DOX-induced Cardiac Damage Research Articles

Artemisia argyi mitigates doxorubicin-induced cardiotoxicity by inhibiting mitochondrial dysfunction through the IGF-IIR/Drp1/GATA4 signaling pathway.

Doxorubicin (DOX) is mostly utilized as a wide range of antitumor anthracycline to treat different cancers. The severe antagonistic impacts of DOX on cardiotoxicity constrain its clinical application. Many mechanisms are involved in cardiac toxicity induced by DOX in the human body. Mitochondria is a central part of fatty acid and glucose metabolism. Thus, impaired mitochondrial metabolism can increase heart failure risk, which can play a vital role in cardiomyocyte mitochondrial dysfunction. This study aimed to assess the possible cardioprotective effect of water-extracted Artemisia argyi (AA) against the side effect of DOX in H9c2 cells and whether these protective effects are mediated through IGF-IIR/Drp1/GATA4 signaling pathways. Although several studies proved that AA extract has benefits for various diseases, its cardiac effects have not yet been identified. The H9c2 cells were exposed to 1μM to establish a model of cardiac toxicity. The results revealed that water-extracted AA could block the expression of IGF-IIR/calcineurin signaling pathways induced by DOX. Notably, our results also showed that AA treatment markedly attenuated Akt phosphorylation and cleaved caspase 3, and the nuclear translocation markers NFATC3 and p-GATA4. Using actin staining for hypertrophy, we determined that AA can reduce the effect of mitochondrial reactive oxygen species and cell size. These findings suggest that water-extracted AA could be a suitable candidate for preventing DOX-induced cardiac damage.

Fasting: A Complex, Double-Edged Blade in the Battle Against Doxorubicin-Induced Cardiotoxicity.

In recent years, there has been a surge in the popularity of fasting as a method to enhance one's health and overall well-being. Fasting is a customary practice characterized by voluntary refraining from consuming food and beverages for a specified duration, ranging from a few hours to several days. The potential advantages of fasting, including enhanced insulin sensitivity, decreased inflammation, and better cellular repair mechanisms, have been well documented. However, the effects of fasting on cancer therapy have been the focus of recent scholarly investigations. Doxorubicin (Dox) is one of the most widely used chemotherapy medications for cancer treatment. Unfortunately, cardiotoxicity, which may lead to heart failure and other cardiovascular issues, has been linked to Dox usage. This study aims to comprehensively examine the possible advantages and disadvantages of fasting concerning Dox-induced cardiotoxicity. Researchers have investigated the potential benefits of fasting in lowering the risk of Dox-induced cardiac damage to solve this problem. Nevertheless, new studies indicate that prolonged alternate-day fasting may adversely affect the heart's capacity to manage the cardiotoxic properties of Dox. Though fasting may benefit overall health, it is essential to proceed cautiously and consider the potential risks in certain circumstances.

Ohwia caudata inhibits doxorubicin-induced cardiotoxicity by regulating mitochondrial dynamics via the IGF-IIR/p-Drp1/PARP signaling pathway.

The most effective drug, doxorubicin (DOX), is widely used worldwide for clinical application as an anticancer drug. DOX-induced cytotoxicity is characterized by mitochondrial dysfunction. There is no alternative treatment against DOX-induced cardiac damage despite intensive research in the present decades. Ohwia caudata has emerged as a potential herbal remedy that prevents from DOX-induced cytotoxicity owing to its pharmacological action of sustaining mitochondrial dynamics by attenuating oxidative stress and inducing cellular longevity. However, its underlying mechanisms are unknown. The novel treatment provided here depends on new evidence from DOX-treated H9c2 cells, which significantly enhanced insulin-like growth factor (IGF) II receptor (IGF-IIR) pathways that activated calcineurin and phosphorylated dynamin-related protein 1 (p-Drp1) at ser616 (p-Drp1[ser616]); cells undergo apoptosis due to these factors, which translocate to mitochondria and disrupt their function and integrity, and in terms of herbal medicine treatment, which significantly blocked these phenomena. Thus, our findings indicate that maintaining integrity of mitochondria is an essential element in lowering DOX-induced cytotoxicity, which further emphasizes that our herbal medicine can successfully block IGF-IIR pathways and could potentially act as an alternative mechanism in terms of cardioprotective against doxorubicin.

The Bach1/HO-1 pathway regulates oxidative stress and contributes to ferroptosis in doxorubicin-induced cardiomyopathy in H9c2 cells and mice.

Doxorubicin (DOX) is one of the most frequently used chemotherapeutic drugs belonging to the class of anthracyclines. However, the cardiotoxic effects of anthracyclines limit their clinical use. Recent studies have suggested that ferroptosis is the main underlying pathogenetic mechanism of DOX-induced cardiomyopathy (DIC). BTB-and-CNC homology 1 (Bach1) acts as a key role in the regulation of ferroptosis. However, the mechanistic role of Bach1 in DIC remains unclear. Therefore, this study aimed to investigate the underlying mechanistic role of Bach1 in DOX-induced cardiotoxicity using the DIC mice in vivo (DOX at cumulative dose of 20mg/kg) and the DOX-treated H9c2 cardiomyocytes in vitro (1μM). Our results show a marked upregulation in the expression of Bach1 in the cardiac tissues of the DOX-treated mice and the DOX-treated cardiomyocytes. However, Bach1-/- mice exhibited reduced lipid peroxidation and less severe cardiomyopathy after DOX treatment. Bach1 knockdown protected against DOX-induced ferroptosis in both in vivo and in vitro models. Ferrostatin-1 (Fer-1), a potent inhibitor of ferroptosis, significantly alleviated DOX-induced cardiac damage. However, the cardioprotective effects of Bach1 knockdown were reversed by pre-treatment with Zinc Protoporphyrin (ZnPP), a selective inhibitor of heme oxygenase-1(HO-1). Taken together, these findings demonstrated that Bach1 promoted oxidative stress and ferroptosis through suppressing the expression of HO-1. Therefore, Bach1 may present as a promising new therapeutic target for the prevention and early intervention of DOX-induced cardiotoxicity.

Exercise preconditioning inhibits doxorubicin-induced cardiotoxicity via YAP/STAT3 signaling

Doxorubicin (DOX) possesses strong anti-tumor effects but is limited by its irreversible cardiac toxicity. The relationship between exercise, a known enhancer of cardiovascular health, and DOX-induced cardiotoxicity has been a focus of recent research. Exercise has been suggested to mitigate DOX's cardiac harm by modulating the Yes-associated protein (YAP) and Signal transducer and activator of transcription 3 (STAT3) pathways, which are crucial in regulating cardiac cell functions and responses to damage. This study aimed to assess the protective role of exercise preconditioning against DOX-induced cardiac injury. We used Sprague–Dawley rats, divided into five groups (control, DOX, exercise preconditioning (EP), EP-DOX, and verteporfin + EP + DOX), to investigate the potential mechanisms. Our findings, including echocardiography, histological staining, Western blot, and q-PCR analysis, demonstrated that exercise preconditioning could alleviate DOX-induced cardiac dysfunction and structural damage. Notably, exercise preconditioning enhanced the nuclear localization and co-localization of YAP and STAT3. Our study suggests that exercise preconditioning may counteract DOX-induced cardiotoxicity by activating the YAP/STAT3 pathway, highlighting a potential therapeutic approach for reducing DOX's cardiac side effects.

Abstract 15483: Mitoquinone Pretreatment Mitigates Dox-induced Systolic Dysfunction in Isolated Rat Hearts

Background: Doxorubicin (DOX) is a common and effective chemotherapeutic agent that can exert cardiotoxicity by inducing oxidative stress in mitochondria, compromising mitochondrial function, and inducing apoptosis. Currently, there is no treatment that targets this mechanism. We previously found that pretreatment with higher concentrations of mitoquinone (MQ), a mitochondrial antioxidant, exerts cardioprotection against DOX in myoblast H9C2 cells. Hypothesis: We hypothesize that MQ pretreatment would preserve cardiac function against DOX in isolated rat hearts. Methods: Cardiac parameters (heart rate (HR) and left ventricular end systolic pressure (LVESP)) were monitored before and after 60 min infusion of DOX, MQ, or DOX with MQ pretreatment (10-15 min) in a Langendorff preparation of male SD rat hearts. TUNEL assay was conducted on heart tissue slides to evaluate apoptosis. The ratio between the final and initial cardiac parameters was calculated and compared among experimental groups. Results: Higher dose DOX (25 μM, n=5) induced a higher reduction in the ratios of LVESP, to 39±5% than lower dose DOX infusion (20 μM, n=3; 74±3%). DOX had no effects on HR. A 60-minute perfusion of higher doses of MQ (1-5 μM, n=7) unexpectedly reduced the ratios of LVESP and HR to 73±12% and 66±5%, respectively. Higher dose MQ (1-2.5 μM, n=6) pretreatment showed similar improvement of LVESP ratio (68±9%) to lower dose MQ (0.25-0.5 μM, n=6; 71±11%). TUNEL assay showed that higher dose DOX caused a higher rate of apoptosis (number of TUNEL-positive nuclei) (326.3±6.4. n=3) than lower dose (214±29.5, n=3). Pretreatment with higher dose MQ prior to DOX 25 μM infusion resulted in a notable reduction of apoptosis (60.3±5, n=6) compared to DOX alone, while lower dose pretreatment reduced apoptosis to 70.5± 0.5 (n=4). Conclusion: This study suggests that infusion of DOX into the heart acutely attenuated cardiac systolic function accompanied with higher apoptotic cell damage. Although prolonged administration of higher doses of MQ suppressed cardiac function, MQ pretreatment for 10-15 minutes mitigated DOX-induced systolic dysfunction and reduced cardiomyocyte apoptosis. In summary, MQ pretreatment may mitigate DOX-induced cardiac damage.

Catalpol Inhibits Autophagy to Ameliorate Doxorubicin-Induced Cardiotoxicity via the AKT-mTOR Pathway.

As a kind of anthracycline, doxorubicin (DOX) is commonly used as an antitumor drug, but its clinical application has been greatly hindered due to its severe cardiotoxicity. Hence, in this study, we investigated the role of catalpol (CTP) and its effect on DOX-induced cardiotoxicity.The cardiac function of mice was evaluated by assessing lactate dehydrogenase, creatine kinase isoenzyme, heart weight to body weight, and heart weight/tibia length levels. Histopathological changes were observed using hematoxylin and eosin staining, and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to examine myocardial apoptosis. Superoxide dismutase (SOD) activity, glutathione (GSH), and malondialdehyde (MDA) levels were measured to confirm the changes in oxidative stress. Western blotting showed the levels of autophagy- and pathway-related proteins. Expression of autophagy marker LC3 was examined using immunofluorescence staining.CTP alleviated DOX-induced cardiac damage in mice. We further observed upregulated SOD and GSH levels, and downregulated MDA level after the CTP treatment in DOX-treated mice, indicating the protective role of CTP against oxidative injury. DOX-induced myocardial apoptosis was also inhibited by CTP treatment in mice. In addition, CTP decreased the levels of Beclin1 and LC3II/LC3I, increased the levels of P62, and activated the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway in DOX-treated mice.CTP ameliorated DOX-induced cardiotoxicity by inhibiting oxidative stress, myocardial apoptosis, and autophagy via the AKT-mTOR pathway.

Boesenbergia rotunda displayed anti-inflammatory, antioxidant and anti-apoptotic efficacy in doxorubicin‐induced cardiotoxicity in rats

This study evaluated the cardioprotective properties of Boesenbergia rotunda extract (BrE) against doxorubicin (DOX) induced cardiotoxicity. Rats received oral gavage of BrE for 28 days and DOX (5 mg/kg/week for 3 weeks). Thereafter the animals were sacrificed, blood and cardiac samples were collected for biochemical, histological and immunohistochemical analyses. The results indicated that BrE attenuated DOX triggered body and cardiac weight loss and prevented against cardiac injury by mitigating histopathological alterations in cardiac tissues as well as serum cardiac function enzymes. BrE significantly reduced serum levels of aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), troponin T (TnT) and creatine kinase-MB (CK-MB) in DOX-treated rats. Furthermore, BrE alleviated cardiotoxicity by reducing DOX instigated oxidative stress and potentiating the level of glutathione, as well as the activities superoxide dismutase and catalase in cardiac tissues. In addition, BrE significantly decreased the characteristic indices of DOX-induced cardiac inflammation and apoptosis. Immuno-histochemical analysis revealed that BrE decreased the stain intensity of p53 and myeloperoxidase (MPO) proteins compared to the DXB alone group. In conclusion, our results indicated that BrE modulated oxidative stress, inflammation and apoptosis to attenuate DOX-induced cardiac damage.

Protective Effect of Omega-3 and the Potential of Toll-like Receptor Gene Expression in Rats with Doxorubicin-induced Cardiac Toxicity

Despite the clinical signs of doxorubicin (DOX)-induced cardiomyopathy, the mechanisms underlying DOX-induced cardiac damage are unknown. Toll-like receptors (TLRs) allow cardiomyocytes to respond to either endogenous or external signals, both of which have the potential to alter the pathophysiological responses to dilated cardiomyopathy. Omega-3 (OMG-3) precursors are highly active metabolites with numerous therapeutic benefits in the prevention and/or treatment of a variety of diseases. Thus, this study was aimed at evaluating the preventive effect of omega-3 on rats suffering from acute DOX-induced cardiotoxicity associated with TLR gene expression. Thirty rats were divided into five equal groups; Group 1 received no treatment, Group 2 received doxorubicin (at a toxic dose of 20 mg/kg), and Groups 3-5 received doxorubicin (20 mg/kg) after receiving OMG-3 at various doses (100, 200, and 400 mg/kg/day, respectively) for 4 weeks. At the end of the experiment, blood samples were obtained from the heart. The real time polymerase chain reaction (RT-PCR) was used to assess TLR2 and TLR4 gene expression. TLR2 had a significantly (p<0.01) elevated fold change in the DOX alone group (28.74+4.95), whereas TLR2 expression was significantly reduced in the OMG-3 pretreated groups (1.633+0.51, 0.733+0.13, and 0.709+0.16, respectively). TLR4 fold change in the DOX alone group was 8.57+1.22, whereas TLR4 expression was significantly reduced in the OMG-3 pretreated groups (100, 200, and 400 mg/kg) with values of 0.809+0.32, 0.852+0.50, and 0.272+0.16, respectively. The findings of this study revealed that OMG-3 decreased doxorubicin-induced cardiotoxicity and showed a significant cardioprotective effect by lowering TLR2 and TLR4 gene expression.

Daidzein ameliorates doxorubicin-induced cardiac injury by inhibiting autophagy and apoptosis in rats.

Backgrounds: Doxorubicin (Dox) is a classical antitumor antibiotic widely restricted for use due to its cardiotoxicity. Daidzein (Daid) is a soy isoflavone that enhances antioxidant enzyme systems and inhibits apoptosis to prevent cardiovascular diseases. In this study, we intended to assess whether Daid protects against Dox-induced cardiotoxicity and explored its underlying mechanisms. Methods: Male Sprague-Dawley (SD) rats were divided into five groups: control (Ctrl), 40 mg per kg per day Daidzein (Daid), 3 mg per kg per week doxorubicin (Dox), 20 mg per kg per day Daidzein + 3 mg per kg per week doxorubicin (Daid20 + Dox) and 40 mg per kg per day Daidzein + 3 mg per kg per week doxorubicin (Daid40 + Dox) groups. Cardiac function assessments, immunohistochemistry (IHC) and immunofluorescence (IF) analyses were initially performed in each group of rats. Secondly, the cell proliferative capacity analysis, AO staining, and LC3 puncta analysis were employed to evaluate the cellular response to Dox in H9c2 cells. Ultimately, the protein expressions of cleaved caspase3, LC3 II, Bcl-2, Bax, Akt, p-Akt, and cyclin D1 were examined by western blotting. Results: Pretreatment with a low dose of Daid rather than a high dose significantly enhanced cardiac function and alleviated histopathological deterioration of cardiomyocytes induced by Dox. Daid downregulated the protein levels of Bax, LC3 II, cleaved caspase3 and p-Akt, while up-regulating Bcl-2 and cyclin D1. The Akt agonist SC79 could invalidate all the protective effects of Daid both in vivo and in vitro. Conclusions: Daid reduced autophagy and apoptosis by inhibiting the PI3K/Akt pathway, thereby protecting the hearts from Dox-induced cardiac damage.

LCZ696 protects against doxorubicin-induced cardiotoxicity by inhibiting ferroptosis via AKT/SIRT3/SOD2 signaling pathway activation.

Doxorubicin (DOX) is an effective and widely used anticancer drug but has limited clinical applicability because of its cardiotoxicity. Ferroptosis plays a key role in DOX-induced cardiac damage and cardiomyocyte cell death. The inhibition of ferroptosis reverses DOX-induced cardiotoxicity (DIC). LCZ696, a first-in-class angiotensin receptor neprilysin inhibitor, protects against DIC. However, the mechanism of action of LCZ696, especially its effect on ferroptosis, is incompletely understood. This study investigates the cardioprotective effects of LCZ696 on DIC in vivo and in vitro.Cardiotoxicity was induced in Wistar rats by tail intravenous injection of 2.5mg/kg DOX once a week for six weeks. Rats and H9c2 cells were treated with or without LCZ696 to determine the cardioprotective role and underlying mechanisms of LCZ696 against DIC. To assess the role of SIRT3 and correlated pathways in ferroptosis, SIRT3 knockout was performed using lentiviral vectors, and AKT was inhibited with LY294002. LCZ696 significantly attenuated DIC by decreasing the concentrations of lipid reactive oxygen species and malondialdehyde and increasing the levels of glutathione peroxidase-4 and reduced glutathione in cells and heart tissues. Moreover, LCZ696 remodeled myocardial structures and improved heart ventricular function in DOX-treated rats. LCZ696 treatment increased SIRT3 expression and deacetylated its target gene SOD2, and these changes were mediated by AKT activation. SIRT3 knockdown and AKT inhibition induced lipid peroxidation and reduced the protective effect of LCZ696 in H9c2 cells. Collectively,LCZ696 prevents DIC by inhibiting ferroptosis via AKT/SIRT3/SOD2 signaling pathway activation. Thus, LZC696 is a potential therapeutic strategy for DIC.

MicroRNAs target the PI3K/Akt/p53 and the Sirt1/Nrf2 signaling pathways in doxorubicin-induced cardiotoxicity.

Doxorubicin (DOX) is used as a chemotherapeutic agent in the treatment of solid tumors. Irreversible cardiotoxicity is the major limitation in the clinical use of DOX. Several microRNAs (miRNAs) with diversified functions are identified that participate in exacerbating or suppressing DOX-induced cardiac damage. The miRNAs are small noncoding regulatory RNAs that modify the expression of the native genes. Studies have demonstrated that miRNAs by modifying the expression of proteins such as PTEN, Akt, and survivin can affect DOX-induced cardiac apoptosis. Moreover, miRNAs can modulate cardiac oxidative stress in DOX treatment through the posttranscriptional regulation of Sirt1, p66shc, and Nrf2 expressions. This manuscript has reviewed the regulation of the PI3K/Akt/p53 and the Sirt1/Nrf2 pathways by miRNAs in DOX-induced cardiotoxicity.

Abstract 10138: Exercise Inhibits Doxorubicin-Induced Cardiotoxicity by Preventing Cardiomyocyte Cellular Senescence

Introduction: Doxorubicin (Dox) induces cardiomyocyte cellular senescence thru proinflammatory mechanisms and by damaging DNA, resulting in Dox-induced cardiotoxicity. Inhibiting cardiomyocyte cellular senescence inhibits Dox-induced cardiac damage. We showed that exercise inhibits both acute and late Dox-induced cardiotoxicity using our juvenile cardiotoxicity mouse model. Here we used p16-3MR transgenic mice to track the induction of senescent cardiomyocytes to determine whether exercise prevents Dox-induced cardiotoxicity by inhibiting senescence. Hypothesis: Cellular senescence plays a key role in Dox-induced cardiotoxicity in the heart following therapy. Exercise prevents Dox-induced cardiotoxicity by inhibiting myocardial cell senescence. Methods: To confirm induction of senescence by Dox in vitro , mouse cardiomyocytes were isolated and treated with 250 nmol/L Dox for 24 h for 24h cells, washed and then analyzed 7 days later for senescent markers. Mice were treated with PBS, Dox (2.5mg/kg 2x/wk for 2 wks), or Dox + exercise (walking 45 min/day at 12 meter/min). Cardiac function was evaluated by echocardiography 24 h after therapy to confirm Dox-induced cardiotoxicity. Heart tissue was collected 14 days after therapy. Tissue sections were analyzed by immunohistochemical and Western immunoblot. Results: Dox-induced senescence was confirmed by a significant increase in β-galactosidase in cardiomyocytes from the in vitro study. Decreased ejection fraction, fractional shortening and F-actin distribution was seen in Dox but not Dox + exercise-treated mice confirming Dox-induced cardiotoxicity and prevention of heart damage by exercise. There was an increase in p16 INK4a expression (signifying senescent cells) in cardiac tissue from Dox-treated but not Dox + exercise-treated mice compared with PBS controls. Conclusion: Taken together our data demonstrated that exercise inhibited Dox-induced cardiotoxicity by inhibiting the induction of cardiomyocyte senescence.

Abstract P1-08-05: Expression of the human APOE4 genotype modulates Doxorubicin cardiotoxicity in mice

Abstract Background: The human apolipoprotein E (APOE) gene has three major allelic variants: APOE2, -E3 and -E4. Carriers of the E4 allele are at increased risk for Alzheimer’s and other age-related neurodegenerative disorders. Recently, APOE4 has been associated with risk for cancer-related cognitive decline following chemotherapy with Doxorubicin (DOX), a frequent component of adjuvant therapy for breast cancer. To date, the mechanisms for APOE4 neurotoxicity are unknown, but may greater vulnerability to oxidative stress. DOX also causes dose-dependent cardiotoxicity, but a role for APOE4 in heart damage has not been previously demonstrated. Objective: To determine whether APOE4 predisposes to DOX-induced damage to the myocardium. Methods: C57Bl/6 mice (5-7 mo and 14-18 mo, male and female) with human APOE3 or APOE4 homozygous knock-in (=hAPOE3 and hAPOE4, respectively) were subjected to a single IP injection of saline (Control) or DOX (10 mg/kg, or 5 weekly injections of 5 mg/kg, =DOX), and monitored between 3-45 days afterward. Left ventricular systolic function was quantitated using echocardiography (Vevo 3100) at baseline, during and at the end of each study. TUNEL assay was used to identify apoptotic cells. Collagen was imaged using Masson’s Trichrome staining. Myocardial protein and RNA were extracted from the left ventricle and was analyzed by immunoblotting, realtime PCR and RNASeq on an Illumina NexGen platform.Results: Aged female APOE4 mice had more myocardial collagen at baseline (8.93±1.23 vs. 6.06±1.44%). Baseline cardiac function were similar at baseline in the 2 mouse strains. Three days after DOX, apoptotic myocytes were more abundant in hAPOE4 vs hAPOE3 hearts (1.2 vs. 4.6 TUNEL+ cells per section, n= 5, p<0.05). After DOX treatment, hAPOE4 mice had greater declines in left ventricular ejection fraction (EF, 46.9 vs 62.1%, p = hAPOE4 vs hAPOE3, p < 0.001). Quantitative PCR, RNASeq and protein studies revealed multiple differences in the cardiac transcriptome between hAPOE3 and hAPOE4 mice both at baseline and after DOX treatment. Baseline expression of MEF2D, a key driver of the myocardial stress response, was increased in hAPOE4 vs APOE3, driven by a 3500x (p = 4.76E-17) in transcript variant 1. Robust differences were seen in expression of c-Jun, JunB, and myosin heavy chain genes Myh6 and Myh7 at baseline and following DOX treatment at day 3. Growth Arrest And DNA Damage Inducible Alpha(GADD45A) gene expression was ~2x higher in hAPOE4 mice at baseline, but was similar to hAPOE3 mice by day 14 after DOX. Protein levels of caspase-1, an essential regulator of inflammation and the innate immune response, increased with age in hAPOE3 mice. In contrast, levels of casp-1 were lower in hAPOE4 and did not increase with age (p= 0.002 for APOE4 vs APOE3 at 17 mo). Conclusions: These findings support a model in which the APOE4 allele increases vulnerability to DOX-induced cardiac damage through mechanisms that involve significant and complex alterations in the response to oxidative stress and/or damage signaling. Studies are ongoing to further elucidate transcriptional differences conferred by APOE4, to analyze substrates downstream of caspase-1 affected by APOE4, and to determine the extent to which APOE4 may contribute to DOX toxicity in patient populations. Citation Format: Guannan Wang, G. William Rebeck, Olga Rodriguez, Marc E. Lippman, Nanette H. Bishopric. Expression of the human APOE4 genotype modulates Doxorubicin cardiotoxicity in mice [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-05.

Cardiac Protective Effect of Kirenol against Doxorubicin-Induced Cardiac Hypertrophy in H9c2 Cells through Nrf2 Signaling via PI3K/AKT Pathways.

Kirenol (KRL) is a biologically active substance extracted from Herba Siegesbeckiae. This natural type of diterpenoid has been widely adopted for its important anti-inflammatory and anti-rheumatic properties. Despite several studies claiming the benefits of KRL, its cardiac effects have not yet been clarified. Cardiotoxicity remains a key concern associated with the long-term administration of doxorubicin (DOX). The generation of reactive oxygen species (ROS) causes oxidative stress, significantly contributing to DOX-induced cardiac damage. The purpose of the current study is to investigate the cardio-protective effects of KRL against apoptosis in H9c2 cells induced by DOX. The analysis of cellular apoptosis was performed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining assay and measuring the modulation in the expression levels of proteins involved in apoptosis and Nrf2 signaling, the oxidative stress markers. Furthermore, Western blotting was used to determine cell survival. KRL treatment, with Nrf2 upregulation and activation, accompanied by activation of PI3K/AKT, could prevent the administration of DOX to induce cardiac oxidative stress, remodeling, and other effects. Additionally, the diterpenoid enhanced the activation of Bcl2 and Bcl-xL, while suppressing apoptosis marker proteins. As a result, KRL is considered a potential agent against hypertrophy resulting from cardiac deterioration. The study results show that KRL not only activates the IGF-IR-dependent p-PI3K/p-AKT and Nrf2 signaling pathway, but also suppresses caspase-dependent apoptosis.

Adiponectin agonist ADP355 ameliorates doxorubicin-induced cardiotoxicity by decreasing cardiomyocyte apoptosis and oxidative stress

Doxorubicin (DOX) is an anthracycline derivative and widely used as an anticancer drug. However, the severe cardiotoxicity of DOX limits its application. ADP355 is an adiponectin-based active peptide with anti-liver fibrosis and atherosclerosis properties. It remains unclear the effects and involved mechanisms of ADP355 in DOX-induced cardiotoxicity. C57BL/6J mice were intraperitoneally injected DOX once a week to induce heart failure while receiving ADP355 treatment daily for 4 weeks. At the end of experiment, blood and heart tissues were collected. We found that ADP355 markedly improved DOX-induced cardiac dysfunction and histopathological damage, and decreased serum creatine kinase, lactate dehydrogenase and hydroxybutyrate dehydrogenase levels. The anti-apoptotic activity of ADP355 was indicated by reduction in TUNEL-positive cells and cleaved caspase-3 expression, along with decreased BCL2-associated X protein/B cell lymphoma 2 (BAX/BCL2) levels in heart tissues. Additionally, ADP355 markedly increased DOX-decreased cell viability by reducing BAX/BCL2, but inhibited reactive oxygen species production in H9c2 cells. Mechanistically, ADP355 attenuated expression of DOX-reduced nuclear factor-erythroid 2-related factor 2 (Nrf2) and superoxide dismutase 2, as well as mRNA levels of Nrf2 downstream targets. Furthermore, ADP355 activated sirtuin 2 and its target genes. In conclusion, we demonstrate that ADP355 alleviates DOX-induced cardiotoxicity by inhibiting myocardial apoptosis and oxidative stress through Nrf2 and sirtuin 2 signaling pathways. These findings suggest that ADP355 can be a promising candidate for the treatment of cardiac dysfunction.

Abstract 5422: Dissecting the mechanism of exercise-mediated protection of Dox-induced cardiotoxicity

Abstract Doxorubicin (Dox) is one of the most effective drugs for the treatment of childhood cancer patients. However, there is a correlation between Dox dose/intensity and cardiotoxicity. We developed a juvenile mouse cardiotoxicity model and showed that Dox treatment affected the cardiac vessel morphology characterized by punctate vessels with decreased pericytes and endothelial cells. Cardiac blood flow and function (as quantified by echocardiography [Echo]) were also compromised. These abnormalities were not seen in mice treated with exercise (Ex) during Dox. We also demonstrated increased c-kit+ cells in the hearts of mice treated with Dox + Ex. To determine the origin of these c-kit+ cells we performed a bone marrow (BM) transplant using GFP+ BM cells from C57BL/6Tg(UBC-GFP)30Scha/J mice into C57BL/6 WT mice treated with Busulfan and anti-CD4 and anti-DC8 Abs. BM engraftment was documented 4 wks post-transplant. Transplanted mice were treated with Dox twice/wk x 2 wks; Dox + Ex, or control (no treatment). Cardiac function was assessed after treatment by Echo and the heart tissues were evaluated by immunohistochemistry (IHC) for cells expressing GFP (denoting cells derived from the BM); GFP+/NG2+ or GFP+/CD31+ (BM cells differentiated into pericytes or endothelial cells respectively). Echo data confirmed decreased cardiac function in Dox but not Dox + Ex-treated mice. Few GFP+ cells were found in the hearts from control or Dox-treated mice. By contrast, mice treated with Dox + Ex showed a significant increase in GFP+ cells indicating that exercise had induced the migration of BM cells into the heart. Co-localization of GFP/NG2 and GFP/CD31 indicated that these BM stem cells had differentiated into both endothelial cells and pericytes, and that these cells were incorporated into the cardiac vessels. Few GFP+cTnI+ cells were observed in the heart tissues from mice treated with Ex + Dox, indicating that the BM stem cells had not differentiated into cardiomyoctes. These studies suggest that acute Dox-induced cardiac damage may be partially mediated by decreased blood flow secondary to damaged vessels with decreased vascular endothelial cells and pericytes. Ex may inhibit this effect by inducing the migration of BM stem cells to repair the damaged vessels. We also showed that Dox therapy induced phosphorylation of MST1/2, LATS1 and YAP, decreased total YAP, and induced cleavage of caspase-3 and PARP. These changes were not seen in Dox + Ex hearts suggesting that Ex protected cardiomyocytes from Dox-induced apoptosis by suppressing Hippo-YAP signaling. Taken together, our data demonstrated that the protective effect of Ex may be mediated by restoring cardiac blood flow which may in turn inhibit Dox-induced Hippo-YAP signaling-mediated cardiomyocyte apoptosis. These data support the concept that exercise interventions have the potential to decrease Dox-induced cardiac damage and morbidity in childhood cancer patients. Citation Format: Rong-Hua Tao, Masato Kobayashi, Fei Wang, Yuanzheng Yang, Eugenie S. Kleinerman. Dissecting the mechanism of exercise-mediated protection of Dox-induced cardiotoxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5422.

Nimbolide prevents myocardial damage by regulating cardiac biomarkers, antioxidantlevel, and apoptosis signaling against doxorubicin-induced cardiotoxicity in rats.

The current work planned to assess the protecting properties of nimbolide against doxorubicin (DOX)-treated myocardial damage. Myocardial damage was produced with2.5 mg/kg of DOX given onalternative days (14 days). Thiobarbituric acid reactive substances (TBARS) levels of alipid peroxidative marker were elevated, whereas reduced body weight, heart weight, blood pressure indices and reduced levels of antioxidants like glutathione-S-transferase, superoxide dismutase, catalase, glutathione peroxidase, glutathione, and glutathione reductase were observed in the heart tissue of DOX-treated animals. DOX-treated animalsshowedaugmented levels of cardiac markers likes monocyte chemotactic protein-1, interferon-gamma, aspartate transferase, creatine kinase, lactate dehydrogenase, creatine kinase-muscle/brain, heart-type fatty acid-binding protein, glycogen phosphorylase isoenzyme BB, transforming growth factor-β, brain natriuretic peptide, myoglobin, and cTnI in serum. Histopathological assessment confirmed the DOX-induced cardiotoxicity. Furthermore, DOX-induced rats showed augmented inflammatory mediators (nuclear factor-κB [NF-kB], tumor necrosis factor-α [TNF-α], and interleukin-1β [IL-1β]) and increased PI3K/Akt signaling proteins (PI3K, p-Bad/Bad, caspase-3, and p-Akt), whereas decreased oxidative markers (HO-1 and NQO-1) and p-PTEN were observed. Nimbolide-supplemented rats showed reduced activity/levels of cardiac markers and TBARS levels in serum and heart tissue. Levels of enzymatic and nonenzymatic antioxidants were augmented in the heart tissue of nimbolide-supplemented rats. Nimbolide influence decreased apoptosis, inflammation, and enhanced antioxidant markers through the modulation of p-Bad/Bad, caspase-3, PI3K, p-Akt, TNF-α, NF-kB, IL-1β, HO-1, NQO-1, and p-PTEN markers. The histopathological explanations were observed to be in line with biochemical analysis. Therefore, the findingof current work wasthat nimbolide has a defensiveeffect onthe myocardium against DOX-induced cardiac tissue damage.

Long-noncoding RNA MALAT1 sponges microRNA-92a-3p to inhibit doxorubicin-induced cardiac senescence by targeting ATG4a.

The clinical application of doxorubicin (Dox) is limited due to its undesirable cardiotoxicity side effects. Cellular senescence plays an important role in Dox-induced cardiotoxicity. Exosomes derived from stem cells showed a therapeutic effect in Dox-induced cardiomyopathy (DIC). Hypoxia-preconditioned exosomes (exosomeHypoxia) display pro-metabolism and pro-survival abilities. Several long-noncoding RNAs/microRNAs act as competing endogenous RNAs (ceRNAs) modulating DIC. No study investigated whether exosomeHypoxia could attenuate DIC through modulating ceRNAs.Treatment of the human adipose–derived mesenchymal stem cells with hypoxia induced lncRNA-MALAT1 accumulation in the secreted exosomes. In addition, the lncRNA-MALAT1 was identified as an exosomal transfer RNA to repress miR-92a-3p expression. Silencing the lncRNA-MALAT1 in MSCs or miR-92a-3p overexpression in cardiomyocytes significantly impaired the rejuvenation induced by exosomeHypoxia. TargetScan and luciferase assay showed that miR-92a-3p targeted the ATG4a 3' untranslated region. Silencing ATG4a blocked the anti-senescent effect of exosomeHypoxia.This study identified the lncRNA-MALAT1 that functioned as ceRNA binding to miR-92a-3p, leading to ATG4a activation, thus improving mitochondrial metabolism. LncRNA-MALAT1/miR-92a-3p/ATG4a partially mediates the cardioprotective roles of exosomeHypoxia in Dox-induced cardiac damage. ExosomeHypoxia may serve as a potential therapeutic target against DIC.

P3115Effect of calcitriol attenuates doxorubicin-induced cardiac dysfunction in mice model: focus on endothelial-to-mesenchymal transition

Abstract Objective Doxorubicin (DOX) is an effective anti-neoplasm drug, but the early and late cardiac toxicity limits its clinical use. The Endothelial-to-mesenchymal transition (EndMT) has been found to involve in the process of heart failure. It's unclear whether EndMT plays a role in DOX-induced cardiomyopathy (DoIC). Calcitriol is an active form Vitamin D3, which blocks the growth of cancer cells via inhibiting Smad pathway. This study investigated the cardiac protective effect of calcitriol via inhibiting of EndMT in DoIC. Methods/Findings C57BL/6 mice and endothelial-specific labeled mice were administered Dox twice weekly for 4 weeks [intraperitoneally (i.p.), 32 mg/kg cumulative dose]and were subsequently treated with/without calcitriol for 12 weeks. The cardiac echography revealed diastolic dysfunction at 13 weeks following the first DOX treatment and was accompanied by increased of myocardial fibrosis and up-regulated pro-fibrotic proteins.(Figure A-C) Calcitriol attenuated DOX-induced myocardial fibrosis, down-regulated pro-fibrotic proteinsand diastolic function. Endothelial fate tracing revealed that endothelium-derived cells contributed DOX-induced cardiac remodelling through EndMT and Calcitriol attenuated this process without attenuating Dox-induced cardiac myocyte and endothelial cell damage.(Figure D) In vitro, we examined if calcitriol would inhibit EndMT and fibroblast-to-myofibroblast transition (FMT) through the Smad pathway. Human umbilical vein endothelial cells (HUVECs) and mouse cardiac fibroblasts were treated with TGF-beta with or without calcitriol. Morphological, immunofluorescence staining, and western blot analyses were carried out to evaluate EndMT and FMT. Calcitriol attenuated EndMT and FMT by inhibiting the Smad2 pathway. Taken together, calcitriol didn't reduced Doxorubicin induced damage of cardiomocyte and endothelial cells. But calcitriol inhibit doxorubicin induced heart failure by attenuating cardiac fibrosis through inhibiting Smad pathway. Conclusion Calcitriol attenuated DOX-induced cardiomyopathy partial through inhibiting EndMT process. Acknowledgement/Funding CMRPG8E0661-3