AP39, a novel mitochondria-targeted hydrogen sulfide donor ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathway

Bin Zhang; Yangxue Li; Ning Liu; Bin Liu

doi:10.1371/journal.pone.0300261

. 2024 Apr 3;19(4):e0300261. doi: 10.1371/journal.pone.0300261

AP39, a novel mitochondria-targeted hydrogen sulfide donor ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathway

Bin Zhang ¹, Yangxue Li ¹, Ning Liu ¹, Bin Liu ^1,^*

Editor: Mohamed Abdel-Daim²

PMCID: PMC10990198 PMID: 38568919

Abstract

Doxorubicin (DOX) is a broad-spectrum, highly effective antitumor agent; however, its cardiotoxicity has greatly limited its use. Hydrogen sulfide (H₂S) is an endogenous gaseous transmitter that exerts cardioprotective effects via the regulation of oxidative stress and apoptosis and maintenance of mitochondrial function, among other mechanisms. AP39 is a novel mitochondria-targeted H₂S donor that, at appropriate concentrations, attenuates intracellular oxidative stress damage, maintains mitochondrial function, and ameliorates cardiomyocyte injury. In this study, DOX-induced cardiotoxicity models were established using H9c2 cells and Sprague–Dawley rats to evaluate the protective effect of AP39 and its mechanisms of action. Both in vivo and in vitro experiments showed that DOX induces oxidative stress injury, apoptosis, and mitochondrial damage in cardiomyocytes and decreases the expression of p-AMPK/AMPK and UCP2. All DOX-induced changes were attenuated by AP39 treatment. Furthermore, the protective effect of AP39 was significantly attenuated by the inhibition of AMPK and UCP2. The results suggest that AP39 ameliorates DOX-induced cardiotoxicity by regulating the expression of AMPK/UCP2.

Introduction

Doxorubicin (DOX), a broad-spectrum anthracycline antineoplastic drug, is widely used for the treatment of leukemia, breast cancer, ovarian cancer, lymphoma, and osteosarcoma [1], and plays an extremely important role in tumor chemotherapy. However, DOX has dose-dependent, cumulative and progressive cardiotoxicity [2], which mainly manifests as arrhythmia, heart failure, myocardial injury, hypertension, and cardiomyopathy. Some studies have shown that the incidence of DOX-induced cardiotoxicity significantly increases with the increase in the total cumulative dose of DOX in a day or in a therapeutic cycle, and the mortality rate after treatment with a single or a cumulative dose of DOX of 5-25mg/kg is 10%-38%. After 2 years of chemotherapy, the mortality rate has been reported to reach 50% [3]. This not only limits the therapeutic dose of DOX, but also greatly affects the quality of life of cancer survivors and may even shorten their life expectancy. Studies have shown that DOX-induced cardiotoxicity is mainly associated with oxidative stress, lipid peroxidation damage, apoptosis and mitochondrial dysfunction. Furthermore, the effects are also reported with inducing inflammation and affecting DNA replication and transcription [4–7]. Currently, the only drug approved by the FDA for the treatment of DOX cardiotoxicity is dexrazoxane, which still has various side effects, including myelotoxicity in patients with soft-tissue sarcoma [8]. Therefore, there is an urgent need to develop or find more safe and effective drugs that may ameliorate DOX cardiotoxicity, explore their mechanisms of action, and translate these agents into clinical applications.

Hydrogen sulfide (H₂S), a gaseous signaling molecule, plays an important role in cardiovascular diseases by ameliorating atherosclerosis and hypertension [9], attenuating myocardial ischemia-reperfusion (I/R) injury [10,11], ameliorating heart failure [12,13], and attenuating myocardial fibrosis [14,15]. This may be related to its ability to regulate oxidative stress, apoptosis, autophagy, inflammation, mitochondrial function, neovascularization, and fibrosis at reasonable concentrations [16,17]. AP39 is a novel mitochondria-targeted H₂S donor that attenuates intracellular oxidative stress at appropriate concentrations while maintaining cell viability, mitochondrial respiration, and mitochondrial DNA integrity [18,19]. It prevents myocardial ischemia-reperfusion injury independently of the cytoplasmic RISK pathway [20], inhibits mitochondrial autophagy, antagonizes cardiomyocyte iron death, and ameliorates myocardial fibrosis in rats with myocardial infarction via the PINK1/Parkin pathway [21]. Supplementation of preservation fluid with AP39 protects heart grafts from long-term ischemic damage and prevents I/R injury in heart transplantation [22].

Adenosine monophosphate-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis and mitochondrial homeostasis. The activation of AMPK modulates cellular metabolism, autophagy, apoptosis, and fibrosis [23]. Uncoupling protein 2 (UCP2) is located within the inner mitochondrial membrane and affects mitochondrial function and metabolism through oxidative phosphorylation uncoupling. AMPK attenuates oxidative stress damage, reduces apoptosis [24], attenuates mitochondrial damage [25], and attenuates inflammatory responses [26] by upregulating UCP2.

DOX causes cardiotoxicity through a variety of possible molecular mechanisms related to AMPK [27], including oxidative stress, mitochondrial damage, and apoptosis, whereas H₂S increases mitochondrial ATP synthesis, induces mitochondrial biogenesis [28], ameliorates oxidative stress, and reduces apoptosis via AMPK [29]. Based on its effects on oxidative stress, apoptosis, and mitochondrial processes, we hypothesized that the exogenous H₂S donor AP39 may attenuate DOX-induced cardiotoxicity. The aim of this study was to assess whether AP39 exerts a protective effect against DOX-induced cardiotoxicity and to investigate its mechanism of action, including its effects on the mitochondrial pathway and AMPK/UCP2.

Materials and methods

Reagents and antibodies

DOX (S1208) was purchased from Selleck (Houston, TX, USA), AP39(HY-126124) was purchased from MCE, Compound C (CC; 171260) and genipin (G4796) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The following primary antibodies for the following proteins were purchased from Cell Signaling Technology (Danvers, MA, USA): Caspase-3(9662, 1:1000), Cleaved Caspase-3 (9664, 1:1000), AMPKα (5831, 1:1000), p-AMPKα (Thr172) (50081, 1:1000), and UCP2 (89326, 1:1000). Primary antibodies for Bax (A0207, 1:1000) and Bcl-2 (A19693, 1:1000) were purchased from ABclonal (Wuhan, China). Small interfering RNA against UCP2 (siUCP2) and its negative control (NC) were synthesized by IBSBIO (Shanghai, China). Lipofectamine 2000(11668500) was purchased from Invitrogen (Waltham, MA, USA). Annexin V-FITC (331200) and SYTOX Red (S34859) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Cell Counting Kit-8(CCK-8)(BA00208) and BCA Protein Assay Kit (C05-02001) were purchased from Bioss (Beijing, China). 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (BB-47053) was purchased from Bestbio (Nanjing, China). The Mitochondrial Membrane Potential Assay Kit with JC-1 (J8030) and H₂S Content Assay Kit (BC2055) were purchased from Solarbio (Beijing, China). The ATP assay kit (A095-1-1) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Superoxide Dismutase (SOD) Activity Assay Kit (AKAO001M), Glutathione Peroxidase (GPX) Activity Assay Kit (AKPR014M), Malondialdehyde (MDA) Content Assay Kit (AKFA013M), Coenzyme II NADP (H) Content Assay Kit (AKCO018M), and Lactate Dehydrogenase (LDH) Activity Assay Kit (AKCO003M) were purchased from Beijing Boxbio Science & Technology Co., Ltd. (Beijing, China). The Rat Troponin T Type 2, Cardiac (TNNT2) ELISA Kit (JL27509), Rat Creatine Kinase MB Isoenzyme (CKMB) ELISA Kit (JL12296), and Rat Brain Natriuretic Peptide (BNP) ELISA Kit (JL11495) were purchased from Jianglai Biology (Shanghai, China). The TUNEL Apoptosis Assay Kits (C1086 and C1091) were purchased from Beyotime (Shanghai, China).

Animals and treatment

The study was approved by the Institutional Committee for the Protection and Utilization of Animals of Jilin University (Approval Number:2023 No.463). All handling of laboratory animals during experiments was in accordance with the Guidelines for the Management and Use of Laboratory Animals published by the National Institutes of Health. Animal studies were conducted in accordance with ARRIVE guidelines.

Male 8 to 10-week-old SPF Sprague–Dawley rats, weighing 300–320 g, were purchased from Yeast Laboratory Animal Technology. The rats were housed individually (one rat in one cage) at the Animal Center of Jilin University, with the room temperature and humidity controlled at 21 ± 1°C and 50–60%, respectively, under a 12h day/night cycle. The animals had free access to water and food, and were acclimatized for 1 week. They were then randomly assigned to groups (10 rats per group) and administered treatments according to different protocols. The groups were as follows: (1) Con;(2) DOX;(3) AP39;(4) DOX+AP39;(5) DOX+AP39+Compound C;(6) DOX+AP39+genipin. The dosing was as follows: The Con group was administered an equal amount of 0.9% NaCl; DOX was administered intraperitoneally once a week for 3 weeks at a dose of 5 mg/kg, resulting in a cumulative dose of 15 mg/kg; AP39 was administered intraperitoneally once every other day at a dose of 50 nmol/kg, starting at the same time as DOX, for 3 weeks; Compound C (171260, Sigma, USA) was administered at a dose of 20mg/kg/d, one day prior to the start of DOX, for 1 week; and genipin was administered for 3 consecutive days prior to the initiation of DOX at a dose of 20 mg/kg/d. The above doses were based on previous study reports [30–33] and experimental data.

Weighing of the animals was performed every 3 days during the experiment, and their mental status, activity status, and any pain or discomfort were also monitored and recorded. The duration of this experiment was 21 days, and no rats died before euthanasia. After 21 days, cardiac ultrasound was performed after isoflurane anesthesia was administered, and then euthanasia was performed by CO₂ inhalation (a total of 60 rats). The above experiments were supervised and directed by the Institutional Committee for the Protection and Utilization of Animals of Jilin University, and all efforts were made to minimize suffering.

Cell culture and treatments

The rat H9c2 cell (191377) was purchased from Beijing Zhongke QC Biotechnology Co. (Beijing, China). DMEM supplemented with 10% fetal bovine serum (Kang Yuan Biology, Tianjin, China) and 1% penicillin and streptomycin (Solarbio, Beijing, China) was used for cell culture in an incubator at 37°C and a 5% CO₂ atmosphere. Different drugs were given to stimulate the cells for 24h according to the experimental protocol including DOX (1 μmol/L), AP39 (100 nmol/L), and CC (10 μmol/L) [34]. To reduce UCP2 expression in vitro, cells were transfected with siUCP2 (50 nmol/L) using the transfection reagent Lipofectamine2000 for 48h, and the effectiveness of transfection was evaluated by qPCR and western blotting.

Cell activity assay

H9c2 cells were inoculated in 96-well plates (4 × 10³/well) and incubated with different concentrations of DOX (0, 0.5, 1, and 2 μmol/L) and AP39 (0, 30, 50, 100, 300, and 500 nmol/L), with a final volume of 100μL in each well. After 24 h, 10 μL of CCK-8 reagent was added to each well, the cells were incubated in the cell incubator for 60 min, and absorbance was measured at 450 nm.

Detection of ROS

H9c2 cells were inoculated in 6-well plates (5 × 10⁴/well), and different stimuli were applied when cells reached approximately 70% confluence. Cells were incubated for 24h in a cell culture incubator. The DCFH-DA probe was diluted with serum-free DMEM at a ratio of 1:1000, hoechst 33342 (C1029, Beyotime, China) at a ratio of 1:100, added to the 6-well plates at 1 mL/well, followed by incubation 37°C in the dark for 20 min. Cells were washed gently with phosphate-buffered saline and images were obtained under a fluorescence microscope. The average fluorescence intensity was evaluated using ImageJ.

Flow cytometry

H9c2 cells were resuspended under different conditions and diluted with 1× Binding Buffer to a concentration of 1 × 10⁶ cells/mL. Then, 100 μL of the cell suspension was used for flow cytometry; briefly, 5 μL of Annexin V-FITC and 5 μL of SYTOX Red were added, samples were incubated at room temperature (25°C) in the dark for 15 min, 400 μL of 1× Binding Buffer was added, and samples were assayed immediately using the flow cytometer (Cytoflex, Beckman).

Western blotting

Total protein was extracted from cell samples and cardiac tissues using RIPA buffer, and the protein concentration was determined using a BCA Kit. Equal concentrations of protein samples were separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA), which were blocked with 5% skim milk powder at room temperature for 60 min. The samples were then incubated with primary antibody overnight at 4°C, followed by incubation with the secondary antibody at room temperature for 1 h. Chemiluminescent color development was performed by adding the developing solution.

Mitochondrial membrane potential assay

Mitochondrial membrane potential was assayed using the JC-1 probe according to the manufacturer’s instructions. When the mitochondrial membrane potential was high, JC-1 aggregated in the mitochondrial matrix and formed a polymer, producing red fluorescence; when the mitochondrial membrane potential was low, JC-1 did not aggregate in the mitochondrial matrix, and the monomers produced green fluorescence. Images were obtained using a fluorescence microscope (Olympus, Japan), and the fluorescence intensity was analyzed using ImageJ. The ratio of red to green fluorescence was used to measure the change in mitochondrial membrane potential.

Quantitative real-time PCR

Total RNA was extracted using TransZol (Transgen Biotech, Beijing, China), and reverse transcription and qRT-PCR were performed according to the instructions provided with the relevant kits. GAPDH was selected as the internal reference gene. The sequences were as follows: UCP2 (F 5’-GCAGTTCTACACCAAGGGCT-3’, R 5’-GGAAGCGGACCTTTACCACA-3’), siUCP2(F 5’-AGAGCACUGUCGAAGC CUACA-3’, R 5’-UAGGCUUCGACAGUGCUCUGG-3’), and GAPDH (F 5’-AGTTCA ACGGCACAGTCAAGGC-3’, R 5’-CGACATACTCAGCACCAGCATCAC-3’). The relative expression was calculated using the 2^-ΔΔCT method.

Oxidative stress and ATP assays

According to the manufacturer’s instructions, oxidative stress levels were measured using SOD, GSH-Px, MDA and NADPH kits, and cellular ATP levels were measured using ATP kits. Absorbance values were measured at different wavelengths using an enzyme meter and analyzed according to the standard curves and corresponding formulas.

ELISA

Cardiomyocyte injury was assessed using ELISA kits for TNNT2, CK-MB, and BNP in rat serum according to the manufacturer’s instructions.

Transmission electron microscopy

Different groups of rat myocardial specimens and different drug-stimulated H9c2 cells were fixed with 2.5% glutaraldehyde phosphate and stained with1% phosphotungstic acid. The mitochondrial ultrastructure was observed and analyzed by using a Transmission Electron Microscope AMT Imaging System (Advanced Microscopy Techniques Co, USA) at magnifications of 5000×, 8000×, and 25000×.

HE and masson staining

Rat myocardial tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 3-μm-thick wax slices. The sections were stained with hematoxylin and eosin (HE), Masson Lichtenstein acidic reagent, and toluidine blue and observed under a light microscope (Olympus, Japan).

TUNEL staining

H9c2 cells were fixed with 4% paraformaldehyde, punched with 0.3% TritonX-100, stained with TUNEL working solution under light-avoidance conditions (37°C for 60min) and stained with DAPI (C1005, Beyotime, China). The cells were then observed under a fluorescence microscope. Rat cardiac muscle tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and then cut into wax slices with a thickness of 3μm. Following this, they were stained with TUNEL and observed under a light microscope.

Statistical analyses

Results

DOX induces H9c2 cell damage

H9c2 cells were stimulated with various concentrations of DOX (0, 0.5, 1, or 2 μmol/L) for 24 h for CCK-8 detection. Exposure to 1 μmol/L DOX for 24 h decreased H9c2 cell viability by approximately 50% (compared with that in the control group), and the DOX-induced decrease in cell viability was dose-dependent. We stimulated H9c2 cells with 1 μmol/L DOX for different durations (0, 6, 12, 24, and 48 h). A CCK-8 assay showed that cell viability decreased by approximately 50% at 24 h. Therefore, we stimulated H9c2 cells with 1 μmol/L DOX for 24 h for subsequent experiments (Fig 1A and 1B).

Fig 1 — (A) and (B) Cell viability determined by CCK-8 assays after treatment with DOX at different concentrations for 24 h and treatment with 1 μmol/L DOX for different times (n = 4 or 5); (C) Representative DCFH-DA images and statistical results (n = 5); (D) SOD, GSH-Px, MDA, and NADPH levels in H9c2 cells (n = 4); (E) Apoptosis rate measured by flow cytometry (n = 3). (F) Representative TUNEL staining images and statistical results (n = 3). Values represent the mean±SD.*p<0.05 vs. Con group,**p<0.01 vs. Con group.

Free radical production is the main cause of cardiomyocyte damage by DOX, and cardiotoxicity occurs progressively with ROS production and lipid peroxidation [35]. As determined using the DCFH-DA probe, DOX increased ROS levels in cardiomyocytes (Fig 1C), resulting in decreased SOD and GSH-Px activity and increased MDA and NADPH levels (Fig 1D), suggesting that DOX causes oxidative stress injury in cardiomyocytes. Flow cytometry and TUNEL staining revealed that the apoptosis rate was significantly higher (p < 0.01) in the DOX group than in the Con group (Fig 1E and 1F), suggesting that DOX caused apoptosis in H9c2 cells.

DOX induces mitochondrial damage in H9c2 cells

Previous studies have shown that DOX can lead to cardiomyocyte apoptosis via endogenous pathways [36], particularly the mitochondrial pathway. Furthermore, DOX can lead to mitochondrial damage [5]. In this study, DOX increased the expression levels of the apoptosis-related protein Bax, decreased expression levels of Bcl-2, and increased expression levels of Cleaved Caspase-3/Caspase-3 (Fig 2A), indicating that DOX promotes apoptosis in cardiomyocytes and its mechanism of action involves mitochondria. We further evaluated mitochondrial membrane potential and ATP levels, revealing that DOX could cause a decrease in mitochondrial membrane potential and ATP levels in cardiomyocytes (Fig 2B and 2C), while mitochondrial damage (mitochondrial structural disorganization, fragmentation, and cristae rupture) was observed by transmission electron microscopy (Fig 2D).

Fig 2 — (A) Western blot detection of apoptosis-related protein levels and statistical results (n = 3); (B) Representative JC-1 images and quantification of fluorescence intensity for JC-1 monomers/aggregates (n = 4); (C) ATP level (n = 4); (D) Representative images of mitochondria in H9c2 cells observed by transmission electron microscopy; (E) Western blot detection of p-AMPK, AMPK, and UCP2 levels and statistical results (n = 3). Values are presented as the mean ± SD. *p<0.05 vs. Con group, **p<0.01 vs. Con group.

We performed AMPK and UCP2 assays. Western blotting showed that DOX treatment resulted in decreased levels of p-AMPK/AMPK and UCP2 in cardiomyocytes (Fig 2E), suggesting that the damage to cardiomyocytes caused by DOX may be related to AMPK/UCP2.

AP39 ameliorates DOX-induced myocardial injury

AP39 has a concentration-dependent effect on mitochondrial activity. At low concentrations (30–100 nmol/L), AP39 stimulates mitochondrial electron transport and cellular bioenergetic functions, and at high concentrations (300 nmol/L), it has an inhibitory effect on mitochondrial activity [19]. Therefore, we first stimulated H9c2 cells with different concentrations of AP39 (0, 30, 50, 100, 300, and 500 nmol/L) for 24 h and performed CCK-8 assays. The results were in accordance with those of previous reports indicating that AP39 at lower concentrations (30–100 nmol/L) does not significantly reduce cell viability. A decrease in cell viability was detected at 300 nmol/L, and a significant decrease in cell viability was detected at 500 nmol/L. Subsequently, we co-stimulated H9c2 cells with 1 μmol/L DOX and different concentrations of AP39 for 24 h. The CCK-8 results showed that the improvement in cell viability was statistically significant at AP39 concentrations of 50 nmol/L and 100 nmol/L, and the improvement was particularly obvious at an AP39 concentration of 100 nmol/L. Owing to this, we chose 100 nmol/L AP39 for subsequent experiments (Fig 3A–3C).

Fig 3 — (A)-(C) Cell viability determined by CCK-8 assays after H9c2 cells were treated with different concentrations of AP39 for 24 h,1 μmol/L DOX and different concentrations of AP39 for 24 h (n = 4); (D) H₂S content in cells of each group (n = 4); (E) Representative DCFH-DA images and statistical results (n = 5); (F) SOD, GSH-Px, MDA, and NADPH levels in H9c2 cells (n = 4); (G) Apoptosis rate measured by flow cytometry (n = 3). (H) Representative TUNEL staining images and statistical results (n = 3). Values are presented as the mean±SD. *p<0.05 vs. Con group, **p<0.01 vs. Con group. #p<0.05 vs. DOX group, ##p<0.01 vs. DOX group.

We measured the intracellular H₂S content under different conditions, demonstrating that DOX stimulation decreases H₂S content in H9c2 cells, and this decrease was attenuated by the exogenous administration of AP39 (Fig 3D). In addition, AP39 significantly ameliorated DOX-induced oxidative stress injury in H9c2 cells, with a significant decrease in intracellular ROS levels (Fig 3E), improvements in SOD and GSH-Px activity, and decreases in MDA and NADPH levels after co-treatment with AP39 compared with corresponding levels in the DOX group (Fig 3F). AP39 ameliorated DOX-induced cardiomyocyte apoptosis, which was significantly lower in the DOX+AP39 group than in the DOX group (Fig 3G and 3H).

AP39 ameliorates DOX-induced mitochondrial damage

We further investigated the mechanisms by which AP39 exerted protective effects against DOX-induced myocardial injury. Western blotting showed that AP39 decreased the expression of the apoptosis-related proteins Bax and Cleaved Caspase-3/Caspase-3 and increased the expression of Bcl-2 (Fig 4A). Additionally, AP39 attenuated the DOX-induced decrease in mitochondrial membrane potential and ATP levels in cardiomyocytes (Fig 4B and 4C). Transmission electron microscopy revealed that mitochondrial damage (i.e., the disorganization of mitochondrial structure, fragmentation, and cristae breakage) was attenuated by AP39 (Fig 4D).

Fig 4 — (A) Western blot detection of apoptosis-related protein levels and statistical results (n = 3 or 4); (B) Representative JC-1 images and quantification of fluorescence intensity for JC-1 monomers/aggregates (n = 4); (C) ATP level (n = 4); (D) Representative images of mitochondria in H9c2 cells observed by transmission electron microscopy; (E) Western blot detection of p-AMPK, AMPK, and UCP2 levels and statistical results (n = 3 or 4). Values are presented as the mean ± SD. *p < 0.05 vs. Con group, **p < 0.01 vs. Con group. #p < 0.05 vs. DOX group, ##p < 0.01 vs. DOX group. NS indicates no significant difference vs. Con group.

As determined by western blotting, cardiomyocyte p-AMPK/AMPK and UCP2 levels were elevated after co-treatment with AP39 and DOX compared to after DOX stimulation alone (Fig 4E), suggesting that the beneficial effect of AP39 on DOX cardiotoxicity may be related to AMPK/UCP2.

Inhibition of AMPK expression limits the beneficial effect of AP39 on DOX-induced cardiotoxicity

To verify whether the beneficial effect of AP39 on DOX cardiotoxicity was related to AMPK, we inhibited AMPK using the AMPK inhibitor Compound C (CC) and demonstrated the effectiveness of CC by western blotting (Fig 5A). As determined by a CCK-8 assay, CC did not influence cell viability (Fig 5B). ROS levels were significantly higher in the DOX+AP39+CC group than in the DOX+AP39 group (Fig 5C). SOD and GSH-Px activities were lower and MDA and NADPH levels were higher in the DOX+AP39+CC group than in the DOX+AP39 group (Fig 5D), suggesting that the inhibition of AMPK expression limited the beneficial effect of AP39 on DOX-induced oxidative stress injury in cardiomyocytes. The apoptosis rate was higher in the DOX+AP39+CC group than in the DOX+AP39 group (Fig 5E and 5F). Western blotting showed that the expression levels of Bax and Cleaved Caspase-3/Caspase-3 were higher and expression levels of Bcl-2 were lower in the DOX+AP39+CC group than in the DOX+AP39 group (Fig 5G), suggesting that the inhibition of AMPK limited the effect of AP39 on DOX-induced apoptosis in cardiomyocytes. Furthermore, the beneficial effects of AP39 on both mitochondrial membrane potential and ATP levels in cardiomyocytes were weakened by the inhibition of AMPK expression (Fig 5H and 5I). These results suggest that AP39 ameliorates DOX-induced cardiotoxicity by regulating AMPK expression.

Fig 5 — (A) Western blot detection of p-AMPK and AMPK levels and statistical results (n = 3); (B) CCK-8 assay of cell viability (n = 4); (C) Representative DCFH-DA images and statistical results (n = 5); (D) SOD, GSH-Px. MDA, and NADPH levels in H9c2 cells (n = 4); (E) Apoptosis rate measured by flow cytometry (n = 3); (F) Representative TUNEL staining images and statistical results (n = 3). (G) Western blot detection of apoptosis-related protein levels and statistical results (n = 3 or 4); (H) Representative JC-1 images and quantification of fluorescence intensity for JC-1 monomers/aggregates (n = 4); (I) ATP level (n = 4); (J) Western blot detection of UCP2 levels and statistical results (n = 3). Values are presented as the mean±SD.*p<0.05,**p<0.01. NS indicates no significant difference vs. Con group.

We further evaluated the regulatory relationship between AMPK and UCP2. Although the down-regulation of UCP2 by DOX was improved by co-treatment with AP39, the expression level of UCP2 in the DOX+AP39+CC group was still significantly lower than that in the DOX+AP39 group (Fig 5J), indicating that the inhibition of AMPK expression suppressed the up-regulation of UCP2 by AP39. These findings suggest that UCP2 may function downstream of AMPK in the regulation of DOX-induced cardiotoxicity by AP39.

AP39 improves DOX-induced cardiotoxicity by preventing the down-regulation of UCP2

To clarify whether the beneficial effect of AP39 on DOX-induced cardiotoxicity was achieved by modulating the expression of UCP2, we inhibited the expression of UCP2 using small interfering RNA and confirmed the effectiveness of transfection by qRT-PCR and western blotting (Fig 6A). CCK-8 results showed that cell viability did not differ significantly in the NC and siUCP2 groups compared with the Con group (Fig 6B). Oxidative stress damage, apoptosis, and mitochondrial damage were not significantly improved in the DOX+AP39+siUCP2 group compared with those in the DOX+AP39 group. In particular, ROS levels were high (Fig 6C), SOD and GSH-Px levels were low, and MDA and NADPH levels were high (Fig 6D). The apoptosis rate remained high, and western blotting showed that Bax and Cleaved Caspase-3/Caspase-3 levels were high and Bcl-2 levels were low (Fig 6E–6G). Mitochondrial membrane potential and ATP levels remained low (Fig 6H and 6I). The above results suggested that the inhibition of UCP2 inhibited the beneficial effect of AP39 on DOX cardiotoxicity, signifying that UCP2 mediates the effects of AP39.

Fig 6 — (A) Western blot analysis of UCP2 (n = 3) and qRT-PCR for UCP2 mRNA levels (n = 3); (B) CCK-8 assay of cell viability (n = 4); (C) Representative DCFH-DA images and statistical results (n = 5); (D) SOD, GSH-Px, MDA, and NADPH levels in H9c2 cells (n = 4); (E) Apoptosis rate measured by flow cytometry (n = 3); (F) Representative TUNEL staining images and statistical results (n = 3). (G) Western blot detection of apoptosis-related protein levels and statistical results (n = 3 or 4); (H) Representative JC-1 images and quantification offluorescence intensity for JC-1 monomers/aggregates (n = 4); (I) ATP levels (n = 4); (J) Western blot detection of p-AMPK and AMPK levels and statistical results (n = 3). Values are presented as the mean±SD.*p<0.05,**p<0.01. NS indicates no significant difference vs. Con group.

We hypothesized that UCP2 acts downstream of AMPK. To verify this, we further evaluated the levels of p-AMPK/AMPK. DOX decreased the expression of p-AMPK/AMPK. AP39 upregulated p-AMPK/AMPK, and the inhibition of UCP2 did not influence the effect of AP39 (Fig 6J). We previously confirmed that the inhibition of AMPK could affect the expression of UCP2; therefore, these findings further demonstrated that UCP2 functions downstream of AMPK.

Collectively, these findings demonstrated that AP39 ameliorates DOX-induced oxidative stress damage, apoptosis, and mitochondrial damage in H9c2 cells by regulating the expression of AMPK/UCP2.

AP39 attenuates DOX-induced cardiotoxicity in rats by regulating the AMPK/UCP2 pathway

To further validate our experimental results, we conducted in vivo experiments in rats. DOX administration resulted in a significant decrease in body weight and an elevated heart/body weight ratio in rats compared with those in the control group (Fig 7A and 7B). Cardiac ultrasound showed a significant decrease in EF%, FS%, and E/A, suggesting that there was a significant decline in cardiac function (Fig 7C). The levels of TNNT2, CK-MB, LDH, and BNP were significantly increased in abdominal aorta blood after DOX administration (Fig 7D), indicating obvious myocardial damage. HE staining of the rat myocardium was observed under an optical microscope; myocardial cells in the DOX group were deformed, broken, and dissolved, with edema, an enlarged myocardial interstitial space, unevenly colored myocardial fibers, and inflammatory cell infiltration. Masson staining showed a disrupted arrangement of cardiomyocytes, obvious increase in blue collagen fibers in the interstitium of the myocardium, and obvious myocardial fibrosis in the DOX group. A large number of TUNEL positive nuclei were observed in the myocardial tissues of TUNEL staining showed DOX group, with different shapes and sizes and uneven distribution (Fig 7E). Mitochondrial swelling, structural disorder, fragmentation, ridge breakage, and vacuole-like degeneration of cardiomyocytes in the DOX group were observed by transmission electron microscopy (Fig 7F). We also tested indexes of serum oxidative stress in rats. SOD and GSH-Px activities were lower and MDA and NADPH levels were higher in the DOX group than in the control group (Fig 7G). Western blotting showed that DOX increased the expression of the apoptosis-related protein Bax, decreased the expression of Bcl-2, increased the expression of Cleaved Caspase-3/Caspase-3, and decreased expression levels of p-AMPK/AMPK and UCP2 (Fig 7H). In the DOX+AP39 group, the toxic effects of DOX were ameliorated to varying degrees, consistent with the results of our in vitro experiments. We also confirmed the mechanism by which AP39 improves DOX cardiotoxicity in vivo by administering the AMPK inhibitor CC and UCP2 inhibitor genipin. Cardiomyocyte injury, oxidative stress injury, mitochondrial injury, and apoptosis, as described above, did not differ significantly in the DOX+AP39+CC and DOX+AP39+genipin groups, which was consistent with results of in vitro experiments. These findings suggest that the beneficial effect of AP39 on DOX-induced cardiotoxicity in rats is achieved by modulating AMPK/UCP2 expression.

Fig 7 — (A) Body weight in different groups of rats (n = 10); (B) Heart/body weight ratio in different groups of rats (n = 10); (C) Representative echocardiographic images and quantitative analysis of EF%, FS%, and E/A (n = 7); (D) Serum TNNT2, CK-MB, LDH, and BNP levels in rats (n = 10); (E) Representative HE, Masson and TUNEL staining images of the rat myocardium;(F) Representative images of mitochondria in rat cardiomyocytes observed by transmission electron microscopy; (G) SOD, GSH-Px, MDA, and NADPH levels in rat serum (n = 10); (H) Westen blot detection of apoptosis-related protein, p-AMPK, AMPK and UCP2 levels and statistical results (n = 3 or 4). Values are presented as the mean±SD.*p<0.05,**p<0.01. NS indicates no significant difference vs. Con group.

Discussion

DOX is a broad-spectrum, highly effective anthracycline-based antitumor drug commonly used to treat different types of tumors. It can significantly improve the survival rate of patients with cancer. However, its severe cardiotoxicity greatly limits its application. It has been shown that anthracyclines are strongly associated with up to 5% left ventricular dysfunction, reduced left ventricular ejection fraction and symptomatic heart failure [37]. The complications of anthracyclines are dose-dependent, with a cumulative drug dose concentration of 400 mg/m² leading to heart failure in 3.5% of patients, and the incidence of cardiotoxicity ranging from 7–16% at a cumulative dose of 550 mg/m², and from 18–48% at a dose of 700 mg/m² [38]. This not only limits the therapeutic dose of DOX, but also greatly affects the quality of the patients and can even shorten their life expectancy. Therefore, there is an urgent need to find drugs that can reduce the cardiotoxicity of DOX. In this study, both in vivo and in vitro experiments demonstrated that the exogenous mitochondria-targeted H₂S donor AP39 could attenuate DOX-induced cardiotoxicity by ameliorating oxidative stress, apoptosis, and mitochondrial damage. Mechanistically, we found that AP39 exerts its protective effects by activating the expression of AMPK/UCP2, and inhibitors of AMPK and UCP2 can attenuate or even eliminate the beneficial effect of AP39. These results clearly indicate that AP39 is promising for the prevention or treatment of DOX cardiotoxicity.

Increasing focus on DOX cardiotoxicity has led to extensive research. Studies have shown that DOX decreases levels of SOD, CAT, and GSH-Px and increases levels of MDA in the rat heart, and the amelioration of oxidative stress injury can ameliorate cardiotoxicity [39,40], consistent with our findings. In our experiments, DOX induced ROS production in H9c2 cardiomyocytes, decreased SOD and GSH-Px activity in cardiomyocytes and rat serum, and increased MDA and NADPH levels, indicating that it induces oxidative stress in cardiomyocytes. DOX can induce cardiomyocyte apoptosis through both endogenous and exogenous pathways [36]. For example, DOX can induce apoptosis and pyroptosis via the Akt/mTOR signaling pathway [41], heat shock proteins (HSP-10, HSP-20, HSP-22, HSP-27, and HSP-60), and lipocalin, and it is possible to reduce the cardiotoxicity of DOX by promoting antiapoptotic activity [42,43], as demonstrated herein. In particular, we found that DOX can significantly increase the apoptosis rate of H9c2 cells, up-regulate Bax and Cleaved Caspase-3/Caspase-3, and down-regulate Bcl-2, suggesting that DOX can induce endogenous apoptosis via the mitochondrial pathway. Compared with other cell types, cardiomyocytes have more mitochondria, and DOX mainly acts on cardiomyocyte mitochondria, interfering with mitochondrial electron transport, leading to the formation of superoxide (O2-) free radicals [44]. DOX induces mitochondrial DNA (mtDNA) mutations and defects along with elevating ROS in mitochondria, and these changes have been implicated in the development of cardiomyopathy [45]. DOX can also induce excessive opening of the mitochondrial permeability transition pore [46] and affect mitochondrial KATP channel activity [47], leading to myocardial injury. In our experiments, DOX decreased mitochondrial membrane potential and ATP levels in cardiomyocytes. Mitochondrial structure disorganization, fragmentation, and cristae rupture were observed. These in vivo and in vitro experiments clearly show that DOX causes structural damage and dysfunction of mitochondria in cardiomyocytes. In recent years, researchers have been actively exploring the mechanism of DOX-induced cardiotoxicity and searching for ways to ameliorate it. Abdel-Daim et al. found that DOX could lead to increased levels of pro-inflammatory factors IL-1β, TNF-α and degenerative changes in myocardial tissues of rats, and allicin was able to attenuate the apoptosis and inflammatory response induced by DOX [48]. Metformin can alleviate DOX-induced cardiac injury by increasing PDGFR expression and decreasing H₂O₂ levels, activating the AMPK signaling pathway [27,49]. Sildenafil, a PDE-5 inhibitor, demonstrated a protective effect in Dox-induced cardiotoxicity by modulating the NO/cyclic GMP, mitochondrial K⁺ATP channel and oxidative stress [50]. In addition, phytochemicals also exhibit cardioprotective effects by exerting antioxidant, anti-inflammatory and anti-apoptotic activities, as well as regulating lipid metabolism and intracellular calcium homeostasis [6].

Hydrogen sulfide (H₂S), initially described as a toxic gas with a rotten egg odor, is similar in nature to nitric oxide (NO) and carbon monoxide (CO), endogenous gaseous signaling molecules in mammals. Increasing studies have shown that it is involved in a variety of pathophysiological processes, such as oxidative stress, inflammation, apoptosis, and angiogenesis; additionally, it plays a protective role in the pathogenesis and progression of cardiovascular diseases [51]. H₂S reduces lipid peroxidation by hydrogen peroxide and superoxide scavenging in a model of isoprenaline-induced myocardial injury [52]. H₂S-mediated activation of Nrf2-dependent pathways leads to the upregulation of genes involved in endogenous antioxidant defense [53]. It protects mitochondrial function by inhibiting respiration, thereby limiting ROS production and reducing mitochondrial uncoupling [54]. Furthermore, H₂S significantly prevents high glucose-induced apoptosis in cardiomyocytes by modulating the expression of Bax and Bcl-2 [55]. AP39, a novel mitochondria-targeted H₂S donor, can ameliorate high-fat-diet-induced liver injury in young rats by attenuating oxidative stress and mitochondrial damage [56]. It can support cellular bioenergetics and prevent Alzheimer’s disease by maintaining mitochondrial function in APP/PS1 mice and neurons [31]. It can prevent 6-hydroxydopamine-induced mitochondrial dysfunction [57]. In this study, both in vivo and in vitro experiments confirmed that exogenous mitochondrial targeting of AP39 ameliorates DOX-induced oxidative stress by decreasing cardiomyocyte ROS levels, elevating SOD and GSH-Px contents, and decreasing MDA and NADPH levels; it improved cardiomyocyte apoptosis by regulating the expression of apoptosis-related proteins, such as Bax, Bcl-2, and Cleaved Caspase-3/ Caspase-3, and improved DOX-induced mitochondrial injury by elevating mitochondrial membrane potential and ATP levels, consistent with results of previous studies on the mechanisms underlying the myocardial protective effects of H₂S or AP39.

Cardiac tissues have high metabolic energy requirements, and growing evidence suggests that AMPK plays a key role as an energy sensor and a major regulator of metabolism in regulating cell survival in vivo and in vitro [58]. In 2005, Tokarska-Schlattner et al. were the first to demonstrate that AMPK inactivation plays an important role in DOX cardiotoxicity [59]. Since then, additional studies have shown that AMPK is closely related to multiple molecular mechanisms underlying DOX-induced cardiomyocyte injury. DOX is able to inhibit the expression and phosphorylation of AMPK proteins in the rat heart via DNA damage-induced Akt signaling, which activates a negative feedback loop of mTOR signaling and leads to cardiac remodeling [60]. DOX can lead to myocardial fibrosis and cardiomyocyte apoptosis in APN-SE mice by inhibiting AMPK expression [61]. Some AMPK activators, such as metformin, statins, resveratrol, and thiazolidinediones, have the potential to prevent DOX cardiotoxicity [62]. Located within the mitochondrial membrane, UCP2 acts as an anion carrier and regulates the transmembrane proton electrochemical gradient in many human tissues; it is involved in a number of processes, including mitochondrial membrane potential, ROS production within the mitochondrial membrane, and calcium homeostasis [63]. UCP2 is involved in the reduction of ROS production and mitochondrial ROS scavenging [64] and can protect cardiomyocytes from oxidative stress by inhibiting ROS production [65]. UCP2 prevents neuronal apoptosis and attenuates brain dysfunction after stroke and traumatic brain injury [66]. It protects the heart from I/R injury by inducing mitochondrial autophagy [67]. Studies on the interaction between AMPK and UCP2 have yielded conflicting results. It has been suggested that UCP2 affects the autophagic process in septic cardiomyopathy via AMPK signaling [68] an regulates cholangiocarcinoma cell plasticity via mitochondrial-AMPK signaling [69]. However, there is substantial evidence that AMPK functions upstream of UCP2. For example, in a model of nonalcoholic fatty liver disease, LB100 regulated UCP2 expression by inhibiting AMPK [34]. Malvidin alleviates mitochondrial dysfunction and ROS accumulation by activating the AMPK-α/UCP2 axis, thereby preventing inflammation and apoptosis in mice with sepsis-associated encephalopathy [26]. Indole sulfate induces oxidative stress and hypertrophy in cardiomyocytes by inhibiting the AMPK/UCP2 signaling pathway [70]. In our experiments, we found that the protective effect of AP39 against DOX cardiotoxicity was mediated by AMPK/UCP2, and the use of AMPK inhibitors affected the expression of UCP2, while the inhibition of UCP2 expression did not have a significant effect on the expression level of AMPK. These findings suggest that AMPK is an upstream signal of UCP2 and regulates the expression of UCP2. The differences in the regulatory relationship between AMPK and UCP2 among studies may be related to differences in disease models, stimuli, and other factors.

Although we confirmed through in vivo and in vitro experiments that AP39 can ameliorate DOX-induced cardiotoxicity by ameliorating oxidative stress, mitochondrial damage, and decreasing apoptosis, and demonstrated that the mechanism of action is related to the modulation of the AMPK/UCP2 pathway by AP39, there are still some limitations to our experiments, which are mainly reflected in the following:(1) The present study clarifies the role of AMPK regulation of UCP2 in the attenuation of DOX cardiotoxicity by AP39; however, the specific mechanism underlying these regulatory effects is not clear. A downstream pathway of AMPK is Sirt1/PGC-1α, and AMPK activates the NAD+-dependent type III deacetylase Sirt1 by increasing the intracellular NAD+/NADH ratio; Sirt1 activation leads to peroxisome proliferation-activated receptor-γ coactivator 1α (PGC-1α) deacetylation and activity regulation, and Sirt1/PGC-1α may be involved in the regulation of UCP2 [24]. Accordingly, the roles of Sirt1/PGC-1α need to be studied further.(2) During our literature review on establishing an animal model of DOX-induced cardiac toxicity, we could not find a relatively uniform standard dose, frequency and cumulative DOX dose. To this end, the dose we used was a comprehensive consideration of various protocols and that used in clinical settings. AP39 is a recently studied mitochondrial targeting H₂S donor, and its application in animal experiments has not been widely reported compared to that of inorganic sulfides such as NaHS; therefore, the dose of AP39 and associated experimental results might be closely related to animal species, age, weight, and experimental environment, and changes in any of these factors might have an impact on experimental findings. (3) Both the in vivo experimental model constructed using Sprague–Dawley rats and the in vitro experimental model of H9c2 cells which simulated cardiomyocytes cannot fully reflect the real pathophysiological changes in the human body, which is a common problem in all experimental studies. We hope that through the continuous development of advanced science and technology such as gene editing, tissue engineering, three-dimensional cell culture, and organoid culture technologies, combined with advancements in bioinformatics, structural biology, biochemistry, and other fields, experimental models will be better optimized to provide a more realistic and effective experimental platform for human disease research. Despite these limitations, our experimental results provide possible therapeutic strategies for DOX cardiotoxicity and support the beneficial effects of AP39. In the future, more in-depth studies can be carried out based on our experimental results to elucidate the specific mechanism of AP39 in improving DOX-induced cardiotoxicity, and discover associated molecular targets, thus providing reliable research to support its use in clinical settings. The findings of this study present the mitochondrial targeted H₂S donor AP39 as a potential therapeutic agent that can improve DOX-induced cardiotoxicity, and possibly having clinical application in treating other cardiovascular diseases.

Conclusions

Taken together, our findings suggest that AP39 ameliorates DOX cardiotoxicity by attenuating oxidative stress, apoptosis, and mitochondrial damage via modulating the expression of AMPK/UCP2. These findings indicate that AP39 is a promising new therapeutic agent for preventing DOX-induced cardiotoxicity.

Supporting information

S1 Raw images. Western blots.

(PDF)

pone.0300261.s001.pdf^{(2.8MB, pdf)}

S1 Raw data

(PDF)

pone.0300261.s002.pdf^{(8.7MB, pdf)}

S1 File. Editing certificate.

(PDF)

pone.0300261.s003.pdf^{(294.4KB, pdf)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by Jilin Province Science and Technology Department (20220303002SF), Jilin Provincial Development and Reform Commission (2022C003), Jilin Province Science and Technology Department (20190905002SF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0300261.r001

Decision Letter 0

Mohamed Abdel-Daim

9 Jan 2024

PONE-D-23-34322AP39 ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathwayPLOS ONE

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Reviewer #1: This study focused on AP39 ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP Pathway. The authors found that both in vivo and in vitro experiments showed that DOX induces oxidative stress injury, apoptosis, and mitochondrial damage in cardiomyocytes and decreases the expression of p-AMPK/AMPK and UCP Pathway. AMPK/AMPK and UCP2.All DOX-induced changes were attenuated by AP39 treatment. Furthermore, the protective effect of AP39 was significantly attenuated by the inhibition of AP39. All DOX-induced changes were attenuated by AP39 treatment. Furthermore, the protective effect of AP39 was significantly attenuated by the inhibition of AMPK and UCP2. AMPK/UCP2. This observation is interesting，but there are some questions as follows:

1. caspase-3 and cleaved caspase-3 are shown in all of the authors' figures, but the results are unusual. Normally, if cleaved caspase-3 is increased, then capspase-3 will be decreased, but in the author's results, caspase-3 is not decreased, and many bands are unchanged or even increased. Could the authors explain why?

2. Fig. 2B Why is there a big difference in cell size between Con and Dox groups?

3. the anatomical position of Fig. 7E is obviously different, please use pictures with the same anatomical position.

4. The author's experimental method is relatively limited, only flow cytometry was used to detect apoptosis, which is not sufficient, it is recommended to use TUNEL or DNA laddering to verify their findings.

5. Fig6C why the last two images have small cell size than the other images?

Major:

Figure 7A shows that the body weight of rats in DOX, DOX+AP39+CC and DOX+AP39+Genipin groups was significantly reduced by more than 25% compared with that of normal rats, suggesting that the rats were in a malignant state, which violated animal ethics. Routinely, if the body weight of an animal model decreases by 20%-25%, it suggests that the animal is in a malignant state and has reached the humane endpoint or experimental terminative indicator, requiring euthanasia, rather than continuation of the experiment. Please check the animal ethics requirements of this journal for compliance.

Reviewer #2:

Experimental Model and Dose Selection: Please further explain the choice of H9c2 cells and Sprague-Dawley rats as experimental models and discuss the limitations of these models in simulating human pathophysiology. Additionally, explain the rationale behind the selected doses and treatment durations for DOX and AP39, ensuring they are relevant to clinical situations.

Data Analysis: Detail the statistical analysis methods used and justify the choice of these methods. Ensure that significance levels and the interpretation of results are accurate and error-free.

Consistency and Reproducibility of Results: Provide additional information about the replicability of the experiments to ensure the reliability and consistency of the results. Describe the details of the control group setup to aid other researchers in replicating the experiment.

Study Limitations: Discuss the limitations of the study, including factors that might affect the conclusions.

Logical Structure: Ensure the paper's clarity and logical flow from the introduction through methods, results, and discussion sections. Provide additional background information on DOX-induced cardiotoxicity to help readers understand its significance and the need for treatment approaches. Enhance the interpretation of experimental results and their relevance. In the discussion, provide future research directions and prospects for potential clinical applications.

Citations and Background Research: Update the references to include the latest and relevant studies to support the research hypothesis and conclusions. Clearly state the motivation and research questions behind the study, and provide background research to illustrate the importance and relevance of the study.

Ethical Compliance: Given the involvement of animal experiments, ensure compliance with all relevant ethical guidelines and clearly state the ethical approval number in the paper.

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Reviewer #1: No

Reviewer #2: Yes: xiong wei

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Attachment

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pone.0300261.s004.pdf^{(1.8MB, pdf)}

PLoS One. 2024 Apr 3;19(4):e0300261. doi: 10.1371/journal.pone.0300261.r002

Author response to Decision Letter 0

29 Jan 2024

Dear Reviewers:

Thank you for your review and for the comments regarding our manuscript entitled “AP39 ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathway” (ID: PONE-D-23-34322). The comments were all valuable and very helpful in revising and improving our paper, as well as improving our research. We have addressed all the comments and have made corrections to the manuscript (highlighted in yellow).We hope that the revised manuscript now meets the requirements of the journal. Our point-by-point responses to all comments are presented below:

Reviewer #1:

1.Caspase-3 and cleaved caspase-3 are shown in all of the authors' figures, but the results are unusual. Normally, if cleaved caspase-3 is increased, then capspase-3 will be decreased, but in the author's results, caspase-3 is not decreased, and many bands are unchanged or even increased. Could the authors explain why?

Response:

Thanks very much for your comment.

This is a very interesting phenomonon that we noticed during the experiment.We repeated several independent experiments and the results were consistent, just as we presented in the manuscript.

As you mentioned, cleaved caspase-3 is derived from caspase-3, and generally speaking, if cleaved caspase-3 increases, then caspase-3 should decrease.For this purpose, we reviewed the literature and found that other studies have reported results similar to ours.Zhang et al. reported that FNDC5 attenuated DOX-induced cardiomyocyte apoptosis by activating AKT, and verified the expressions of caspase-3 and cleaved caspase-3 in their study. The expression of caspase-3 did not decrease due to the increase of cleaved caspase-3, which was a similar occurrence to our experimental results[1].In another study by Shabaan et al., the authors found that in DOX-induced apoptosis in Wistar rat cardiomyocytes, immunohistochemical staining of rat left ventricular tissues showed a significant elevation in caspase-3 expression [2].Further,Wang et al. found that ropivacaine promoted apoptosis by activating caspase-3 in Bel 7402 and HLE hepatocellular carcinoma cell lines, and although only the expression of cleaved caspase-3 was quantitatively analyzed in their study, both caspase-3 and cleaved caspase-3 were elevated in the reported WB bands [3].

Based on these findings and our results, we may speculate that in our experimental model, the total amount of caspase-3 was not constant but increased, and the expression of cleaved caspase-3 that was sheared off after activation is also increased, so that there will be an increase in cleaved caspase-3 in the WB bands at the same time as a constant or even a slight increase in caspase-3.However, some studies have reported a similar occurrence to what you have proposed, including a study by Wu et al [4], in which caspase-3 expression was decreased when cleaved caspase-3 expression was increased, but the animal species, experimental model, and method of drug administration in their experiments all differed from ours, and these may be factors that led to the contradicting observations.Taken together, we believe that it would be more appropriate to assess the level of apoptosis by using the ratio of cleaved caspase-3/caspase-3.

2. Fig. 2B Why is there a big difference in cell size between Con and Dox groups?

Response:

Thank you very much for your comment.

In Fig 2B, a JC-1 fluorescent probe was used to detect the mitochondrial membrane potential. When the mitochondrial membrane potential was high, JC-1 aggregated in the mitochondrial matrix and presented red fluorescence. When the mitochondrial membrane potential was low, JC-1 existed as monomers and presented green fluorescence.We considered that the reason for the difference in cell size images between the Con group and DOX groups might be related to the difference in fluorescence intensity. We noticed this occurrence during the study,when we took pictures of cells in different groups under fluorescence microscope, our microscope magnification and fluorescence excitation time were consistent; however, the stronger the fluorescence intensity, the bigger the cells looked and vice versa. This was also the case in Fig 6C as you asked in Question 5. We have attached a picture of JC-1 detection with nucleus staining below as an illustration.Since the JC-1 results mainly compare the intensity of red-green fluorescence, the results we present in the paper do not have merged nuclei.

3. the anatomical position of Fig. 7E is obviously different, please use pictures with the same anatomical position.

Response:

Thank you very much for your comment.

We have rephotographed the tissue samples under the microscope and revised some of the images in Fig 7E.

Response:

Thank you very much for your comment.

We performed western blotting to determine Bax,Bcl-2,caspase-3 and cleaved caspase-3 levels and flow cytometry to determine the apoptosis.We also supplemented both in vivo and in vitro experiments with TUNEL to detect apoptosis, as described in Fig 1F,Fig 3H,Fig 5F,Fig 6F and Fig 7E.

5. Fig6C why the last two images have small cell size than the other images?

Response:

Thank you very much for your comment.

Fig 6C shows the ROS levels in H9c2 cells detected by DCFH-DA fluorescent probe. The higher the intracellular ROS levels, the stronger the green fluorescence intensity.The magnification and fluorescence exposure time of the six images in this group were consistent. The reason why the cell sizes of the last two images are small is because of the different fluorescence intensity between different groups. The stronger the fluorescence intensity, the larger the cells looked and vice versa. Following your question, we realized that the existing images could confuse readers; therefore, we re-tested the ROS levels and performed nucleus staining at the same time, thus making it a clearer comparison between different groups. The results are depicted in Fig 1C,Fig 3E,Fig 5C and Fig 6C.

Major:

Response:

Thank you very much for your comment.

Animal ethics are crucial for conducting animal experiments,and our animal study was approved and supervised by the Animal Protection and Utilization Institutional Committee of Jilin University (Animal Ethics Approval number:2023 No. 463).The end points of our study based on the Animal ethics approval regulations were as follows:if there was a significant weight loss in rats, and the decrease exceeded 25%, DOX was to be stopped to avoid further damage from drug dose accumulation, and the general status of rats was to be carefully evaluated; If there was an obvious state of pain, near-death, or serious adverse damage, experimental euthanasia was to be immediately performed; If the general state of the rats was acceptable, the state of the animal was be continuously observed without the administration of DOX.

In our study, we noticed a reduction in animal body weight of nearly 25% on the 18th day. However, the general state of the rats was good in three groups. The rats did not appear to be in an obvious state of near-death or obvious pain, the intake of food and water was reduced but could freely be obtained at a normal frequency, and the activity level was reduced but was still good compared with that of rats in the Con group at the same time period. At this point, we had completed all DOX dosing;therefore,we continued to observe the rats for the remaining 3 days.

We love and respect experimental animals.We believe that they have the same right to live and they also experience emotions such as joy and sorrow, pain, and fear. During the study, we treated all animals with kindness, comfort, and minimized their stress, anxiety, and pain,and the whole process is supervised by the Animal Protection and Utilization Institutional Committee of Jilin University.

[References]

1、Zhang X,Hu C,Kong CY,et al.FNDC5 alleviates oxidative stress and cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity via activating AKT.Cell Death & Differentiation (2020) 27:540–555.doi:org/10.1038/s41418- 019-0372-z.

2、Shabaana DA, Mostafab N, El-Desokyb MM, and Arafat EA.Coenzyme Q10 protects against doxorubicin-induced cardiomyopathy via antioxidant and anti-apoptotic pathway.TISSUE BARRIERS. 2023, VOL. 11, NO. 1, e2019504 (14 pages). doi:10.1080/ 21688370.2021.2019504.

3、Wang WT, Zhu MY, Xu ZX,et al.Ropivacaine promotes apoptosis of hepatocellular carcinoma cells through damaging mitochondria and activating caspase-3 activity.Biol Res. 2019 Jul 12;52(1):36. doi:10.1186/s40659-019 -0242-7.

4、Wu XT, Wang LJ,Wang K,et al.ADAR2 increases in exercised heart and protects against myocardial infarction and doxorubicin-induced cardiotoxicity. Mol Ther. 2022 Jan 5; 30(1): 400–414.doi: 10.1016/j.ymthe. 2021.07.004.

Reviewer #2:

1.Experimental Model and Dose Selection: Please further explain the choice of H9c2 cells and Sprague-Dawley rats as experimental models and discuss the limitations of these models in simulating human pathophysiology. Additionally, explain the rationale behind the selected doses and treatment durations for DOX and AP39, ensuring they are relevant to clinical situations.

Response:

Thanks very much for your comment.

(1)Reasons and limitations of choosing H9c2 cells and Sprague-Dawley rats as experimental models

In our opinion, it is reasonable to select H9c2 cells and Sprague-Dawley rats as experimental models.H9c2 rat cardiomyocyte is a subclone of the original cloned cell line derived from embryonic BD1X rat heart tissue. It is an in vitro cell model that can be used in place of cardiomyocytes, and can simulate the physiological characteristics of cardiomyocytes.The Sprague-Dawley rat is a common experimental animal model, and its heart structure and function are similar to that of humans. However, these models still have some limitations in simulating human pathophysiology.For example, H9c2 cells cannot fully reproduce the complexity of human cardiomyocytes in vivo under in vitro culture conditions, and lack the regulation of the nervous and endocrine systems. Further,the cells may lose some original biological characteristics after repeated passage. Although Sprague-Dawley rats can simulate the physiological function of the human heart, there are still differences between Sprague-Dawley rats and humans in genetic background, physiological response, metabolism and other aspects, and it is difficult to overcome the influences of species differences. The above limitations are included in our Discussion section.

(2)Dosing and timing of DOX and AP39

The choice of dose and timing of DOX:

In vitro: We used DOX at different concentrations (0.5, 1, 2μM) to stimulate H9c2 cells for 24h, and then conducted CCK-8 cell activity detection.DOX stimulated cell activity was dose-dependent; when DOX concentration was 1μM, H9c2 cells were stimulated for 24h, and cell activity decreased to about 50%.We then used 1μM DOX to stimulate H9c2 cells for different times (6h, 12h, 24h, 48h), and found that the cell activity gradually decreased with the extension of the stimulation time, and after 24h of stimulation, the cell activity decreased to about 50%. Therefore, we selected the experimental condition of stimulating H9c2 cells with 1μM DOX for 24h.These results are reflected in Fig 1A and 1B.

In vivo: There are different literature reports on DOX dosing, and it has been reported that the cumulative dose of DOX will not induce heart failure in rats when it is less than 10mg/kg [1].According to current reports, the most commonly used administration modes in animal experimental models of DOX-induced cardiotoxicity are: DOX 5mg/kg every three days, intraperitoneally (ip),total 20 mg/kg; DOX 6mg/kg once every other day, ip, total 18mg/kg; DOX 5mg/kg/w, ip, total 15mg/kg; DOX 20mg/kg/ day for 5 consecutive days, total volume 100mg/kg; DOX 4mg/kg/w, ip, total 16mg/kg; DOX 2.5mg/kg/w, ip, total 15mg/kg.There is no uniform dosing and frequency of administration.In clinical treatment, patients receiving chemotherapy generally use DOX for several consecutive times in a few months, with a therapeutic dose of 50-75mg/m2 each time, and the cumulative maximum therapeutic dose is about 450mg/m2, which is equivalent to 12mg/kg[2]. Therefore, summarizing the above, a dose of 5mg/kg/w, three times, accumulating to 15mg/kg was chosen for this experiment.

The choice of dosage and timing of AP39:

In vitro: According to relevant reports, AP39 exerts a protective effect on cells at a lower concentration (30 and 100 nM), but at a higher concentration (300 nM), the protective effect may become damaging, and a moderate concentration of AP39(100 nM) can reduce intracellular oxidative stress and maintain cell viability and mitochondrial DNA integrity [3].Therefore, we stimulated H9c2 cardiomyocytes with different concentrations of AP39 (30,50,100,300,500nM) for 24h, and stimulated H9c2 cells with different concentrations of AP39 and 1μM DOX for 24h. The CCK-8 kit was used to detect cell activity. The results showed that, AP39 at 100nM could ameliorate the DOX-induced decline in H9c2 cell activity to the greatest extent, as shown in Fig 3A-C. Therefore, 100 nM of AP39 was selected as the experimental concentration.

In vivo: According to existing reports, AP39 dosing in animal experiments are as follows:0.1mg/kg/d, ip, for 7 weeks; 50nmol/kg/d, ip, for 4 weeks; 100nmol/kg/d,ip, for 6 weeks. It can be seen that there is no relatively uniform dose and frequency of administration. Since AP39 is not used in clinical application, a clinical dosing reference is not available. Therefore, in our pre-experiments, we first tried AP39 at a dose of 50nmol/kg/d, ip,for 4 weeks, but the rats in the DOX+AP39 group continuously died within 2 weeks, and some of the rats in AP39 group also died. Although the remaining rats were in a normal state, their cardiac function was decreased based on echocardiography. We then tried AP39 at a dose of 50nmol/kg, once every other day, ip, for 3 weeks and AP39 25nmol/kg, once every other day, ip, for 3 weeks. Echocardiography results showed the latter dose had no toxic effect; however, it did not improve cardiac function. With the AP39 50 nmol/kg regimen, rats in the AP39 group had no obvious cardiac function damage, and those in the DOX+AP39 group had significantly improved cardiac function compared with those in the DOX group. Owing to these findings, we chose this as the dosing regimen for experimentation.

2.Data Analysis: Detail the statistical analysis methods used and justify the choice of these methods. Ensure that significance levels and the interpretation of results are accurate and error-free.

Response:

Thanks very much for your comment.

All statistical analyses were performed using GraphPad Prism 9.0. Datas are expressed as the mean ± standard deviation (SD). Comparisons between two groups were performed using Student's t-test and comparisons among multiple groups were performed using one-way ANOVA followed by Tukey's post hoc test.Statistically different at p<0.05,statistically significant at p<0.01.All data used in statistical analyses were obtained from three or more independent repeated experiments.According to your suggestions, we have carefully checked the data statistics involved in the whole paper again to ensure that the significance level is expressed correctly, and also checked all descriptions of the results in the paper in detail to ensure that the interpretation of the results is accurate.

3.Consistency and Reproducibility of Results: Provide additional information about the replicability of the experiments to ensure the reliability and consistency of the results. Describe the details of the control group setup to aid other researchers in replicating the experiment.

Response:

Thanks very much for your comment.

According to your suggestions, we have re-checked and included the detailed information of all instruments and reagents used in the study, and improved the descriptions of different group settings and different drug administration schemes, which are specifically reflected in the Materials and Methods section.

4.Study Limitations: Discuss the limitations of the study, including factors that might affect the conclusions.

Response:

Thanks very much for your comment.

We have included a detailed discussion on the limitations of the study and the factors that may affect the observed results in the Discussion section. All specific changes have been highlighted on Page 29-30 line 627-659.

5.Logical Structure: Ensure the paper's clarity and logical flow from the introduction through methods, results, and discussion sections. Provide additional background information on DOX-induced cardiotoxicity to help readers understand its significance and the need for treatment approaches. Enhance the interpretation of experimental results and their relevance. In the discussion, provide future research directions and prospects for potential clinical applications.

Response:

Thanks very much for your comment.

We have reviewed and revised all parts of the article and adjusted the structural order of some paragraphs to ensure that the logical structure is clear, correct and complete.In addition, we improved the relevant background of DOX-induced cardiac toxicity in the Introduction and Discussion sections. Its high incidence in clinical treatment and poor prognosis do not only affect the therapeutic dose of DOX, but also affect the quality of life of cancer survivors and even shorten their life expectancy.The only FDA-approved drug that can be used to treat DOX-induced cardiotoxicity, dexrazoxane, exhibits side effects.Therefore, it is cardinal to find a drug that can improve DOX cardiotoxicity, explore its mechanism of action, and translate it into clinical application.

We also re-examined and refined the interpretation of the experimental results and their correlation. In the Discussion, we put forward our thoughts on the future research directions and prospects of potential clinical application. All specific changes have been highlighted.

6.Citations and Background Research: Update the references to include the latest and relevant studies to support the research hypothesis and conclusions. Clearly state the motivation and research questions behind the study, and provide background research to illustrate the importance and relevance of the study.

Response:

Thanks very much for your comment.

According to your suggestions, we have revised the Introduction and Discussion sections to add relevant researches from recent years to better support our hypothesis and conclusion. Explanations for the importance and relevance of the research context have also been strengthened. All specific changes have been highlighted.

7.Ethical Compliance: Given the involvement of animal experiments, ensure compliance with all relevant ethical guidelines and clearly state the ethical approval number in the paper.

Response:

Thanks very much for your comment.

The study was approved by the Institutional Committee for the Protection and Utilization of Animals of Jilin University(Approval Number:2023 No. 463).All handling of laboratory animals during experiments was in accordance with the Guidelines for the Management and Use of Laboratory Animals published by the National Institutes of Health. Animal studies were conducted in accordance with ARRIVE guidelines. We have included this in the Animals and treatment section.

[References]

1、Jensen RA,Acton EM,Peters H.Doxorubicin cardiotoxicity in the rat: comparison of electrocardiogram,transmembrane potential,and structuraleffect [J].J Cardiovasc Pharmacol,1984,6(1):186-200.

Yi X,Bekeredjian R,DeFilippis NJ,et al.Transcriptional analysis of doxorubicin-induced cardiotoxicity [J].Am J Physiol Heart Cir Physiol, 2006, 290(3):H1098-H1102.doi：10.1152/ajpheart.00832.2005.

5、Szczesny B, Módis K, Yanag K,et al. AP39 [10-oxo-10-(4-(3-thioxo-3H-1,2 -dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], a mitochondrially targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro.Nitric Oxide. 2014 September 15; 41: 120–130. doi:10.1016/j.niox. 2014.04.008.

In all, we appreciate for your warm work earnestly, and we revised our paper point-by-point. Once again, thank you very much for your comments and suggestions.

Kind Regards,

Bin Zhang,Yangxue Li,Ning Liu and Bin Liu

Attachment

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pone.0300261.s005.docx^{(1.5MB, docx)}

PLoS One. doi: 10.1371/journal.pone.0300261.r003

Decision Letter 1

Mohamed Abdel-Daim

18 Feb 2024

PONE-D-23-34322R1AP39 ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathwayPLOS ONE

Dear Dr. Liu,

Please submit your revised manuscript by Apr 03 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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Academic Editor

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Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: (No Response)

Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: I suggest title modification to explain what is AP39?

The suggested title will be as follows.

AP39, a novel mitochondria-targeted hydrogen sulfide donor ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathway.

Pathogenesis of DOX cardiotoxicity should be covered in depth in introduction and discussion sections with reference to the possible mechanisms of potential cardioprotective agents. The following references might be helpful.

https://doi.org/10.1016/j.biopha.2017.04.033

https://doi.org/10.1007/s00280-017-3413-7

https://doi.org/10.3389/fphar.2019.00635

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Reviewer #2: No

Reviewer #3: Yes: ZEINAB MAHASNEH

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PLoS One. 2024 Apr 3;19(4):e0300261. doi: 10.1371/journal.pone.0300261.r004

Author response to Decision Letter 1

20 Feb 2024

Dear editor and dear reviewers:

Thank you for your letter and the reviewers’ comments regarding our manuscript titled “AP39 ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathway” (ID: PONE-D-23-34322R1). The comments were all valuable and very helpful in amending and improving our paper, as well as our research. We have addressed all the comments and have made corrections to the manuscript (highlighted in yellow).We hope that the revised manuscript now meets the requirements of PLOS ONE. Our point-by-point responses to all comments are presented below:

Reviewer #3:

1.I suggest title modification to explain what is AP39?

Response:

Thank you very much for your comment.

We have changed the title to ”AP39, a novel mitochondria-targeted hydrogen sulfide donor ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathway”as you suggested.

2. Pathogenesis of DOX cardiotoxicity should be covered in depth in introduction and discussion sections with reference to the possible mechanisms of potential cardioprotective agents. The following references might be helpful.

https://doi.org/10.1016/j.biopha.2017.04.033

https://doi.org/10.1007/s00280-017-3413-7

https://doi.org/10.3389/fphar.2019.00635

Response:

Thank you very much for your comment.

Based on your suggestions and regarding the literature you provided, we have further discussed and improved the information concerning the pathogenesis of DOX-induced cardiotoxicity in the Introduction and Discussion sections,referencing the possible mechanisms of potential cardioprotective agents. All specific changes have been highlighted.

We appreciate your earnest work reviewing our manuscript, which we have revised point-by-point per your suggestions. Once again, thank you very much for your comments and suggestions.

Kind Regards,

Bin Zhang,Yangxue Li,Ning Liu and Bin Liu

Attachment

Submitted filename: Response to Reviewers.docx

pone.0300261.s007.docx^{(14.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0300261.r005

Decision Letter 2

Mohamed Abdel-Daim

26 Feb 2024

AP39, a novel mitochondria-targeted hydrogen sulfide donor ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathway

PONE-D-23-34322R2

Dear Dr. Liu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mohamed Abdel-Daim, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0300261.r006

Acceptance letter

Mohamed Abdel-Daim

21 Mar 2024

PONE-D-23-34322R2

PLOS ONE

Dear Dr. Liu,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Raw images. Western blots.

(PDF)

pone.0300261.s001.pdf^{(2.8MB, pdf)}

S1 Raw data

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pone.0300261.s002.pdf^{(8.7MB, pdf)}

S1 File. Editing certificate.

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pone.0300261.s003.pdf^{(294.4KB, pdf)}

Attachment

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pone.0300261.s004.pdf^{(1.8MB, pdf)}

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pone.0300261.s005.docx^{(1.5MB, docx)}

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pone.0300261.s006.docx^{(11.5KB, docx)}

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pone.0300261.s007.docx^{(14.5KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

AP39, a novel mitochondria-targeted hydrogen sulfide donor ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathway

Bin Zhang

Yangxue Li

Ning Liu

Bin Liu

Roles

Abstract

Introduction

Materials and methods

Reagents and antibodies

Animals and treatment

Cell culture and treatments

Cell activity assay

Detection of ROS

Flow cytometry

Western blotting

Mitochondrial membrane potential assay

Quantitative real-time PCR

Oxidative stress and ATP assays

ELISA

Transmission electron microscopy

HE and masson staining

TUNEL staining

Statistical analyses

Results

DOX induces H9c2 cell damage

Fig 1. DOX induces H9c2 cell damage.

DOX induces mitochondrial damage in H9c2 cells

Fig 2. DOX induces mitochondrial damage in H9c2 cells.

AP39 ameliorates DOX-induced myocardial injury

Fig 3. AP39 ameliorates DOX-induced myocardial injury.

AP39 ameliorates DOX-induced mitochondrial damage

Fig 4. AP39 ameliorates DOX-induced mitochondrial damage.

Inhibition of AMPK expression limits the beneficial effect of AP39 on DOX-induced cardiotoxicity

Fig 5. Inhibition of AMPK expression limits the beneficial effect of AP39 on DOX-induced cardiotoxicity.

AP39 improves DOX-induced cardiotoxicity by preventing the down-regulation of UCP2

Fig 6. AP39 improves DOX-induced cardiotoxicity by preventing the down-regulation of UCP2.

AP39 attenuates DOX-induced cardiotoxicity in rats by regulating the AMPK/UCP2 pathway

Fig 7. AP39 ameliorates DOX-induced cardiotoxicity in rats by regulating the AMPK/UCP2 pathway.

Discussion

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Mohamed Abdel-Daim

Roles

Author response to Decision Letter 0

Decision Letter 1

Mohamed Abdel-Daim

Roles

Author response to Decision Letter 1

Decision Letter 2

Mohamed Abdel-Daim

Roles

Acceptance letter

Mohamed Abdel-Daim

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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