Cardioprotective Effects of the GRK2 Inhibitor Paroxetine on Isoproterenol-Induced Cardiac Remodeling by Modulating NF-κB Mediated Prohypertrophic and Profibrotic Gene Expression

Figures

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  Figure 1. Effect of paroxetine on ISO-induced cardiac injury, hypertrophic, and fibrotic markers. (A) Representative pictures of hearts from control, CH, and treated rats (treated with 5 mg/kg/day paroxetine or fluoxetine). (B) HW/BW ratio as an indicator of cardiac hypertrophy (n = 6). Serum levels of cardiac injury biomarkers Tn-I (n = 4) (C) and CK-MB (n = 6) (D), cardiac hypertrophic biomarker BNP (n = 5) (E), and cardiac fibrotic marker hydroxyproline (n = 4) (F) in cardiac tissues. All data are expressed as mean ± SEM. Differences between groups were determined using one-way ANOVA followed by Dunnett’s post hoc test. Statistically significant changes compared to CH (untreated) rats are shown as * p < 0.05, ** p < 0.01, **** p < 0.0001. CH: cardiac hypertrophy untreated; CH + P: cardiac hypertrophy pretreated with paroxetine; CH + F: cardiac hypertrophy pretreated with fluoxetine.

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  Figure 2. Effect of paroxetine on cardiac myocytes morphology and fibrosis development in hypertrophic heart. (A) H&E-stained cardiac muscle fascicle and fibers showing no inflammatory cell infiltration in the normal control group, inflammatory cell infiltration, mainly histiocytes with few lymphocytes (black arrow), in the CH (untreated) group, no inflammatory cell infiltration in the group pretreated with paroxetine (CH + P), and inflammatory cell infiltration, mainly histiocytes with few lymphocytes (black arrow) in the group pretreated with fluoxetine (CH + F) (20× power field). (B) Masson’s trichrome-stained cardiac muscle showing no fibrosis in the normal control group, moderate fibrosis (highlighted by blue stain) in the CH (untreated) group, mild and focal fibrosis (blue stain) in the CH + P group, and moderate fibrosis (highlighted by blue stain) in the CH + F group (20× power field). (C) H&E-stained rat thoracic aorta cross sections showing the three aortic layers (intima, media, and adventitia) intact with no inflammatory cell infiltration (4× power field) in the normal control. CH, CH + P, and CH + F groups. CH: cardiac hypertrophy untreated; CH + P: cardiac hypertrophy…

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  Figure 3. Effect of paroxetine on ISO-induced NF-κB and inflammatory biomarkers. (A) Level of NFκB(p105) in myocardium tissues increased with ISO treatment (CH group) and was prevented by paroxetine pre-treatment (CH + P group) (n = 6 per group). Serum levels of inflammatory biomarkers IL6 (n = 4) (B) and CRP (n = 6) (C) increased with ISO treatment (CH group) and were prevented by paroxetine and fluoxetine pre-treatment (CH + P and CH + F groups). All data are expressed as mean ± SEM. Differences between groups were detected using one-way ANOVA followed by Dunnett’s post hoc test. Statistically significant differences between groups are shown as * p < 0.05, *** p < 0.001. CH: cardiac hypertrophy untreated; CH + P: cardiac hypertrophy pretreated with paroxetine; CH + F: cardiac hypertrophy pretreated with fluoxetine; NFκB: Nuclear factor-kappa-light-chain-enhancer of activated B-cells; IL6: interleukin 6; CRP: C-reactive protein.

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  Figure 4. Effect of paroxetine on GRK2, pIκBα, and pNF-κBp(105) expression. (A) Photomicrographs showing normal GRK2 expression in the control group, increased expression in the CH (untreated) group (black arrow), reduced GRK2 expression in the CH + P group, and no marked change in the increased GRK2 expression in the CH + F group (black arrow). (B) Photomicrographs showing normal pIκBα expression in the control group, increased expression in the CH (untreated) group showing (black arrow), reduced expression in the CH + P group, and mild reduction of the increased pIκBα expression in the CH + F group (black arrow). (C) Photomicrographs showing normal pNFκBp(105) in the control group, increased expression in the CH (untreated) group (black arrow), and reduced expression in the CH + P and CH + F (black arrow). (magnification 400×). Semi-quantification results for GRK2 (D), pIκBα (E), and pNF-κBp(105) (F) immunoreactivity (using ImageJ software). Data are expressed as mean ± SEM (n = 3 different analyses per group). Statistical analyses were performed using one-way ANOVA and Tukey’s multiple comparison post hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the CH group. CH: cardiac…

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  Figure 5. Effect of paroxetine on prohypertrophic and profibrotic gene expression. Myocardium tissue gene expression of GRK2 (A), NFκB (p65) subunit (B), and IκBα (C). Expression of prohypertrophic and profibrotic genes, namely ANP (D), α-SMA (E), TGF-β1 (F), and Smad3 (G). All data are expressed as mean ± SEM (n ≥ 3). Differences between groups were detected using a one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. Statistically significant differences between groups are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CH, cardiac hypertrophy untreated; CH + P, cardiac hypertrophy pretreated with paroxetine; CH + F, cardiac hypertrophy pretreated with fluoxetine.

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  Figure 6. Schematic diagram representing the design and timeline of the experimental procedure. After 7 days of adaption, 24 rats were divided randomly into four groups (n = 6 per group): The control group, cardiac hypertrophy untreated group (CH), cardiac hypertrophy pretreated with paroxetine group (CH + P), and cardiac hypertrophy pretreated with fluoxetine group (CH + F). At the end of the experiment, serum samples were collected, and heart tissue samples were harvested.