20 μM, FCCP at 15 μM, and a mixture of rotenone and antimycin A each at 10 µM. Oxygen consumption rates were recorded to measure mitochondrial respiration parameters including basal respiration, ATP-linked respiration, maximal respiration, proton leak, and non-mitochondrial respiration in the LIMA segments. Total protein content in each sample was determined using the Bradford assay according to the manufacturer’s instructions. Oxygen consumption rate (OCR) values were normalized to protein content and are presented as pmol O2/min/mg protein.
Adenine nucleotide and nicotinamide adenine dinucleotide measurements
Tissue nucleotide concentrations were determined using high-performance liquid chromatography (HPLC) as described earlier (; ). Briefly, LIMA sections (n = 3) were snap-frozen and homogenized in ice-cold perchloric acid to extract nucleotides. After neutralization, samples were centrifuged, and the supernatant was subjected to HPLC analysis. Adenosine triphosphate (ATP), diphosphate (ADP) and monophosphate (AMP) as well as guanosine triphosphate (GTP) and diphosphate (GDP) and oxidized (NAD+) and reduced (NADH) nicotinamide adenine dinucleotide were measured. As adenosine 5′-diphosphoribose (ADPR) is the major product formed by acidic cleavage of NADH during the tissue extraction, the measured ADPR level reflected NADH concentration. Quantification was performed using external standards, and results were normalized to total protein content in sample.
Results
We successfully established an ex vivo protocol for measuring mitochondrial respiration in human LIMA tissue using the Seahorse XF Flex Analyzer. Histological analysis confirmed preservation of vascular structure in prepared LIMA segments (Figure 2).
FIGURE 2
Real-time OCR measurements demonstrated that mitochondrial respiration can be reliably assessed in intact human arterial tissue using the Seahorse XF Flex 3D platform (Figure 3). The applied protocol enabled reproducible measurement of key bioenergetic parameters, including basal respiration, ATP-linked respiration and maximal respiratory capacity.
FIGURE 3
In parallel, HPLC analysis confirmed the presence of measurable levels of nucleotides in LIMA tissue (Figure 4). Nucleotide profiling demonstrated preserved energy status of the analyzed samples, supporting the functional integrity of the vascular tissue under ex vivo conditions. Combined assessment of mitochondrial respiration and nucleotide concentrations provides complementary insight into vascular bioenergetics at both functional and metabolic levels.
FIGURE 4
Discussion
In this study, we demonstrate the feasibility of real-time ex vivo assessment of mitochondrial respiration in human LIMA grafts using the Seahorse XF Flex 3D Analyzer. To our knowledge, this is the first application of the Seahorse XF Flex 3D platform for bioenergetic analysis in intact human vascular tissue.
To date, the assessment of mitochondrial function in the vascular system has been primarily performed in isolated endothelial or vascular smooth muscle cells as well as intact vessels from experimental animal models (; ; ). These approaches have provided important insights into vascular bioenergetics, but they do not fully capture the structural and cellular complexity of intact human vascular tissue.
The application of the Seahorse XF Flex 3D platform to structurally preserved human arterial segments therefore represents a methodological advancement, enabling bioenergetic measurements under conditions that more closely resemble the in vivo environment. The ability to measure oxygen consumption rate in real time in intact human vessels provides a novel tool for studying vascular bioenergetics in clinically relevant settings.
In this study, we complemented functional assessment of mitochondrial respiration with biochemical quantification of nucleotide levels using HPLC. While extracellular flux analysis provides dynamic information on mitochondrial activity, nucleotide profiling reflects the energetic state of the tissue. Importantly, the integration of these approaches allows simultaneous assessment of functional mitochondrial activity, cellular energy and redox status for a more comprehensive evaluation of vascular bioenergetics.
The left internal mammary artery, widely used in CABG due to its superior long-term patency, represents an ideal model for translational studies of vascular function (). Despite its clinical importance, its bioenergetic profile has been poorly characterized. The methodological approach presented here provides a new opportunity to investigate metabolic features of clinically relevant human vascular grafts. Thus, the integration of mitochondrial respiration measurements with nucleotide analysis may be particularly valuable in future studies aimed at identifying metabolic alterations associated with vascular dysfunction or patient comorbidities.
Importantly, the ability to assess mitochondrial respiration in human LIMA tissue may have potential clinical implications for evaluating graft quality. Although LIMA is widely regarded as the gold standard conduit in CABG, variability in endothelial function and metabolic status between patients may influence graft performance and long-term patency. Clinical studies have shown that both biochemical and patient-specific factors significantly affect short- and long-term outcomes following cardiac surgery (INFLACOR study) (). However, current intraoperative assessment of graft quality is largely limited to anatomical and flow-based measurements, which do not provide insight into the metabolic condition of the vessel (). Ex vivo bioenergetic profiling using this approach may therefore offer a complementary functional readout reflecting mitochondrial integrity and cellular viability within the vascular wall. Such an approach may help identify subtle alterations in vascular metabolism associated with patient comorbidities, such as diabetes or oxidative stress. In the future, this methodology could contribute to the development of biomarkers of graft quality and support personalized strategies in coronary artery bypass grafting.
Limitations
This study represents a proof-of-concept analysis based on a single human LIMA and should therefore be considered preliminary. The findings demonstrate technical feasibility rather than providing generalizable biological conclusions. In addition, variability related to tissue handling, preparation and positioning within the Seahorse platform may influence the measurements and requires further standardization. Future studies involving larger patient cohorts are necessary to validate the robustness, reproducibility, and potential clinical relevance of this approach.
Statements
Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
Ethics statement
The studies involving humans were approved by Bioethics Committee for Scientific Research at the Medical University of Gdansk. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
Author contributions
Conceptualization, AB, AŁ, MH, and BK-Z; methodology, AB, IW, MP, RS, and BK-Z; validation, AB and BK-Z; investigation, AB, AŁ, MP, AK, MK, MB, and BK-Z; writing - original draft preparation, AB, AŁ, AK, and BK-Z; writing - review and editing, MK, MP, MH, MB, RS, and BK-Z; visualization, AB, MP, IW, and BK-Z; supervision, BK-Z; project administration, BK-Z. All authors contributed to the article and approved the submitted version.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This research was supported by the National Science Centre in Poland (task grant no. 2023/51/B/NZ4/03017).
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Acknowledgements
We would like to express our appreciation for the support of the “Excellence Initiative – Research University” program, which played a key role in fostering interdisciplinary collaboration and promoting scientific excellence at the Medical University of Gdańsk, Poland.
References
1
ArslanhanG.Taşdemir MeteM. E.SargınM.BaştopçuM.SelçukN.AylinŞ. A.et al (2026). SYNTAX score affects LIMA graft flow dynamics in coronary artery bypass surgery. Turk Gogus Kalp Damar Cerrahisi Derg.34, 19–26. 10.4274/TJTCS.2025.27038
2
BrandM. D.NichollsD. G. (2011). Assessing mitochondrial dysfunction in cells. Biochem. J.435, 297–312. 10.1042/BJ20110162
3
DivakaruniA. S.ParadyseA.FerrickD. A.MurphyA. N.JastrochM. (2014). Analysis and interpretation of microplate-based oxygen consumption and pH data. Methods Enzymol.547, 309–354. 10.1016/B978-0-12-801415-8.00016-3
4
EmmertM. Y.BonattiJ.CaliskanE.GaudinoM.GrabenwögerM.GrapowM. T.et al (2024). Consensus Statement—Graft treatment in cardiovascular bypass graft surgery. Front. Cardiovasc. Med.11, 1285685. 10.3389/FCVM.2024.1285685
5
FangF.WangE.YangH.ZhaoT.WangQ.ZhangZ.et al (2025). Reprogramming mitochondrial metabolism and epigenetics of macrophages via miR-10a liposomes for atherosclerosis therapy. Nat. Commun.16 (1), 9117. 10.1038/s41467-025-64201-8
6
GroschnerL. N.Waldeck-WeiermairM.MalliR.GraierW. F. (2012). Endothelial mitochondria—less respiration, more integration. Pflügers Archiv - Eur. J. Physiology464 (1), 63–76. 10.1007/S00424-012-1085-Z
7
KaraśA.BarA.PandianK.JasztalA.KuryłowiczZ.Kutryb-ZającB.et al (2024). Functional deterioration of vascular mitochondrial and glycolytic capacity in the aortic rings of aged mice. Geroscience46, 3831–3844. 10.1007/S11357-024-01091-6
8
KarthikS.FabriB. M. (2006). Left internal mammary artery usage in coronary artery bypass grafting: a measure of quality control. Ann. R. Coll. Surg. Engl.88, 367–369. 10.1308/003588406X98667
9
KowalikM. M.LangoR.SiondalskiP.ChmaraM.BrzezińskiM.LewandowskiK.et al (2018). Clinical, biochemical and genetic risk factors for 30-day and 5-year mortality in 518 adult patients subjected to cardiopulmonary bypass during cardiac surgery - the INFLACOR study. Acta Biochim. Pol.65, 241–250. 10.18388/ABP.2017_2361
10
ShadrinI. Y.HolmesD. R.BehfarA. (2023). Left internal mammary artery as an endocrine organ: insights into graft biology and long-term impact following coronary artery bypass grafting. Mayo Clin. Proc.98, 150–162. 10.1016/j.mayocp.2022.10.003
11
SmolenskiR. T.LachnoD. R.LedinghamS. J. M.YacoubM. H. (1990). Determination of sixteen nucleotides, nucleosides and bases using high-performance liquid chromatography and its application to the study of purine metabolism in hearts for transplantation. J. Chromatogr. B Biomed. Sci. Appl.527, 414–420. 10.1016/S0378-4347(00)82125-8
12
WalczakI.TarnawskaM.StawarskaK.BraczkoA.PaterekA.RolskiF.et al (2026). Endothelial cell senescence and mitochondrial dysfunction in vascular ageing. Ageing Res. Rev.118, 103119. 10.1016/J.ARR.2026.103119
13
XieX.ChowdhuryS. R.SangleG.ShenG. X. (2010). Impact of diabetes-associated lipoproteins on oxygen consumption and mitochondrial enzymes in porcine aortic endothelial cells. Acta Biochim. Pol.57, 393–398. 10.18388/ABP.2010_2423
14
XuX.PangY.FanX. (2025). Mitochondria in oxidative stress, inflammation and aging: From mechanisms to therapeutic advances. Signal Transduct. Target. Ther.10 (1), 190. 10.1038/s41392-025-02253-4
15
YuE. P. K.ReinholdJ.YuH.StarksL.UrygaA. K.FooteK.et al (2017). Mitochondrial respiration is reduced in atherosclerosis, promoting necrotic core formation and reducing relative fibrous cap thickness. Arterioscler. Thromb. Vasc. Biol.37, 2322–2332. 10.1161/ATVBAHA.117.310042
Summary
Keywords
CABG, ex vivo model, LIMA, mitochondrial respiration, seahorse XF flex 3D
Citation
Braczko A, Łoś A, Piotrowska M, Kawecka A, Walczak I, Krysiak M, Hellmann M, Brzeziński M, Smoleński RT and Kutryb-Zając B (2026) Mitochondrial respiration and nucleotide profiling in human left internal mammary artery from CABG: a novel real-time ex vivo approach. Acta Biochim. Pol. 73:16877. doi: 10.3389/abp.2026.16877
Received
30 April 2026
Revised
08 July 2026
Accepted
14 July 2026
Published
27 July 2026
Volume
73 - 2026
Edited by
Grzegorz Wegrzyn, University of Gdansk, Poland
Reviewed by
Ewa Piotrowska, University of Gdansk, Poland
Katarzyna Tońska, University of Warsaw, Poland
Updates
Copyright
© 2026 Braczko, Łoś, Piotrowska, Kawecka, Walczak, Krysiak, Hellmann, Brzeziński, Smoleński and Kutryb-Zając.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Barbara Kutryb-Zając, b.kutryb-zajac@gumed.edu.pl
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