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Xenotransplantation of mitochondrial electron transfer enzyme, Ndi1, in myocardial reperfusion injury.

Perry CN, Huang C, Liu W, Magee N, Carreira RS, Gottlieb RA - PLoS ONE (2011)

Bottom Line: Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane.The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56 kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping.Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT
A significant consequence of ischemia/reperfusion (I/R) is mitochondrial respiratory dysfunction, leading to energetic deficits and cellular toxicity from reactive oxygen species (ROS). Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane. Damage to complex I leads to superoxide production which further damages complex I as well as other proteins, lipids and mtDNA. The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56 kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping. Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone. Expression of Ndi1 in HL-1 cardiomyocytes and in neonatal rat ventricular myocytes protected the cells from simulated ischemia/reperfusion (sI/R), accompanied by lower ROS production, and preservation of ATP levels and NAD+/NADH ratios. We next generated a fusion protein of Ndi1 and the 11aa protein transduction domain from HIV TAT. TAT-Ndi1 entered cardiomyocytes and localized to mitochondrial membranes. Furthermore, TAT-Ndi1 introduced into Langendorff-perfused rat hearts also localized to mitochondria. Perfusion of TAT-Ndi1 before 30 min no-flow ischemia and up to 2 hr reperfusion suppressed ROS production and preserved ATP stores. Importantly, TAT-Ndi1 infused before ischemia reduced infarct size by 62%; TAT-Ndi1 infused at the onset of reperfusion was equally cardioprotective. These results indicate that restoring NADH oxidation and electron flow at reperfusion can profoundly ameliorate reperfusion injury.

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TAT-Ndi1 is cardioprotective in the Langendorff-perfused rat heart                            model of ischemia/reperfusion.A. Rat hearts were perfused with or without TAT-Ndi1 for 20                            min prior to 30 min ischemia and 2 hour reperfusion. Frozen sections                            were stained with TTC. TAT-Ndi1 reduced infarct size                            61.5%±8.01. Mean and S.D. from at least 5 hearts per                            condition. (*p<0.05). B. Perfusate collected prior                            to ischemia (baseline) and 15 min following onset of reperfusion.                            Creatine kinase release was reduced 51.6%±9.8 following                            ischemia/reperfusion in hearts perfused with TAT-Ndi1. Mean and S.D.                            from at least 4 hearts per condition. (**p<0.01).                                C. Hearts were subjected to 30 min ischemia and                            perfused with or without TAT-Ndi1 at the onset of reperfusion. Hearts                            were reperfused for 2 hours. Sections were stained with TTC                            (representative image, n = 5). TAT-Ndi1 reduced                            infarct size 67.1%±17.1. Mean and S.D. from at least 5                            hearts per condition (*p<0.05).
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pone-0016288-g007: TAT-Ndi1 is cardioprotective in the Langendorff-perfused rat heart model of ischemia/reperfusion.A. Rat hearts were perfused with or without TAT-Ndi1 for 20 min prior to 30 min ischemia and 2 hour reperfusion. Frozen sections were stained with TTC. TAT-Ndi1 reduced infarct size 61.5%±8.01. Mean and S.D. from at least 5 hearts per condition. (*p<0.05). B. Perfusate collected prior to ischemia (baseline) and 15 min following onset of reperfusion. Creatine kinase release was reduced 51.6%±9.8 following ischemia/reperfusion in hearts perfused with TAT-Ndi1. Mean and S.D. from at least 4 hearts per condition. (**p<0.01). C. Hearts were subjected to 30 min ischemia and perfused with or without TAT-Ndi1 at the onset of reperfusion. Hearts were reperfused for 2 hours. Sections were stained with TTC (representative image, n = 5). TAT-Ndi1 reduced infarct size 67.1%±17.1. Mean and S.D. from at least 5 hearts per condition (*p<0.05).

Mentions: Given the ability of TAT-Ndi1 to overcome the effects of complex I damage and preserve mitochondrial integrity, we wanted to determine the capacity of Ndi1 to protect against I/R injury ex vivo. Rat hearts were perfused 20 min with or without TAT-Ndi1 prior to 30 min global no-flow ischemia and 2 hr reperfusion. TAT-Ndi1 reduced infarct size by 62%±8.1, based on TTC staining (Figure 7A). Creatine kinase release was reduced by 51.6%±3.02 following I/R in hearts perfused with TAT-Ndi1 (Figure 7B).


Xenotransplantation of mitochondrial electron transfer enzyme, Ndi1, in myocardial reperfusion injury.

Perry CN, Huang C, Liu W, Magee N, Carreira RS, Gottlieb RA - PLoS ONE (2011)

TAT-Ndi1 is cardioprotective in the Langendorff-perfused rat heart                            model of ischemia/reperfusion.A. Rat hearts were perfused with or without TAT-Ndi1 for 20                            min prior to 30 min ischemia and 2 hour reperfusion. Frozen sections                            were stained with TTC. TAT-Ndi1 reduced infarct size                            61.5%±8.01. Mean and S.D. from at least 5 hearts per                            condition. (*p<0.05). B. Perfusate collected prior                            to ischemia (baseline) and 15 min following onset of reperfusion.                            Creatine kinase release was reduced 51.6%±9.8 following                            ischemia/reperfusion in hearts perfused with TAT-Ndi1. Mean and S.D.                            from at least 4 hearts per condition. (**p<0.01).                                C. Hearts were subjected to 30 min ischemia and                            perfused with or without TAT-Ndi1 at the onset of reperfusion. Hearts                            were reperfused for 2 hours. Sections were stained with TTC                            (representative image, n = 5). TAT-Ndi1 reduced                            infarct size 67.1%±17.1. Mean and S.D. from at least 5                            hearts per condition (*p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3038860&req=5

pone-0016288-g007: TAT-Ndi1 is cardioprotective in the Langendorff-perfused rat heart model of ischemia/reperfusion.A. Rat hearts were perfused with or without TAT-Ndi1 for 20 min prior to 30 min ischemia and 2 hour reperfusion. Frozen sections were stained with TTC. TAT-Ndi1 reduced infarct size 61.5%±8.01. Mean and S.D. from at least 5 hearts per condition. (*p<0.05). B. Perfusate collected prior to ischemia (baseline) and 15 min following onset of reperfusion. Creatine kinase release was reduced 51.6%±9.8 following ischemia/reperfusion in hearts perfused with TAT-Ndi1. Mean and S.D. from at least 4 hearts per condition. (**p<0.01). C. Hearts were subjected to 30 min ischemia and perfused with or without TAT-Ndi1 at the onset of reperfusion. Hearts were reperfused for 2 hours. Sections were stained with TTC (representative image, n = 5). TAT-Ndi1 reduced infarct size 67.1%±17.1. Mean and S.D. from at least 5 hearts per condition (*p<0.05).
Mentions: Given the ability of TAT-Ndi1 to overcome the effects of complex I damage and preserve mitochondrial integrity, we wanted to determine the capacity of Ndi1 to protect against I/R injury ex vivo. Rat hearts were perfused 20 min with or without TAT-Ndi1 prior to 30 min global no-flow ischemia and 2 hr reperfusion. TAT-Ndi1 reduced infarct size by 62%±8.1, based on TTC staining (Figure 7A). Creatine kinase release was reduced by 51.6%±3.02 following I/R in hearts perfused with TAT-Ndi1 (Figure 7B).

Bottom Line: Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane.The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56 kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping.Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT
A significant consequence of ischemia/reperfusion (I/R) is mitochondrial respiratory dysfunction, leading to energetic deficits and cellular toxicity from reactive oxygen species (ROS). Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane. Damage to complex I leads to superoxide production which further damages complex I as well as other proteins, lipids and mtDNA. The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56 kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping. Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone. Expression of Ndi1 in HL-1 cardiomyocytes and in neonatal rat ventricular myocytes protected the cells from simulated ischemia/reperfusion (sI/R), accompanied by lower ROS production, and preservation of ATP levels and NAD+/NADH ratios. We next generated a fusion protein of Ndi1 and the 11aa protein transduction domain from HIV TAT. TAT-Ndi1 entered cardiomyocytes and localized to mitochondrial membranes. Furthermore, TAT-Ndi1 introduced into Langendorff-perfused rat hearts also localized to mitochondria. Perfusion of TAT-Ndi1 before 30 min no-flow ischemia and up to 2 hr reperfusion suppressed ROS production and preserved ATP stores. Importantly, TAT-Ndi1 infused before ischemia reduced infarct size by 62%; TAT-Ndi1 infused at the onset of reperfusion was equally cardioprotective. These results indicate that restoring NADH oxidation and electron flow at reperfusion can profoundly ameliorate reperfusion injury.

Show MeSH
Related in: MedlinePlus