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Cited by 119Transplantation: Methods and OutcomesCardiac Ischemia and ReperfusionOrgan Transplantation Techniques and Outcomes

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The mitochondria-targeted anti-oxidant MitoQ decreases ischemia-reperfusion injury in a murine syngeneic heart transplant model

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Dare, Anna J., Logan, Angela, Prime, Tracy A., Rogatti, Sebastian, Goddard, Martin, Bolton, Eleanor M., Bradley, J. Andrew, Pettigrew, Gavin J., Murphy, Michael P., Saeb-Parsy, Kourosh·2015·The Journal of Heart and Lung Transplantation
DOI 10.1016/j.healun.2015.05.007PMC PMC4626443
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Abstract

BACKGROUND: Free radical production and mitochondrial dysfunction during cardiac graft reperfusion is a major factor in post-transplant ischemia-reperfusion (IR) injury, an important underlying cause of primary graft dysfunction. We therefore assessed the efficacy of the mitochondria-targeted anti-oxidant MitoQ in reducing IR injury in a murine heterotopic cardiac transplant model. METHODS: Hearts from C57BL/6 donor mice were flushed with storage solution alone, solution containing the anti-oxidant MitoQ, or solution containing the non-anti-oxidant decyltriphenylphosphonium control and exposed to short (30 minutes) or prolonged (4 hour) cold preservation before transplantation. Grafts were transplanted into C57BL/6 recipients and analyzed for mitochondrial reactive oxygen species production, oxidative damage, serum troponin, beating score, and inflammatory markers 120 minutes or 24 hours post-transplant. RESULTS: MitoQ was taken up by the heart during cold storage. Prolonged cold preservation of donor hearts before IR increased IR injury (troponin I, beating score) and mitochondrial reactive oxygen species, mitochondrial DNA damage, protein carbonyls, and pro-inflammatory cytokine release 24 hours after transplant. Administration of MitoQ to the donor heart in the storage solution protected against this IR injury by blocking graft oxidative damage and dampening the early pro-inflammatory response in the recipient. CONCLUSIONS: IR after heart transplantation results in mitochondrial oxidative damage that is potentiated by cold ischemia. Supplementing donor graft perfusion with the anti-oxidant MitoQ before transplantation should be studied further to reduce IR-related free radical production, the innate immune response to IR injury, and subsequent donor cardiac injury.

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Figure 1 Experimental system. (A) MitoQ uptake and protection during transplantation. Donor hearts were flushed with preservation solution and then stored at 4°C. MitoQ was taken up into the donor heart, accumulating several hundredfold within mitochondria. After cold preservation, the heart was transplanted into the abdomen of the recipient and allowed to reperfuse. (B) The donor heart was removed from the donor. (C and D) The heart was flushed to clear the vasculature of blood and was then placed in preservation solution. (E) The heart was transplanted heterotopically into the abdomen of the anesthetized recipient. A midline abdominal incision in the recipient was made and the inferior vena cava and were aorta exposed and clamped proximally and distally to allow bloodless vascular anastomoses. The donor heart was grafted to the recipient vessels as follows: donor pulmonary artery to recipient inferior vena cava and donor aorta to recipient aorta. (F) The clamps were removed and the graft was revascularized.
Figure 1 Experimental system. (A) MitoQ uptake and protection during transplantation. Donor hearts were flushed with preservation solution and then stored at 4°C. MitoQ was taken up into the donor heart, accumulating several hundredfold within mitochondria. After cold preservation, the heart was transplanted into the abdomen of the recipient and allowed to reperfuse. (B) The donor heart was removed from the donor. (C and D) The heart was flushed to clear the vasculature of blood and was then placed in preservation solution. (E) The heart was transplanted heterotopically into the abdomen of the anesthetized recipient. A midline abdominal incision in the recipient was made and the inferior vena cava and were aorta exposed and clamped proximally and distally to allow bloodless vascular anastomoses. The donor heart was grafted to the recipient vessels as follows: donor pulmonary artery to recipient inferior vena cava and donor aorta to recipient aorta. (F) The clamps were removed and the graft was revascularized.CC BY · PubMed Central
Figure 2 Mitochondria-targeted compounds can be delivered during hypothermic organ preservation. (A) Uptake of [3H]-methyltriphenylphosphonium (mTPP) into isolated mitochondria under hypothermic and normothermic conditions, with or without carbonylcyanide p-trifluoromethoxy phenylhydrazone (FCCP; n = 3). The accumulation ratio (ACR) was calculated as the amount of mTPP in the mitochondria (nmol/mg protein) divided by that in the supernatant (nmol/µ1) after pelleting the mitochondria. (B) Loading of the heart graft before transplantation with MitoQ was assessed by measuring tissue levels by liquid chromatography tandem mass spectrometry (LC-MS/MS). MitoQ was administered by intravenous injection into the donor mouse 15 minutes before organ procurement or during flushing and short (30 minutes) or prolonged (4 hours) cold ischemia time (CIT) preservation of the donor hearts (n = 3–4). CSS, cold static storage. (C) To understand MitoQ uptake into the donor heart, hearts were flushed with MitoQ and stored in standard CSS solution or were flushed with standard solution and stored in CSS solution supplemented with MitoQ for 30 minutes or 4 hours (n = 3) **p < 0.01, ***p < 0.001 determined by 1-way analysis of variance. Data are mean ± standard error of the mean.
Figure 2 Mitochondria-targeted compounds can be delivered during hypothermic organ preservation. (A) Uptake of [3H]-methyltriphenylphosphonium (mTPP) into isolated mitochondria under hypothermic and normothermic conditions, with or without carbonylcyanide p-trifluoromethoxy phenylhydrazone (FCCP; n = 3). The accumulation ratio (ACR) was calculated as the amount of mTPP in the mitochondria (nmol/mg protein) divided by that in the supernatant (nmol/µ1) after pelleting the mitochondria. (B) Loading of the heart graft before transplantation with MitoQ was assessed by measuring tissue levels by liquid chromatography tandem mass spectrometry (LC-MS/MS). MitoQ was administered by intravenous injection into the donor mouse 15 minutes before organ procurement or during flushing and short (30 minutes) or prolonged (4 hours) cold ischemia time (CIT) preservation of the donor hearts (n = 3–4). CSS, cold static storage. (C) To understand MitoQ uptake into the donor heart, hearts were flushed with MitoQ and stored in standard CSS solution or were flushed with standard solution and stored in CSS solution supplemented with MitoQ for 30 minutes or 4 hours (n = 3) **p < 0.01, ***p < 0.001 determined by 1-way analysis of variance. Data are mean ± standard error of the mean.CC BY · PubMed Central
Figure 3 MitoQ protects against ischemia-reperfusion injury after heterotopic cardiac transplantation in mice. (A) Quantification of myocardial damage 120 minutes post-transplantation by serum troponin I after short (30 minutes) and prolonged (4 hours) cold ischemia time (CIT) preservation, with or without treatment with MitoQ (n = 4–5). (B) Quantification of myocardial damage 24 hours post-transplantation by serum troponin I after short (30 minutes) and prolonged (4 hours) cold preservation, with or without treatment with MitoQ or decyltriphenylphosphonium (dTPP; n = 4–5). Note the 10-fold decrease in the scale compared with panel A. Blinded assessment of heart function (C) 120 minutes and (D) 24 hours after transplant by beating score (n = 4–5). *p < 0.05, **p < 0.01, determined by 1-way analysis of variance with Bonferroni post-test. Data are mean ± standard error of the mean.
Figure 3 MitoQ protects against ischemia-reperfusion injury after heterotopic cardiac transplantation in mice. (A) Quantification of myocardial damage 120 minutes post-transplantation by serum troponin I after short (30 minutes) and prolonged (4 hours) cold ischemia time (CIT) preservation, with or without treatment with MitoQ (n = 4–5). (B) Quantification of myocardial damage 24 hours post-transplantation by serum troponin I after short (30 minutes) and prolonged (4 hours) cold preservation, with or without treatment with MitoQ or decyltriphenylphosphonium (dTPP; n = 4–5). Note the 10-fold decrease in the scale compared with panel A. Blinded assessment of heart function (C) 120 minutes and (D) 24 hours after transplant by beating score (n = 4–5). *p < 0.05, **p < 0.01, determined by 1-way analysis of variance with Bonferroni post-test. Data are mean ± standard error of the mean.CC BY · PubMed Central
Figure 4 Prolonged cold preservation results in increased mitochondrial reactive oxygen species (ROS) and oxidative damage within the graft 24 hours after transplant, and MitoQ reduces this oxidative damage. (A) The MitoP/MitoB ratio, which increases in response to elevated mitochondrial H2O2, was measured within the heart graft in vivo at 24 hours post-transplant after short and prolonged cold ischemia time (CIT) preservation (n = 4). (B) Mitochondrial DNA (mtDNA) damage in the heart graft 24 hours after transplantation. Decreased mtDNA amplification is detected by quantitative polymerase chain reaction (qPCR) using a mitochondrial long target relative to a short target, which reflects damage. mtDNA amplification levels from heart grafts exposed to short or prolonged cold preservation, with or without MitoQ, have been normalized to healthy control mouse heart mtDNA amplification (n = 4). (C) Assessment of oxidative damage to proteins measured by protein carbonyl formation in heart grafts 24 hours after transplantation (n = 3–4). *p < 0.05, **p < 0.01, ***p < 0.001 determined by 1-way analysis of variance. Data are mean ± standard error of the mean.
Figure 4 Prolonged cold preservation results in increased mitochondrial reactive oxygen species (ROS) and oxidative damage within the graft 24 hours after transplant, and MitoQ reduces this oxidative damage. (A) The MitoP/MitoB ratio, which increases in response to elevated mitochondrial H2O2, was measured within the heart graft in vivo at 24 hours post-transplant after short and prolonged cold ischemia time (CIT) preservation (n = 4). (B) Mitochondrial DNA (mtDNA) damage in the heart graft 24 hours after transplantation. Decreased mtDNA amplification is detected by quantitative polymerase chain reaction (qPCR) using a mitochondrial long target relative to a short target, which reflects damage. mtDNA amplification levels from heart grafts exposed to short or prolonged cold preservation, with or without MitoQ, have been normalized to healthy control mouse heart mtDNA amplification (n = 4). (C) Assessment of oxidative damage to proteins measured by protein carbonyl formation in heart grafts 24 hours after transplantation (n = 3–4). *p < 0.05, **p < 0.01, ***p < 0.001 determined by 1-way analysis of variance. Data are mean ± standard error of the mean.CC BY · PubMed Central
Figure 5 Prolonged cold preservation increases the systemic pro-inflammatory response after transplantation, which is ameliorated by MitoQ. (A) After 120 minutes of reperfusion there was a significant increase in interleukin (IL)-1β from 30 minutes to 4 hours of cold ischemia time (CIT) preservation (p = 0.004, 1-way analysis of variance [ANOVA]); however, treatment with MitoQ did not significantly reduce IL-1β (not significant, Bonferroni post-testing). (B) After 120 minutes there was a significant difference increase in IL-6 from 30 minutes to 4 h CIT (p < 0.05, Bonferroni post-testing). Treatment with MitoQ significantly reduced serum IL-6 for 4 h CIT (p < 0.05, Bonferroni post-testing). (C) After 120 minutes of reperfusion, there was a significant increase in chemokine (C-X-C motif) ligand 1 (CXCL-1) from 30 minutes to 4 hours CIT (p = 0.006, 1-way ANOVA). Treatment with MitoQ during 30 minutes of CIT and 4 hours of CIT significantly reduced serum CXCL-1 (p < 0.05, Bonferroni post-testing). (D) There were no significant differences in the levels of the anti-inflammatory cytokine IL-10 at 120 minutes of reperfusion with CIT or MitoQ treatment (not significant, 1-way ANOVA). (E) At 24 hours of reperfusion, serum IL-6 remained significantly elevated in the 4-hour CIT control group compared with 30 minutes of CIT (p < 0.01, 1-way ANOVA, Bonferroni post-testing), whereas serum IL-1β and CXCL-1 had normalized in all groups at 24 hours after reperfusion (data not shown). Treatment with MitoQ prevented the increase in IL-6 caused by 4 hours of CIT when measured 24 hours after reperfusion (p < 0.01, 1-way ANOVA, Bonferroni post-testing). For all experiments n = 4–5. *p < 0.05, **p < 0.01. Data are mean ± standard error of the mean.
Figure 5 Prolonged cold preservation increases the systemic pro-inflammatory response after transplantation, which is ameliorated by MitoQ. (A) After 120 minutes of reperfusion there was a significant increase in interleukin (IL)-1β from 30 minutes to 4 hours of cold ischemia time (CIT) preservation (p = 0.004, 1-way analysis of variance [ANOVA]); however, treatment with MitoQ did not significantly reduce IL-1β (not significant, Bonferroni post-testing). (B) After 120 minutes there was a significant difference increase in IL-6 from 30 minutes to 4 h CIT (p < 0.05, Bonferroni post-testing). Treatment with MitoQ significantly reduced serum IL-6 for 4 h CIT (p < 0.05, Bonferroni post-testing). (C) After 120 minutes of reperfusion, there was a significant increase in chemokine (C-X-C motif) ligand 1 (CXCL-1) from 30 minutes to 4 hours CIT (p = 0.006, 1-way ANOVA). Treatment with MitoQ during 30 minutes of CIT and 4 hours of CIT significantly reduced serum CXCL-1 (p < 0.05, Bonferroni post-testing). (D) There were no significant differences in the levels of the anti-inflammatory cytokine IL-10 at 120 minutes of reperfusion with CIT or MitoQ treatment (not significant, 1-way ANOVA). (E) At 24 hours of reperfusion, serum IL-6 remained significantly elevated in the 4-hour CIT control group compared with 30 minutes of CIT (p < 0.01, 1-way ANOVA, Bonferroni post-testing), whereas serum IL-1β and CXCL-1 had normalized in all groups at 24 hours after reperfusion (data not shown). Treatment with MitoQ prevented the increase in IL-6 caused by 4 hours of CIT when measured 24 hours after reperfusion (p < 0.01, 1-way ANOVA, Bonferroni post-testing). For all experiments n = 4–5. *p < 0.05, **p < 0.01. Data are mean ± standard error of the mean.CC BY · PubMed Central
Figure 6 This schematic summarizes the conclusions from this work. Increasing the duration of cold ischemia exacerbates the production of mitochondrial reactive oxygen species (mtROS) upon reperfusion of the donor heart within the recipient. This ROS production leads to mitochondrial oxidative damage that in turn causes the cell death and inflammation that underlies delayed graft function and primary graft failure. Infusing the mitochondria-targeted anti-oxidant MitoQ into the donor heart before storage decreases this oxidative damage and the subsequent inflammatory response. IL, interleukin; mtDNA, mitochondrial DNA.
Figure 6 This schematic summarizes the conclusions from this work. Increasing the duration of cold ischemia exacerbates the production of mitochondrial reactive oxygen species (mtROS) upon reperfusion of the donor heart within the recipient. This ROS production leads to mitochondrial oxidative damage that in turn causes the cell death and inflammation that underlies delayed graft function and primary graft failure. Infusing the mitochondria-targeted anti-oxidant MitoQ into the donor heart before storage decreases this oxidative damage and the subsequent inflammatory response. IL, interleukin; mtDNA, mitochondrial DNA.CC BY · PubMed Central

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Authors
Dare, A. J., Logan, A., Prime, T. A., Rogatti, S., Goddard, M., Bolton, E. M., Bradley, J. A., Pettigrew, G. J., Murphy, M. P., & Saeb-Parsy, K.
Affiliations
  • University of Cambridge (GB)
  • National Health Service (GB)
Corresponding author
Murphy, Michael P.
ORCID
Venue
The Journal of Heart and Lung Transplantation
Publisher
Elsevier BV
Published
2015-11
Volume
34
Issue
11
Pages
1471-1480
ISSN
1053-2498 (ISSN-L)
DOI
10.1016/j.healun.2015.05.007
PMC
PMC4626443
OpenAlex
W1788023720
Cited by
119
References
48
Citations by year
2026: 10 · 2025: 19 · 2024: 13 · 2023: 8 · 2022: 7 · 2021: 14 · 2020: 13 · 2019: 7 · 2018: 11 · 2017: 8 · 2016: 9
FWCI
9.41
Type
journal article
Language
en
Open access
Licence
Locations
  • The Journal of Heart and Lung Transplantation (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Health Sciences › Medicine › Surgery › Transplantation: Methods and Outcomes
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiac Ischemia and Reperfusion
  • Health Sciences › Medicine › Surgery › Organ Transplantation Techniques and Outcomes
Keywords
MitoQ, mitochondria-targeted antioxidants, ischemia-reperfusion injury, heart transplantation, primary graft dysfunction, oxidative stress, mitochondrial reactive oxygen species, cold ischemia, donor heart preservation, mitochondrial DNA damage, inflammatory cytokines
MeSH terms
Animals, Antioxidants, Disease Models, Animal, Female, Male, Mice, Inbred C57BL, Mitochondria, Heart, Organ Preservation, Organophosphorus Compounds, Ubiquinone, Reperfusion Injury, Heart Transplantation, Free Radical Scavengers, Rats, Wistar, Reactive Oxygen Species, Oxidative Stress, Micronutrients, Mice, Rats, Primary Graft Dysfunction

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