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

IPC triggers Parkin translocation to mitochondria in vivo.

<p><b>A.</b> Mice were subjected to IPC consisting of 3 cycles of 5 min coronary artery ligation alternating with 5 min reperfusion. Heavy membrane fractions prepared from heart were probed for Parkin. Cytochrome oxidase subunit III (COX3) was used to normalize mitochondrial loading. <b>B.</b> Quantification of Parkin translocation to mitochondria is shown (*p<0.05, n = 5). <b>C.</b> Mice were subjected to 3 cycles of IPC in vivo. Cryosections of hearts were examined to visualize Parkin translocation to mitochondria by immunolabeling with antibodies to Tom70 (green) and Parkin (red). White line indicates segment used for pseudo-line scan analysis. Representative images from the risk zone of sham-operated (CON) and preconditioned (IPC) hearts are shown. <b>D.</b> Pseudo-line scan analysis of mitochondria (Tom70, solid green line) and endogenous Parkin (dotted red line) in cryosections from control (CON) and preconditioned (IPC) mouse hearts demonstrates translocation of Parkin to the mitochondria after IPC.</p

FCCP induces Parkin translocation to mitochondria in HL-1 cardiomyocytes and mitochondrial fractions in Langendorff-perfused rat hearts.

<p><b>A.</b> HL-1 cardiomyocytes were treated with the mitochondrial uncoupler FCCP (10 µM) or vehicle (ethanol, CON) for 1 hour, then fixed and immunolabeled for Tom70 (green) and Parkin (red). Boxes outline fields that were enlarged (at right) to show details of mitochondrial structure and colocalization. <b>B.</b> Cells were then scored for significant colocalization. Over 100 cells from sequential fields were assessed for each group (*p<0.03, n = 4). <b>C.</b> Isolated rat hearts were subjected to continuous perfusion with 100 nM FCCP or vehicle (ethanol, CON) for 5 minutes after stabilization with KHB. Mitochondrial fractions were probed for Parkin. <b>D.</b> Quantification of Parkin in mitochondrial fractions from CON and FCCP treated hearts (*p<0.05, n = 4).</p

Depletion of p62 attenuates sI-induced mitochondrial loss.

<p><b>A.</b> HL-1 cells were treated with scrambled siRNA (siControl) or siRNA corresponding to p62 (sip62) and labeled with antibodies to Tom70 (green) and p62 (red). <b>B.</b> HL-1 cells treated with siRNA were subjected to sI for 60 min and mitochondrial mass was quantified by fluorescence intensity of the mitochondrial marker Tom70. Over 100 cells were assessed for each group and the experiment was performed 3 times. Error bars represent SEM of the 3 experiments (ANOVA: *p<0.001, **p<0.05, ***p<0.005). <b>C.</b> Shown is a representative deconvolved image of HL-1 cells transfected with mCherry-Parkin (red) and subjected to sI for 30 min and probed for Tom70 (green). <b>D.</b> Mitochondrial content in mCherry-Parkin-transfected cells was assessed by fluorescence intensity of Tom70, and the percentage of cells showing substantial mitochondrial loss (similar to the cell depicted here in the center of the field) was scored for non-ischemic cells (CON) and time points of 15, 30 and 45 min of simulated ischemia. A minimum of 100 transfected cells were scored for each time point (ANOVA: *p<0.01, **p<0.001 versus CON, n = 4). Error bars represent SEM of the 4 experiments. <b>E.</b> Mitochondrial content per cell was assessed in HL-1 cardiomyocytes subjected to 60 min of sI. The total amount of green (Tom70, outer mitochondrial membrane marker), and red (COX4, inner mitochondrial membrane marker) fluorescence intensity per unit area within each cell were measured (ImageJ). <b>F.</b> For each condition, 100 cells were scored in sequential fields (*p<0.01, n = 4).</p

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

Translocation of p62 to mitochondria is induced by IPC.

<p><b>A.</b> Cryosectioned hearts from WT mice sham operated (CON) or subject to IPC were immunolabeled for p62 and Tom70. Colocalization was assessed via PDM value images using Image J software where high colocalization scores are shown in green. <b>B.</b> WT and Parkin^−/− (KO) mice were subjected to sham surgery (CON) or IPC, and mitochondria were probed for p62 and normalized to Tom70. <b>C.</b> Quantification of p62 translocation is shown (*p<0.05, n = 5).</p

p62 translocates to mitochondria in HL-1 cells subjected to simulated ischemia (sI).

<p><b>A.</b> HL-1 cells were subjected to sI for 30 or 60 min, then fixed and immunolabeled for Tom70 (green) and p62 (red). Boxes outline fields that were enlarged (at right) to show details of mitochondrial structure and colocalization. <b>B.</b> p62 Western blot of mitochondrial fractions from HL-1 cardiomyocytes subjected to 60 min sI. <b>C.</b> Quantification of Western blots is shown (Student's T-test: *p<0.02, n = 3).</p

Parkin mediates cytoprotection in HL-1 cells subjected to simulated ischemia and reperfusion (sI/R).

<p><b>A.</b> HL-1 cells were treated with siRNA for Parkin or control siRNA and lysates were probed for Parkin protein. Equal protein loading is shown by blotting for actin. <b>B.</b> Cells treated with siRNA were subjected to sI/R (60 min simulated ischemia and 60 min reperfusion) and cell death was determined by measuring LDH release into the culture medium (Student's T-Test: *p<0.05, **p<0.001, n = 6). <b>C.</b> HL-1 cells were incubated in normal media for 60 min, subjected to sI/R, or preconditioned (PreC) with 30 min sI and 60 min recovery in normal media before being subjected to sI/R. Cell culture supernatants during the final 60 min reperfusion were assayed for LDH release to determine cell death. (*p<0.005, and **p<0.01, n = 3).</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