Selective ROCK2 inhibition reduces microvascular obstruction but does not reduce myocardial infarction after ischaemia and reperfusion

Figures

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  Fig. 1. RNAscope analysis demonstrated that ROCK1 mRNA expression was greater than ROCK2 mRNA in rat coronary artery and myocardium. Sections were co-labelled with DAPI (E, K, blue) and an antibody to TAGLN (B, H, red), a marker of vascular smooth muscle cells (VSMC) in the coronary arteries. (A–F) ROCK2 mRNA (D, J, purple) is more abundant than ROCK1 mRNA (C, I, green) in coronary artery VSMC (thin arrow) (* P = 0.03, n = 4 rats). (G–L) Higher magnification of a region of panels A–E shows that ROCK2 mRNA is expressed more highly than ROCK1 throughout the myocardium (thick arrow) (*** P = 0.0006, n = 4 rats). (M) ROCK2 mRNA (purple) is highly expressed in a section of rat aorta (NB: elastin fibre autofluorescence appears green in this image). Scale bars 100 μm (A, C, G) or 50 μm (B, D). Student's unpaired t-test. Mean and SEM shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 2. Effects of fasudil on cardiac ischaemia and reperfusion (I/R) injury in rats. (A,B). Representative images of heart slices from hearts following I/R, from rats treated with vehicle or fasudil, and stained for haemorrhage microvascular obstruction or infarct. Blue indicates non-ischaemia area (Evan's blue stained). (C–F) Fasudil (10 mg/kg) significantly reduced IS% (C), MVO% (D) and IMH% (E). There was no significant difference in the area at risk (AAR as % left ventricle) between groups (F). Mean ± SEM is indicated. Students unpaired t-test, * P < 0.05; ** P < 0.01, n = 6 hearts per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 3. A. Fasudil induced relaxation of aortic rings in an organ bath experiment, which was partly dependent on nitric oxide synthase. Fasudil (10−9–10−2.5 M) induced dose-dependent vasodilation, EC50 = 10–5.0 M. Fasudil was less potent than sodium nitroprusside (SNP, EC50 = 10–7.9) & Acetylcholine (EC50 = 10–7.1). L-NAME significantly decreased potency of fasudil to EC50 = 10–4.5 M. SNP n = 5, acetylcholine n = 5, fasudil +vehicle n = 8, fasudil +L-NAME n = 8. See methods for description of statistical analysis. All groups were significantly different from each other (see P values in Supplementary table). (B) Emax of vascular relaxation by fasudil was not affected by 100 μm L-NAME reduced (ns = non-significant by Students paired t-test), n = 8 aortae. Mean ± SEM is indicated. Fasudil (10−9–10−2.5 M) induced dose-dependent vasodilation, EC50 = 10–5.0 M. Fasudil was less potent than sodium nitroprusside (SNP, EC50 = 10–7.9) & Acetylcholine (EC50 = 10–7.1). L-NAME significantly decreased potency of fasudil to EC50 = 10–4.5 M. SNP n = 5, acetylcholine n = 5, fasudil +vehicle n = 8, fasudil +L-NAME n = 8. See methods for description of statistical analysis. All groups were significantly…

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  Fig. 4. KD025 had no apparent effect on aortic ring relaxation of blood pressure, while Fasudil decreased blood pressure in rats in vivo. A: KD025 had no vasodilatory effect on PE-pre-constricted aortic rings ex vivo (n = 5 aortae). B,C: Mean arterial blood pressure (B) and heart rate (C) were measured in anaesthetized rats, following during ischaemia and reperfusion. Rats were administered either vehicle (n = 6 rats), 3 mg/kg fasudil (n = 6 rats) or 100 mg/kg KD025 (KD, n = 7 rats) prior to reperfusion. By 2-way ANOVA and Tukey post test, BP transiently decreased significantly only in the fasudil group (* P < 0.05, ** P < 0.01, *** P < 0.001 vs vehicle).

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  Fig. 5. Although KD025 did not reduce infarct size in vivo after ischaemia and reperfusion, fasudil significantly reduced microvascular obstruction (MVO), and both KD025 and Fasudil decreased intramyocardial haemorrhage (IMH). 3 mg/kg Fasudil, 20 mg/kg KD025 or 100 mg/kg KD025 (a selective ROCK2 inhibitor) were administered i.p. 15 min prior to reperfusion in rats subject to 30 min cardiac ischaemia and 2 h reperfusion. At the end of the experiment, infarct size (A), MVO (B) or IMH (C) were assessed as percentage of the area at risk (AAR). AAR is presented relative to the left ventricular (LV) area (D). Statistics by 1-way ANOVA with Tukey post test. * P < 0.05. ** P < 0.01. Mean ± SEM indicated.

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  Fig. 6. Infarct size (IS expressed as percentage of area at risk or AAR) was not significantly different in WT and HET mice subject to 40 min cardiac ischaemia and 120 min reperfusion in vivo. Representative images of heart slices (A) and infarct size (B). Mean and SEM are indicated. Analysis by Students unpaired t-test (n = 6 mice per group).