Sacubitril/valsartan attenuated myocardial inflammation, fibrosis, apoptosis and promoted autophagy in doxorubicin-induced cardiotoxicity mice via regulating the AMPKα-mTORC1 signaling pathway

Sacubitril/valsartan attenuated myocardial inflammation, fibrosis, apoptosis and promoted autophagy in doxorubicin-induced cardiotoxicity mice via regulating the AMPKα-mTORC1 signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sacubitril/valsartan attenuated myocardial inflammation, fibrosis, apoptosis and promoted autophagy in doxorubicin-induced cardiotoxicity mice via regulating the AMPKα-mTORC1 signaling pathway Feng Hu, Senbo Yan, Lin Li, Xiaoxia Qiu, Xinghe Lin, Weiwei Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4603884/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Sep, 2024 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted 7 You are reading this latest preprint version Abstract Background This study aimed to investigate the potential cardio-protective effects of sacubitril/valsartan (Sac/Val) in mice with doxorubicin (DOX)-induced cardiomyopathy, a common manifestation of cancer therapy-related cardiac dysfunction (CTRCD) associated with DOX. Methods A total of 24 mice were equally classified into 4 groups; control group, DOX (total 24 mg/kg), Sac/Val (80 mg/kg), and Sac/Val + DOX (Sac/Val was given from seven day before doxorubicin administration). Neonatal rat ventricular myocytes was exposed to 5 µM of DOX for 6 h in vitro to mimic the in vivo conditions. A variety of techniques were used to investigate cardiac inflammation, fibrosis, apoptosis, and autophagy, including western blot, real time quantitative PCR (RT-qPCR), immunohistochemistry, and fluorescence. Results Mice with Dox-induced cardiotoxicity displayed impaired systolic and diastolic function, characterized by elevated levels of cardiac inflammation, fibrosis, cardiomyocyte hypertrophy, apoptosis, and autophagy inhibition in the heart. Treatment with Sac/Val partially reversed these effects. In comparison to the control group, the protein expression of NLRP3, caspase-1, Collagen I, bax, cleaved caspase-3, and P62 were significantly increased, while the protein expression of bcl-2 and LC3-II were significantly decreased in the myocardial tissues of the Dox-induced cardiomyopathy group. The administration of Sac/Val demonstrated the potential to partially reverse alterations in protein expression within the myocardium of mice with Dox-induced cardiotoxicity by modulating the AMPKα-mTORC1 signaling pathway and suppressing oxidative stress. Additionally, Sac/Val treatment may mitigate Dox-induced apoptosis and inhibition of autophagy in primary cardiomyocytes. Conclusion Sac/Val seems to be cardio-protective against Dox-induced cardiotoxicity in pretreatment mice model. These findings could be attributed to the anti-inflammatory, antioxidant, anti-apoptotic and de-autophagy effects of Sac/Val through regulation of the AMPKα-mTORC1 signaling pathway. Doxorubicin cardiotoxicity sacubitril/valsartan apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Recent advancements in targeted therapies and enhanced screening techniques have shown promising results in improving cancer prognosis. Despite the effectiveness of anthracycline chemotherapy as a fundamental component of cancer treatment, its utilization has been associated with a higher incidence of cancer therapy-related cardiac dysfunction (CTRCD), characterized by a significant decrease in left ventricular ejection fraction (LVEF) of at least 10% or a reduction in LVEF to less than 50% 1 . In a study involving 2625 patients who received anthracycline treatment, with a median follow-up time of 5.2 years post-chemotherapy, the incidence of chemotherapy-related cardiac dysfunction (CTRC) was found to be 9% 2 . Among a total of 12,500 breast cancer patients, the cumulative rates of CTRCD at the first and fifth years were 1.20% and 4.30%, respectively, in patients treated with anthracycline alone, as opposed to 6.20% and 20.10%, respectively, in patients who received a combination regimen of anthracycline and trastuzumab 3 . According to a retrospective study from Thailand, patients treated with anthracycline and trastuzumab had a higher risk profile for CTRCD 4 . Myocardial ultrastructural abnormalities, accompanied by irreversible cardiac dysfunction, were identified as the predominant evidence of anthracycline-induced cardiotoxicity. The main mechanism underlying this phenomenon was attributed to oxidative stress damage 5 . Over the past six decades since the discovery of anthracyclines, significant attention has been devoted to basic science and clinical trials research investigating both its antitumour effects and cardiotoxic mechanisms. A study found that sixty-four percent of patients with chemotherapy-related cardiotoxicity (CTRCD) who were treated with anthracyclines and initiated therapy with enalapril and carvedilol within 1–2 months of detecting left ventricular ejection fraction (LVEF) impairment experienced complete or partial recovery, in contrast to a lack of response when treatment was delayed until 6 months later 6 . Patients with heart failure and reduced ejection fraction (HFrEF) have significantly benefited from the introduction of angiotensin receptor-neprilysin inhibitor (ARNI). The distinctive dual neuroendocrine regulatory mechanism of sacubitril/valsartan (Sac/Val) is characterized by the inhibition of neprilysin by LBQ657, which leads to the augmentation of natriuretic peptide levels, and the inhibition of the renin-angiotensin-aldosterone system by valsartan through the blockade of angiotensin II type 1 receptors (AT1R) 7 . In comparison to angiotensin-converting enzyme inhibitors (ACEIs), Sac/Val has demonstrated superior efficacy in reducing cardiovascular mortality and hospitalizations due to heart failure in patients with heart failure 8 . Although cancer patients were not initially excluded from enrollment in the PARADIGM-HF study, they were ultimately not enrolled 9 . Patients with anthracycline-related cardiomyopathy who were treated with Sac/Val showed improvements in cardiac function and NT-proBNP levels, consistent with findings from previous observational studies 10 . Prior research has demonstrated the potential of Sac/Val to mitigate doxorubicin-induced cardiotoxicity and enhance cardiac function 11 – 15 . However, the precise molecular mechanisms responsible for these beneficial effects remain unclear. In this study, we aimed to investigate whether prophylactic treatment with Sac/Val could preserve cardiac function in a mouse model of Dox-induced cardiomyopathy through modulation of the AMPK-mTORC1 signaling pathway. 2. Materials and methods 2.1. Ethics Statements In this study, male C57BL/6N mice were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd (Beijing, China). All animal experiments were conducted in accordance with the National Institutes of Health (NIH) policies outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Fujian Medical University Union Hospital (2024KJT011). 2.2. Mice model of DOX‑induced cardiomyopathy Doxorubicin hydrochloride was purchased from Sigma-Aldrich (#D1515) and Sac/Val complex was purchased from Novartis Pharma AG Co., Ltd. Thirty-two mice were randomly divided into four groups (n = 8) as shown in Table 1 ; the phosphate buffer saline (PBS); Saline group (CONTROL), PBS; Sac/Val group (Sac/Val), Dox; Saline group (DOX), and Dox; Sac/Val group (Sac/Val + DOX). The mice in the CONTROL and DOX groups were administered an equal volume of perioral 0.9% saline for 42 days. The mice in the Sac/Val and Sac/Val + DOX groups were administered perioral Sac/Val (80 mg/kg sacubitril + valsartan 1/1 complex) 12 in 0.9% saline by gavage for 42 days. On the seventh, fourteenth, 21st day in the DOX and Sac/Val + DOX group, one hour after the perioral saline administration, single dose intraperitoneal (IP) Dox [8 mg/(kg.wk)] was administered 16 . In mouse chronic heart failure in vivo models, cumulative doses of Dox up to 24 mg/kg have been shown to cause left ventricular systolic dysfunction 16 – 18 . Correspondingly, on the seventh, fourteenth, 21st day in the Control group, an equal volume of PBS was IP injected in the CONTROL and Sac/Val group. Table 1 Study protocol. Groups 1-6th day 7th day 8-13th day 14th day 15-20th day 21st day 22-42nd day PBS; Saline group Saline (PO) Saline (PO) PBS (IP) Saline (PO) Saline (PO) PBS (IP) Saline (PO) Saline (PO) PBS (IP) Saline (PO) PBS; Sac/Val group Sac/Val (PO) Saline (PO) PBS (IP) Sac/Val (PO) Saline (PO) PBS (IP) Sac/Val (PO) Saline (PO) PBS (IP) Sac/Val (PO) Dox; Saline group Saline (PO) Saline (PO) Doxorubicin (IP) Saline (PO) Saline (PO) Doxorubicin (IP) Saline (PO) Saline (PO) Doxorubicin (IP) Saline (PO) Dox; Sac/Val group Sac/Val (PO) Saline (PO) Doxorubicin (IP) Sac/Val (PO) Saline (PO) Doxorubicin (IP) Sac/Val (PO) Saline (PO) Doxorubicin (IP) Sac/Val (PO) Note: Doses of treatment: 0.9% saline PO; Sac/Val PO (80 mg/kg); doxorubicin IP [8 mg/(kg.wk)]. Dox - doxorubicin, IP - intraperitoneal, PO - perioral, Sac/Val - sacubitril/valsartan. All analyses were conducted after a 42-day period. The mice were anesthetized via intraperitoneal injection of sodium pentobarbital (50 mg/kg) and subsequently euthanized through bloodletting. Following euthanization, the heart was perfused and certain tissues were preserved in 4% paraformaldehyde for histological examination. The remaining tissues were promptly frozen for subsequent expression analysis. 2.3. Echocardiography Transthoracic echocardiography was conducted utilizing a 30-MHz linear array ultrasound transducer (MS-400, VisualSonics Inc.) while administering 2% isoflurane. The papillary muscles were visualized through M-mode echocardiography using a short-axis view of the parasternal aspect of the heart. Left ventricular (LV) internal diameters were assessed during both diastole and systole, with left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) being automatically computed. A tissue Doppler ultrasound was used to measure the E′ and A′ peaks inside the mitral valve orifice. The E′/A′ ratio was calculated as an indirect measure of diastolic function. 2.4. Histological studies Following euthanasia of the rats, one-half of each heart ventricle was preserved in formalin and subsequently embedded in paraffin. Hematoxylin and eosin (H&E) as well as Masson's trichrome staining were utilized to evaluate myocardial morphology and inflammation in consecutive 4 mm thick tissue sections. Semi-quantitative analysis of the stained tissues was conducted using Image-Pro plus 6.0 software under a light microscope (Olympus, Japan). 2.5. Immunohistochemistry Antigens were extracted from paraffin-embedded cardiac sections utilizing EDTA antigen retrieval buffer (pH 8.0) following deparaffinization and rehydration procedures. Subsequently, a 3% bovine serum albumin block was administered to rehydrated slides for a duration of 30 minutes, followed by the application of P62 rabbit antibody (#ab109012, Abcam, 1:400) overnight at 4°C. The slides were then subjected to incubation with a secondary goat anti-rabbit antibody (#5220 − 0336, SeraCare Inc., USA, 1:200) in conjunction with avidin-biotin complex and horseradish peroxidase subsequent to PBS washing. 2.6. Cardiomyocyte size staining To assess cardiomyocyte size, rehydrated slides were treated with 3% bovine serum albumin for 30 minutes and subsequently incubated overnight at 4°C with FITC-conjugated wheat germ agglutinin (WGA) (#L4895, Sigma, USA). Cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, #C0065, Solarbio, Beijing), and fluorescent microscopes (Olympus, Tokyo) were employed for visualization of the stained sections. 2.7. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay Apoptotic cardiomyocytes in paraffin sections were identified using a TUNEL detection kit (#6432344001, Roche, USA) under a light microscope (Leica DM 4000 B; Leica, Wetzlar, Germany). Myocardial cytoskeleton colocalization with the anti-actin antibody (#23660-1-AP, Proteintech, Wuhan, 1:100) was observed. Slides were then treated with the goat anti-rabbit secondary antibody (FITC conjugate, #SA00003-2, Proteintech, Wuhan, 1:100). Four regions were randomly selected from each digitized image, and the number of apoptotic and healthy nuclei was quantified. The apoptotic index was calculated as the number of TUNEL-positive nuclei/total number of nuclei 14 19 . 2.8. Evaluation of electron microscopy The fixation process involved prefixing 1 mm 3 heart tissues for 4 hours at 4 degrees Celsius with 2.5% glutaraldehyde immediately following tissue extraction from the left ventricle. Subsequently, the tissues were fixed at room temperature in 1% osmium tetroxide after rinsing with PBS. Dehydrated sections were then cut on an ultramicrotome (Leica UC 7, Leica) and stained with lead citrate and uranyl acetate. The ultrastructure of the autophagic vacuoles was examined using transient electron microscopy (TECNAI G2 20 TWIN, FEI), which is considered the gold standard for analyzing autophagy. 2.9. Detection of superoxide production Dihydroethidium (DHE, #810253P, Sigma-Aldrich) staining was employed on frozen LV tissue (4 µm sections) to assess superoxide production. Fluorescence was detected using a fluorescent microscope (Olympus, Tokyo) with excitation and emission wavelengths of 488 nm and 568 nm, respectively. 2.10. Cell culture It was described previously that Neonatal Sprague-Dawley rats (SD) were used to isolate ventricular myocytes (NRVMs) 19 . For Sac/Val pre-treatment following Dox-induced cardiomyocyte toxicity, NRVMs were pre-treated with 10 µM and 20 µM each of valsartan and LCZ696 for 12 h and then treated with 5 µM of DOX for 6 h 15 . Similar to animal experimental classification, an equal volume of PBS was incubated with NRVMs for 12 h in the CONTROL and Sac/Val group. 2.11. Invitro ROS production measurement Reactive oxygen species (ROS) were quantified utilizing a 2′,7′-Dichlorofluorescin diacetate (DCF-DA) reagent (35845, Sigma, USA) in this study. Various reagents were administered to neonatal rat ventricular myocytes (NRVMs) cultured in six-well plates for the duration of 18 hours. Following a 30-minute incubation at 37°C, the culture medium was replaced with serum-free medium containing 10 µm DCF-DA 19 . Fluorescent intensity was measured using excitation/emission wavelengths of 488/525 nm on a flow cytometer. 2.12. Cell viability detection Cell viability was assessed using a Cell Counting Kit-8 (CCK-8, #CA1210, Solarbio, China) by incubating 10 mol CCK-8 solutions with NRVMs for one hour under standard incubation conditions. Viability was quantified by measuring the relative optical density of treated cells compared to untreated controls using a microplate reader (BioRad, USA). 2.13. Fluorescence‑activated cell sorting (FACS) analysis Fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) were utilized for the identification of apoptotic cells through the application of an apoptosis detection kit (KGA108, KeyGEN BioTECH, China) following the manufacturer's instructions 19 . NRVMs were resuspended in binding buffer and subsequently incubated with FITC-annexin V and PI at room temperature for approximately ten minutes. Fluorescence measurements were conducted using a flow cytometer (BD Biosciences) equipped with a FACS flow cytometer (BD Biosciences). 2.14. Real time quantitative PCR (RT-qPCR) The Trizol reagent (Invitrogen, Carlsbad, CA) was employed for total RNA extraction, followed by cDNA synthesis using the Prime Script RT reagent kit (Takara). Real-time qPCR was conducted using the StepOnePlus Real-Time PCR System (Applied Biosystems) in this study. Supplementary Table 1 provides a comprehensive list of primer sequences utilized for assessing relative gene expression levels with GAPDH serving as the reference gene. 2.15. Western blot analysis Radiation immunoprecipitation (RIPA) buffer (#R0010, Solarbio, Beijing) was utilized for the homogenization and lysis of heart tissue or NRVMs prior to electrotransfer onto PVDF membranes (Millipore, USA). Subsequently, the membranes were blocked in TBST buffer and incubated with primary antibodies (Supplementary Table 2) overnight at -4°C. Immunoreactive bands were then detected by incubating with a secondary antibody (Boster, Wuhan, China) conjugated with horseradish peroxidase (HRP) and visualized using a chemiluminescence system (ECL, GE Healthcare Bio-Sciences). 2.16. Statistical analysis Analysis of Variance (ANOVA) was employed for the examination of data involving multiple comparisons, with post hoc tests such as the Least Significant Difference (LSD) test assuming equal variances, and Dunnett's T3 test in cases where equal variances were not assumed. The study utilized Graphpad Prism 8.0 software (GraphPad Software Inc., CA, USA) and SPSS version 26 (IBM, Armonk, New York) for statistical analysis. A significance level of P < 0.05 was deemed statistically significant. 3. Results 3.1. Sac/Val treatment inhibited myocardium inflammation In comparison to the control groups, mice with Dox-induced cardiotoxicity demonstrated inflammatory cell infiltration in the myocardium as observed through H&E staining (Fig. 1 A). Mononuclear macrophages were the predominant cell type infiltrating the myocardial interstitium. Treatment with Sac/Val was shown to decrease this inflammatory infiltration in the myocardium. The mRNA levels of inflammatory cytokines, including IL-1β, IFN-γ, TNF-α, and MCP-1, were significantly elevated in the myocardium of mice with Dox-induced cardiotoxicity in the DOX groups compared to the CONTROL group (p < 0.01 for all comparisons, Fig. 1 B-E). The mRNA levels of the aforementioned inflammatory cytokines were notably reduced in the Sac/Val + DOX group compared to the DOX group (p < 0.05 for all comparisons, as shown in Fig. 1 B-E). Additionally, western blot analysis revealed an increase in the protein expression of NLRP3 and Caspase-1 in the myocardium of mice with Dox-induced cardiotoxicity compared to the CONTROL group, with Sac/Val treatment partially restoring the protein expression of NLRP3 and Caspase-1 in the cardiotoxic hearts (Fig. 2 A-D). These findings suggested that Sac/Val treatment could ameliorate Dox-induced cardiotoxicity by the deregulation of myocardial inflammation. 3.2. Sac/Val treatment inhibited cardiac fibrosis In comparison to the control groups, mice with Doxorubicin-induced cardiotoxicity displayed notable collagen matrix deposition in the myocardium as evidenced by Masson's trichrome staining. Subsequent treatment with Sacubitril/Valsartan (Sac/Val) was found to mitigate this myocardial fibrosis (see Fig. 3 A-B). Additionally, western blot analysis revealed that the protein expression of Collagen I in the myocardium of Doxorubicin-induced cardiotoxic mice was elevated compared to the control groups, with Sac/Val treatment partially restoring Collagen I protein expression in the cardiotoxic hearts (see Fig. 3 C-D). The mRNA levels of fibrotic factors, including α-SMA, Collagen I, and Collagen III, were found to be significantly elevated in the myocardium of mice with Dox-induced cardiotoxicity in the DOX groups compared to the CONTROL group (p < 0.01 for all comparisons, Fig. 2 E-G). Conversely, the mRNA levels of these fibrotic factors were significantly reduced in the Sac/Val + DOX group compared to the DOX group (p < 0.05 for all comparisons, Fig. 3 E-G). These results indicated that Sac/Val treatment may mitigate Dox-induced cardiotoxicity by suppressing cardiac fibrosis. 3.3. Sac/Val treatment inhibited cardiomyocyte hypertrophy In comparison to the control groups, mice with Dox-induced cardiotoxicity exhibited a significantly larger cardiomyocyte size in the myocardium as indicated by WGA staining (Fig. 4 A-B ) and an increased ratio of heart weight to body weight ( Fig. 4 C). Treatment with Sac/Val was found to mitigate this cardiomyocyte hypertrophy and heart weight to body weight ratio (Fig. 4 A-C). The mRNA levels of cardiac fetal reactivation genes, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (MHC), were found to be significantly elevated in the myocardium of mice with Dox-induced cardiotoxicity in the DOX groups compared to the CONTROL group (P < 0.05 for all comparisons, Fig. 4 D-F). Conversely, the mRNA levels of these cardiomyocyte hypertrophy factors were significantly reduced in the Sac/Val + DOX group compared to the DOX group (p < 0.05 for all comparisons, Fig. 4 D-F). These findings suggested that Sac/Val treatment could ameliorate Dox-induced cardiotoxicity by the inhibiting myocardial hypertrophy. 3.4. Sac/Val treatment improved myocardial apoptosis In comparison to the control groups, the myocardium of mice with Dox-induced cardiotoxicity exhibited increased protein expression of pro-apoptotic bax, as well as a notable increase in cleaved caspase-3 and cleaved caspase-9, and a significant decrease in anti-apoptotic bcl-2 (Fig. 5 A-F). Subsequent treatment with Sac/Val partially reversed the alterations in expression of apoptosis-related proteins in the myocardium of Dox-induced cardiotoxicity mice (Fig. 5 A-F). Dox-induced cardiotoxicity mice showed a higher proportion of apoptosis in cardiac myocytes according to TUNEL staining as compared to that in controls, which was partly reversed by Sac/Val treatment (Fig. 6 A /B ). These findings suggested that Sac/Val treatment improved myocardial apoptosis in Dox-induced cardiotoxicity mice. 3.5. Sac/Val treatment promoted myocardial autophagy To determine the effects of Sac/Val treatment on Dox-induced myocardial lessened autophagy, we performed immune-histochemical analysis of P62 protein to assess cardiomyocyte autophagy. Dox-induced cardiotoxicity mice showed a higher proportion of P62 positive cells as shown by immunohistochemistry as compared to that in controls, which was ameliorated by Sac/Val treatment (Fig. 7 A /B ). Western blotting showed that compared to the control arms, the autophagy related protein expression of P62 increased significantly, of ULK1 and LC3-II decreased obviously in the myocardium of Dox-induced cardiotoxicity mice, which was also reversed by T Sac/Val treatment (Fig. 7 C-G). The ultrastructural morphologies of the hearts were observed by transmission electron microscopy. The Dox-induced cardiotoxicity mice showed a lower proportion of autophagic-like vesicles as compared to that in controls, which was reversed by Sac/Val treatment ( Supplementary Fig. 1A/B ). These findings suggested that Sac/Val treatment promoted myocardial lessened autophagy in Dox-induced cardiotoxicity mice. 3.6. Sac/Val treatment improved systolic and diastolic function Echocardiographic analysis of cardiac function in mice treated with Doxorubicin revealed a reduction in left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), indicating a deterioration in cardiac systolic function compared to control mice. This impairment was mitigated by treatment with Sacubitril/Valsartan (Sac/Val), as shown in Fig. 8 A /B . Additionally, a significant decrease in the ratio of E verus A and E' verus A', indicative of worsened cardiac diastolic function, was observed in Doxorubicin-treated mice. This diastolic dysfunction was reversed by Sac/Val treatment, as illustrated in Fig. 8 C-F. 3.7. Changes in AMPKα-mTORC1 signaling pathway Mice with Dox-induced cardiotoxicity demonstrated increased protein expression of p-AMPKα in heart homogenates compared to controls, a change that was reversed by Sac/Val treatment (Fig. 9 A-B). Additionally, these mice exhibited decreased protein expression levels of p-mTOR(Ser2448), Raptor, p-S6K1(Thr389), and p-4EBP1(Thr37/46) in heart homogenates compared to controls, which were also reversed by Sac/Val treatment (Fig. 9 C-G). Besides, Dox-induced cardiotoxicity mice exhibited an increased oxidative stress level in myocardium according to DHE staining as compared to that in controls, which was partly reversed by Sac/Val treatment ( Supplementary Fig. 2A/B ). 3.8. Sac/Val treatment defended against Dox–induced apoptosis and autophagy inhibition in primary cardiomyocytes In comparison to the control group, the viability of cardiomyocytes exhibited a significant decrease following stimulation with Dox, a decrease that was partially ameliorated by treatment with Sac/Val (Fig. 10 A). Similarly, the apoptosis level of primary cardiomyocytes significantly increased after exposure to Dox, but was partially mitigated by Sac/Val treatment (Fig. 10 B /C ). Additionally, the protein expression levels of cleaved Caspase-3 increased significantly, while the ratio of Bcl-2/Bax decreased in primary cardiomyocytes following Dox stimulation, with partial restoration observed after treatment with Sac/Val (Fig. 10 D /F/G ). The protein expression levels of P62 significantly increased and LC3-II significantly decreased in primary cardiomyocytes after Dox stimulation, compared to controls. This effect was partially reversed by Sac/Val treatment, as shown in Fig. 10 E /H/I . These findings indicate that Sac/Val treatment may partially restore the Dox-induced apoptosis and autophagy inhibition in primary cardiomyocytes. 3.9. Sac/Val treatment defended against Dox–induced apoptosis and autophagy inhibition in primary cardiomyocytes via regulating the AMPKα-mTORC1signaling pathway As illustrated in Fig. 11 (A/B) , the levels of reactive oxygen species (ROS) in primary cardiomyocytes significantly increased following 24 hours of Dox stimulation compared to controls, a change that was mitigated by prior incubation with Sac/Val. Additionally, Dox stimulation resulted in an elevated protein expression of p-AMPKα in primary cardiomyocytes, an effect that was partially attenuated by treatment with Sac/Val (Fig. 11 C /D ). In comparison to the control group, the protein expression levels of p-mTOR (Ser2448), Raptor, p-S6K1 (Thr389), and p-4EBP1 (Thr37/46) were notably reduced following Dox stimulation in primary cardiomyocytes, with partial restoration observed after Sac/Val treatment (Fig. 11 E-I). These findings indicate that Sac/Val treatment may mitigate Dox-induced apoptosis and autophagy inhibition in primary cardiomyocytes by modulating the AMPKα-mTORC1 signaling pathway. 4. Discussion As advancements in cancer treatment lead to increased survival rates, the long-term cardiovascular side effects of chemotherapy, particularly anthracyclines used in the treatment of various cancers such as breast cancer, have become a significant concern due to their dose-dependent cardiotoxicity 1 . To effectively address the development of chemotherapy-related cardiotoxicity (CTRCD), it is imperative to gain a comprehensive understanding of the underlying mechanisms. Among the various factors contributing to anthracycline-induced cardiotoxicity, lipid peroxidation of the cell membrane emerges as a primary cause. The generation of reactive oxygen species through iron-dependent pathways is identified as the predominant source of anthracycline-induced cardiotoxicity 5 . Research indicates that inhibition of anthracycline exacerbates the production of reactive oxygen species and disrupts mitochondrial biogenesis 20 . Willis et al 21 demonstrated subacute anthracycline-induced myocyte atrophy in both mice and humans. MuRF1 (muscle-specific ubiquitin ligase muscle ring finger-1) was required for doxorubicin-induced cardiac atrophy in mice. In this study, we examined the prophylactic properties of Sac/Val in mitigating Dox-induced cardiotoxicity and elucidated the potential underlying mechanisms. Our findings demonstrate that pre-treatment with Sac/Val can mitigate myocardial inflammation, fibrosis, and apoptosis, while promoting autophagy and improving heart function in mice with Dox-induced cardiotoxicity through modulation of the AMPKα-mTORC1 signaling pathway. These results offer a molecular rationale for the inhibition of apoptosis by Sac/Val via regulation of the AMPKα-mTORC1 signaling pathway in the context of Dox-induced cardiotoxicity. In the context of cardio-oncology, treatment-induced cardiotoxicity poses a significant risk to patient health. Sac/Val regimens are recommended for managing this complication. A retrospective case study documented successful outcomes with Sac/Val treatment in two individuals with anthracycline-related cardiomyopathy and heart failure with reduced ejection fraction (HFrEF) who had previously shown poor responses to conventional evidence-based medications 22 . Both patients experienced improvements in heart failure symptoms, normalization of NT-proBNP levels, and avoided rehospitalization for their condition 22 . In their study, Canale et al . 23 presented a case series involving four patients diagnosed with cancer therapy-related cardiac dysfunction (CTRCD) and severe heart failure with reduced ejection fraction (HFrEF). The patients received Sacubitril/Valsartan (Sac/Val) treatment while wearing an automatic defibrillator until their cardiac function normalized. A subsequent study conducted by researchers in six Spanish hospitals with specialized cardio-oncology clinics followed up on 67 cancer survivors, the majority of whom had received anthracycline-based chemotherapy 24 . Among patients with HFrEF, Sac/Val therapy was found to be well-tolerated and associated with improvements in NT-proBNP levels, NYHA functional class, and echocardiographic findings 24 . Renato et al. 25 reported anthracycline cardiomyopathy was treated with Sac/Val in two clinical cases where symptoms and echocardiographic parameters improved in response to the treatment. Ana et al. 26 evaluated ten consecutive patients suffering from cardiotoxicity-related HFrEF were evaluated by comprehensive multiparametric cardiac magnetic resonance (CMR) for the therapeutic effect of Sac/Val. When Sac/Val was administered, LV volumes were markedly reduced and LVEF was significantly improved. NYHA functional class also improved in association with a marked decrease in NT-proBNP concentration 26 . LV dysfunction within CTRCD is partly restorable, but this strongly dependeds on timely treatment with Sac/Val 26 . After failing to respond to conventional evidence-based drug therapy, Sac/Val was introduced to 28 patients with breast cancer and refractory cardiotoxicity-related HFrEF 27 . The NYHA cardiac function grade, six-minute walking distance, LVEF, LV diastolic function, LV end-diastolic diameter, and mitral regurgitation assessment significantly improved after captopril or valsartan was replaced with ARNI. While several small observational studies have found that Sac/Val improves cardiac structure and function in CTRCD patients, large-scale prospective clinical trials are needed to confirm these findings. Studies on the efficacy of Sac/Val in mitigating doxorubicin-induced cardiotoxicity in animal experimental models are limited. Following administration of doxorubicin, mice exhibited impaired heart function, abnormal mitochondrial structure, and compromised respiratory function, all of which were significantly improved with Sac/Val treatment 11 . Additionally, it is suggested that sacubitril/valsartan may enhance dynamin-related protein 1 (Drp1)-mediated mitochondrial dysfunction caused by doxorubicin 11 . In a preclinical model of prophylactic treatment, Sacubitril/Valsartan demonstrated efficacy in mitigating oxidative stress damage, inflammation, and apoptosis associated with doxorubicin-induced heart failure and arrhythmia 12 . Furthermore, compared to doxorubicin alone, Sacubitril/Valsartan attenuated matrix metalloproteinase (MMP) activity in rats, thereby safeguarding against doxorubicin-induced cardiac systolic dysfunction and left ventricular remodeling 13 . Rats administered with Sacubitril/Valsartan exhibited significant amelioration of doxorubicin-induced cardiac dysfunction through the downregulation of endoplasmic reticulum stress and apoptosis-related proteins 14 . The mitigation of cardiotoxicity induced by doxorubicin in rat hearts and H9C2 cardiomyocytes was achieved through the reduction of oxidative stress by Sac/Val 15 . These findings suggest that the cardiotoxic effects of doxorubicin may have been attenuated by the anti-inflammatory, anti-apoptotic, and antioxidant properties of Sac/Val. Based on the data presented, it is suggested that the cardiotoxic effects induced by DOX may have been mitigated by the anti-inflammatory, anti-apoptotic, and antioxidant properties 28 . Activation of autophagy at a moderate level may support cellular energy and nutrient provision, thereby safeguarding cardiomyocytes 28 . Notably, our findings demonstrate for the first time that Sac/Val treatment could ameliorate autophagy suppression in mice with DOX-induced cardiotoxicity. AMPK, a heterotrimeric enzyme, plays a crucial role in regulating cardiac energy homeostasis 29 . The serine/threonine-specific protein kinase, mTOR, consists of two distinct multi-complexes, mTORC1, which is involved in regulating cardiac autophagy in response to oxidative stress 19 . The deficiency of soluble epoxide hydrolase has been shown to reduce myocardial lipid accumulation by enhancing AMPK-mTORC mediated autophagy 29 . Our study demonstrates that Sac/Val exhibits cardioprotective effects against Dox-induced cardiotoxicity through its anti-apoptotic and de-autophagy properties, which are mediated by the regulation of the AMPKα-mTORC1 signaling pathway. This study is subject to several limitations. Future research is required to establish the generalizability of the findings in a murine model to human subjects. Moreover, a deeper understanding of anthracycline-induced cardiomyopathy could be achieved through experimentation with human cardiomyocyte models. Additionally, the lack of comparison between Sac/Val and valsartan in the experiments prevents a definitive conclusion regarding whether the beneficial effects of Sac/Val are solely attributed to valsartan or if sacubitril also plays a role. 5. Conclusion This study advanced our understanding of the molecular pathways involved in anthracycline-induced cardiomyopathy. The administration of Sac/Val was shown to alleviate myocardial inflammation, fibrosis, apoptosis, enhance autophagy, and improve cardiac function in mice with Dox-induced cardiotoxicity by reducing oxidative stress and regulating the AMPKα-mTORC1 signaling pathway. Declarations Conflict of interest All authors declare that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Funding This study were supported by grants from the talent start-up capital program of Fujian Medical University Union Hospital (2023XH027), the science and technology innovation joint fund project of Fujian Provincial Science and Technology Department (2023Y9183), the Fujian Provincial Natural Science Foundation. Author Contribution (I) Conception and design: Weiwei Wang and Xinghe Lin; (II) Administrative support: Xinghe Lin; (III) Provision of study materials: Li Lin; (IV) Collection and assembly of data: Feng Hu and Xiaoxia Qiu; (V) Data analysis and interpretation: Senbo Yan; (VI) Manuscript writing: Feng Hu and Senbo Yan; (VII); Final approval of manuscript: All authors. Acknowledgements None Availability of data and material The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. References Michel L, Schadendorf D, Rassaf T (2020) Oncocardiology: new challenges, new opportunities. Herz 45:619–625 Cardinale D, Colombo A, Bacchiani G, Tedeschi I, Meroni CA, Veglia F, Civelli M, Lamantia G, Colombo N, Curigliano G, Fiorentini C, Cipolla CM (2015) Early detection of anthracycline cardiotoxicity and improvement with heart failure therapy. 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Heart Fail Rev 26:1159–1173 Cardinale D, Colombo A, Lamantia G, Colombo N, Civelli M, De Giacomi G, Rubino M, Veglia F, Fiorentini C, Cipolla CM (2010) Anthracycline-induced cardiomyopathy: clinical relevance and response to pharmacologic therapy. J Am Coll Cardiol 55:213–220 Docherty KF, Vaduganathan M, Solomon SD, McMurray JJV (2020) Sacubitril/Valsartan: Neprilysin Inhibition 5 Years After PARADIGM-HF. JACC Heart Fail 8:800–810 Zheng Y, Huang S, Xie B, Zhang N, Liu Z, Tse G, Liu T (2023) Cardiovascular Toxicity of Proteasome Inhibitors in Multiple Myeloma Therapy. Curr Probl Cardiol 48:101536 McMurray JJ, Packer M, Desai AS, Gong J, Lefkowitz MP, Rizkala AR, Rouleau JL, Shi VC, Solomon SD, Swedberg K, Zile MR (2014) Angiotensin-neprilysin inhibition versus enalapril in heart failure. N Engl J Med 371:993–1004 Duraes AR, de Souza Lima Bitar Y, Neto MG, Mesquita ET, Chan JS, Tse G, Liu T, Bocchi EA, Biondi-Zoccai G, Roever L (2022) Effectiveness of sacubitril-valsartan in patients with cancer therapy-related cardiac dysfunction: a systematic review of clinical and preclinical studies. Minerva Med 113:551–557 Xia Y, Chen Z, Chen A, Fu M, Dong Z, Hu K, Yang X, Zou Y, Sun A, Qian J, Ge J (2017) LCZ696 improves cardiac function via alleviating Drp1-mediated mitochondrial dysfunction in mice with doxorubicin-induced dilated cardiomyopathy. J Mol Cell Cardiol 108:138–148 Dindaş F, Güngör H, Ekici M, Akokay P, Erhan F, Doğduş M, Yılmaz MB (2021) Angiotensin receptor-neprilysin inhibition by sacubitril/valsartan attenuates doxorubicin-induced cardiotoxicity in a pretreatment mice model by interfering with oxidative stress, inflammation, and Caspase 3 apoptotic pathway. 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Sci Rep 12:4930 Asselin CY, Lam A, Cheung DYC, Eekhoudt CR, Zhu A, Mittal I, Mayba A, Solati Z, Edel A, Austria JA, Aukema HM, Ravandi A, Thliveris J, Singal PK, Pierce GN, Niraula S, Jassal DS (2020) The Cardioprotective Role of Flaxseed in the Prevention of Doxorubicin- and Trastuzumab-Mediated Cardiotoxicity in C57BL/6 Mice. J Nutr 150:2353–2363 Yu W, Qin X, Zhang Y, Qiu P, Wang L, Zha W, Ren J (2020) Curcumin suppresses doxorubicin-induced cardiomyocyte pyroptosis via a PI3K/Akt/mTOR-dependent manner. Cardiovasc diagnosis therapy 10:752–769 Nicol M, Sadoune M, Polidano E, Launay JM, Samuel JL, Azibani F, Cohen-Solal A (2021) Doxorubicin-induced and trastuzumab-induced cardiotoxicity in mice is not prevented by metoprolol. ESC Heart Fail 8:928–937 Hu F, Lin C (2024) TRPM2 knockdown attenuates myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice via regulating the MEK/ERK and mTORC1 signaling pathway. Mol Cell Biochem Zhang S, Liu X, Bawa-Khalfe T, Lu LS, Lyu YL, Liu LF, Yeh ET (2012) Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat Med 18:1639–1642 Willis MS, Parry TL, Brown DI, Mota RI, Huang W, Beak JY, Sola M, Zhou C, Hicks ST, Caughey MC, D'Agostino RB Jr., Jordan J, Hundley WG, Jensen BC (2019) Doxorubicin Exposure Causes Subacute Cardiac Atrophy Dependent on the Striated Muscle-Specific Ubiquitin Ligase MuRF1. Circulation Heart Fail 12:e005234 Sheppard CE, Anwar M (2019) The use of sacubitril/valsartan in anthracycline-induced cardiomyopathy: A mini case series. J Oncol Pharm practice: official publication Int Soc Oncol Pharm Practitioners 25:1231–1234 Canale ML, Coviello K, Solarino G, Del Meglio J, Simonetti F, Venturini E, Camerini A, Maurea N, Bisceglia I, Tessa C, Casolo G (2022) Case Series: Recovery of Chemotherapy-Related Acute Heart Failure by the Combined Use of Sacubitril Valsartan and Wearable Cardioverter Defibrillator: A Novel Winning Combination in Cardio-Oncology. Front Cardiovasc Med 9:801143 Martín-Garcia A, López-Fernández T, Mitroi C, Chaparro-Muñoz M, Moliner P, Martin-Garcia AC, Martinez-Monzonis A, Castro A, Lopez-Sendon JL, Sanchez PL (2020) Effectiveness of sacubitril-valsartan in cancer patients with heart failure. ESC Heart Fail 7:763–767 De Vecchis R, Paccone A (2020) A case series about the favorable effects of sacubitril/valsartan on anthracycline cardiomyopathy. SAGE open Med case Rep 8:2050313x20952189 Martín-García A, Díaz-Peláez E, Martín-García AC, Sánchez-González J, Ibáñez B, Sánchez PL (2020) Myocardial function and structure improvement with sacubitril/valsartan in cancer therapy-induced cardiomyopathy. Revista Esp de cardiologia (English ed) 73:268–269 Gregorietti V, Fernandez TL, Costa D, Chahla EO, Daniele AJ (2020) Use of Sacubitril/valsartan in patients with cardio toxicity and heart failure due to chemotherapy. Cardio-oncology (London England) 6:24 Ajoolabady A, Chiong M, Lavandero S, Klionsky DJ, Ren J (2022) Mitophagy in cardiovascular diseases: molecular mechanisms, pathogenesis, and treatment. Trends Mol Med 28:836–849 Wang L, Zhao D, Tang L, Li H, Liu Z, Gao J, Edin ML, Zhang H, Zhang K, Chen J, Zhu X, Wang D, Zeldin DC, Hammock BD, Wang J, Huang H (2021) Soluble epoxide hydrolase deficiency attenuates lipotoxic cardiomyopathy via upregulation of AMPK-mTORC mediated autophagy. J Mol Cell Cardiol 154:80–91 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableLegends.doc Supplementaryfigure2.tif Supplementary Figure 2: Detection of superoxide production according to dihydroethidium (DHE) staining in HFD/STZ-induced diabetic mice. (A) Representative DHE staining in the myocardium from the different groups (magnification =50x). (B) Corresponding statistic analysis of DHE staining in A (n=5 per group). Dox-induced cardiotoxicity mice exhibited an increased oxidative stress level in myocardium according to DHE staining as compared to that in controls, which was partly reversed by Sac/Val treatment. The data are represented as the means ± SE; *P < 0.05, **P < 0.01. Supplementaryfigure1.tif Supplementary Figure Legends Supplementary Figure 1: Autophagosomes detected by transmission electron microscopy in HFD/STZ-induced diabetic mice. (A) Representative transmission electron microscopy images of the left ventricular tissues from the different groups; arrows indicate autophagic vacuoles. (n=4 per group). (B) Quantitative analysis of the numbers autophagosomes in different groups. The data are represented as the means ± SE; *P < 0.05. Cite Share Download PDF Status: Published Journal Publication published 20 Sep, 2024 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted Editorial decision: Revision requested 12 Aug, 2024 Reviews received at journal 07 Aug, 2024 Reviewers agreed at journal 29 Jul, 2024 Reviewers invited by journal 04 Jul, 2024 Editor assigned by journal 04 Jul, 2024 Submission checks completed at journal 19 Jun, 2024 First submitted to journal 19 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4603884","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":324888707,"identity":"bf33018f-73a8-43fb-893a-3dabe42c4cb3","order_by":0,"name":"Feng Hu","email":"","orcid":"","institution":"Fujian Medical University Union Hospital, Fujian Cardiovascular Medical Center, Fujian Institute of Coronary Artery Disease, Fujian Cardiovascular Research Center","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Hu","suffix":""},{"id":324888709,"identity":"30976fb4-bdc8-4da5-80ee-ff7688d28238","order_by":1,"name":"Senbo Yan","email":"","orcid":"","institution":"Fujian Medical University Union Hospital, Fujian Cardiovascular Medical Center, Fujian Institute of Coronary Artery Disease, Fujian Cardiovascular Research Center","correspondingAuthor":false,"prefix":"","firstName":"Senbo","middleName":"","lastName":"Yan","suffix":""},{"id":324888710,"identity":"361e198c-4674-4a9a-94ab-e0a2a53b437f","order_by":2,"name":"Lin Li","email":"","orcid":"","institution":"Fujian Medical University Union Hospital, Fujian Cardiovascular Medical Center, Fujian Institute of Coronary Artery Disease, Fujian Cardiovascular Research Center","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Li","suffix":""},{"id":324888711,"identity":"73963a0b-b723-4786-88bf-bca93f7205cc","order_by":3,"name":"Xiaoxia Qiu","email":"","orcid":"","institution":"Fujian Medical University Union Hospital, Fujian Cardiovascular Medical Center, Fujian Institute of Coronary Artery Disease, Fujian Cardiovascular Research Center","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxia","middleName":"","lastName":"Qiu","suffix":""},{"id":324888712,"identity":"085348ee-0e28-44e3-8cf7-85f06263a037","order_by":4,"name":"Xinghe Lin","email":"","orcid":"","institution":"Fujian Medical University Union Hospital, Fujian Cardiovascular Medical Center, Fujian Institute of Coronary Artery Disease, Fujian Cardiovascular Research Center","correspondingAuthor":false,"prefix":"","firstName":"Xinghe","middleName":"","lastName":"Lin","suffix":""},{"id":324888713,"identity":"67fcdec6-5429-4c5f-813c-8725d0bec35b","order_by":5,"name":"Weiwei Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYFACxgYGBgMQg/nAgQ8VpGlhSzw44wxp1vEYH+ZtIUKdwfHmNokPBYfl5aN7PhzgbWCQ5xc7QEDLmYNtkjMMDhtuvHN2wwHJHQyGM2cn4NdidiOx7TaPwW3GjTNyNxwwPMOQYHCbkJb7D9tu/zG4bb9xRs6DA4ltxGi5wdh2m8HgduJ8iRyGAweJ0WJ/JrH9Z4/B/+QNEmkGBxvOSBD2i2T78ccGP/6k2c6fkfz4858KG3l+aQJa4MDgAJiSIFI5CMg3kKB4FIyCUTAKRhYAAMArUKTT+jQqAAAAAElFTkSuQmCC","orcid":"","institution":"Fujian Medical University Union Hospital, Fujian Cardiovascular Medical Center, Fujian Institute of Coronary Artery Disease, Fujian Cardiovascular Research Center","correspondingAuthor":true,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-06-19 07:11:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4603884/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4603884/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11010-024-05117-7","type":"published","date":"2024-09-20T15:57:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61350661,"identity":"9b41fd8b-8770-4032-831f-53b507b49b53","added_by":"auto","created_at":"2024-07-29 18:43:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11157193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInflammatory cell infiltration in the myocardium.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of inflammatory cell infiltration into the myocardium according to H\u0026amp;Estaining from the different groups (n=5 per group,magnification =200x). (B-E) The mRNA expression of inflammatory cytokines including IL-1β, IFN-γ, TNF-α and MCP-1, was determined by quantitative RT-PCR in the myocardium from the different groups (n=6 per group). The values were normalized to the housekeeping gene GAPDH. The data are represented as the means ± SE; * P \u0026lt;0.05, ** P \u0026lt;0.01, *** P \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/a5d2ce7606ed50ec1cda7bae.png"},{"id":61350056,"identity":"971d3e7f-400f-41d7-a59a-3796c14ab515","added_by":"auto","created_at":"2024-07-29 18:35:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":326087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMyocardium inflammation Dox-induced cardiotoxicity mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative western blot image of NLRP3 in the myocardium from the different groups. The α-tubulin was used as a loading control. (B) Corresponding densitometric analysis of blots in A (n=6 per group). (C) Representative western blot image of Caspase-1 in the myocardium from the different groups. The β-actin was used as a loading control. (D) Corresponding densitometric analysis of blots in C (n=6 per group). The data are represented as the means ± SE; * P \u0026lt;0.05, ** P \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/0ae8387ae531ccafb2a4d606.png"},{"id":61350658,"identity":"39376fed-0ddd-4c26-a5c7-0b87b1895e6c","added_by":"auto","created_at":"2024-07-29 18:43:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7153849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCardiac fibrosis in Dox-induced cardiotoxicity mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of collagen matrix deposition in the myocardium according to Masson’s trichrome staining from the different groups (magnification =50x). (B) Corresponding statistic analysis of cardiac fibrosis in A (n=5 per group). (C) Representative western blot image of Collagen I in the myocardium from the different groups. The GAPDH was used as a loading control. (D) Corresponding densitometric analysis of blots in C (n=6 per group). (E-G) The mRNA expression of fibrotic factors such as α-SMA, Collagen I and Collagen Ⅲ, was determined by quantitative RT-PCR in the myocardium from the different groups (n=4 per group). The values were normalized to the housekeeping gene GAPDH. The data are represented as the means ± SE; * P \u0026lt;0.05, ** P \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/318837722d16b292a8e74089.png"},{"id":61350059,"identity":"49b9a9ff-9caf-413c-8fe2-73110172a086","added_by":"auto","created_at":"2024-07-29 18:35:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4232832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCardiomyocyte hypertrophy in Dox-induced cardiotoxicity mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative staining images of wheat germ agglutinin (WGA) in mice hearts from the different groups. (B) Corresponding statistic analysis of cardiomyocyte size used WGA staining in A (n=5 per group). (C) The ratio of mouse heart weight versus body weight from the different groups (n=8 per group). (D-F) The mRNA expression of ANP, BNP and β-MHC detected by RT-qPCR in the myocardium from the different groups (n=6 per group). The values were normalized to the housekeeping gene GAPDH. The data are represented as the means ± SE; * P \u0026lt;0.05, ** P \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/d115a493fd06dea67d62302f.png"},{"id":61350058,"identity":"91ec8bdb-87c1-452a-a43d-b30fae89f14c","added_by":"auto","created_at":"2024-07-29 18:35:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":608977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe apoptosis related proteins in Dox-induced cardiotoxicity mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative western blot image of Bax and Bcl-2 in the myocardium from the different groups. (B) Corresponding densitometric analysis of blots in A (n=6 per group). (C) Representative western blot image of cleaved caspase-3 in the myocardium from the different groups. (D) Corresponding densitometric analysis of blots in C (n=6 per group). (E) Representative western blot image of cleaved caspase-9 in the myocardium from the different groups. (F) Corresponding densitometric analysis of blots in E (n=6 per group). The α-tubulin was used as a loading control. The data are represented as the means ± SE; * P \u0026lt;0.05, ** P \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/175964adba9f1b72a2bc7be5.png"},{"id":61350660,"identity":"ff1043a2-9f65-41b7-b428-031ea13bc1bc","added_by":"auto","created_at":"2024-07-29 18:43:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8294469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMyocardial apoptosis detected by the TUNEL staining.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of TUNEL staining in the myocardium from the different groups. The α-actin staining was used for colocalization of cardiomyocytes and DAPI was used for nuclear staining. (B) Corresponding statistic analysis of cardiomyocyte apoptosis in A (n=5 per group). The data are represented as the means ± SE; *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/4cf7afd2156b87a9d99dd97e.png"},{"id":61350659,"identity":"aa41f490-b21d-4f62-a32f-2be406df5c23","added_by":"auto","created_at":"2024-07-29 18:43:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7036720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSac/Val treatment promoted myocardial autophagy in Dox-induced cardiotoxicity mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunohistochemical analysis of autophagy substrates P62 protein in mice hearts from the different groups (magnification =200x). (B) Corresponding statistic analysis of P62 in A (n=5 per group). (C) Representative western blot image of P62 in the myocardium in each group. The α-tubulin was used as a loading control. (D) Corresponding densitometric analysis of blots in C (n=6 per group). (E) Heart homogenates were analyzed by western blot using an antibody against LC3 Ⅱ/Ⅰ and ULK1 proteins. The α-tubulin was used as a loading control. ((F/G) Corresponding densitometric analysis of blots in E (n=6 per group). The data are represented as the means ± SE; *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/fc8b7784654c79d750ba0ddd.png"},{"id":61350068,"identity":"3fc41df4-6f02-45e1-b4e0-a8ae5df6fb09","added_by":"auto","created_at":"2024-07-29 18:35:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2008946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCardiac function measured by echocardiography.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images by M-mode echocardiography in mice hearts from the different groups. (B) Echocardiography analysis showing cardiac systolic dysfunction assessed by LVEF and LVFS (n=5 per group). (C) The ratio of the peak mitral valve blood velocity during early diastolic period (E) verus peak mitral valve blood velocity during late diastolic period (A). (D) The diastolic dysfunction assessed by the ratio of E verus A (n=5 per group). (E) Representative images by pulsed wave Doppler echocardiography in mice hearts from the different groups. (F) The diastolic dysfunction assessed by ratio of diastolic mitral annulus velocities (E' verus A') (E) (n=5 per group). The data are represented as the means ± SE; *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/d489e81023a989eb79c5cb9d.png"},{"id":61350067,"identity":"0bfc1384-57e2-4dde-b256-914c9d766073","added_by":"auto","created_at":"2024-07-29 18:35:28","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":345184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in AMPKα-mTORC1 signaling pathway in Dox-induced cardiotoxicity mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative western blot analyses of p-AMPKα and AMPKα in mice heart homogenates from the different groups. (B) Corresponding densitometric analysis of blots in A (n=6 per group). (C) Representative western blot analyses of p-mTOR (Ser2448) and mTOR, Raptor, p-S6K1 (Thr389) and S6K1, and p-4EBP1 (Thr37/46) and 4EBP1 in miceheart homogenates from the different groups. (D-G) Corresponding densitometric analysis of blots in C (n=6 per group). The β-actin was used as a loading control. The data are represented as the means ± SE; *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/d23d1809941129eff29db4c6.png"},{"id":61350063,"identity":"e88ea66d-d479-446b-9175-246d4ca3efc0","added_by":"auto","created_at":"2024-07-29 18:35:28","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1063952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSac/Val treatment defended against Dox–induced apoptosis and autophagy inhibition in primary cardiomyocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor Sac/Val pre-treatment following Dox-induced cardiomyocyte toxicity, NRVMswere pre-treated with 10 μM and 20 μM each of valsartan and LCZ696 for 12 h and then treated with 5 µM of DOX for 6 h. An equal volume of PBS was incubated with NRVMs for 12 h in the CONTROL and Sac/Val group. (A) The cell viability was measured by cell counting kit-8. Compared to that in controls, the viability of cardiomyocytes significantly decreased after Dox stimulation, which was partially restored by Sac/Val treatment. (B/C) The apoptosis in different treatment groups was detected by flow cytometry and corresponding statistic analysis. Compared to that in controls, the apoptosis level of primary cardiomyocytes significantly increased after Dox stimulation, which was partially reversed by Sac/Val treatment. (D) Representative western blots of Bcl-2, Bax and cleaved Caspase-3 protein in primary cardiomyocytes from the different groups. The α-tubulin was used as a loading control. (E) Representative western blots of P62 and LC3 protein in primary cardiomyocytes from the different groups. The β-actin was used as a loading control. (F/G) Corresponding densitometric analysis of blots in D. Compared to that in controls, the protein expression level of cleaved Caspase-3 significantly increased, the ratio of Bcl-2/Bax significantly decreased, in primary cardiomyocytes after Dox stimulation, which was partially restored by Sac/Val treatment. (H/I) Corresponding densitometric analysis of blots in E. Compared to that in controls, the protein expression level of P62 significantly increased, of LC3-II significantly decreased in primary cardiomyocytes after Dox stimulation, which was partially reversed by Sac/Val treatment. The cell experiment was repeated independently for three times. The data are represented as the means ± SE; *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/78b3c731c55db01e86e34856.png"},{"id":61350065,"identity":"1f9a2826-bae1-45af-bc1e-1b02f52466bf","added_by":"auto","created_at":"2024-07-29 18:35:28","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":949851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSac/Val treatment defended against Dox–induced apoptosis and autophagy inhibition via regulating the AMPKα-mTORC1 signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A/B) Intracellular ROS production in primary cardiomyocytes was detected with DCF-DA reagent and corresponding statistic analysis. Compared to that in controls, ROS levels significantly increased after Dox stimulation for 24 hours in primary cardiomyocytes, which was decreased by pre-incubation with Sac/Val. (C/D) Representative blots of p-AMPKα and AMPKα in Dox-stimulated primary cardiomyocytes and corresponding densitometric analysis. The β-actin was used as a loading control. Dox stimulation exhibited an up-regulated protein expression level of p-AMPKα in primary cardiomyocytes, which was partly reversed by Sac/Val treatment. (E-I) Representative blots of p-mTOR (Ser2448), Raptor, p-S6K1 (Thr389), and p-4EBP1 (Thr37/46) in Dox-stimulated primary cardiomyocytes and corresponding densitometric analysis. The β-actin was used as a loading control. Compared to that in controls, the proteinexpression levels of p-mTOR (Ser2448), Raptor, p-S6K1 (Thr389), and p-4EBP1 (Thr37/46) were significantly decreased after Dox stimulation in primary cardiomyocytes, which was partly restored by Sac/Val treatment. The cell experiment was repeated independently for three times. The data are represented as the means ± SE; *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/5f868ac5cdb73625a444696d.png"},{"id":65104019,"identity":"dfc9cdd2-040e-4231-b625-6b7b64de6161","added_by":"auto","created_at":"2024-09-23 16:10:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":48083556,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/5548f532-4160-4a57-b18b-a27cfd86e98e.pdf"},{"id":61350055,"identity":"2dbabb92-a248-4517-841f-122acf3ec805","added_by":"auto","created_at":"2024-07-29 18:35:27","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":63488,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableLegends.doc","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/02eb2acbcb9927dc50cb8caa.doc"},{"id":61350061,"identity":"9c32aa51-9064-4c43-9fc0-9873465d86bd","added_by":"auto","created_at":"2024-07-29 18:35:28","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15182736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2:\u003c/strong\u003e \u003cstrong\u003eDetection of superoxide production according to dihydroethidium (DHE) staining in HFD/STZ-induced diabetic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative DHE staining in the myocardium from the different groups (magnification =50x). (B) Corresponding statistic analysis of DHE staining in A (n=5 per group). Dox-induced cardiotoxicity mice exhibited an increased oxidative stress level in myocardium according to DHE staining as compared to that in controls, which was partly reversed by Sac/Val treatment. The data are represented as the means ± SE; *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Supplementaryfigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/e2a03fc86bca255c9d50cbc1.tif"},{"id":61350062,"identity":"d7073b98-2d48-4746-ae32-75795e5dc7f0","added_by":"auto","created_at":"2024-07-29 18:35:28","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7879064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure Legends\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1:\u003c/strong\u003e \u003cstrong\u003eAutophagosomes detected by transmission electron microscopy in HFD/STZ-induced diabetic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative transmission electron microscopy images of the left ventricular tissues from the different groups; arrows indicate autophagic vacuoles. (n=4 per group). (B) Quantitative analysis of the numbers autophagosomes in different groups. The data are represented as the means ± SE; *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Supplementaryfigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4603884/v1/86727c3aba725fa3246d0563.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sacubitril/valsartan attenuated myocardial inflammation, fibrosis, apoptosis and promoted autophagy in doxorubicin-induced cardiotoxicity mice via regulating the AMPKα-mTORC1 signaling pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRecent advancements in targeted therapies and enhanced screening techniques have shown promising results in improving cancer prognosis. Despite the effectiveness of anthracycline chemotherapy as a fundamental component of cancer treatment, its utilization has been associated with a higher incidence of cancer therapy-related cardiac dysfunction (CTRCD), characterized by a significant decrease in left ventricular ejection fraction (LVEF) of at least 10% or a reduction in LVEF to less than 50% \u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn a study involving 2625 patients who received anthracycline treatment, with a median follow-up time of 5.2 years post-chemotherapy, the incidence of chemotherapy-related cardiac dysfunction (CTRC) was found to be 9% \u003csup\u003e2\u003c/sup\u003e. Among a total of 12,500 breast cancer patients, the cumulative rates of CTRCD at the first and fifth years were 1.20% and 4.30%, respectively, in patients treated with anthracycline alone, as opposed to 6.20% and 20.10%, respectively, in patients who received a combination regimen of anthracycline and trastuzumab \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. According to a retrospective study from Thailand, patients treated with anthracycline and trastuzumab had a higher risk profile for CTRCD \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMyocardial ultrastructural abnormalities, accompanied by irreversible cardiac dysfunction, were identified as the predominant evidence of anthracycline-induced cardiotoxicity. The main mechanism underlying this phenomenon was attributed to oxidative stress damage \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Over the past six decades since the discovery of anthracyclines, significant attention has been devoted to basic science and clinical trials research investigating both its antitumour effects and cardiotoxic mechanisms.\u003c/p\u003e \u003cp\u003eA study found that sixty-four percent of patients with chemotherapy-related cardiotoxicity (CTRCD) who were treated with anthracyclines and initiated therapy with enalapril and carvedilol within 1\u0026ndash;2 months of detecting left ventricular ejection fraction (LVEF) impairment experienced complete or partial recovery, in contrast to a lack of response when treatment was delayed until 6 months later \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Patients with heart failure and reduced ejection fraction (HFrEF) have significantly benefited from the introduction of angiotensin receptor-neprilysin inhibitor (ARNI).\u003c/p\u003e \u003cp\u003eThe distinctive dual neuroendocrine regulatory mechanism of sacubitril/valsartan (Sac/Val) is characterized by the inhibition of neprilysin by LBQ657, which leads to the augmentation of natriuretic peptide levels, and the inhibition of the renin-angiotensin-aldosterone system by valsartan through the blockade of angiotensin II type 1 receptors (AT1R) \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In comparison to angiotensin-converting enzyme inhibitors (ACEIs), Sac/Val has demonstrated superior efficacy in reducing cardiovascular mortality and hospitalizations due to heart failure in patients with heart failure \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although cancer patients were not initially excluded from enrollment in the PARADIGM-HF study, they were ultimately not enrolled \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Patients with anthracycline-related cardiomyopathy who were treated with Sac/Val showed improvements in cardiac function and NT-proBNP levels, consistent with findings from previous observational studies \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrior research has demonstrated the potential of Sac/Val to mitigate doxorubicin-induced cardiotoxicity and enhance cardiac function \u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, the precise molecular mechanisms responsible for these beneficial effects remain unclear. In this study, we aimed to investigate whether prophylactic treatment with Sac/Val could preserve cardiac function in a mouse model of Dox-induced cardiomyopathy through modulation of the AMPK-mTORC1 signaling pathway.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Ethics Statements\u003c/h2\u003e\n \u003cp\u003eIn this study, male C57BL/6N mice were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd (Beijing, China). All animal experiments were conducted in accordance with the National Institutes of Health (NIH) policies outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Fujian Medical University Union Hospital (2024KJT011).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Mice model of DOX‑induced cardiomyopathy\u003c/h2\u003e\n \u003cp\u003eDoxorubicin hydrochloride was purchased from Sigma-Aldrich (#D1515) and Sac/Val complex was purchased from Novartis Pharma AG Co., Ltd. Thirty-two mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;8) as shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; the phosphate buffer saline (PBS); Saline group (CONTROL), PBS; Sac/Val group (Sac/Val), Dox; Saline group (DOX), and Dox; Sac/Val group (Sac/Val\u0026thinsp;+\u0026thinsp;DOX). The mice in the CONTROL and DOX groups were administered an equal volume of perioral 0.9% saline for 42 days. The mice in the Sac/Val and Sac/Val\u0026thinsp;+\u0026thinsp;DOX groups were administered perioral Sac/Val (80 mg/kg sacubitril\u0026thinsp;+\u0026thinsp;valsartan 1/1 complex) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e in 0.9% saline by gavage for 42 days. On the seventh, fourteenth, 21st day in the DOX and Sac/Val\u0026thinsp;+\u0026thinsp;DOX group, one hour after the perioral saline administration, single dose intraperitoneal (IP) Dox [8 mg/(kg.wk)] was administered \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In mouse chronic heart failure \u003cem\u003ein vivo\u003c/em\u003e models, cumulative doses of Dox up to 24 mg/kg have been shown to cause left ventricular systolic dysfunction \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Correspondingly, on the seventh, fourteenth, 21st day in the Control group, an equal volume of PBS was IP injected in the CONTROL and Sac/Val group.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStudy protocol.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1-6th day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e7th day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e8-13th day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e14th day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e15-20th day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e21st day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e22-42nd day\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePBS; Saline group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) PBS (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) PBS (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) PBS (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePBS; Sac/Val group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSac/Val (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) PBS (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSac/Val (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) PBS (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSac/Val (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) PBS (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSac/Val (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDox; Saline group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) Doxorubicin (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) Doxorubicin (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) Doxorubicin (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDox; Sac/Val group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSac/Val (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) Doxorubicin (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSac/Val (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) Doxorubicin (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSac/Val (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaline (PO) Doxorubicin (IP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSac/Val (PO)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"8\"\u003e\n \u003cp\u003eNote: Doses of treatment: 0.9% saline PO; Sac/Val PO (80 mg/kg); doxorubicin IP [8 mg/(kg.wk)].\u003c/p\u003e\n \u003cp\u003eDox - doxorubicin, IP - intraperitoneal, PO - perioral, Sac/Val - sacubitril/valsartan.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAll analyses were conducted after a 42-day period. The mice were anesthetized via intraperitoneal injection of sodium pentobarbital (50 mg/kg) and subsequently euthanized through bloodletting. Following euthanization, the heart was perfused and certain tissues were preserved in 4% paraformaldehyde for histological examination. The remaining tissues were promptly frozen for subsequent expression analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Echocardiography\u003c/h2\u003e\n \u003cp\u003eTransthoracic echocardiography was conducted utilizing a 30-MHz linear array ultrasound transducer (MS-400, VisualSonics Inc.) while administering 2% isoflurane. The papillary muscles were visualized through M-mode echocardiography using a short-axis view of the parasternal aspect of the heart. Left ventricular (LV) internal diameters were assessed during both diastole and systole, with left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) being automatically computed. A tissue Doppler ultrasound was used to measure the E\u0026prime; and A\u0026prime; peaks inside the mitral valve orifice. The E\u0026prime;/A\u0026prime; ratio was calculated as an indirect measure of diastolic function.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Histological studies\u003c/h2\u003e\n \u003cp\u003eFollowing euthanasia of the rats, one-half of each heart ventricle was preserved in formalin and subsequently embedded in paraffin. Hematoxylin and eosin (H\u0026amp;E) as well as Masson\u0026apos;s trichrome staining were utilized to evaluate myocardial morphology and inflammation in consecutive 4 mm thick tissue sections. Semi-quantitative analysis of the stained tissues was conducted using Image-Pro plus 6.0 software under a light microscope (Olympus, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Immunohistochemistry\u003c/h2\u003e\n \u003cp\u003eAntigens were extracted from paraffin-embedded cardiac sections utilizing EDTA antigen retrieval buffer (pH 8.0) following deparaffinization and rehydration procedures. Subsequently, a 3% bovine serum albumin block was administered to rehydrated slides for a duration of 30 minutes, followed by the application of P62 rabbit antibody (#ab109012, Abcam, 1:400) overnight at 4\u0026deg;C. The slides were then subjected to incubation with a secondary goat anti-rabbit antibody (#5220\u0026thinsp;\u0026minus;\u0026thinsp;0336, SeraCare Inc., USA, 1:200) in conjunction with avidin-biotin complex and horseradish peroxidase subsequent to PBS washing.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Cardiomyocyte size staining\u003c/h2\u003e\n \u003cp\u003eTo assess cardiomyocyte size, rehydrated slides were treated with 3% bovine serum albumin for 30 minutes and subsequently incubated overnight at 4\u0026deg;C with FITC-conjugated wheat germ agglutinin (WGA) (#L4895, Sigma, USA). Cell nuclei were stained with 4\u0026prime;, 6-diamidino-2-phenylindole (DAPI, #C0065, Solarbio, Beijing), and fluorescent microscopes (Olympus, Tokyo) were employed for visualization of the stained sections.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay\u003c/h2\u003e\n \u003cp\u003eApoptotic cardiomyocytes in paraffin sections were identified using a TUNEL detection kit (#6432344001, Roche, USA) under a light microscope (Leica DM 4000 B; Leica, Wetzlar, Germany). Myocardial cytoskeleton colocalization with the anti-actin antibody (#23660-1-AP, Proteintech, Wuhan, 1:100) was observed. Slides were then treated with the goat anti-rabbit secondary antibody (FITC conjugate, #SA00003-2, Proteintech, Wuhan, 1:100). Four regions were randomly selected from each digitized image, and the number of apoptotic and healthy nuclei was quantified.\u003c/p\u003e\n \u003cp\u003eThe apoptotic index was calculated as the number of TUNEL-positive nuclei/total number of nuclei \u003csup\u003e14 19\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. Evaluation of electron microscopy\u003c/h2\u003e\n \u003cp\u003eThe fixation process involved prefixing 1 mm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e heart tissues for 4 hours at 4 degrees Celsius with 2.5% glutaraldehyde immediately following tissue extraction from the left ventricle. Subsequently, the tissues were fixed at room temperature in 1% osmium tetroxide after rinsing with PBS. Dehydrated sections were then cut on an ultramicrotome (Leica UC 7, Leica) and stained with lead citrate and uranyl acetate. The ultrastructure of the autophagic vacuoles was examined using transient electron microscopy (TECNAI G2 20 TWIN, FEI), which is considered the gold standard for analyzing autophagy.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. Detection of superoxide production\u003c/h2\u003e\n \u003cp\u003eDihydroethidium (DHE, #810253P, Sigma-Aldrich) staining was employed on frozen LV tissue (4 \u0026micro;m sections) to assess superoxide production. Fluorescence was detected using a fluorescent microscope (Olympus, Tokyo) with excitation and emission wavelengths of 488 nm and 568 nm, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10. Cell culture\u003c/h2\u003e\n \u003cp\u003eIt was described previously that Neonatal Sprague-Dawley rats (SD) were used to isolate ventricular myocytes (NRVMs) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. For Sac/Val pre-treatment following Dox-induced cardiomyocyte toxicity, NRVMs were pre-treated with 10 \u0026micro;M and 20 \u0026micro;M each of valsartan and LCZ696 for 12 h and then treated with 5 \u0026micro;M of DOX for 6 h \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Similar to animal experimental classification, an equal volume of PBS was incubated with NRVMs for 12 h in the CONTROL and Sac/Val group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11. Invitro ROS production measurement\u003c/h2\u003e\n \u003cp\u003eReactive oxygen species (ROS) were quantified utilizing a 2\u0026prime;,7\u0026prime;-Dichlorofluorescin diacetate (DCF-DA) reagent (35845, Sigma, USA) in this study. Various reagents were administered to neonatal rat ventricular myocytes (NRVMs) cultured in six-well plates for the duration of 18 hours. Following a 30-minute incubation at 37\u0026deg;C, the culture medium was replaced with serum-free medium containing 10 \u0026micro;m DCF-DA \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Fluorescent intensity was measured using excitation/emission wavelengths of 488/525 nm on a flow cytometer.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.12. Cell viability detection\u003c/h2\u003e\n \u003cp\u003eCell viability was assessed using a Cell Counting Kit-8 (CCK-8, #CA1210, Solarbio, China) by incubating 10 mol CCK-8 solutions with NRVMs for one hour under standard incubation conditions. Viability was quantified by measuring the relative optical density of treated cells compared to untreated controls using a microplate reader (BioRad, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.13. Fluorescence‑activated cell sorting (FACS) analysis\u003c/h2\u003e\n \u003cp\u003eFluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) were utilized for the identification of apoptotic cells through the application of an apoptosis detection kit (KGA108, KeyGEN BioTECH, China) following the manufacturer\u0026apos;s instructions \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. NRVMs were resuspended in binding buffer and subsequently incubated with FITC-annexin V and PI at room temperature for approximately ten minutes. Fluorescence measurements were conducted using a flow cytometer (BD Biosciences) equipped with a FACS flow cytometer (BD Biosciences).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e2.14.\u003c/em\u003e Real time quantitative PCR (RT-qPCR)\u003c/h2\u003e\n \u003cp\u003eThe Trizol reagent (Invitrogen, Carlsbad, CA) was employed for total RNA extraction, followed by cDNA synthesis using the Prime Script RT reagent kit (Takara). Real-time qPCR was conducted using the StepOnePlus Real-Time PCR System (Applied Biosystems) in this study. \u003cem\u003eSupplementary Table\u0026nbsp;1\u003c/em\u003e provides a comprehensive list of primer sequences utilized for assessing relative gene expression levels with GAPDH serving as the reference gene.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e2.15. Western blot analysis\u003c/h2\u003e\n \u003cp\u003eRadiation immunoprecipitation (RIPA) buffer (#R0010, Solarbio, Beijing) was utilized for the homogenization and lysis of heart tissue or NRVMs prior to electrotransfer onto PVDF membranes (Millipore, USA). Subsequently, the membranes were blocked in TBST buffer and incubated with primary antibodies (Supplementary Table\u0026nbsp;2) overnight at -4\u0026deg;C. Immunoreactive bands were then detected by incubating with a secondary antibody (Boster, Wuhan, China) conjugated with horseradish peroxidase (HRP) and visualized using a chemiluminescence system (ECL, GE Healthcare Bio-Sciences).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e2.16. Statistical analysis\u003c/h2\u003e\n \u003cp\u003eAnalysis of Variance (ANOVA) was employed for the examination of data involving multiple comparisons, with post hoc tests such as the Least Significant Difference (LSD) test assuming equal variances, and Dunnett\u0026apos;s T3 test in cases where equal variances were not assumed. The study utilized Graphpad Prism 8.0 software (GraphPad Software Inc., CA, USA) and SPSS version 26 (IBM, Armonk, New York) for statistical analysis. A significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was deemed statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.1. Sac/Val treatment inhibited myocardium inflammation\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eIn comparison to the control groups, mice with Dox-induced cardiotoxicity demonstrated inflammatory cell infiltration in the myocardium as observed through H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Mononuclear macrophages were the predominant cell type infiltrating the myocardial interstitium. Treatment with Sac/Val was shown to decrease this inflammatory infiltration in the myocardium. The mRNA levels of inflammatory cytokines, including IL-1β, IFN-γ, TNF-α, and MCP-1, were significantly elevated in the myocardium of mice with Dox-induced cardiotoxicity in the DOX groups compared to the CONTROL group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for all comparisons, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E). The mRNA levels of the aforementioned inflammatory cytokines were notably reduced in the Sac/Val\u0026thinsp;+\u0026thinsp;DOX group compared to the DOX group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all comparisons, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E). Additionally, western blot analysis revealed an increase in the protein expression of NLRP3 and Caspase-1 in the myocardium of mice with Dox-induced cardiotoxicity compared to the CONTROL group, with Sac/Val treatment partially restoring the protein expression of NLRP3 and Caspase-1 in the cardiotoxic hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). These findings suggested that Sac/Val treatment could ameliorate Dox-induced cardiotoxicity by the deregulation of myocardial inflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.2. Sac/Val treatment inhibited cardiac fibrosis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eIn comparison to the control groups, mice with Doxorubicin-induced cardiotoxicity displayed notable collagen matrix deposition in the myocardium as evidenced by Masson's trichrome staining. Subsequent treatment with Sacubitril/Valsartan (Sac/Val) was found to mitigate this myocardial fibrosis (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Additionally, western blot analysis revealed that the protein expression of Collagen I in the myocardium of Doxorubicin-induced cardiotoxic mice was elevated compared to the control groups, with Sac/Val treatment partially restoring Collagen I protein expression in the cardiotoxic hearts (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). The mRNA levels of fibrotic factors, including α-SMA, Collagen I, and Collagen III, were found to be significantly elevated in the myocardium of mice with Dox-induced cardiotoxicity in the DOX groups compared to the CONTROL group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for all comparisons, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G). Conversely, the mRNA levels of these fibrotic factors were significantly reduced in the Sac/Val\u0026thinsp;+\u0026thinsp;DOX group compared to the DOX group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all comparisons, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-G). These results indicated that Sac/Val treatment may mitigate Dox-induced cardiotoxicity by suppressing cardiac fibrosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.3. Sac/Val treatment inhibited cardiomyocyte hypertrophy\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eIn comparison to the control groups, mice with Dox-induced cardiotoxicity exhibited a significantly larger cardiomyocyte size in the myocardium as indicated by WGA staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B\u003cem\u003e)\u003c/em\u003e and an increased ratio of heart weight to body weight \u003cem\u003e(\u003c/em\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Treatment with Sac/Val was found to mitigate this cardiomyocyte hypertrophy and heart weight to body weight ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). The mRNA levels of cardiac fetal reactivation genes, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (MHC), were found to be significantly elevated in the myocardium of mice with Dox-induced cardiotoxicity in the DOX groups compared to the CONTROL group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all comparisons, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). Conversely, the mRNA levels of these cardiomyocyte hypertrophy factors were significantly reduced in the Sac/Val\u0026thinsp;+\u0026thinsp;DOX group compared to the DOX group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all comparisons, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). These findings suggested that Sac/Val treatment could ameliorate Dox-induced cardiotoxicity by the inhibiting myocardial hypertrophy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.4. Sac/Val treatment improved myocardial apoptosis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eIn comparison to the control groups, the myocardium of mice with Dox-induced cardiotoxicity exhibited increased protein expression of pro-apoptotic bax, as well as a notable increase in cleaved caspase-3 and cleaved caspase-9, and a significant decrease in anti-apoptotic bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F). Subsequent treatment with Sac/Val partially reversed the alterations in expression of apoptosis-related proteins in the myocardium of Dox-induced cardiotoxicity mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F). Dox-induced cardiotoxicity mice showed a higher proportion of apoptosis in cardiac myocytes according to TUNEL staining as compared to that in controls, which was partly reversed by Sac/Val treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cem\u003e/B\u003c/em\u003e). These findings suggested that Sac/Val treatment improved myocardial apoptosis in Dox-induced cardiotoxicity mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Sac/Val treatment promoted myocardial autophagy\u003c/h2\u003e \u003cp\u003eTo determine the effects of Sac/Val treatment on Dox-induced myocardial lessened autophagy, we performed immune-histochemical analysis of P62 protein to assess cardiomyocyte autophagy. Dox-induced cardiotoxicity mice showed a higher proportion of P62 positive cells as shown by immunohistochemistry as compared to that in controls, which was ameliorated by Sac/Val treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cem\u003e/B\u003c/em\u003e). Western blotting showed that compared to the control arms, the autophagy related protein expression of P62 increased significantly, of ULK1 and LC3-II decreased obviously in the myocardium of Dox-induced cardiotoxicity mice, which was also reversed by T Sac/Val treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe ultrastructural morphologies of the hearts were observed by transmission electron microscopy. The Dox-induced cardiotoxicity mice showed a lower proportion of autophagic-like vesicles as compared to that in controls, which was reversed by Sac/Val treatment (\u003cem\u003eSupplementary Fig.\u0026nbsp;1A/B\u003c/em\u003e). These findings suggested that Sac/Val treatment promoted myocardial lessened autophagy in Dox-induced cardiotoxicity mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Sac/Val treatment improved systolic and diastolic function\u003c/h2\u003e \u003cp\u003eEchocardiographic analysis of cardiac function in mice treated with Doxorubicin revealed a reduction in left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), indicating a deterioration in cardiac systolic function compared to control mice. This impairment was mitigated by treatment with Sacubitril/Valsartan (Sac/Val), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u003cem\u003e/B\u003c/em\u003e. Additionally, a significant decrease in the ratio of E verus A and E' verus A', indicative of worsened cardiac diastolic function, was observed in Doxorubicin-treated mice. This diastolic dysfunction was reversed by Sac/Val treatment, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-F.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Changes in AMPKα-mTORC1 signaling pathway\u003c/h2\u003e \u003cp\u003eMice with Dox-induced cardiotoxicity demonstrated increased protein expression of p-AMPKα in heart homogenates compared to controls, a change that was reversed by Sac/Val treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-B). Additionally, these mice exhibited decreased protein expression levels of p-mTOR(Ser2448), Raptor, p-S6K1(Thr389), and p-4EBP1(Thr37/46) in heart homogenates compared to controls, which were also reversed by Sac/Val treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC-G). Besides, Dox-induced cardiotoxicity mice exhibited an increased oxidative stress level in myocardium according to DHE staining as compared to that in controls, which was partly reversed by Sac/Val treatment (\u003cem\u003eSupplementary Fig.\u0026nbsp;2A/B\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Sac/Val treatment defended against Dox\u0026ndash;induced apoptosis and autophagy inhibition in primary cardiomyocytes\u003c/h2\u003e \u003cp\u003eIn comparison to the control group, the viability of cardiomyocytes exhibited a significant decrease following stimulation with Dox, a decrease that was partially ameliorated by treatment with Sac/Val (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). Similarly, the apoptosis level of primary cardiomyocytes significantly increased after exposure to Dox, but was partially mitigated by Sac/Val treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB\u003cem\u003e/C\u003c/em\u003e). Additionally, the protein expression levels of cleaved Caspase-3 increased significantly, while the ratio of Bcl-2/Bax decreased in primary cardiomyocytes following Dox stimulation, with partial restoration observed after treatment with Sac/Val (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD\u003cem\u003e/F/G\u003c/em\u003e). The protein expression levels of P62 significantly increased and LC3-II significantly decreased in primary cardiomyocytes after Dox stimulation, compared to controls. This effect was partially reversed by Sac/Val treatment, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eE\u003cem\u003e/H/I\u003c/em\u003e. These findings indicate that Sac/Val treatment may partially restore the Dox-induced apoptosis and autophagy inhibition in primary cardiomyocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e3.9. Sac/Val treatment defended against Dox\u0026ndash;induced apoptosis and autophagy inhibition in primary cardiomyocytes via regulating the AMPKα-mTORC1signaling pathway\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e \u003cem\u003e(A/B)\u003c/em\u003e, the levels of reactive oxygen species (ROS) in primary cardiomyocytes significantly increased following 24 hours of Dox stimulation compared to controls, a change that was mitigated by prior incubation with Sac/Val. Additionally, Dox stimulation resulted in an elevated protein expression of p-AMPKα in primary cardiomyocytes, an effect that was partially attenuated by treatment with Sac/Val (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eC\u003cem\u003e/D\u003c/em\u003e). In comparison to the control group, the protein expression levels of p-mTOR (Ser2448), Raptor, p-S6K1 (Thr389), and p-4EBP1 (Thr37/46) were notably reduced following Dox stimulation in primary cardiomyocytes, with partial restoration observed after Sac/Val treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eE-I). These findings indicate that Sac/Val treatment may mitigate Dox-induced apoptosis and autophagy inhibition in primary cardiomyocytes by modulating the AMPKα-mTORC1 signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAs advancements in cancer treatment lead to increased survival rates, the long-term cardiovascular side effects of chemotherapy, particularly anthracyclines used in the treatment of various cancers such as breast cancer, have become a significant concern due to their dose-dependent cardiotoxicity \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo effectively address the development of chemotherapy-related cardiotoxicity (CTRCD), it is imperative to gain a comprehensive understanding of the underlying mechanisms. Among the various factors contributing to anthracycline-induced cardiotoxicity, lipid peroxidation of the cell membrane emerges as a primary cause. The generation of reactive oxygen species through iron-dependent pathways is identified as the predominant source of anthracycline-induced cardiotoxicity \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Research indicates that inhibition of anthracycline exacerbates the production of reactive oxygen species and disrupts mitochondrial biogenesis \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Willis \u003cem\u003eet al\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e demonstrated subacute anthracycline-induced myocyte atrophy in both mice and humans. MuRF1 (muscle-specific ubiquitin ligase muscle ring finger-1) was required for doxorubicin-induced cardiac atrophy in mice.\u003c/p\u003e \u003cp\u003eIn this study, we examined the prophylactic properties of Sac/Val in mitigating Dox-induced cardiotoxicity and elucidated the potential underlying mechanisms. Our findings demonstrate that pre-treatment with Sac/Val can mitigate myocardial inflammation, fibrosis, and apoptosis, while promoting autophagy and improving heart function in mice with Dox-induced cardiotoxicity through modulation of the AMPKα-mTORC1 signaling pathway. These results offer a molecular rationale for the inhibition of apoptosis by Sac/Val via regulation of the AMPKα-mTORC1 signaling pathway in the context of Dox-induced cardiotoxicity.\u003c/p\u003e \u003cp\u003eIn the context of cardio-oncology, treatment-induced cardiotoxicity poses a significant risk to patient health. Sac/Val regimens are recommended for managing this complication. A retrospective case study documented successful outcomes with Sac/Val treatment in two individuals with anthracycline-related cardiomyopathy and heart failure with reduced ejection fraction (HFrEF) who had previously shown poor responses to conventional evidence-based medications \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Both patients experienced improvements in heart failure symptoms, normalization of NT-proBNP levels, and avoided rehospitalization for their condition \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn their study, Canale \u003cem\u003eet al\u003c/em\u003e. \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e presented a case series involving four patients diagnosed with cancer therapy-related cardiac dysfunction (CTRCD) and severe heart failure with reduced ejection fraction (HFrEF). The patients received Sacubitril/Valsartan (Sac/Val) treatment while wearing an automatic defibrillator until their cardiac function normalized. A subsequent study conducted by researchers in six Spanish hospitals with specialized cardio-oncology clinics followed up on 67 cancer survivors, the majority of whom had received anthracycline-based chemotherapy \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Among patients with HFrEF, Sac/Val therapy was found to be well-tolerated and associated with improvements in NT-proBNP levels, NYHA functional class, and echocardiographic findings \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Renato \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e reported anthracycline cardiomyopathy was treated with Sac/Val in two clinical cases where symptoms and echocardiographic parameters improved in response to the treatment. Ana \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e evaluated ten consecutive patients suffering from cardiotoxicity-related HFrEF were evaluated by comprehensive multiparametric cardiac magnetic resonance (CMR) for the therapeutic effect of Sac/Val. When Sac/Val was administered, LV volumes were markedly reduced and LVEF was significantly improved. NYHA functional class also improved in association with a marked decrease in NT-proBNP concentration \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. LV dysfunction within CTRCD is partly restorable, but this strongly dependeds on timely treatment with Sac/Val \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. After failing to respond to conventional evidence-based drug therapy, Sac/Val was introduced to 28 patients with breast cancer and refractory cardiotoxicity-related HFrEF \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The NYHA cardiac function grade, six-minute walking distance, LVEF, LV diastolic function, LV end-diastolic diameter, and mitral regurgitation assessment significantly improved after captopril or valsartan was replaced with ARNI. While several small observational studies have found that Sac/Val improves cardiac structure and function in CTRCD patients, large-scale prospective clinical trials are needed to confirm these findings.\u003c/p\u003e \u003cp\u003eStudies on the efficacy of Sac/Val in mitigating doxorubicin-induced cardiotoxicity in animal experimental models are limited. Following administration of doxorubicin, mice exhibited impaired heart function, abnormal mitochondrial structure, and compromised respiratory function, all of which were significantly improved with Sac/Val treatment \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Additionally, it is suggested that sacubitril/valsartan may enhance dynamin-related protein 1 (Drp1)-mediated mitochondrial dysfunction caused by doxorubicin \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In a preclinical model of prophylactic treatment, Sacubitril/Valsartan demonstrated efficacy in mitigating oxidative stress damage, inflammation, and apoptosis associated with doxorubicin-induced heart failure and arrhythmia \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Furthermore, compared to doxorubicin alone, Sacubitril/Valsartan attenuated matrix metalloproteinase (MMP) activity in rats, thereby safeguarding against doxorubicin-induced cardiac systolic dysfunction and left ventricular remodeling \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Rats administered with Sacubitril/Valsartan exhibited significant amelioration of doxorubicin-induced cardiac dysfunction through the downregulation of endoplasmic reticulum stress and apoptosis-related proteins \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The mitigation of cardiotoxicity induced by doxorubicin in rat hearts and H9C2 cardiomyocytes was achieved through the reduction of oxidative stress by Sac/Val \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These findings suggest that the cardiotoxic effects of doxorubicin may have been attenuated by the anti-inflammatory, anti-apoptotic, and antioxidant properties of Sac/Val.\u003c/p\u003e \u003cp\u003eBased on the data presented, it is suggested that the cardiotoxic effects induced by DOX may have been mitigated by the anti-inflammatory, anti-apoptotic, and antioxidant properties \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Activation of autophagy at a moderate level may support cellular energy and nutrient provision, thereby safeguarding cardiomyocytes \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Notably, our findings demonstrate for the first time that Sac/Val treatment could ameliorate autophagy suppression in mice with DOX-induced cardiotoxicity.\u003c/p\u003e \u003cp\u003eAMPK, a heterotrimeric enzyme, plays a crucial role in regulating cardiac energy homeostasis \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The serine/threonine-specific protein kinase, mTOR, consists of two distinct multi-complexes, mTORC1, which is involved in regulating cardiac autophagy in response to oxidative stress \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The deficiency of soluble epoxide hydrolase has been shown to reduce myocardial lipid accumulation by enhancing AMPK-mTORC mediated autophagy \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Our study demonstrates that Sac/Val exhibits cardioprotective effects against Dox-induced cardiotoxicity through its anti-apoptotic and de-autophagy properties, which are mediated by the regulation of the AMPKα-mTORC1 signaling pathway.\u003c/p\u003e \u003cp\u003eThis study is subject to several limitations. Future research is required to establish the generalizability of the findings in a murine model to human subjects. Moreover, a deeper understanding of anthracycline-induced cardiomyopathy could be achieved through experimentation with human cardiomyocyte models. Additionally, the lack of comparison between Sac/Val and valsartan in the experiments prevents a definitive conclusion regarding whether the beneficial effects of Sac/Val are solely attributed to valsartan or if sacubitril also plays a role.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study advanced our understanding of the molecular pathways involved in anthracycline-induced cardiomyopathy. The administration of Sac/Val was shown to alleviate myocardial inflammation, fibrosis, apoptosis, enhance autophagy, and improve cardiac function in mice with Dox-induced cardiotoxicity by reducing oxidative stress and regulating the AMPKα-mTORC1 signaling pathway.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eAll authors declare that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study were supported by grants from the talent start-up capital program of Fujian Medical University Union Hospital (2023XH027), the science and technology innovation joint fund project of Fujian Provincial Science and Technology Department (2023Y9183), the Fujian Provincial Natural Science Foundation.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e(I) Conception and design: Weiwei Wang and Xinghe Lin; (II) Administrative support: Xinghe Lin; (III) Provision of study materials: Li Lin; (IV) Collection and assembly of data: Feng Hu and Xiaoxia Qiu; (V) Data analysis and interpretation: Senbo Yan; (VI) Manuscript writing: Feng Hu and Senbo Yan; (VII); Final approval of manuscript: All authors.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNone\u003c/p\u003e\u003ch2\u003eAvailability of data and material\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMichel L, Schadendorf D, Rassaf T (2020) Oncocardiology: new challenges, new opportunities. Herz 45:619\u0026ndash;625\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCardinale D, Colombo A, Bacchiani G, Tedeschi I, Meroni CA, Veglia F, Civelli M, Lamantia G, Colombo N, Curigliano G, Fiorentini C, Cipolla CM (2015) Early detection of anthracycline cardiotoxicity and improvement with heart failure therapy. Circulation 131:1981\u0026ndash;1988\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBowles EJ, Wellman R, Feigelson HS, Onitilo AA, Freedman AN, Delate T, Allen LA, Nekhlyudov L, Goddard KA, Davis RL, Habel LA, Yood MU, McCarty C, Magid DJ, Wagner EH (2012) Risk of heart failure in breast cancer patients after anthracycline and trastuzumab treatment: a retrospective cohort study. J Natl Cancer Inst 104:1293\u0026ndash;1305\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoodee J, Sookprasert A, Sanguanboonyaphong P, Chanthawong S, Seateaw M, Subongkot S (2021) An Exploration of Heart Failure Risk in Breast Cancer Patients Receiving Anthracyclines with or without Trastuzumab in Thailand: A Retrospective Study. Clin Pract 11:484\u0026ndash;493\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaleh Y, Abdelkarim O, Herzallah K, Abela GS (2021) Anthracycline-induced cardiotoxicity: mechanisms of action, incidence, risk factors, prevention, and treatment. Heart Fail Rev 26:1159\u0026ndash;1173\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCardinale D, Colombo A, Lamantia G, Colombo N, Civelli M, De Giacomi G, Rubino M, Veglia F, Fiorentini C, Cipolla CM (2010) Anthracycline-induced cardiomyopathy: clinical relevance and response to pharmacologic therapy. J Am Coll Cardiol 55:213\u0026ndash;220\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDocherty KF, Vaduganathan M, Solomon SD, McMurray JJV (2020) Sacubitril/Valsartan: Neprilysin Inhibition 5 Years After PARADIGM-HF. JACC Heart Fail 8:800\u0026ndash;810\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng Y, Huang S, Xie B, Zhang N, Liu Z, Tse G, Liu T (2023) Cardiovascular Toxicity of Proteasome Inhibitors in Multiple Myeloma Therapy. Curr Probl Cardiol 48:101536\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcMurray JJ, Packer M, Desai AS, Gong J, Lefkowitz MP, Rizkala AR, Rouleau JL, Shi VC, Solomon SD, Swedberg K, Zile MR (2014) Angiotensin-neprilysin inhibition versus enalapril in heart failure. N Engl J Med 371:993\u0026ndash;1004\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuraes AR, de Souza Lima Bitar Y, Neto MG, Mesquita ET, Chan JS, Tse G, Liu T, Bocchi EA, Biondi-Zoccai G, Roever L (2022) Effectiveness of sacubitril-valsartan in patients with cancer therapy-related cardiac dysfunction: a systematic review of clinical and preclinical studies. Minerva Med 113:551\u0026ndash;557\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia Y, Chen Z, Chen A, Fu M, Dong Z, Hu K, Yang X, Zou Y, Sun A, Qian J, Ge J (2017) LCZ696 improves cardiac function via alleviating Drp1-mediated mitochondrial dysfunction in mice with doxorubicin-induced dilated cardiomyopathy. J Mol Cell Cardiol 108:138\u0026ndash;148\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDindaş F, G\u0026uuml;ng\u0026ouml;r H, Ekici M, Akokay P, Erhan F, Doğduş M, Yılmaz MB (2021) Angiotensin receptor-neprilysin inhibition by sacubitril/valsartan attenuates doxorubicin-induced cardiotoxicity in a pretreatment mice model by interfering with oxidative stress, inflammation, and Caspase 3 apoptotic pathway. Anatol J Cardiol 25:821\u0026ndash;828\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoutagy NE, Feher A, Pfau D, Liu Z, Guerrera NM, Freeburg LA, Womack SJ, Hoenes AC, Zeiss C, Young LH (2020) Spinale FG and Sinusas AJ. Dual Angiotensin Receptor-Neprilysin Inhibition With Sacubitril/Valsartan Attenuates Systolic Dysfunction in Experimental Doxorubicin-Induced Cardiotoxicity. JACC CardioOncology 2:774\u0026ndash;787\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim BS, Park IH, Lee AH, Kim HJ, Lim YH, Shin JH (2022) Sacubitril/valsartan reduces endoplasmic reticulum stress in a rat model of doxorubicin-induced cardiotoxicity. Arch Toxicol 96:1065\u0026ndash;1074\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyoshi T, Nakamura K, Amioka N, Hatipoglu OF, Yonezawa T, Saito Y, Yoshida M, Akagi S, Ito H (2022) LCZ696 ameliorates doxorubicin-induced cardiomyocyte toxicity in rats. Sci Rep 12:4930\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsselin CY, Lam A, Cheung DYC, Eekhoudt CR, Zhu A, Mittal I, Mayba A, Solati Z, Edel A, Austria JA, Aukema HM, Ravandi A, Thliveris J, Singal PK, Pierce GN, Niraula S, Jassal DS (2020) The Cardioprotective Role of Flaxseed in the Prevention of Doxorubicin- and Trastuzumab-Mediated Cardiotoxicity in C57BL/6 Mice. J Nutr 150:2353\u0026ndash;2363\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu W, Qin X, Zhang Y, Qiu P, Wang L, Zha W, Ren J (2020) Curcumin suppresses doxorubicin-induced cardiomyocyte pyroptosis via a PI3K/Akt/mTOR-dependent manner. Cardiovasc diagnosis therapy 10:752\u0026ndash;769\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicol M, Sadoune M, Polidano E, Launay JM, Samuel JL, Azibani F, Cohen-Solal A (2021) Doxorubicin-induced and trastuzumab-induced cardiotoxicity in mice is not prevented by metoprolol. ESC Heart Fail 8:928\u0026ndash;937\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu F, Lin C (2024) TRPM2 knockdown attenuates myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice via regulating the MEK/ERK and mTORC1 signaling pathway. Mol Cell Biochem\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Liu X, Bawa-Khalfe T, Lu LS, Lyu YL, Liu LF, Yeh ET (2012) Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat Med 18:1639\u0026ndash;1642\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWillis MS, Parry TL, Brown DI, Mota RI, Huang W, Beak JY, Sola M, Zhou C, Hicks ST, Caughey MC, D'Agostino RB Jr., Jordan J, Hundley WG, Jensen BC (2019) Doxorubicin Exposure Causes Subacute Cardiac Atrophy Dependent on the Striated Muscle-Specific Ubiquitin Ligase MuRF1. Circulation Heart Fail 12:e005234\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSheppard CE, Anwar M (2019) The use of sacubitril/valsartan in anthracycline-induced cardiomyopathy: A mini case series. J Oncol Pharm practice: official publication Int Soc Oncol Pharm Practitioners 25:1231\u0026ndash;1234\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCanale ML, Coviello K, Solarino G, Del Meglio J, Simonetti F, Venturini E, Camerini A, Maurea N, Bisceglia I, Tessa C, Casolo G (2022) Case Series: Recovery of Chemotherapy-Related Acute Heart Failure by the Combined Use of Sacubitril Valsartan and Wearable Cardioverter Defibrillator: A Novel Winning Combination in Cardio-Oncology. Front Cardiovasc Med 9:801143\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;n-Garcia A, L\u0026oacute;pez-Fern\u0026aacute;ndez T, Mitroi C, Chaparro-Mu\u0026ntilde;oz M, Moliner P, Martin-Garcia AC, Martinez-Monzonis A, Castro A, Lopez-Sendon JL, Sanchez PL (2020) Effectiveness of sacubitril-valsartan in cancer patients with heart failure. ESC Heart Fail 7:763\u0026ndash;767\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Vecchis R, Paccone A (2020) A case series about the favorable effects of sacubitril/valsartan on anthracycline cardiomyopathy. SAGE open Med case Rep 8:2050313x20952189\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;n-Garc\u0026iacute;a A, D\u0026iacute;az-Pel\u0026aacute;ez E, Mart\u0026iacute;n-Garc\u0026iacute;a AC, S\u0026aacute;nchez-Gonz\u0026aacute;lez J, Ib\u0026aacute;\u0026ntilde;ez B, S\u0026aacute;nchez PL (2020) Myocardial function and structure improvement with sacubitril/valsartan in cancer therapy-induced cardiomyopathy. Revista Esp de cardiologia (English ed) 73:268\u0026ndash;269\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGregorietti V, Fernandez TL, Costa D, Chahla EO, Daniele AJ (2020) Use of Sacubitril/valsartan in patients with cardio toxicity and heart failure due to chemotherapy. Cardio-oncology (London England) 6:24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjoolabady A, Chiong M, Lavandero S, Klionsky DJ, Ren J (2022) Mitophagy in cardiovascular diseases: molecular mechanisms, pathogenesis, and treatment. Trends Mol Med 28:836\u0026ndash;849\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Zhao D, Tang L, Li H, Liu Z, Gao J, Edin ML, Zhang H, Zhang K, Chen J, Zhu X, Wang D, Zeldin DC, Hammock BD, Wang J, Huang H (2021) Soluble epoxide hydrolase deficiency attenuates lipotoxic cardiomyopathy via upregulation of AMPK-mTORC mediated autophagy. J Mol Cell Cardiol 154:80\u0026ndash;91\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Doxorubicin, cardiotoxicity, sacubitril/valsartan, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-4603884/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4603884/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThis study aimed to investigate the potential cardio-protective effects of sacubitril/valsartan (Sac/Val) in mice with doxorubicin (DOX)-induced cardiomyopathy, a common manifestation of cancer therapy-related cardiac dysfunction (CTRCD) associated with DOX.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 24 mice were equally classified into 4 groups; control group, DOX (total 24 mg/kg), Sac/Val (80 mg/kg), and Sac/Val\u0026thinsp;+\u0026thinsp;DOX (Sac/Val was given from seven day before doxorubicin administration). Neonatal rat ventricular myocytes was exposed to 5 \u0026micro;M of DOX for 6 h \u003cem\u003ein vitro\u003c/em\u003e to mimic the \u003cem\u003ein vivo\u003c/em\u003e conditions. A variety of techniques were used to investigate cardiac inflammation, fibrosis, apoptosis, and autophagy, including western blot, real time quantitative PCR (RT-qPCR), immunohistochemistry, and fluorescence.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMice with Dox-induced cardiotoxicity displayed impaired systolic and diastolic function, characterized by elevated levels of cardiac inflammation, fibrosis, cardiomyocyte hypertrophy, apoptosis, and autophagy inhibition in the heart. Treatment with Sac/Val partially reversed these effects. In comparison to the control group, the protein expression of NLRP3, caspase-1, Collagen I, bax, cleaved caspase-3, and P62 were significantly increased, while the protein expression of bcl-2 and LC3-II were significantly decreased in the myocardial tissues of the Dox-induced cardiomyopathy group. The administration of Sac/Val demonstrated the potential to partially reverse alterations in protein expression within the myocardium of mice with Dox-induced cardiotoxicity by modulating the AMPKα-mTORC1 signaling pathway and suppressing oxidative stress. Additionally, Sac/Val treatment may mitigate Dox-induced apoptosis and inhibition of autophagy in primary cardiomyocytes.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eSac/Val seems to be cardio-protective against Dox-induced cardiotoxicity in pretreatment mice model. These findings could be attributed to the anti-inflammatory, antioxidant, anti-apoptotic and de-autophagy effects of Sac/Val through regulation of the AMPKα-mTORC1 signaling pathway.\u003c/p\u003e","manuscriptTitle":"Sacubitril/valsartan attenuated myocardial inflammation, fibrosis, apoptosis and promoted autophagy in doxorubicin-induced cardiotoxicity mice via regulating the AMPKα-mTORC1 signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 18:35:22","doi":"10.21203/rs.3.rs-4603884/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-12T17:12:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-07T07:31:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127996477101585689428709939554619647895","date":"2024-07-29T04:40:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-04T20:34:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-04T19:24:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-19T10:29:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2024-06-19T07:10:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"39c3022b-6848-4381-87f6-52a56825b76d","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:01:57+00:00","versionOfRecord":{"articleIdentity":"rs-4603884","link":"https://doi.org/10.1007/s11010-024-05117-7","journal":{"identity":"molecular-and-cellular-biochemistry","isVorOnly":false,"title":"Molecular and Cellular Biochemistry"},"publishedOn":"2024-09-20 15:57:29","publishedOnDateReadable":"September 20th, 2024"},"versionCreatedAt":"2024-07-29 18:35:22","video":"","vorDoi":"10.1007/s11010-024-05117-7","vorDoiUrl":"https://doi.org/10.1007/s11010-024-05117-7","workflowStages":[]},"version":"v1","identity":"rs-4603884","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4603884","identity":"rs-4603884","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}