Vardenafil Alleviates Doxorubicin-Induced Cardiotoxicity Associated with Restoration of the AMPK/SIRT1 Signaling Pathway

Figures

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  Figure 1. Experimental design of the prophylactic treatment protocol. Rats received oral vardenafil (Var, 3 mg/kg/day) or carvedilol (Carv, 30 mg/kg/day) once daily for 28 consecutive days, with treatment initiated 14 days before the first doxorubicin (Dox) injection and continued throughout the Dox administration period. Dox-induced cardiotoxicity was established by six intraperitoneal injections of Dox (2.5 mg/kg each) administered on alternate days from Day 14 to Day 24. On Day 28, animals were sacrificed, and blood and heart tissues were collected for serum biochemical, histopathological, immunohistochemical, ELISA, and qRT-PCR analyses.

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  Figure 2. Var protected against Dox-induced myocardial injury in the DIC rat model. Effect of Var against DIC was evaluated in the different groups by (A) heart/body weight ratio, (B) serum creatine kinase (CK) activity (U/L), (C) serum creatine kinase MB (CK-MB) activity (U/L), (D) serum lactate dehydrogenase (LDH) activity (U/L), (E) serum creatinine level (mg/dL), (F) blood urea nitrogen (BUN) level (mg/dL), and (G) histopathological investigation of heart tissue stained by H&E. * indicates prominent interstitial, blue arrow indicates perivascular edema, and black arrows indicate marked vacuolation of muscle fibers. Magnifications ×100: bar 100 and ×400: bar 50. Data are presented as mean ± SD (n = 6 biological replicates per group, where each biological replicate represents one animal). Statistical significance denoted as ** p ≤ 0.01, and *** p ≤ 0.001.

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  Figure 3. Vardenafil improves left ventricular function in Dox-induced cardiotoxicity. (A) Echocardiography was performed at day 28 after initiating the treatment protocol; (B) IVSd: interventricular septal thickness in diastole; (C) IVSs: interventricular septal thickness in systole; (D) LVIDd: left ventricular internal diameter at end-diastole; (E) EF: ejection fraction; (F) FS: fractional shortening. Data are presented as mean ± SD (n = 6 biological replicates per group, where each biological replicate represents one animal). Statistical significance denoted as * p < 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

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  Figure 4. Regulation of AMPK activity by Var. The interaction of Var with AMPK was examined through molecular docking and protein expression analysis. Docking simulations were performed by fitting Var into the binding pockets of AMPK chains E and F, followed by evaluation of AMPK protein expression levels. (A) Amino acid residues involved in the interaction within AMPK chain E, visualized using Discovery Studio Visualizer. (B) Hydrophobic interactions between Var and the binding pocket of AMPK chain E, visualized with Chimera. (C) Amino acid residues contributing to the interaction within AMPK chain F, visualized using Discovery Studio Visualizer. (D) Hydrophobic contacts between Var and the binding site of AMPK chain F, visualized with Chimera. (E) Western blot analysis of AMPK protein expression across experimental groups (n = 3 biological replicates per group, where each biological replicate represents one animal). (F) Level of cardiac p-AMPK protein (pg/mg protein) by ELISA (n = 6 biological replicates per group, where each biological replicate represents one animal). Data are presented as mean ± SD. Statistical significance denoted as *** p ≤ 0.001, ** p ≤ 0.01.

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  Figure 5. Var upregulated SIRT1 and PPARγ in Dox-treated rats. (A) The levels of cardiac SIRT1 (ng/mg protein) via ELISA. (B) The levels of cardiac PPARγ (pg/mg protein) via ELISA. (C) Cardiac PPARγ mRNA expression was determined by qRT-PCR. Data are presented as mean ± SD (n = 6 biological replicates per group, where each biological replicate represents one animal). Statistical significance denoted as * p < 0.05, and *** p ≤ 0.001.

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  Figure 6. Var attenuated Dox-induced oxidative stress and modulated Nrf2/HO-1 signaling pathway. The antioxidant activity of Var was assessed by measuring: (A) level of cardiac MDA (nmol/g. tissue) spectrophotometry, (B) level of cardiac GSH (nmol/g. tissue) spectrophotometry, (C) level of cardiac Nrf2 protein (ng/mg protein) by ELISA, (D) level of HO-1 protein by immunohistochemistry. Positive immunoreactivity is indicated by black arrows. Tissue sections were counterstained with Mayer’s hematoxylin. Data are presented as Mean ± SD (n = 6 biological replicates per group, where each biological replicate represents one animal), with statistical significance denoted as (*** p ≤ 0.001, ** p ≤ 0.01, and * p < 0.05).

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  Figure 7. Var mitigated Dox-triggered fibrosis and apoptosis in the DIC rat model. Antifibrotic and anti-apoptotic activities of Var in Dox-treated rats were evaluated by the measurement of: (A) TGF-β protein expression by immunohistochemistry, (B) Caspase-3 protein expression by immunohistochemistry. Positive immunoreactivity is indicated by black arrows. Sections were counterstained with Mayer’s hematoxylin. Data are presented as mean ± SD (n = 6 biological replicates per group, where each biological replicate represents one animal). Statistical significance represented as *** p ≤ 0.001, and * p < 0.05, scale bar 50 µm.

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  Figure 8. Proposed mechanism underlying the cardioprotective effects of vardenafil against doxorubicin-induced cardiotoxicity based on the present findings and published literature. Vardenafil treatment was associated with restoration of the p-AMPK/SIRT1 signaling pathway, accompanied by enhanced Nrf2 activation, upregulation of HO-1 and PPARγ, attenuation of oxidative stress, and reduced myocardial injury. The downstream effects on mitochondrial function and apoptosis represent a proposed mechanistic model derived from integration of the present findings with previous literature. Molecules enclosed within gray dashed boxes and marked with a red asterisk (*) (PGC-1α, Bax, cytochrome c, and caspase-9), together with their associated gray dashed arrows, were not experimentally evaluated in the present study and are included based on previously published evidence to illustrate potential downstream mechanisms. In contrast, green/red solid boxes and arrows represent experimentally measured findings obtained in the present study.