Antiedemic Effect of the Myosin Light Chain Kinase Inhibitor PIK7 in the Rat Model of Myocardial Ischemia Reperfusion Injury

Figures

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  Figure 1. Protocol of the experimental study.

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  Figure 2. Effects of 100 µM PIK7 on the thrombin-induced EA.hy926 endothelial monolayer hyperpermeability and myosin RLC phosphorylation in EA.hy926 endothelial cells. (a) The levels of the thrombin-induced myosin regulatory light chain (RLC) phosphorylation in EA.hy926 cells were calculated based on sequential visualization of myosin RLC monophosphorylated at Ser19 (P-RLC), diphosphorylated at Thr18/Ser19 (PP-RLC), and total RLC on Western blots of endothelial cell samples using an external standard mixture containing RLC/P-RLC/PP-RLC, as described in [23]. Representative Western blots are shown. Bars below represent the sum of mono- and di-phosphorylated (activated) myosin RLC before (0 min) or 20 min after thrombin (Thr) administration normalized by the total RLC content in each sample. Data are presented as means ± SD, n = 4. (b) Effect of PIK7 on the thrombin-stimulated 70 kDa FITC-dextran permeability across the EA.hy926 endothelial cell monolayer. Cultured cells were preincubated or not with PIK7 for 60 min before 100 nM thrombin was added at 0 min. Cells in control were left untreated by PIK7 or thrombin. Data are presented as means ± SD, n = 5. p < 0.05 for all PIK7 data points…

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  Figure 3. Dynamics of the level of mean arterial pressure during the experiment. Data are presented as median (Me) and interquartile range (Q1; Q3).

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  Figure 4. Dynamics of heart rate level during the experiment. Data are presented as median (Me) and interquartile range (Q1; Q3).

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  Figure 5. Effect of PIK7 on mean arterial pressure.

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  Figure 6. Duration of intravenous administration of sodium nitroprusside (60 μg/kg).

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  Figure 7. Myocardial infarction size. Sizes of area at risk (a) and area of necrosis (b).

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  Figure 8. Comparison of sizes of no-reflow zones at the beginning and at the end of reperfusion in the same heart in the CNT and PIK7 2.5 groups. Representative images of ICG-fluorescence (ICG-0′) and ThS-fluorescence (ThS) in transverse sections of hearts from CNT (a) and PIK7 2.5 (b) groups. Dotted lines are the boundaries of the no-reflow (NR) zone. (c) Comparison of sizes of no-reflow zones obtained by planimetric analysis of fluorescence images in transverse heart slices. Comparative analysis of ICG- and ThS-fluorescence images in the SNP group revealed a trend toward an increase in the size of the no-reflow zone between the beginning of reperfusion (ICG-0′) and the end (ThS-120′). (d) Representative images of apical slices of the same heart stained with Evans blue: in white light (left) and in ThS-fluorescence (right). ThS was injected at the second minute of reperfusion (ThS-2′); planimetric analysis allows confirmation of the primary no-reflow observed by ICG staining. Scale bar: 1 mm.

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  Figure 9. Comparison of no-reflow zone area sizes between groups with late (ICG-90′) ICG administration. Representative images of ICG-fluorescence (ICG-90′) and ThS-fluorescence (ThS) in transverse sections of hearts from CNT (a) and PIK7 2.5 (b) groups. Dotted lines are the boundaries of the no-reflow (NR) zone. (c) Comparison of no-reflow zone sizes at the first minutes of reperfusion (ICG-0′ subgroup) and 90 min of reperfusion (ICG-90′ subgroup) obtained by ICG-fluorescence image analysis. (d) Comparison of no-reflow zone sizes at the end of the second hour of reperfusion (ThS) obtained by analyzing ThS-fluorescence images. Scale bar: 1 mm.

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  Figure 10. Effect of sodium nitroprusside administered during the first minutes of reperfusion on ICG-0′ fluorescence intensity in the myocardial infarction zone. Representative images of ICG-fluorescence (ICG-0′) and ThS-fluorescence (ThS) in transverse slices of hearts from CNT (a) and SNP (b) groups. (b) The area between the dashed lines bounding the anatomical risk zone is divided into 6 equal sectors: two border sectors (BZ-1 and BZ-2) and four inner sectors (S1–S4). Each sector is divided into three grid cells: (1) subepicardial (Subep.), (2) intramural (Intr.), and (3) subendocardial (Suben.). Ref. is the red reference line in the reference sector plotted at an equal distance from the border sectors. (c) and (d) ICG- and ThS-fluorescence intensity plots along scan line 3 from epicardium to endocardium and reference lines from CNT (a) and SNP (b) slice images, respectively. The gray “White” line is drawn from TTC-stained slice images. The three arrows in the two graphs indicate the fluorescence intensity levels (ICG or ThS) in the three myocardial layers and the “+” or “−” signs indicate the ratio to the reference fluorescence intensity level (ICG ref. or ThS ref.). NR—no-reflow,…

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  Figure 11. Comparison of contrast between sectors of the intramural layer of the left ventricular wall in the risk zone and the remote zone (interventricular septum) at different time points. (a,b) ICG-fluorescence intensity in the intramural layer of apical (a) and midline (b) slices in groups with early ICG administration (ICG-0′). (c,d) Intensity of ThS-fluorescence in the intramural layer of apical (c) and medial (d) slices in groups with early ICG administration (ICG-0′). (e,f) ICG-fluorescence intensity in the intramural layer of apical (e) and midline (f) slices in groups with delayed administration of ICG (ICG-90′). (g,h) Intensity of ThS-fluorescence in the intramural layer of apical (g) and medial (h) slices in groups with late ICG administration (ICG-90′). BZ-1 and BZ-2 are border sectors of the risk zone (Figure 9b); S1, S2, S3, and S4 are inner sectors. *—statistically significant difference (p < 0.05) with the same cell in the control group.

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  Figure 12. Representative electronograms of myocardial samples taken from the intramural layer of the central sectors of the zone at risk from control rats (a,c,e) and PIK7 2.5 rats (b,d,f) at 10 min of reperfusion. (a) Black arrows show mitochondria with rupture of the outer membrane. (c,d) Arrows point to the extravasated erythrocyte; short thick arrows—basement membrane. (e) Arrows point to the open interendothelial space with a membrane bleb (mb) adjacent to it. (f) Two pentagonal arrows point to protrusions of edematous endothelium into the capillary lumen containing two erythrocytes: Ec—endotheliocyte; thin arrow—capillary lumen (the lumen of a capillary in which two erythrocytes are stuck together); Er—erythrocytes; PV—pinocytic vesicles, short arrows; short thick arrows—basement membrane; M—mitochondria; cf—collagen fibers; CMc—cardiomyocyte. Electronograms were taken with a transmission electron microscope HITACHI7800 at magnifications of 52,000 (a), 65,000 (b), 20,000 (c), 16,000 (d), 52,000 (e) and 39,000 (f).