The effect of zofenopril on the cardiovascular system of spontaneously hypertensive rats treated with the ACE2 inhibitor MLN-4760

Figures

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  Fig. 1. Equilibrium concentration of angiotensin peptides (a-d). The effect of MLN and ZOFE treatments on the equilibrium plasma renin activity (e) and activity of soluble RAS enzymes determined from angiotensin product/substrate (pmol/L/pmol/L) ratios in plasma (f,g). PRA – plasma renin activity, ACE-S – soluble angiotensin converting enzyme, ALT-S – total alternative renin angiotensin system activity, SHR – spontaneously hypertensive rats (n = 10), SHR + MLN – spontaneously hypertensive rats treated by MLN-4760 (n = 10), SHR + ZOFE – spontaneously hypertensive rats treated by zofenopril (n = 10), SHR + MLN + ZOFE – spontaneously hypertensive rats treated by MLN-4760 and zofenopril (n = 10). The results are presented as the mean ± S.E.M., and differences between groups were analyzed by two-way ANOVA, * p < 0.05, *** p < 0.001 vs. SHR, ++ p < 0.01, +++ p < 0.001 vs. SHR + MLN

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  Fig. 2. The effect of MLN and ZOFE treatment on left ventricular ejection fraction (LVEF) (a), left ventricular fractional shortening (LVFS) (b), the ratio of the diastolic transmitral peak early and late filling velocities (E/A ratio) (c), and the ratio of the diastolic transmitral peak early filling velocity and the maximal velocity of early diastolic wall movement wave at the level of mitral annulus (E/Em ratio) (d). SHR – spontaneously hypertensive rats (n = 8), SHR + MLN – spontaneously hypertensive rats treated by MLN-4760 (n = 8), SHR + ZOFE – spontaneously hypertensive rats treated by zofenopril (n = 8), SHR + MLN + ZOFE – spontaneously hypertensive rats treated by MLN-4760 and zofenopril (n = 8). The results are presented as the mean ± S.E.M., and differences between groups were analyzed by two-way ANOVA, * p < 0.05 vs. SHR, + p < 0.05 vs. SHR + MLN

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  Fig. 3. The effect of MLN and ZOFE treatment on the integrated pressure responses of the cardiovascular system. The changes in mean blood pressure (∆MAP) were induced by infusion of acetylcholine (1 µg/kg, a), noradrenaline (1 µg/kg, b), NOsynthase inhibitor (L-NAME, 30 mg/kg, c), ACE inhibitor captopril (10 mg/kg, e) and H2S scavenger bismuth(III) subsalicylate (BSC, 0.25 µg/kg, (f). The effect of acetylcholine (1 µg/kg) was also investigated before and after pretreatment with L-NAME (30 mg/kg, (d). SHR – spontaneously hypertensive rats (n = 8), SHR + MLN – spontaneously hypertensive rats treated by MLN-4760 (n = 8), SHR + ZOFE – spontaneously hypertensive rats treated by zofenopril (n = 8), SHR + MLN + ZOFE – spontaneously hypertensive rats treated by MLN-4760 and zofenopril (n = 8). The results are presented as the mean ± S.E.M., and differences between groups were analyzed by two-way ANOVA, *** p < 0.001 vs. SHR, + p < 0.05 vs. SHR + MLN, # p < 0.05, ## p < 0.01 vs. before L-NAME

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  Fig. 4. The effect of MLN and ZOFE treatment on the endothelium dependent vasorelaxation of the thoracic aorta isolated from spontaneously hypertensive rats (SHR, n = 10), spontaneously hypertensive rats treated by MLN-4760 (SHR + MLN, n = 10), spontaneously hypertensive rats treated by zofenopril (SHR + ZOFE, n = 10), spontaneously hypertensive rats simultaneously treated by MLN-4760 and zofenopril (SHR + MLN + ZOFE, n = 10). The results are presented as the mean ± S.E.M., and differences between groups were analyzed by three-way ANOVA, + p < 0.05 vs. SHR + MLN

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  Fig. 5. The effect of treatment with zofenopril on the participation of NO/NOS in endothelium dependent vasorelaxation in thoracic aortas isolated from rats without (a) or with (b) MLN-4760. SHR – untreated spontaneously hypertensive rats (n = 10); SHR + MLN – spontaneously hypertensive rats treated by MLN-4760 (n = 9); SHR + ZOFE – spontaneously hypertensive rats treated by zofenopril (n = 8); SHR + MLN + ZOFE – spontaneously hypertensive rats treated by MLN-4760 and zofenopril (n = 8). +L-NAME – aortic rings incubated with NG-nitro-L-arginine methylester. The results are presented as the mean ± S.E.M., and differences between groups were analyzed by three-way ANOVA, # p < 0.05, ### p < 0.001 vs. the respective group without L-NAME, §§§ p < 0.001 vs. SHR + L-NAME, >>> p < 0.001 vs. SHR + MLN + L-NAME

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  Fig. 6. The effect of treatment with zofenopril on the participation of endogenous H2S in endothelium dependent vasorelaxation in thoracic aortas isolated from rats without (a) or with (b) MLN-4760. SHR – untreated spontaneously hypertensive rats (n = 10); SHR + MLN – spontaneously hypertensive rats treated by MLN-4760 (n = 9); SHR + ZOFE – spontaneously hypertensive rats treated by zofenopril (n = 8); SHR + MLN + ZOFE – spontaneously hypertensive rats treated by MLN-4760 and zofenopril (n = 8). +BSC– aortic rings incubated with bismuth(III) subsalicylate. The results are presented as the mean ± S.E.M., and differences between groups were analyzed by three-way ANOVA, # p < 0.05 vs. the respective group without BSC

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  Fig. 7. The effect of treatment with zofenopril on the participation of Mas receptors in endothelium dependent vasorelaxation in thoracic aortas isolated from rats without (a) or with (b) MLN-4760. SHR – untreated spontaneously hypertensive rats (n = 10); SHR + MLN – spontaneously hypertensive rats treated by MLN-4760 (n = 9); SHR + ZOFE – spontaneously hypertensive rats treated by zofenopril (n = 8); SHR + MLN + ZOFE – spontaneously hypertensive rats treated by MLN-4760 and zofenopril (n = 8). +A799 – aortic rings incubated with A-799 trifluoroacetate salt. The results are presented as the mean ± S.E.M., and differences between groups were analyzed by three-way ANOVA, ## p < 0.01, ### p < 0.001 vs. the respective group without A799p, §§§ p < 0.001 vs. SHR + A799

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  Fig. 8. The protein expression of ACE2 (a), Mas receptor (b) eNOS (c) nNOS (d), iNOS (e), CSE (f) and CBS (g) in left ventricle isolated form spontaneously hypertensive rats (SHR, n = 8), spontaneously hypertensive rats treated by MLN-4760 (SHR + MLN, n = 8), spontaneously hypertensive rats treated by zofenopril (SHR + ZOFE, n = 8), spontaneously hypertensive rats treated by MLN-4760 and zofenopril (SHR + MLN + ZOFE, n = 8). The results are presented as the mean ± S.E.M., and differences between groups were analyzed by two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. SHR, ++ p < 0.01, +++ p < 0.001 vs. SHR + MLN, § p < 0.05, §§ p < 0.01 vs. SHR + ZOFE

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  Fig. 9. The mRNA levels of the Ace2 (a), Mas1 (b), Nos3 (c), Nos1 (d), Nos2 (e) and Cbs (f) genes in aortic tissue isolated from spontaneously hypertensive rats (SHR, n = 8), spontaneously hypertensive rats treated by MLN-4760 (SHR + MLN, n = 8), spontaneously hypertensive rats treated by zofenopril (SHR + ZOFE, n = 8), spontaneously hypertensive rats treated by MLN-4760 and zofenopril (SHR + MLN + ZOFE, n = 8). The results are presented as the mean ± S.E.M., and differences between groups were analyzed by two-way ANOVA, § p < 0.05 vs. SHR + ZOFE

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  Fig. 10. The protein expression of ACE2 (a), Mas receptor (b) eNOS (c) nNOS (d), iNOS (e), CSE (f) and CBS (g) in aortic tissue isolated form spontaneously hypertensive rats (SHR, n = 8), spontaneously hypertensive rats treated by MLN-4760 (SHR + MLN, n = 8), spontaneously hypertensive rats treated by zofenopril (SHR + ZOFE, n = 8), spontaneously hypertensive rats treated by MLN-4760 and zofenopril (SHR + MLN + ZOFE, n = 8). The results are presented as the mean ± S.E.M., and differences between groups were analyzed by two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. SHR, ++ p < 0.01, +++ p < 0.001 vs. SHR + MLN, § p < 0.05, §§§ p < 0.001 vs. SHR + ZOFE

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  Fig. 11. The distribution of CSE (orange color) and CBS (green color) enzymes shown using fluorescent stained of 10 μm thick cryosections of thoracic aorta tissue isolated from spontaneously hypertensive rats (SHR), spontaneously hypertensive rats treated by MLN-4760 (SHR + MLN), spontaneously hypertensive rats treated by zofenopril (SHR + ZOFE), spontaneously hypertensive rats treated by MLN-4760 and zofenopril (SHR + MLN + ZOFE). E – endothelium, M – tunica media. Blue color shows cell nuclei (stained by 4´,6´- diamidino-2-phenylindole, DAPI)

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  Fig. 12. The total NOS activity and superoxide production in left ventricle (a,c) and aortic tissue (b,d). SHR – spontaneously hypertensive rats (n = 8), SHR + MLN – spontaneously hypertensive rats treated by MLN-4760 (n = 8), SHR + ZOFE - spontaneously hypertensive rats treated by zofenopril (n = 8), SHR + MLN + ZOFE - spontaneously hypertensive rats simultaneously treated by MLN-4760 and zofenopril (n = 8). The results are presented as the mean ± S.E.M., and differences between groups were analyzed by two-way ANOVA, * p < 0.05, *** p < 0.001 vs. SHR, +++ p < 0.001 vs. SHR + MLN, § p < 0.05 vs. SHR + ZOFE