Correlations between cardiomyocyte progenitors and endogenous IGF-1/HGF mRNA expressions in the different groups.

<p>The scatter plots on logged data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036804#pone-0036804-g005" target="_blank">Fig 5A, B and E</a> reveal that Nkx2.5 is positively correlated with c-Kit, Isl1 and HGF expression. c-Kit is also positively correlated to IGF-1 expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036804#pone-0036804-g005" target="_blank">Fig 5C</a>) and there is a borderline relationship to HGF expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036804#pone-0036804-g005" target="_blank">Fig 5D</a>). As shown in fig F, Isl1 is positively correlated with IGF-1 expression. In figure; R = Pearson correlation coefficient.</p

Immunohistochemical analysis of Nkx2.5, c-Kit and Isl1 cardiac progenitors.

<p><b>Section A:</b> The distribution of Nkx2.5+ cells in the myocardium of the different groups together with semi-quantitative analysis. Figures (a-f) represent: 9.5 weeks human fetal heart as a positive control (a), followed by left ventricular regions from the; control (b), pregnancy (c), myocardial infarction (d), ischemia-reperfusion (e) and ischemia-reperfusion+growth factors (f) groups, respectively. The delineated areas in figure b-f represent a higher magnification of that region. Bars in panel (a-f) = 100 µm. Figure (g) demonstrates the relative number of Nkx2.5+ cells in relation to the total number of cells in the different groups. Data is presented as mean± SD. *<i>P</i><0.05. <b>Section B</b>: Representative stainings of c-Kit+cells in: adult rat kidney as positive control (a), followed by the left ventricular regions from the ischemia-reperfusion+growth factors (b, d) and myocardial infarction groups (e). The delineated area in figure (b) is magnified (100 x) in figure (c). In figures (b-e), red arrows indicate c-Kit+ cells. Bars in figures (a, b) = 100 µm and in figures (c-e) = 50 µm. Figure (f) demonstrates the percentage of c-Kit+cells in relation to the total number of cells in the different groups. Data is presented as mean ± SD. <i>*P<0.05 and **P<0.001. </i><b>Section C</b>. Representative stainings of Isl1+ cells in whole heart cytospins from (c) pregnancy, (d) myocardial infarction, (e) ischemia-reperfusion and (f) ischemia-reperfusion+growth factors groups. Figures (a) and (b) represent the positive controls; whole rat embryo ED13 and insulin cell line-1E respectively. Delineated areas in figure c, d, e and f represent a higher magnification of that region. Bars in figures (a–f) = 100µm. In figure; ED: embryonic day; MI: myocardial infarction; IR: ischemia-reperfusion; IR+GF: ischemia-reperfusion+growth factors.</p

Quantitative RT-PCR showing the endogenous expression of IGF-1 and HGF mRNA at two weeks in the different groups.

<p>The IGF-1 and HGF mRNA expression is related to the expression of GAPDH. Fig A and C, show the mean whole heart expression of IGF-1 and HGF in the different groups, while in fig B and D the growth factor expression in each sub-domain is demonstrated. Number of animals is 7–10 per group (see flow chart in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036804#pone-0036804-g001" target="_blank">Fig 1</a>). In figure; MI: myocardial Infarction; IR: ischemia-reperfusion; IR+GF: ischemia-reperfusion+ growth factors. RV: Right ventricle, LV: Left ventricle, OFT: Outflow tract and INF: Peri-infarction and Ischemia region respectively. Data is presented as mean± SD. *<i>P</i><0.05 and **<i>P</i><0.001 vs. Control group. In fig. C and D the mRNA expression of each region is related to the corresponding region of the control group.</p

Quantitative RT-PCR showing the time-dependent distribution of Isl1 mRNA in the different groups.

<p>The Isl1 mRNA expression in the different groups was related to the expression of GAPDH. Fig A shows the mean of the whole heart Isl1 mRNA expression in each group. In fig B, the distribution of Isl1 mRNA between each region is demonstrated at 2 weeks. Number of animals is 7–10 per group (see flow chart in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036804#pone-0036804-g001" target="_blank">Fig 1</a>). In figure; MI: myocardial infarction; IR: ischemia-reperfusion; IR+GF: ischemia-reperfusion+ growth factors. RV: Right ventricle, LV: Left ventricle, OFT: Outflow tract and Peri-INF/Ischemia: Peri-infarction and Ischemia region respectively. Data is presented as mean± SD. *<i>P</i><0.05 vs. Control group, ***<i>P</i><0.05 vs. pregnancy group, ****<i>P</i><0.05 vs. MI (2 weeks) group and † <i>P</i><0.05, 1 week vs. 2 weeks. In fig. B the mRNA expression of each region is related to the corresponding region of the control group.</p

Mast cell distribution and Western blot analysis.

<p>Fig A-C: Two weeks after the intervention, mast cells were detected in whole heart cytospins from the different groups using toluidineblue staining. As seen in fig A and B, it is easy to detect the mast cells as red-purple cells. Fig C shows a semi-quantitative analysis of the distribution of mast cells among the different groups. Four whole heart cytospins from two rats per group were counted in the light microscope. Data is presented as mean± SD. *<i>P</i><0.05 vs. Control group and **<i>P</i><0.05 vs. MI (2 weeks) group. Fig D: The protein expression for c-Kit, Isl1 and Nkx2.5 is related to the GAPDH expression in each group at 2 weeks. The Isl1 protein expression showed a band at 39 kDa and another band was consistently observed at 80 kDa. Experiments were run in triplicates (n = 2 per group). In figure; MI: myocardial infarction; IR: ischemia-reperfusion; IR+GF: ischemia-reperfusion+ growth factors. Bars in panel (A) = 25 µm and in panel (B) = 50 µm.</p

Quantitative RT-PCR showing the time-dependent distribution of the Nkx2.5 early cardiomyocyte progenitor in the different groups.

<p>The Nkx2.5 mRNA expression in the different groups was related to the expression of GAPDH. Fig A shows the mean of the whole heart Nkx2.5 mRNA expression in each group. In fig B, the distribution of Nkx2.5 mRNA between each region of the myocardial infarction group at 1 week is demonstrated. In fig C, the Nkx2.5 mRNA distribution between each region is demonstrated at 2 weeks. Number of animals is 7–10 per group (see flow chart in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036804#pone-0036804-g001" target="_blank">Fig 1</a>). In figure; MI: myocardial infarction; IR: ischemia-reperfusion; IR+GF: ischemia-reperfusion+ growth factors. RV: Right ventricle, LV: Left ventricle, OFT: Outflow tract and Peri-INF/Ischemia: Peri-infarction and Ischemia region respectively. Data is presented as mean ± SD. *<i>P</i><0.05 vs. Control group, ** <i>P</i><0.001 vs. Control group, ***<i>P</i><0.05 vs. pregnancy group, ****<i>P</i><0.05 vs. MI (2 weeks) group and † <i>P</i><0.05, 1 week vs. 2 weeks and †† <i>P</i><0.05 vs. MI (1 week) group. In fig B and C the mRNA expression of each region is related to the corresponding region of the control group. </p

Flow chart showing the experimental study design.

<p>In figure; RV: Right ventricle, LV: Left ventricle, OFT: Outflow tract, RT-PCR: Real Time Polymerase Chain Reaction and ICC: Immunocytochemistry.</p

Quantitative RT-PCR showing the time-dependent distribution of c-Kit mRNA in the different groups.

<p>The c-Kit mRNA expression in the different groups is related to the expression of GAPDH. Fig A shows the mean of the whole heart c-Kit mRNA expression in each group. In fig B, the distribution of the c-Kit mRNA between each region at 2 weeks is demonstrated. Number of animals is 7–10 per group (see flow chart in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036804#pone-0036804-g001" target="_blank">Fig 1</a>). In figure; MI: myocardial Infarction; IR: ischemia-reperfusion; IR+GF: ischemia-reperfusion+ growth factors. RV: Right ventricle, LV: Left ventricle, OFT: Outflow tract and Peri-INF/Ischemia: Peri-infarction and Ischemia region respectively. Data is presented as mean ± SD. *<i>P</i><0.05 vs. Control group, ** <i>P<</i>0.001 vs. Control group, ***<i>P<</i>0.05 vs. pregnancy group, ****<i>P<</i>0.05 vs. MI (2 weeks) group and † <i>P<0.05</i> 1 week vs. 2 weeks. In fig B, each region is related to corresponding region of the control, pregnancy, myocardial infarction and ischemia-reperfusion w/o growth factors.</p