Rap1a Overlaps the AGE/RAGE Signaling Cascade to Alter Expression of α-SMA, p-NF-κB, and p-PKC-ζ in Cardiac Fibroblasts Isolated from Type 2 Diabetic Mice

Figures

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  Figure 1. Diabetic conditions and exogenous AGEs caused a shift in expression of proteins associated with the RAGE signaling cascade. Cardiac fibroblasts were isolated from non-diabetic, diabetic, non-diabetic RKO, and diabetic RKO mice hearts and were either untreated or treated with exogenous AGEs (0.5 mg/mL). Protein expression was assessed for (A,B) α-SMA (42 kDA), (C,D) p-NF-κB (65 kDa), and (E,F) SOD-1 (23 kDa) in cardiac fibroblasts. Expression data were normalized to β-tubulin (55 kDa) protein expression and mean ± SEM are depicted on graph (n = 8–12). Representative western blot images are shown above graphs, but are not displayed as a continuous blot due to running order on the blot not aligning with graphical order of samples. However, original western blot images are available at DOI:10.6084/m9.figshare.12625409. A two-way ANOVA followed by a Fisher’s protected least significant difference post hoc determined significance. (* p < 0.05, ** p < 0.01, *** p < 0.001).

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  Figure 2. Treatment with exogenous AGEs and EPAC induced an increase in Rap1a protein expression. Cardiac fibroblasts were isolated from non-diabetic and diabetic mice hearts. Fibroblasts were either untreated, treated with a single pharmacological modifier, or treated in combination with EPAC (100 µM), EPAC+AGE (100 µM and 0.5 mg/mL, respectively), Rap1a siRNA (100 nM), or Rap1a siRNA + AGE (100 nM and 0.5 mg/mL, respectively). Rap1a (21 kDa) protein expression was assessed in (A) non-diabetic and (B) diabetic fibroblasts. Expression of Rap1a was normalized to β-tubulin (55 kDa) and mean ± SEM are depicted (n = 5–11). Representative western blot images are shown above graphs but are not displayed as a continuous blot due to running order on the blot not aligning with graphical order of samples. However, original western blot images are available at DOI:10.6084/m9.figshare.12625409. Statistical analysis consisted of one-way ANOVA followed by a Fisher’s protected least significant difference post hoc (* p < 0.5, ** p < 0.01, *** p < 0.001).

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  Figure 3. Increased AGE/RAGE signaling and reduced Rap1a expression induced a decrease in α-SMA expression. Cardiac fibroblasts were isolated from (A) non-diabetic, (B) diabetic, and (C) Rap1a KO mice hearts. Fibroblasts were either untreated, treated with a single pharmacological modifier, or treated in combination with EPAC (100 µM), EPAC + AGE (100 µM and 0.5 mg/mL, respectively), Rap1a siRNA (100 nM), or Rap1a siRNA + AGE (100 nM and 0.5 mg/mL, respectively). α-SMA (42 kDa) expression was assessed in cardiac fibroblasts. Expression of α-SMA was normalized to β-tubulin (55 kDa) and mean ± SEM are depicted on graphs (n = 5–11). Data depicted for exogenous AGE treatment group for non-diabetic and diabetic fibroblasts were previously presented Figure 1. Representative western blot images are shown above graphs but are not displayed as a continuous blot due to running order on the blot not aligning with graphical order of samples. However, original western blot images are available at DOI:10.6084/m9.figshare.12625409. Statistical analysis consisted of a one-way ANOVA followed by a Fisher’s protected least significant difference post hoc (* p < 0.5, ** p < 0.01, *** p < 0.001, **** p <…

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  Figure 4. The presence of Rap1a mediated an AGE/RAGE induced increase in p-NF-κB expression. Cardiac fibroblasts isolated from (A) non-diabetic, (B) diabetic, and (C) Rap1a KO hearts were either untreated or treated with pharmacological modifiers, either solo or in combination: EPAC (100 µM), EPAC + AGE (100 µM and 0.5 mg/mL, respectively), Rap1a siRNA (100 nM), or Rap1a siRNA + AGE (100 nM and 0.5 mg/mL, respectively). Phosphorylation of NF-κB (65 kDa) was assessed and protein expression was normalized to β-tubulin (55 kDa) expression. Data depicted for exogenous AGE treatment group for non-diabetic and diabetic fibroblasts were previously presented Figure 1. Representative western blot images are shown above graphs but are not displayed as a continuous blot due to running order on the blot not aligning with graphical order of samples. However, original western blot images are available at DOI:10.6084/m9.figshare.12625409. Graph depicts mean ± SEM with n = 5–9 and statistical analysis consisted of one-way ANOVA followed by a Fisher’s protected least significant difference post hoc (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

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  Figure 5. Reduced Rap1a expression caused increased SOD-1 expression. (A) Non-diabetic, (B) diabetic, and (C) Rap1a KO cardiac fibroblasts were isolated and treated with different combinations of EPAC (100 µM), AGE (0.5 mg/mL), and Rap1a siRNA (100 nM). SOD-1 (23 kDa) protein expression was normalized to β-tubulin (55 kDa) protein expression. Data depicted for exogenous AGE treatment group for non-diabetic and diabetic fibroblasts were previously presented Figure 1. Representative western blot images are shown above graphs but are not displayed as a continuous blot due to running order on the blot not aligning with graphical order of samples. However, original western blot images are available at DOI:10.6084/m9.figshare.12625409. Mean ± SEM are depicted in graphs (n = 7–9) with significance determined by one-way ANOVA followed by a Fisher’s protected least significant difference post hoc (* p < 0.05, *** p < 0.001, **** p < 0.0001).

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  Figure 6. AGE/RAGE signaling induced elevated levels of hydrogen peroxide in non-diabetic and Rap1a KO cardiac fibroblasts. Cardiac fibroblasts isolated from (A) non-diabetic, (B) diabetic, and (C) Rap1a KO mice were treated with different combinations of pharmacological modifiers: EPAC (100 µM), exogenous AGEs (0.5 mg/mL), and Rap1a siRNA (100 nM). Cell lysates were collected and assessed for concentration of hydrogen peroxide. Values graphed are mean ± SEM (n = 4–13) and a one-way ANOVA followed by a Fisher’s protected least significant difference post hoc was conducted to determine significance (* p < 0.05 and ** p < 0.01).

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  Figure 7. Rap1a mediated AGE/RAGE induced ERK1/2 activation in cardiac fibroblasts. Cardiac fibroblasts isolated from (A) non-diabetic, (B) diabetic, and (C) Rap1a KO mouse hearts were treated with pharmacological modifiers either solo or in combination: EPAC (100 µM), exogenous AGEs (0.5 mg/mL), and Rap1a siRNA (100 nM). Protein expression of p-ERK1/2 (42 and 44 kDa) was normalized to total ERK1/2 (44 and 42 kDa, respectively) expression. Representative western blot images are shown above graphs but are not displayed as a continuous blot due to running order on the blot not aligning with graphical order of samples. However, original western blot images are available at DOI:10.6084/m9.figshare.12625409. Values graphed represent mean ± SEM (n = 6–10) with an one-way ANOVA followed by a Fisher’s protected least significant difference post hoc determining significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

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  Figure 8. Rap1a modified p-PKC-ζ expression both dependently and independently of AGE/RAGE signaling. Total protein was isolated from (A) non-diabetic, (B) diabetic, and (C) Rap1a KO cardiac fibroblasts. Pharmacological modifiers EPAC (100 µM), exogenous AGEs (0.5 mg/mL), and Rap1a siRNA (100 nM) were added to fibroblasts either solo or in combination. p-PKC-ζ (72 kDa) protein expression was normalized to β-tubulin (55 kDa) protein expression. Representative western blot images are shown above graphs but are not displayed as a continuous blot due to running order on the blot not aligning with graphical order of samples. However, original western blot images are available at DOI:10.6084/m9.figshare.12625409. Mean ± SEM are displayed in the graphs (n = 5–10) and significance was determined with a one-way ANOVA followed by a Fisher’s protected least significant difference post hoc (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

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  Figure 9. Inhibition of p-PKC-ζ caused a decreased in RAGE associated proteins which was further attenuated with Rap1a siRNA treatment. Cardiac fibroblasts were isolated from non-diabetic, diabetic, and Rap1a KO mice. Cells were treated either with EPAC (100 µM), EPAC + ps PKC-ζ (100 µM and 1 µg/mL, respectively), Rap1a siRNA (100 nM), or Rap1a siRNA + ps PKC-ζ (100 nM and 1 µg/mL, respectively), followed by collection of total protein. (A–C) α-SMA (42 kDa), (D–F) p-NF-κB (65 kDa), and (G–I) SOD-1 (23 kDa) protein expression was assessed. Protein expression was normalized to β-tubulin (55 kDa) protein expression and mean ± SEM were depicted in graph (n = 6–9). Representative western blot images are shown above graphs but are not displayed as a continuous blot due to running order on the blot not aligning with graphical order of samples. However, original western blot images are available at DOI:10.6084/m9.figshare.12625409. Data depicted for EPAC and Rap1a siRNA treatment groups were previously presented Figure 3, Figure 4 and Figure 5. Significance was assessed using a one-way ANOVA followed by a Fisher’s protected least significant difference post hoc (* p < 0.05, ** p < 0.01, *** p <…

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  Figure 10. Diagram depicting the relationship between Rap1a and the AGE/RAGE signaling cascade. Potential interactions between proteins are shown with arrows with directionality of impact being denoted with arrow orientation. Solid line arrows indicate connections that are supported by evidence presented within this manuscript, while dash line arrows show connections that are supported within the literature.