Cyclic GMP-Dependent Regulation of Vascular Tone and Blood Pressure Involves Cysteine-Rich LIM-Only Protein 4 (CRP4)

Figures

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  Figure 1. The LIM-only protein CRP4 status of VSMCs affects the NO-GC/cGMP/cGKI axis. (A) Immunofluorescence (IF) staining of CRP4 WT and global CRP4 KO aortic cryosections demonstrate expression of CRP4 and cGKI in the medial layer of the aorta. As expected, CRP4 was not detectable in CRP4 KO tissue, while the IF signal of cGKI was weaker in the absence of CRP4. (B) Western Blot analysis of protein lysates obtained from CRP4 WT and CRP4 KO aorta (C) confirmed a significantly lower cGKI protein abundance in CRP4 KO aorta (B1,C1), whereas the expression of NO-GCβ1 was not altered (B2,C2). Heat shock protein 60 (HSP60) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as loading controls. Data represent means ± SEM with n = 8 per genotype (cGKI) and n = 4 per genotype (NO-GCβ1), *** p < 0.001, two-tailed students t-test. (D,E) Autoradiography reveals incorporation of 33P from (γ-33P)-ATP into CRP4 only in 8-pCTP-cGMP (100 µM) stimulated aortic tissue expressing cGKI. Data represent means ± SEM with n = 6 per genotype and condition, * p < 0.05, two-tailed students t-test. (F–H) In VSMCs (P0), cGKI protein levels did not differ between genotypes at baseline or upon short-term…

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  Figure 2. NO-GC/cGMP oppose agonist-induced (Ca2+)i signals through CRP4. Ratiometric Fura-2 AM Ca2+ measurements were performed in norepinephrine (NE) (2 µM) stimulated CRP4 WT and KO VSMCs (P0) in the presence or absence of cGMP-elevating compounds or 8-Br-cGMP. (A–C) NE-induced (Ca2+)i transients were decreased by 8-Br-cGMP, applied 5 min prior to NE, in both genotypes, albeit the decrease in peak height was significantly greater in CRP4 WT VSMCs. The percentage peak height in (C) was calculated as the quotient of the NE-induced second peak (+8-Br-cGMP) in relation to the first peak (without 8-Br-cGMP). Data represent means ± SEM with n = 3 (8–9 cells) per genotype, * p < 0.05, *** p < 0.001, two-way ANOVA, Bonferroni’s multiple comparisons test used as a post hoc test. (D,E) Pre-incubation (5 min) with the NO-donor diethylamine NONOate (DEA/NO) (10 µM) reduced NE-induced (Ca2+)i peak height in CRP4 WT and in KO VSMCs with significant differences between genotypes (F). Data represent means ± SEM with n = 5–6 (9–11 cells) per genotype, * p < 0.05, *** p < 0.001, two-way ANOVA with Bonferroni’s multiple comparisons test used as a post hoc test. (G–I) Cinaciguat (25 nM) reduced NE-elicited…

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  Figure 3. Lack of CRP4 facilitates Ca2+ desensitization mechanisms and cGMP-mediated effects on vascular tone.(A) Maximal contraction (delta F (mN)) elicited by Prostaglandin F2α (PGF2α) (7.5 µM) was significantly reduced in CRP4 KO vs. CRP4 WT aortic rings equilibrated in an organ bath. Data represent means ± SEM with n = 8–12 per genotype, * p < 0.05, two-tailed students t-test. (B–D) Dose-dependent relaxation of aortic ring segments was examined upon precontraction with PGF2α for (B) cinaciguat (0.1 nM to 1 µM), (C) glyceryl trinitrate (1 nM to 10 mM) and (D) carbachol (1 nM to 10 mM)). Continuously, maximal vasorelaxation was significant improved in CRP4 KO aortic ring segments. Relaxation is expressed as percent in comparison to the maximal contraction induced by PGF2α. Data points represent means ± SEM with n = 4 per genotype for cinaciguat, n = 5–6 per genotype for glyceryl trinitrate, and n = 6–9 per genotype for carbachol. * p < 0.05 or ** p < 0.01 represent the statistical difference in responses between the presence, and the absence of cinaciguat, glyceryl trinitrate, or carbachol to PGF2α contracted rings, two-tailed students t-test, two-way ANOVA with an uncorrected Fisher’s…

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  Figure 4. cGMP-mediated blood pressure regulation involves CRP4. (A) As previously reported [57], basal systolic BP (SBP) of CRP4 KO mice was significantly lower by 13.1 ± 3.15 mmHg than in CRP4 WT mice. * p < 0.05 represents the statistical difference between WT and CRP4 KO, two-tailed students t-test. (B) The NO-GC activator cinaciguat (100 µg/kg bodyweight, i.p.) induced a significantly higher decrease in SBP in CRP4 KO animals. Data points represent means ± SEM with n = 8 per genotype, * p < 0.05, repeated-measure ANOVA with an uncorrected Fisher’s LSD used as a post hoc test. (C–D) Effects of NO-donor sodium nitroprusside (SNP) (2.5 mg/kg bodyweight, i.p.) and the endothelium-dependent vasorelaxant carbachol (0.5 mg/kg bodyweight, i.p.) on SBP were more transient than cinaciguat and resulted in a much faster drop of SBP with consistently significant lower values for CRP4 KO mice. Data points represent means ± SEM with n = 7–8 per genotype for SNP; n = 5 per genotype for carbachol, * p < 0.05, repeated-measure ANOVA with an uncorrected Fisher’s LSD was used as a post hoc test. (E) Inhibition of endogenous NO-synthase activity by L-NAME (100 mg/kg bodyweight, i.p.) increased SBP to a…

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  Figure 5. Synopsis of the altered signaling in CRP4 null VSMCs and vessels. Lack of CRP4 leads to discrete changes in total protein levels of NO-GCβ1, cGKI, and MYPT1 and it causes alteration in the phosphorylation status of pVASP (Ser-239), pMYPT1 (Thr-696) and pMLC (Ser-19), which promotes the Ca2+ desensitization of the contractile apparatus independently from the actual (Ca2+)i concentration. Combined, these effects are indicative for a disinhibited activity of the NO-GC/cGKI pathway. This results in an improved vascular relaxation and lower BP in mice globally lacking CRP4 (further explanations are given in the main text).