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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 overcomes effects of complex I dysfunction.A. ATP levels in rat heart tissue +/− TAT-Ndi1                            following 30 min ischemia and 15min reperfusion or without treatment                            (veh). TAT-Ndi1 prevents depletion of ATP stores in I/R hearts.                            (n = 4, *p<0.05,                            ***p<0.0005). B. Dihydroethidium stained 1mm                            rat heart sections +/−TAT-Ndi1 following 30 min no-flow                            ischemia and 15 min reperfusion. TAT-Ndi1 reduces superoxide production                            following I/R (representative image, n = 3).                                C. Total free MDA levels normalized to total protein in                            hearts perfused 20 min with or without TAT-Ndi1 and subjected to 30 min                            ischemia and 15 min reperfusion or perfused constantly with vehicle                            (n = 3, **p<0.005). D.                                NAD+/NADH ratios from rat hearts perfused 15 min                            +/− TAT-Ndi1 then subjected to 30 min ischemia and 15 min                            reperfusion or perfused continuously with vehicle                            (n = 4, *p<0.05, **p<0.005).
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pone-0016288-g005: TAT-Ndi1 overcomes effects of complex I dysfunction.A. ATP levels in rat heart tissue +/− TAT-Ndi1 following 30 min ischemia and 15min reperfusion or without treatment (veh). TAT-Ndi1 prevents depletion of ATP stores in I/R hearts. (n = 4, *p<0.05, ***p<0.0005). B. Dihydroethidium stained 1mm rat heart sections +/−TAT-Ndi1 following 30 min no-flow ischemia and 15 min reperfusion. TAT-Ndi1 reduces superoxide production following I/R (representative image, n = 3). C. Total free MDA levels normalized to total protein in hearts perfused 20 min with or without TAT-Ndi1 and subjected to 30 min ischemia and 15 min reperfusion or perfused constantly with vehicle (n = 3, **p<0.005). D. NAD+/NADH ratios from rat hearts perfused 15 min +/− TAT-Ndi1 then subjected to 30 min ischemia and 15 min reperfusion or perfused continuously with vehicle (n = 4, *p<0.05, **p<0.005).

Mentions: To determine the effect of TAT-Ndi1 on I/R-induced energetic deficits and oxidative damage, we perfused rat hearts with TAT-Ndi1 followed by global no-flow ischemia and 15 min reperfusion. I/R results in a 50% reduction of ATP content, but TAT-Ndi1 prevents ATP depletion (Figure 5A). Dihydroethidium-stained sections of hearts subjected to I/R showed that TAT-Ndi1 transduction reduced superoxide production (Figure 5B) and lipid peroxidation (Figure 5C). Administration of TAT-Ndi1 shifted the NAD+/NADH ratio towards NAD+ under basal conditions and following I/R. TAT-Ndi1 increased the ratio 3-fold over I/R conditions without TAT-Ndi1 (Figure 5D).


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 overcomes effects of complex I dysfunction.A. ATP levels in rat heart tissue +/− TAT-Ndi1                            following 30 min ischemia and 15min reperfusion or without treatment                            (veh). TAT-Ndi1 prevents depletion of ATP stores in I/R hearts.                            (n = 4, *p<0.05,                            ***p<0.0005). B. Dihydroethidium stained 1mm                            rat heart sections +/−TAT-Ndi1 following 30 min no-flow                            ischemia and 15 min reperfusion. TAT-Ndi1 reduces superoxide production                            following I/R (representative image, n = 3).                                C. Total free MDA levels normalized to total protein in                            hearts perfused 20 min with or without TAT-Ndi1 and subjected to 30 min                            ischemia and 15 min reperfusion or perfused constantly with vehicle                            (n = 3, **p<0.005). D.                                NAD+/NADH ratios from rat hearts perfused 15 min                            +/− TAT-Ndi1 then subjected to 30 min ischemia and 15 min                            reperfusion or perfused continuously with vehicle                            (n = 4, *p<0.05, **p<0.005).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3038860&req=5

pone-0016288-g005: TAT-Ndi1 overcomes effects of complex I dysfunction.A. ATP levels in rat heart tissue +/− TAT-Ndi1 following 30 min ischemia and 15min reperfusion or without treatment (veh). TAT-Ndi1 prevents depletion of ATP stores in I/R hearts. (n = 4, *p<0.05, ***p<0.0005). B. Dihydroethidium stained 1mm rat heart sections +/−TAT-Ndi1 following 30 min no-flow ischemia and 15 min reperfusion. TAT-Ndi1 reduces superoxide production following I/R (representative image, n = 3). C. Total free MDA levels normalized to total protein in hearts perfused 20 min with or without TAT-Ndi1 and subjected to 30 min ischemia and 15 min reperfusion or perfused constantly with vehicle (n = 3, **p<0.005). D. NAD+/NADH ratios from rat hearts perfused 15 min +/− TAT-Ndi1 then subjected to 30 min ischemia and 15 min reperfusion or perfused continuously with vehicle (n = 4, *p<0.05, **p<0.005).
Mentions: To determine the effect of TAT-Ndi1 on I/R-induced energetic deficits and oxidative damage, we perfused rat hearts with TAT-Ndi1 followed by global no-flow ischemia and 15 min reperfusion. I/R results in a 50% reduction of ATP content, but TAT-Ndi1 prevents ATP depletion (Figure 5A). Dihydroethidium-stained sections of hearts subjected to I/R showed that TAT-Ndi1 transduction reduced superoxide production (Figure 5B) and lipid peroxidation (Figure 5C). Administration of TAT-Ndi1 shifted the NAD+/NADH ratio towards NAD+ under basal conditions and following I/R. TAT-Ndi1 increased the ratio 3-fold over I/R conditions without TAT-Ndi1 (Figure 5D).

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