TAT-Ndi1 overcomes effects of complex I dysfunction.

<p><b>A</b>. 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>B</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). <b>C</b>. 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). <b>D</b>. 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).</p

Mitochondrial integrity and function is preserved by TAT-Ndi1.

<p><b>A</b>. The absorbance of cardiac mitochondrial suspension from rat heart tissue was measured in the presence or absence of TAT-Ndi1. Hearts were perfused +/− TAT-Ndi1 for 20 min prior to isolating mitochondria. TAT-Ndi1 protects against calcium-induced mitochondrial swelling and this inhibition is abolished by Ndi1 inhibitor, flavone (representative trace, n = 4). <b>B</b>. Slope and V_max of mitochondrial swelling are reduced in mitochondria with TAT-Ndi1 (n = 4, p<0.05). <b>C</b>. Oxygen consumption of mitochondria isolated from rat hearts with or without TAT-Ndi1 and subjected to I/R (I/R+TAT-Ndi1:double line, I/R alone: dashed line) or constantly perfused (Con:thick line, +TAT-Ndi1:thin line). Oxygen levels were continuously monitored using a platinum Clark-type oxygen electrode. Changes of O<b>₂</b> concentration in chamber are shown with administration of treatments indicated (n = 4, representative trace). <b>D</b>. Rate of oxygen consumption following addition of complex I substrates palmitoyl-L-carnitine/malate and ADP (1mM final) prior to (black bars) and following (grey bars) addition of rotenone (*p<0.05). Mitochondria were isolated from hearts +/− TAT-Ndi1 subjected to I/R or constantly perfused (control and Ndi1 alone).</p

Generation of TAT-Ndi1 and expression <i>in vitro</i>.

<p><b>A</b>. Map of TAT-Ndi1 construct generated from inserting full length NDI1 gene (1,539bp) from pHook(NDI1) into the 6xHis-TAT-HA cloning vector. <b>B</b>. Lysates of adult rat ventricular myocytes were transduced with TAT-Ndi1 at 500nM in complete maintenance media for 20 min. Cell lysates were probed with anti-HA antibody to detect TAT-Ndi1. <b>C</b>. Adult cardiac myocytes (first and second rows) and HL-1 cells (third row) were transduced with TAT-Ndi1 at 500nM for 1 or 15 min as indicated, fixed and double-labeled with affinity-purified rabbit antibody to <i>S. cerevisiae</i> Ndi1 and mouse monoclonal cytochrome <i>c</i> antibody.</p

TAT-Ndi1 is cardioprotective in the Langendorff-perfused rat heart model of ischemia/reperfusion.

<p><b>A</b>. 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>B</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). <b>C</b>. 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).</p

Ndi1-mediated cytoprotection following simulated ischemia reperfusion is specific.

<p><b>A</b>. pHook(Ndi1) and pDsRed2-mito were transiently transfected into HL-1 cells. Ndi1 (stained with anti-HA antibody) co-localized with mitochondria (DsRed-mito) (merged). <b>B</b>. Ndi1-transfected and empty pHook-transfected HL-1 cells and neonatal rat cardiomyocytes were subjected to 2 hours simulated ischemia and 24 hours reperfusion. Cell death was scored by permeability to Yo-Pro-1 stain and only transfected cells were scored. (≥250 cells scored per experiment, n = 3, *p<0.05). <b>C</b>. Neonatal rat cardiomyocytes transfected with empty pHook or pHook(Ndi1) were subjected to 2 hours simulated ischemia and 24 hours reperfusion. Pretreatment with Ndi1-inhibitor flavone abolished the cytoprotective effect of Ndi1 expression. Cell death was scored by permeability to Yo-Pro-1 stain and only transfected cells were scored. (≥250 cells scored per experiment, n = 3, **p<0.005).</p