Morphological changes and LDH release induced by rCPB in pEC.

<p>(A) pEC were either left untreated (white) or exposed to rCPB or staurosporine (200 nM) for 4 h. Cell morphology was visualized using phase contrast microscopy. (B) Changes in total cell number (determined using automated cell counter) of pEC after 2, 4 and 16 h incubation with control medium, rCPB at indicated concentrations, neutralized rCPB using monoclonal anti-CPB antibodies, or staurosporine. Bar graphs represent the mean ± SEM of n = 3–6 independent experiments. Statistical difference to control cells was assessed by 1-way ANOVA and Dunnnet post-hoc test. *P<0.05, **P<0.01, †P<0.001. (C) The supernatants of pEC cultures from B were analyzed for LDH activity to determine LDH release after different times of exposure. Bar graph shows summary of results from 3 to 6 independent experiments expressed as percentage of activity compared to lysed control cells ( = 100%). Statistical difference to non-treated control cells was assessed by 1-way ANOVA and Dunnnet post-hoc test. *P<0.05, **P<0.01, †P<0.001.</p

rCPB-induced efflux of K⁺ and accumulation of intracellular Ca²⁺.

<p>(A) Incubation of confluent pEC with 30 ng/ml rCPB resulted in rapid and significant reduction of intracellular K⁺. rCPB preincubated with mAb-CPB did not cause K⁺ efflux. Values represent means ± SEM from 3 independent experiments. Statistical difference to cells treated with neutralized toxin ( = control) was assessed by 2-way ANOVA and Bonferroni multiple comparisons test. *P<0.05, **P<0.01, ****P<0.0001. (B) Intracellular calcium ([Ca²⁺]_i) levels were measured by Fluo-4 fluorescence in the presence of 3 mM extracellular CaCl₂. Results are expressed as mean ± SEM from 3 independent experiments.</p

Effect of QVD and nectrostatin-1 on CPB-induced cell death.

<p>(A) Representative cytograms of cells exposed to 30 ng/ml of rCPB in the absence or presence of QVD (50 µM) (at 16 h) or necrostatin-1 (Nec-1, 50 µM) (at 4 h) (B) Bar graphs show quantitative summary of FACS analysis from 4 to 6 independent experiments. Results are expressed as mean ± SD. Same key as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064644#pone-0064644-g002" target="_blank">Figure 2B</a>. Statistical difference to control cells was assessed by non-parametric Kruskal-Wallis and Dunn’s post-hoc test. *P<0.05, **P<0.01, †P<0.001. (C) Bar graphs show % inhibition of LDH release of cells treated with QVD or Nec-1 compared to cells treated with toxin only (control) and represent results from 3 to 5 independent experiments. Statistical difference to control was assessed by 1-way ANOVA and Dunnnet post-hoc test, †P<0.001.</p

pEC exposed to rCPB do not exhibit apoptotic but typical features of necrotic cell death.

<p>(A) Presence of cleaved caspase 3 was assessed by Western blotting at different time points after exposure to 30 ng/ml rCPB or staurosporine. Tubulin was used as loading control. Representative results of 3 independent experiments. (B) Internucleosomal DNA fragmentation was assessed after 16 h of exposure to rCPB. Staurosporine was used as a control for apoptosis, while incubation with 5 mM of H₂O₂ or freeze thawing served as controls for necrosis. Representative results of 3 independent experiments. (C) Intracellular ATP levels were determined at the indicated time points. Results represent mean ± SEM of 3 independent experiments. Statistical difference to untreated control cells was assessed by 2-way ANOVA and Bonferroni multiple comparisons test. *P<0.05, **P<0.01, †P<0.001, ††P<0.0001. (D) Cytoplasmic translocation of HMGB-1 (red) was detectable by immunofluorescence in pEC already 30 min after exposure to 30 ng/ml rCPB. Control cells incubated with toxin-free medium exhibit typical nuclear localization. Nuclear counterstain with Hoechst 33258 (blue). (E) Electron microscopic image of pEC 6 h after incubation with 30 ng/ml rCPB. Cells exhibit small irregular clumps of chromatin abutting to the nuclear membrane (asterisk), swelling of cell organelles with disappearance of the elongated mitochondria with cristae (arrows), and plasma membrane discontinuities (arrowhead).</p

Characterization of rCBP-induced cell death by FACS analysis.

<p>pEC were either left untreated or exposed to 30 or 250 ng/ml of rCPB, or staurosporine, detached from the culture dishes by gentle trypsinization, and analyzed by FACS for PI uptake and GFP-annexin V staining at 2, 4 and 16 h of incubation. (A) Representative cytograms and bar graphs (B) in which cells in the lower left quadrant (PI/annexin V double-negative) were considered to be viable (white), cells in the upper left quadrant (PI single-positive) to be necrotic (gray), cells in the lower right quadrant (annexin V single-positive) to be apoptotic (light gray), and cells in the upper right quadrant (PI/annexin double-positive) to be either necrotic or secondary necrotic after apoptosis (dark gray). Bar graphs show quantitative summary of FACS analysis from 3 to 6 independent experiments. Results are expressed as mean ± SD. Statistical difference to control cells was assessed by non-parametric Kruskal-Wallis and Dunn’s post-hoc test. *P<0.05, **P<0.01, †P<0.001.</p

Attenuation of rCPB-induced necrosis by high extracellular K⁺, chelation of intracellular Ca²⁺, and calpain inhibition.

<p>(A) Suppression of potassium efflux by high K⁺ medium (140 mM) significantly inhibited rCPB-induced LDH release from pEC at 4 h, and still reduced LDH release at 16 h, when cells were exposed to 30 ng/ml rCPB. (B) Chelation of intracellular Ca²⁺ with BAPTA-AM (2 or 20 µM) or treatment with cyclosporin A (200 nM) markedly inhibited rCPB induced LDH release at 4 h, and to a lesser extend at 16 h post-exposure. LDH release of rCPB-treated cells in absence or presence of increasing concentrations of PD150606 (C) or calpeptin (D) was measured at 4 and 16 h. Bar graphs show summary of results from 2 to 5 independent experiments, which are expressed as percentage ± SEM relative to rCPB-treated cells in the absence of inhibitor or presence of low K⁺ medium ( = control). Statistical difference to control was assessed by 1-way ANOVA and Dunnnet post-hoc test. *P<0.05, **P<0.01. u = untreated cells, t = treated cells.</p

Detection of myocardial necrosis, endothelial dysfunction and inflammation in blood serum.

<p>Blood serum samples obtained at baseline, 50min after ischemia, 10min, 1hour and 2hours after reperfusion were analyzed. for myocardial necrosis by measuring cardiac troponin-I. Endothelial dysfunction was analyzed by measuring CD31 protein levels and inflammation was analyzed by measuring MCP-1, C3b/c and C5b9 levels.</p

Tissue staining for antibody, complement deposition and damage due to ROS.

<p>Tissue samples were obtained from different sections of the myocardium including area not at ischemic risk (ANR), viable ischemic tissue (VIT) and necrotic ischemic tissue (NIT). The samples were then stained for IgG antibody deposition, C3b/c and C5b9 complement deposition. Damage to the myocardial tissue due to reactive oxygen species was analyzed by staining for 4-hydroxynonenal.</p

Myocardial area at risk and necrosis.

<p>Myocardial area at ischemic risk was compared by expressing the area at ischemic risk as percentage of the left ventricle. Myocardial necrosis was determined by TTC staining and expressed as percentage of the area at ischemic risk.</p

Left ventricular function.

<p>Left ventricular function.</p