Ambrisentan attenuates cisplatin-related mitochondrial dysfunction in the heart via regulation of p53 and NF-κB signaling

Figures

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  Fig. 1. Ambrisentan diminishes cisplatin-induced apoptosis and inflammatory responses in H9c2 cells. Cells were treated with cisplatin (CP, 10 µM), ambrisentan (Amb, 1 µM), or pre-treated with ambrisentan followed by cisplatin exposure. Vehicle-treated cells served as the control group. (A) Cell viability was assessed using the MTT assay. (B) Apoptotic activity was measured using a caspase-3/7 luminescence assay. (C) TNFα levels were quantified using ELISA to assess the inflammatory response. (D) mRNA expressions of genes associated with apoptosis (Bcl-2, BAX) and inflammatory responses (TNFα, IL6) were determined by RT-qPCR. (E) Protein levels of Bcl-2 and TNFα were examined by western blotting. Data are expressed as mean ± SD (N = 4). *p < 0.05 vs control, #p < 0.05 vs cisplatin-treated group.

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  Fig. 2. Ambrisentan suppresses mitochondrial malfunctions caused by cisplatin. Cisplatin (CP, 10 µM) and ambrisentan (Amb, 1 µM) were administered either individually or in combination, with cells receiving ambrisentan pre-treatment prior to subsequent exposure to cisplatin. Vehicle-treated cells served as controls. (A) Mitochondrial ROS levels were assessed using MitoSOX Red staining (20 × ; scale bar: 50 µm), with DAPI counterstaining the nuclei (blue). (B) Intracellular ROS levels were measured using the DCFH-DA fluorescence assay. (C) Relative mRNA levels of mitochondrial fission (DNM1, FIS1), fusion (OPA1, MFN1), and biogenesis (NRF1, PGC1α) markers were determined by RT-qPCR. (D) Protein expressions of NRF1, DNM1, and OPA1 were examined using western blotting. Data are expressed as mean ± SD (N = 4). *p < 0.05 vs control, #p < 0.05 vs cisplatin-treated group.

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  Fig. 3. Ambrisentan preserves mitochondrial structural integrity in cisplatin-treated H9c2 cells. Treatment conditions included exposure of cells to cisplatin (CP, 10 µM), ambrisentan (Amb, 1 µM), or a sequential regimen in which cells were pre-treated with ambrisentan before cisplatin. Vehicle-exposed cells served as the control. Mitochondrial morphology was assessed by staining with MitoTracker Red CMXRos and imaging via structured illumination microscopy (SIM). (A) Representative images showing various mitochondrial morphologies, including long tubular, short tubular, large spherical, and small spherical shapes. (B) Representative images of mitochondrial morphology for each treatment group (63 × ; scale bar: 0.25 µm). (C, D) Mitochondrial morphological features, including area, perimeter, and length, were quantified using ImageJ software. Data are expressed as mean ± SD (N = 4). *p < 0.05 vs control, #p < 0.05 vs cisplatin-treated group.

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  Fig. 4. Ambrisentan boosts mitochondrial glycolytic bioenergetics in H9c2 cells exposed to cisplatin. Cells were treated with cisplatin (CP, 10 µM), ambrisentan (Amb, 1 µM), or pre-treated with ambrisentan before exposure to cisplatin. Vehicle-treated cells served as the control group. (A, C) Mitochondrial respiration was examined using the Seahorse XF Mito Stress Test to evaluate OCR parameters, including basal and maximal respiration, ATP production, and spare respiratory capacity. (B, D) Glycolytic activity was investigated using the Seahorse XF Glycolysis Stress Test to track ECAR, which reflects glycolysis and glycolytic capacity. (E) Protein expression levels of ATP5A and pro-survival signaling markers (p-Akt/Akt and p-Erk1/2/Erk1/2) were quantified via western blotting. Data are expressed as mean ± SD (N = 4). *p < 0.05 vs control, #p < 0.05 vs cisplatin-treated group.

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  Fig. 5. Role of the p53 in ambrisentan-mediated cardioprotection against cisplatin-induced H9c2 cell dysfunctions. Cells were pretreated with pifithrin-α (PIFα, p53 inhibitor, 20 µM) or Nutlin-3 (NUT3, p53 activator, 10 µM), followed by ambrisentan (Amb, 1 µM). Cells were then exposed to cisplatin (CP, 10 µM) for the indicated time. Vehicle-exposed cells were used as the control group. (A) Cell viability was assessed using the MTT assay. (B) Apoptotic activity was quantified via a caspase-3/7 assay. (C) TNFα secretion was measured using ELISA. (D, E) Mitochondrial and intracellular ROS levels were examined using MitoSOX Red (20 × ; scale bar: 50 µm) and DCFH-DA, respectively. (F, G) Relative mRNA expression of genes involved in mitochondrial biogenesis (NRF1, PGC1α), mitochondrial dynamics (OPA1, MFN1, DNM1, FIS1), apoptosis (Bcl-2, BAX), and inflammation (TNFα, IL6) was analyzed by RT-qPCR. (H) Western blot analyses were performed to assess protein levels of mitochondrial markers, apoptotic regulators, and inflammatory proteins. Data are expressed as mean ± SD (N = 4). *p < 0.05 vs control, #p < 0.05 vs cisplatin-treated group.

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  Fig. 6. Role of the NF-κB underlying cardioprotective effects of ambrisentan in H9c2 cells stressed by cisplatin. Cells first received either JSH (NF-κB inhibitor, 10 µM) or NA1 (NF-κB activator, 5 µM), prior to incubation with ambrisentan (Amb, 1 µM); subsequently, cisplatin (CP, 10 µM) was applied. Vehicle-treated cells served as controls. (A) Cell viability was determined using the MTT assay. (B) Caspase-3/7 luminescence assay was used to measure apoptotic activity. (C) Inflammatory TNFα levels were quantified using ELISA. (D, E) MitoSOX Red staining (20 × ; scale bar: 50 µm) was employed to assess mitochondrial ROS levels, while intracellular ROS levels were evaluated using DCFH-DA fluorescence staining. (F, G) RT-qPCR was performed to analyze the relative mRNA expression of genes related to mitochondrial biogenesis (NRF1, PGC1α), mitochondrial dynamics (OPA1, MFN1, DNM1, FIS1), apoptosis (Bcl-2, BAX), and inflammation (TNFα, IL6). (H) Expression levels of proteins related to mitochondrial functions, apoptosis, and inflammatory response were assessed by western blotting. Data are presented as mean ± SD (N = 4). *p < 0.05 vs control, #p < 0.05 vs cisplatin-treated group.

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  Fig. 7. Ambrisentan regulates cellular bioenergetics via p53 and NF-κB pathways in cisplatin-treated H9c2 cells. Cells were pre-treated with ambrisentan (Amb, 1 µM) in combination with p53 (PIFα or NUT3) or NF-κB (JSH or NA1) pathway modulators before being exposed to cisplatin (CP, 10 µM). Vehicle-challenged cells served as the control group. (A, C, E, G) Mitochondrial respiration was evaluated using the Seahorse XF Mito Stress Test to measure OCR parameters, including basal and maximal respiration, ATP production, and spare respiratory capacity. (B, D, F, H) Glycolytic activity was assessed using the Seahorse XF Glycolysis Stress Test to monitor ECAR, reflecting glycolysis and glycolytic capacity. Data are expressed as mean ± SD (N = 4). *p < 0.05 vs control, #p < 0.05 vs cisplatin-treated group.

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  Fig. 8. Mechanistic Overview of Ambrisentan-Mediated Cardioprotection Against Cisplatin-Induced Toxicity in H9c2 Cardiomyoblasts. In cisplatin-induced cardiotoxicity, pre-treatment with ambrisentan inhibits the binding of ET-1 to the ETA receptor, thereby activating downstream pro-survival Akt and Erk signaling. Consequently, this cascade attenuates the activity of p53 and NF-κB, thereby ameliorating mitochondrial dysfunction through the promotion of mitochondrial fusion (OPA1, MFN1), stimulation of biogenesis (NRF1, PGC1α), and enhancement of ATP synthesis (ATP5A), while simultaneously suppressing mitochondrial fission (DNM1, FIS1) and reducing ROS generation. Restoration of mitochondrial homeostasis is further supported by increased mitochondrial tubularity, as well as improved OCR and ECAR. In addition, ambrisentan upregulates anti-apoptotic marker Bcl-2 as well as downregulates pro-apoptotic (BAX, caspase-3/7) and inflammatory markers (TNFα, IL-6), thereby preserving cell viability compromised by cisplatin. Solid lines denote confirmed interactions, while dashed lines indicate hypothetical interactions. Red arrows illustrate cisplatin-induced cardiotoxic pathways, whereas green…