Distinct Effects of Rap1 Subtype A GTPase Deficiency on the Male Mouse Heart

Figures

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  Figure 1. Characterization and validation of Rap1A gene expression in Rap1A wild-type and Rap1A-deficient mice. (A) Representative PCR results showing bands for identification of wild-type (1358 bp only), heterozygous (1358 bp and 1107 bp) and knockout (1107 bp only) for the Rap1A gene [23]. (B) Representative RT-PCR results showing endogenous gene expression of the glycolytic pathway enzyme glyceraldehyde 3-phosphate dehydrogenase (Gapdh), Rap1A and Rap1B isoforms in left ventricle tissue in wild-type and Rap1A knockout mice. (C) Representative RT-qPCR results of the Rap1A gene in the left ventricle showing no mRNA expression in Rap1A-deficient mice (indicated by arrow). (D) Representative RT-qPCR results of the Rap1B isoform gene in the left ventricle showing no differences in mRNA expression in wild-type and Rap1A-deficient mice. Data are represented as Mean ± S.E.M., where n = 8 for Rap1A WT and n = 8 for Rap1A KO mice. p-values are reported as ns = not significant using a t-test.

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  Figure 2. Effect of Rap1A deficiency on heart and left ventricle. (A) Rap1A-deficient mice displayed considerably smaller hearts and visible intercellular gaps with H&E stain, as compared to control mice (scale bar, 10× objective = 200 µm, 40× objective = 50 µm). (B) The heart weights of Rap1A knockout mice were significantly reduced compared to the age-matched littermates (*** p < 0.0007, n = 12). (C,D) The ratio of heart weight to body weight and heart weight to tibia length was also found to be significantly reduced in Rap1A knockout mice ((HW/BW, ** p < 0.006, n = 12), (HW/TB, *** p < 0.0003, n = 12)). (E) The left ventricle weights of Rap1A-deficient mice differ significantly from those of control mice (* p < 0.01, n = 12). (F,G) The ratio of left ventricle to body weight and left ventricle to tibia length was significantly reduced in Rap1A-deficient mice ((LV/BW, * p < 0.01, n = 12), (LV/TB, ** p < 0.005, n = 12)). Data are represented as Mean ± S.E.M and p-values are reported as * p < 0.05, ** p < 0.001 and *** p < 0.0001 using a t-test.

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  Figure 3. Loss of Rap1A leads to reduced collagen type I and III deposition in LV. (A) Representative micrographs of Masson’s trichrome-stained sections of the left ventricle. Blue area represents collagen and red represents myocardium. Scale bar = 100 µm. (B) Quantification of interstitial total collagen deposited in stained left ventricle tissue sections by ImageJ as the ratio of quantified collagen to LV area (** p < 0.003, n = 5 for each group). For collagen quantification, bright-field photographs of 10–15 randomly selected sites per left ventricular section were taken at 200× magnification. Analysis of mRNA levels by RT-qPCR showed significantly decreased mRNA levels of (C) collagen type I (** p < 0.001, n = 8–9) and (D) collagen type III (* p < 0.02, n = 8) in Rap1A-deficient mice. The graph represents expression as fold change (2−ΔΔCt). Data are represented as Mean ± S.E.M., and p-values are reported as * p < 0.05, and ** p < 0.001 using a t-test.

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  Figure 4. NanoLC-MS/MS analysis of differentially expressed proteins (DEPs) in the left ventricle of Rap1A knockout mice. (A) Schematic diagram illustrating various filtration steps to remove false positives. (B) A total of 376 DEPs were identified by setting the fold-change criteria of less than 0.8 (p-value < 0.05) for most downregulated proteins, and fold change greater than 1.1 (p-value < 0.05) for most upregulated proteins. The red bar shows downregulated DEPs, and the green bar shows upregulated DEPs. (C) Venn diagram showcasing 51 and 67 exclusive proteins identified in wild-type and Rap1A knockout left ventricle tissue, respectively, with 883 common proteins identified in both. (D) Volcano plot for DEPs screening based on LFQ intensities. The Y-axis shows log10-transformed p-values, while the X-axis shows the scale of differences in protein expression. The DEPs were screened using the set criteria of fold change >1.1 or <0.8 with the p-value < 0.05. Most significantly upregulated proteins are plotted on the right side, and significantly downregulated proteins are plotted on the left side, while the common proteins are shown in the middle of the plot with no significant change in…

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  Figure 5. Functional classification and pathway analysis of upregulated DEPs. (A) Gene ontology enrichment results for the differentially expressed proteins using Fisher’s exact test obtained by the Panther online tool. The DEPs were annotated into three independent ontologies in the GO database, including biological process, molecular function and cellular components. (B) Enriched pathway identification of DEPs. Enriched KEGG pathways were identified using the STRING database.

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  Figure 6. Loss of Rap1A leads to increased production of myosin heavy chain isoforms in LV. Analysis of mRNA expression by RT-qPCR showed significantly increased expression of (A) myosin heavy chain 7 (Myh7, * p < 0.04, n = 8) (B) alpha-actinin-2 (Actn2, * p < 0.04, n = 7–8) and (C) myosin heavy chain 6 (Myh6, * p < 0.01, n = 8–9) in left ventricle of Rap1A deficient mice. The graph represents expression as fold change (2−ΔΔCt). Data are represented as Mean ± S.E.M., and p-values are reported as * p < 0.05 using a t-test.

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  Figure 7. Acute cardiac stress induction in Rap1A-deficient mice. (A) Schematic timeline of subcutaneous isoproterenol (ISO) induced acute stress in wild-type (WT) and Rap1A knockout (KO) mice. Wild-type and Rap1A-deficient mice underwent treatment with daily subcutaneous (SC) isoproterenol injections (Rap1A-KO (n = 17) and WT (n = 13)) and PBS/ascorbic acid (carrier, Rap1A-KO (n = 11) and WT (n = 11)) for 14 days, followed by morphometric and profibrotic gene expression analysis of left ventricular tissue. (B) Kaplan–Meier survival curve of ISO-treated (purple, red) and untreated (green, yellow) Rap1A WT and Rap1A KO mice. Survival distributions were compared using the log-rank (Mantel–Cox) test (χ2 = 6.568, df = 3, p = 0.0870), which demonstrated a drop in percent survival from 100% to 82% in ISO-treated Rap1A KO; however, this observation remained statistically insignificant. (C,D) Absolute heart weights and heart weight to tibia length ratio indicated a significant increase upon acute stress induction in wild-type and Rap1A knockout mice; however, the Rap1A-deficient mice displayed an overall reduction in HW and HW/TB ratio with or without stress compared with wild-type mice. No…

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  Figure 8. Effect of acute stress imposition in Rap1A knockout left ventricle tissues. (A) Representative images of isoproterenol-treated left ventricle tissue captured at 200× magnification. Scale bar = 100 µm. Collagen deposition, visualized by blue staining (captured by bright-field photographs of 10–15 randomly selected sites per left ventricular section), was increased in PBS/Asc acid-treated Rap1A wild-type mice compared with Rap1A-null mice. The collagen percentage represents the ratio of total collagen detected to heart area. Following isoproterenol treatment, Rap1A wild-type mice demonstrated significantly increased collagen accumulation, while Rap1A knockout mice showed comparatively less collagen deposition. (B) Quantitative analysis by ImageJ software showed a significant decrease in collagen in ISO-treated Rap1A knockout mice compared to control. Overall, Rap1A knockout mice displayed reduced collagen accumulation in acute stress and without stress conditions compared with wild-type mice, while no significant genotype × treatment interaction was observed (pinteraction < 0.7191). Data are represented as Mean ± S.E.M where two-way ANOVA was performed followed by a Bonferroni post…

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  Figure 9. mRNA expression profile of profibrotic gene markers in Rap1A-deficient left ventricle upon acute stress. (A) Collagen type I (Col1a), (B) collagen type III (Col3a), and (C) alpha-smooth muscle actin (Acta2) were significantly downregulated in left ventricles of ISO-treated Rap1A knockout when compared to the ISO-treated Rap1A wild-type group. Noticeably, Col1a and Acta2 were significantly reduced in PBS-treated knockout groups compared to the PBS-treated wild-type group. A significant genotype × treatment interaction was observed for Col1a (p < 0.0001), whereas no significant interaction was observed for Col3a and Acta2 (p < 0.0776 and p < 0.1055, respectively). (D,E) The mRNA expression of connective tissue growth factor (Ctgf) and matrix metalloproteinase 9 (MMP9) was decreased in PBS- and ISO-treated knockout groups versus wild-type groups; however, the difference in expression remained insignificant. (F,G) Increased mRNA expression of Srf and Mrtfa was observed in ISO treated tissues compared to untreated controls in both wild-type and knockout groups. No significant genotype × treatment interaction was observed for Ctgf (p < 0.7586), Mmp9 (p < 0.3242), Srf (p < 0.5174) and…

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  Figure 10. Proposed model illustrating the potential pathway underlying the cardiac profibrotic phenotype and its possible association with Rap1A (adapted from ref. [28]). The involvement of the Rap1A-RhoA/ROCK-actin signaling axis is proposed based on previous studies demonstrating regulation of cytoskeletal dynamics by Rap1A and is presented as a potential mechanistic link requiring future experimental validation.