Knocking Out Rap1a Attenuates Cardiac Remodeling and Fibrosis in a Male Murine Model of Angiotensin II-Induced Hypertension

Figures

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  Figure 1. (A) No significant differences were observed between Saline and AngII treatments in any genotype. (B) Significant differences in ventricular weights (VW) were observed between Saline and AngII treated mice for the WT genotype. There were no significant differences observed in RAGE KO and RapKO mice. (C) When ventricular weights (VW) were normalized to body weight (BW) significant differences were observed between Saline and AngII treated mice in WT and RAGE KO genotypes. There were no significant differences observed in RapKO mice. (D) AngII reduced blood glucose in all genotypes; however, these differences were not significantly different from Saline treated mice. Data are presented as means ± standard deviation (SD). n = 6/genotype/treatment. p values were indicated on graphs; * p < 0.05; Saline vs. AngII treatment within genotype. p values were determined by 2-way ANOVA with Sidak’s multiple comparisons test post hoc analysis.

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  Figure 2. Two-way RM ANOVA analyses revealed that AngII significantly influenced both (A) systolic (p = 0.0019) and (B) diastolic (p = 0.0002) left ventricular anterior wall (LVAW) measurements. For systolic values, there was a significant genotype × treatment interaction (p = 0.0233). Similarly, diastolic measurements showed a significant interaction (p = 0.0381), suggesting differential responses across conditions. These findings highlight AngII treatment as the primary determinant of LVAW function, with variations depending on genotype. Data are presented as means ± standard error of the mean (SEM). n = 6/genotype/treatment. p values were indicated on graphs. * p < 0.05. Two-way RM ANOVA with Sidak’s multiple comparisons test post hoc analysis was performed. Representative images of M-mode recordings can be found in Supplementary Figure S1.

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  Figure 3. Analysis of left ventricular posterior wall (LVPW) measurements was conducted. (A) Systolic LVPW measurements showed no significant effects of the genotype (p = 0.7865), AngII treatment (p = 0.6976), or their interaction (p = 0.3644). (B) Diastolic LVPW measurements similarly exhibited no significant main effects for genotype (p = 0.7829), AngII treatment (p = 0.2232), or their interaction (p = 0.3143). Data are presented as means ± standard error of the mean (SEM). n = 6/genotype/treatment. Two-way RM ANOVA with Sidak’s multiple comparisons test post hoc analysis was performed. No significance was found between genotypes or treatment. Representative images of M-mode recordings can be found in Supplementary Figure S1.

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  Figure 4. Analysis of the effects of genotype, AngII treatment, and their interaction on (A) systolic volume, (B) diastolic volume, (C) end-systolic volumes adjusted for body weight and (D) end-diastolic volumes adjusted for body weight. No significant effects were found for genotype (p > 0.05), AngII treatment (p > 0.05), or the genotype × AngII treatment interaction (p > 0.05) in any analysis. Data are presented as means ± standard error of the mean (SEM). n = 6/genotype/treatment. Two-way RM ANOVA with Sidak’s multiple comparisons test post hoc analysis was performed. No significance was found between genotypes or treatments. Representative images of M-mode recordings can be found in Supplementary Figure S1.

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  Figure 5. The results from echocardiographic functional analysis of the left ventricle for (A) stroke volume (SV), (B) ejection fraction (%EF), (C) fractional shortening (%FS), and (D) cardiac output (CO). No significant effects were found for genotype, AngII treatment, or their interaction in the ejection fraction, fractional shortening, and cardiac output analyses (p > 0.05). Genotype in the SV analysis was the only factor showing a significant effect (p = 0.0429). Data are presented as means ± standard error of the mean (SEM). n = 6/genotype/treatment. p values were indicated on graphs; p > 0.05; Saline vs. AngII treatment within genotype. p values were determined by 2-way RM ANOVA with Sidak’s multiple comparisons test post hoc analysis. Representative images of M-mode recordings can be found in Supplementary Figure S1.

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  Figure 6. Analysis of left ventricular (LV) function showed (A) MV E (mm/s) had no significant effects of genotype, AngII treatment, or their interaction. (B) MV A (mm/s) was significantly influenced by AngII treatment (p = 0.0182), while genotype and their interaction had no significant effects. (C) MV E/A ratio also showed no significant effects from either factor or their interaction on LV function. These findings suggest that AngII treatment plays a significant role in modulating LV function, independent of genotype. Data are presented as means ± standard error of the mean (SEM). n = 6/genotype/treatment. p values were indicated on graphs; Saline vs. AngII treatment within genotype. p values were determined by 2-way ANOVA with Sidak’s multiple comparisons test post hoc analysis. No significance was found between genotypes or treatment. Representative images of M-mode recordings can be found in Supplementary Figure S1.

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  Figure 7. Representative 20× fluorescence images showing total collagen (FITC, 488/520 nm; green) in left-ventricular sections from WT, RAGE KO, and RapKO mice treated with saline or AngII. Sections were fixed in 4% paraformaldehyde and imaged under identical exposure settings using a Nikon Eclipse fluorescence microscope (Nikon Instruments Inc., Melville, NY, USA) using triggered FITC exposure and acquisition with a digital CMOS camera (Nikon Instruments Inc., Melville, NY, USA). Bright field images of PASR were also captured at 20×. Collagen content was quantified in ImageJ as percent fluorescent area per field (15–35 fields per heart; scale bar = 100 µm or 20 µm). Data are presented as means ± standard error of the mean (SEM); p values were determined by 1 way ANOVA with Tukey’s post hoc analysis ** p < 0.01, *** p < 0.001.