Binding of ANT with SUR/VDAC1.

<p>Co-immunoprecipitation assay was performed to determine binding of ANT with SUR 2 (A), VDAC 1 (VDAC) (B), and VDAC1 with ANT (C) using the Dynabeads Protein G method (Invitrogen). The experiments were conducted in the PMPs obtained from the sham and sepsis ARVMs (N = 3 in each treatment group). <b>D</b>. Colocalization of ANT with VDAC1 was performed using a fluorescent microscope in the purified mitochondrial fraction obtained from the sham and septic ARVMs. Representative photomicrographs (Magnification, 10×; Scale bar = 100 µm) were treated with anti-rabbit VDAC1 and anti-goat ANT antibodies to observe their co-localization in the mitochondrial fractions. The co-localized expression of VDAC1 (555 nm, red fluorescence, left panel), and ANT (488 nm, green fluorescence, middle panel) was observed in yellowish-orange fluorescence (right panel).</p

Effect of 5HD, administered at the time of sham/sepsis induction, on cardiodynamics and TNF-α levels in the rat.

<p>The effect of 5HD (100 µg/100 µL) on the cardiac output, CO (<b>A</b>); average ejection fraction, EF (<b>B</b>); fractional shortening, FS (<b>C</b>) measured using Vivid I at baseline (basal), 6 and 24 hr post-treatment by Alzet pump (<b>D</b>); the representative m-mode echocardiography recorded to calculate Simpson's ejection fraction in the rat. (<b>E</b>) the effect of 5HD on the concentration of TNF-α in the myocardial supernatant obtained from the heart collected at 24 hr post-sepsis in the rat. *P≤0.05 compared to the basal values and # P≤0.05 compared to the sepsis group at respective time points. (N = 6 in each group).</p

Effect of 5HD on ARVM contractility.

<p>The effect of 5HD on percent peak shortening in sham and septic ARVMs at 12 hr post-incubation in the culture medium (N = at least 50 ARVMs isolated from 5 rat hearts in each treatment group). The data are expressed as mean ± SEM. *p≤0.05 compared to the respective vehicle treatment group; and # p≤0.05 compared to the respective vehicle treatment group; $ p≤0.05 compared to the respective NE-treated sham/sepsis groups.</p

Effect of 5HD, administered at 6 hr post-sham/sepsis induction, on hemodynamics and survival in the rat.

<p>The effect of 5HD on (<b>A</b>) % survival at 48 hr and (<b>B</b>) MAP at 0, 6, 12, 24 and 48 hr post-Alzet pump (containing 5HD or saline) placement. *P≤0.05 compared to the 0 hr values and # P≤0.05 compared to the sepsis group at respective time points (N = 10 in each group).</p

Effect of 5HD, administered at the time of sham/sepsis induction, on hemodynamics and survival in the rat.

<p>The effect of 5HD on the, (<b>A</b>) rectal temperature, (<b>B</b>) survival and (<b>C</b>) Mean Arterial Pressure, MAP of the septic rats. Sham (N = 10), sepsis (N = 10); sepsis+5HD (N = 7). The values in the sepsis group at 48 and 72 hr include only two surviving animals.*P≤0.05 compared to the sham group and # P≤0.05 compared to the sepsis group at respective time points.</p

Effect of 5HD on VDAC1, release of cytochrome C and Bax.

<p>The effect of 5HD on the expression of mitochondrial VDAC1 (<b>A</b>), cytosolic Cytochome C (<b>B</b>) and Bax (<b>C</b>) in the sham and septic ARVMs (N = 4 in each treatment group). *P<0.05 compared to the respective vehicle-treated sham and sepsis groups; #P<0.05 compared to the respective sham treatment groups. $P<0.05 compared to the respective sham/sepsis NE group.</p

Effect of 5HD on mitochondrial membrane potential (ΔΨm) in ARVMs.

<p>Mitochondrial ΔΨm was examined in sham and septic ARVMs and visualized using a fluorescent microscope. Representative photomicrographs (magnification 40×; scale bar, 75 µm) of sham and septic ARVMs treated in the presence and absence of 5HD are stained with JC-1 reagent. Lane 1 exhibits the ARVMs studied under a light microscope. The red (JC-1 aggregates in the mitochondria; lane 2) and green (JC-1 monomers in cytoplasm; lane 3) fluorescence was recorded and merged using an image software (lane 4). The white arrowheads depict the location of mitochondria in the ARVMs (which were zoomed 10 times and shown in the boxedsquare).</p

Ultrastructural changes in the mitochondrial membrane in LV tissue.

<p><b>A</b>. Ultrastructural changes in the mitochondrial cristae (using TEM) in the left ventricular tissue section (N = 5 in each treatment group) obtained from sham and septic rat hearts(6 and 12 hr post-sepsis). The mitochondrial cristae deformation was seen in the purified mitochondrial preparation from the septic rat left ventricular tissue (6 hr post-sepsis). B. The lower panel represents the magnified image of selected mitochondria in the purified mitochondrial preparation (red box, upper panel). Arrows points to the mitochondrial cristae deformation (RED) with balloon expansion (YELLOW). Magnification, 40 k×; Scale bar = 0.1 µm.</p

Survival of Sprague Dawley rats (250 to 300 g) treated with single intravenous bolus infusion of LeTx (0.03 mg PA+0.015 mg LF) or EdTx (0.15 mg PA+0.075 mg EF) and monitored for two weeks.

<p>These are the same doses used for telemetry and ECHO experiments. N = 12 for each experiment. Kaplan-Meier graphs prepared.</p

Boxplots at different times post anthrax toxin bolus iv infusion of echocardiographic parameters:

<p>A. LeTx treated rats at zero, one and two hr post-infusion, velocity of propagation was 24±5, 34±7 and 46±17 cm/sec, respectively, with significant difference (P = 0.05 at one hr compared to controls and P = 0.007 at two hr compared to controls); B. LeTx treated rats at zero and one hr post-infusion, left ventricular diastolic area was 0.98±0.07 and 1.15±0.06, respectively, with significant difference (P = 0.01); C. LeTx treated rats at zero and one hr post-infusion, left ventricular systolic area was 0.79±0.08 and 0.98±0.10 cm, respectively, with significant difference (P = 0.02); D. EdTx treated rats at zero, one and two hr post-infusion, heart rate was 326±49, 371±28, and 393±10 beats per min, respectively, with significant difference (P = 0.05 at one hr and P = 0.001 at two hr compared to controls). The box represents the middle 50% of the data. The line through the box represents the median. The line (whiskers) extending from the box represent the upper and lower 25% of the data. The line of each plot connects the means of the sample.</p