Doxorubicin-induced Cardiotoxicity In Mice Research Articles

The NEDD8 activating enzyme inhibitor MLN4924 mitigates doxorubicin-induced cardiotoxicity in mice

Doxorubicin (DOX) is a widely utilized chemotherapeutic agent in clinical oncology for treating various cancers. However, its clinical use is constrained by its significant side effects. Among these, the development of cardiomyopathy, characterized by cardiac remodeling and eventual heart failure, stands as a major concern following DOX chemotherapy. In our current investigation, we have showcased the efficacy of MLN4924 in mitigating doxorubicin-induced cardiotoxicity through direct inhibition of the NEDD8-activating enzyme, NAE. MLN4924 demonstrated the ability to stabilize mitochondrial function post-doxorubicin treatment, diminish cardiomyocyte apoptosis, alleviate oxidative stress-induced damage in the myocardium, enhance cardiac contractile function, mitigate cardiac fibrosis, and impede cardiac remodeling associated with heart failure. At the mechanistic level, MLN4924 intervened in the neddylation process by inhibiting the NEDD8 activating enzyme, NAE, within the murine cardiac tissue subsequent to doxorubicin treatment. This intervention resulted in the suppression of NEDD8 protein expression, reduction in neddylation activity, and consequential manifestation of cardioprotective effects. Collectively, our findings posit MLN4924 as a potential therapeutic avenue for mitigating doxorubicin-induced cardiotoxicity by attenuating heightened neddylation activity through NAE inhibition, thereby offering a viable and promising treatment modality for afflicted patients.

Protective effect of diindolylmethane-enriched dietary cabbage against doxorubicin-induced cardiotoxicity in mice.

Chemotherapy with doxorubicin (Dox) can lead to cardiotoxic effects, presenting a major complication in cancer therapy. Diindolylmethane (DIM), derived from cruciferous vegetables like cabbage, exhibits numerous health benefits. However, its clinical application is limited because of low bioavailability and suboptimal natural concentrations in dietary sources. To address this limitation, we developed a processing methodology, specifically fermentation and boiling, to enhance DIM levels in cabbage. High-performance liquid chromatography (HPLC) analysis revealed a threefold DIM increase in fermented cabbage and a substantial ninefold increase in fermented-boiled cabbage compared to raw cabbage. To evaluate the clinical implications, we formulated a DIM-enriched diet and administered it to mice undergoing Dox treatment. Our in vivo results revealed that Dox treatment led to cardiotoxicity, manifested by changes in body and heart weight, increased mortality, and severe myocardial tissue degeneration. Dietary administration of the DIM-enriched diet enhanced antioxidant defenses and inhibited apoptosis in the cardiac tissue by interfering with mitoptosis and increasing antioxidant enzyme expression. Interestingly, we found that the DIM-enriched diet inhibited the nuclear translocation of NF-kB in cardiac tissue, thereby downregulating the expression of inflammatory mediators such as TNF-α and IL-6. Further, the DIM-enriched diet significantly reduced serum cardiac injury markers elevated by Dox treatment. These results suggest that the DIM-enriched cabbage diet can serve as a complementary dietary intervention for cancer patients undergoing chemotherapy. Further, our research highlights the role of plant-based diets in reducing treatment side effects and improving the quality of life for cancer patients.

Abstract 12348: Targeted Degradation of Topoisomerase 2b by Dexrazoxane for Prevention of Doxorubicin-Induced Cardiotoxicity: Dose and Time Course Study in Human

Introduction: Doxorubicin is known to induce cardiotoxicity through topoisomerase 2b (Top2b), whereas doxorubicin poisons topoisomerase 2a (Top2a) to kill cancer cells. When Top2b was genetically deleted in the cardiomyocytes of adult mice, doxorubicin-induced cardiomyopathy was prevented. Pharmacological degradation of Top2b by dexrazoxane can also prevent doxorubicin-induced cardiotoxicity in mice. However, it is not known whether dexrazoxane can induce degradation of Top2b in human. Hypothesis: Targeted degradation of Top2b by dexrazoxane can be achieved in humans. Methods: Top2b protein level in peripheral blood mononuclear cells (PBMC) were assessed serially after intravenous dexrazoxane infusion in human volunteers. The effectiveness of dexrazoxane on Top2b degradation and the time course of Top2b degradation were determined. Blood was drawn before dexrazoxane infusion, hourly after infusion up to 12 hours, at 24 hours, and at 48 hours. PBMCs were isolated using SepMate tube and fractionated into nuclear and cytosolic fractions. The nuclear fractions were used in Western blot analysis. Immunoblotting was carried out with rabbit anti-Top2b, anti-Top2a, or anti-Lamin B1 antibodies, followed by Peroxidase AffiniPure goat anti-rabbit IgG. The ratio of the intensity of the Top2b and Lamin B1 bands were normalized by setting the pre-ratio to 1. Results: One hour after dexrazoxane infusion, Top2b level in leukocytes was reduced to 14%-21% of baseline. Dexrazoxane-induced degradation of Top2b persisted for up to 12 hours and returned to baseline 24 hours after dexrazoxane infusion. Importantly, Top2a level did not vary significantly following dexrazoxane infusion. There were no serious adverse reactions in all participants. Conclusions: Human PBMC can be used as a surrogate to follow Top2b degradation. Degradation of Top2b can be achieved with dexrazoxane dose as low as 100 mg/M 2 (one-fifth of the current FDA recommended dose when used in conjunction with doxorubicin). Thus, early dexrazoxane followed by delayed-administration of doxorubicin can be studied as a new strategy to prevent doxorubicin-induced cardiotoxicity by depleting Top2b. Moreover, dexrazoxane did not affect Top2a, thus preserving doxorubicin’s efficacy.

The Effect of Aerobic Training and Nanomicelle Curcumin Supplementation on MDA and BAX And BCL2 Activity Levels on Cardiotoxicity of Mice Infected with Breast Cancer in the Phase of Mani with Doxorubicin

Background: Doxorubicin is a universal drug in the treatment of all types of cancers, which is limited due to its many side effects. Therefore, the present study has investigated the effect of aerobic exercise and the use of curcumin nano micelles on the activity levels of BAX, BCL2, and MDA concentration in doxorubicin-induced cardiotoxicity in mice with breast cancer.
 Methods: In this research, which was of a fundamental type and controlled experimentally, 24BalbC female rats were randomly divided into 4 groups: doxorubicin, doxorubicin patient and exercise, doxorubicin patient and nano curcumin supplement and doxorubicin patient, exercise and supplement. Breast cancer induction was done with MC4-L2 cells. Doxorubicin 2mg/kg was injected subcutaneously 6 times on day 28, 21, 14, 7, 1 and 35. The aerobic program consisted of 5 sessions per week for 42 days with an increase in the speed of the treadmill every two weeks including 14, 16, and 18 meters per minute and the use of nano curcumin at a dose of 100mg/k. The collected data were analyzed with a two-way analysis of variance with a significance level of 0.05 in the tests.
 Results: 6 weeks of aerobic training had no effect on BCL2 activity and MDA concentration, but it significantly decreased BAX activity. Taking nano curcumin supplement for 6 weeks decreased BAX activity and MDA concentration and significantly increased BCL2 activity. Conclusion 6 A week of aerobic training along with nano curcumin supplementation did not affect BAX activity and MDA concentration, but it caused an increase in BCL2 activity (p value < 0.05).
 Conclusion: Aerobic exercise and the use of curcumin nano micelle supplement separately or at the same time can be effective in reducing the cardiac toxicity of doxorubicin in patients with breast cancer.

Assessing the cardioprotective effect of necrosulfonamide in doxorubicin-induced cardiotoxicity in mice.

This study aimed to determine the cardioprotective effect of necrosulfonamide (NSA), a pyroptosis and necroptosis inhibitor, against acute doxorubicin cardiotoxicity. Fifteen male mice were divided into three groups (n=5/group). Cardiotoxicity was induced by a single intraperitoneal injection of 20 mg/kg of DOX on the 3rd day of the experiment. The control group received daily intraperitoneal (i.p.) injections of 5% DMSO for five consecutive days. The second group, the DOX group, received a single i.p. injection of 20 mg/kg DOX on the third day of the experiment. The third group, the DOX plus necrosulfonamide (NSA) group, received DOX injections like the second group and 5 mg/kg of NSA i.p. daily for five days, starting two days before the DOX injection. At the end of the study, animals were euthanized, and blood and tissue samples were collected. Various parameters, including cardiac troponin I (cTnI), TNF-α, IL-1β, caspase-1, glutathione peroxidase-4 (GPX-4), and hemeoxygenase 1 (Hmox-1), were measured using ELISA. Cardiac expression of the NF-κB gene was determined by RT-qPCR. A histopathological assessment of myocardial lesions was also performed. DOX administration significantly increased serum cTnI levels and tissue inflammatory biomarkers (TNF-α, IL-1β, caspase-1) while reducing tissue antioxidant enzymes (GPX-4, Hmox-1). In addition, it significantly increased nuclear factor-κB (NF-κB) gene expression compared to the control (about 10.5-fold elevation). Histopathological analysis revealed marked vacuolization and necrosis. However, pretreatment with NSA dramatically altered these findings, with serum cTnI levels significantly lower in this group compared to DOX. Inflammatory indicators decreased, and antioxidant enzymes were restored to varying degrees. NSA pretreatment downregulated NF-κβ gene expression and preserved near-normal myocardial morphology. Our results showed that NSA protected against DOX-induced cardiotoxicity, an effect likely mediated by its anti-pyroptotic, anti-necroptotic, and antioxidant properties.

Hyperhomocysteinaemia Promotes Doxorubicin-Induced Cardiotoxicity in Mice.

Doxorubicin, a widely used chemotherapeutic drug in clinical oncology, causes a series of cardiac side effects referred to as doxorubicin-induced cardiotoxicity. Hyperhomocysteinaemia is an independent risk factor for multiple cardiovascular diseases. However, whether hyperhomocysteinaemia contributes to doxorubicin-induced cardiotoxicity is currently unknown. In this study, we explored the pathogenic effects of hyperhomocysteinaemia induced by dietary methionine supplementation (2% wt/wt in rodent chow) in a mouse model of doxorubicin-induced cardiotoxicity. Our data showed that methionine supplementation doubled serum homocysteine levels, inducing mild hyperhomocysteinaemia. Doxorubicin at a cumulative dosage of 25 mg/kg body weight led to significant weight loss and severe cardiac dysfunction, which were further exacerbated by methionine-induced mild hyperhomocysteinaemia. Doxorubicin-induced cardiac atrophy, cytoplasmic vacuolisation, myofibrillar disarray and loss, as well as cardiac fibrosis, were also exacerbated by methionine-induced mild hyperhomocysteinaemia. Additional folic acid supplementation (0.006% wt/wt) prevented methionine-induced hyperhomocysteinaemia and inhibited hyperhomocysteinaemia-aggravated cardiac dysfunction and cardiomyopathy. In particular, hyperhomocysteinaemia increased both serum and cardiac oxidative stress, which could all be inhibited by folic acid supplementation. Therefore, we demonstrated for the first time that hyperhomocysteinaemia could exacerbate doxorubicin-induced cardiotoxicity in mice, and the pathogenic effects of hyperhomocysteinaemia might at least partially correlate with increased oxidative stress and could be prevented by folic acid supplementation. Our study provides preliminary experimental evidence for the assessment of hyperhomocysteinaemia as a potential risk factor for chemotherapy-induced cardiotoxicity in cancer patients.

Rifampicin efficacy against doxorubicin-induced cardiotoxicity in mice

BackgroundThe toxic effect of doxorubicin on the heart limits its clinical usage in cancer therapy. This work intended to investigate, for the first time, the efficacy of rifampicin administration against doxorubicin-induction of cardiotoxicity in mice. Forty adult male albino mice were distributed into four sets: Control, Doxorubicin, Doxorubicin + Rifampicin 0.107, and Doxorubicin + Rifampicin 0.214, with n = 10 for each. Heart histopathology and biochemical assays for heart function tests [creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), cardiac troponin I (cTnI), atrial natriuretic peptide (ANP), and vascular endothelial growth factor (VEGF)], oxidative stress [malondialdehyde (MDA) and superoxide dismutase (SOD)], and minerals [phosphorus, sodium, potassium, and calcium] were done.ResultsDoxorubicin-induced cardiotoxicity using a total dose of 15 mg/kg was confirmed histologically. Cardiomyocytes showed congestion, necrosis, edema, and inflammatory cell infiltration. Biochemically, elevations in LDH, CK, and AST activities, p < 0.001, as well as increases in cTnI and ANP levels, p < 0.001, increased oxidative stress (MDA, p < 0.001), high minerals (Na, K, p < 0.001, P, p < 0.01, and Ca, p < 0.05), with reduced VEGF concentration, p < 0.001, and low antioxidant (SOD, p < 0.001) were observed in the Doxorubicin group compared to control. Co-treatment with rifampicin significantly (p < 0.001) reduced the increased oxidative stress, high Na and K, increased LDH, CK, AST, cTnI, and ANP, and elevated the low SOD toward the normal ranges. Our histological data supported our biochemical data; rifampicin dose 0.214 mg/kg showed better improvements than dose 0107.ConclusionsOur results demonstrated that rifampicin could help protect the body against doxorubicin-induced cardiotoxicity through its antioxidative effect.

Leucine zipper protein 1 prevents doxorubicin-induced cardiotoxicity in mice

ObjectiveDoxorubicin (DOX) is commonly used for chemotherapy; however, its clinical value is extremely dampened because of the fatal cardiotoxicity. Leucine zipper protein 1 (LUZP1) plays critical roles in cardiovascular development, and this study is designed for determining its function and mechanism in DOX-induced cardiotoxicity. MethodsCardiac-specific Luzp1 knockout (cKO) and transgenic (cTG) mice received a single or repeated DOX injections to establish acute and chronic cardiotoxicity. Biomarkers of inflammation, oxidative damage and cell apoptosis were evaluated. Transcriptome and co-immunoprecipitation analysis were used to screen the underlying molecular pathways. Meanwhile, primary cardiomyocytes were applied to confirm the beneficial effects of LUZP1 in depth. ResultsLUZP1 was upregulated in DOX-injured hearts and cardiomyocytes. Cardiac-specific LUZP1 deficiency aggravated, while cardiac-specific LUZP1 overexpression attenuated DOX-associated inflammation, oxidative damage, cell apoptosis and acute cardiac injury. Mechanistic studies revealed that LUZP1 ameliorated DOX-induced cardiotoxicity through activating 5′-AMP-activated protein kinase (AMPK) pathway, and AMPK deficiency abolished the cardioprotection of LUZP1. Further findings suggested that LUZP1 interacted with protein phosphatase 1 to activate AMPK pathway. Moreover, we determined that cardiac-specific LUZP1 overexpression could also attenuate DOX-associated chronic cardiac injury in mice. ConclusionLUZP1 attenuates DOX-induced inflammation, oxidative damage, cell apoptosis and ventricular impairment through regulating AMPK pathway, and gene therapy targeting LUZP1 may provide novel therapeutic approached to treat DOX-induced cardiotoxicity.

Targeting Energy Protection as a Novel Strategy to Disclose Di’ao Xinxuekang against the Cardiotoxicity Caused by Doxorubicin

Doxorubicin (DOX) can induce myocardial energy metabolism disorder and further worsen heart failure. "Energy protection" is proposed as a new cardiac protection strategy. Previous studies have found that Di'ao Xinxuekang (DXXK) can improve doxorubicin-induced cardiotoxicity in mice by inhibiting ferroptosis. However, there are very few studies associating DXXK and energy protection. This study aims to explore the "energy protection" effect of DXXK on cardiotoxicity induced by DOX. A DOX-induced cardiotoxicity model established in rats and H9c2 cells are used to analyze the therapeutic effects of DXXK on serum indexes, cardiac function indexes and cardiac histopathology. The metabonomic methods were used to explore the potential mechanism of DXXK in treating DOX-induced cardiotoxicity. In addition, we also observed the mitochondrial- and autophagy-related indicators of myocardial cells and the mRNA expression level of the core target regulating energy-metabolism-related pathways. Our results indicated that DXXK can improve cardiac function, reduce myocardial enzymes and alleviate the histological damage of heart tissue caused by DOX. In addition, DXXK can improve mitochondrial damage induced by DOX and inhibit excessive autophagy. Metabonomics analysis showed that DOX can significantly affects the pathways related to energy metabolism of myocardial cells, which are involved in the therapeutic mechanism of DXXK. In conclusion, DXXK can treat DOX-induced cardiotoxicity through the AMPK-mediated energy protection pathway.

Untargeted metabolomics reveals the combination effects and mechanisms of Huangqi-fuzi herb-pair against doxorubicin-induced cardiotoxicity

Ethnopharmacological relevanceQifu decoction (QFD) is a famous traditional Chinese medicine (TCM) composed of Astragali Radix (HuangQi) and Aconiti Lateralis Radix Praeparaia (Fuzi), which can alleviate doxorubicin (DOX)-induced cardiotoxicity (DIC). However, its protective mechanism remains obscured. Aim of the studyThe present study aimed to uncover the cardioprotective mechanism and the synergistic effect of QFD against DIC in mice. Materials and methodsThe cardioprotective activity of QFD against DIC was assessed by electrocardiogram, serum biochemical assays and histopathology. Mass spectrometry-based metabolomic approach was conducted to elucidate the preventive mechanisms of QFD, HuangQi decoction (HQD), and Fuzi decoction (FZD) against DIC. QFD, HQD, FZD-targeted metabolic pathways were identified and compared to investigate the synergistic mechanism of QFD by computational systems analysis. Quantitative real-time PCR (qRT-PCR) was further employed to validate the key metabolic pathways at the level of the gene. ResultsThe electrocardiogram combined with the biochemical analysis and histopathology showed that the protection effects were sorted as QFD > HQD ≈ FZD. A total of 41 metabolites contributing to DIC were identified in the mice serum, among which 32, 12 and 10 metabolites were significantly reverted by QFD, HQD and FZD, respectively. Metabolic pathway analysis revealed that DOX perturbed 12 metabolic pathways, and QFD, HQD, and FZD-treated groups could significantly reverse 12, 7 and 6 metabolic pathways of these 12 metabolic pathways. Metabolic pathway and qRT-PCR revealed that QFD could protect DIC mainly by regulating energy metabolism, amino acids metabolism, arachidonic acid metabolism and glycerophospholipid metabolism, and HQD and FZD mutually reinforced each other. ConclusionThese evidences revealed that QFD was a promising drug candidate for DIC by maintaining metabolic homeostasis. Meanwhile, this work provided a useful approach for evaluating the efficacy and the synergistic effects of TCMs against cardiomyopathy.

Potential cardioprotective effects of Amentoflavone in doxorubicin-induced cardiotoxicity in mice

Doxorubicin (DOX) is an available chemotherapeutic drug for treating various tumors. However, its effectiveness is limited by cardiotoxicity. Amentoflavone (AMF), a natural biflavonoid separated from Cycas thouarsii ethyl acetate fraction, displays promising anticancer, anti-inflammatory, and antioxidant effects. Thus, our research aims to explore whether AMF could boost cardioprotective effects against DOX cardiotoxicity and reveal the potential underlying mechanisms of cardioprotection. Mice were classified into four groups; Normal control, Untreated DOX group, and DOX groups treated with AMF (40 and 80 mg/kg, respectively) intraperitoneal injection daily for four days before doxorubicin administration and for additional three days following DOX administration to assess cardiotoxicity. Echocardiography showed that AMF 80 treated group was protected from DOX cardiotoxicity. Additionally, it alleviated histopathological structural alterations and effectively restored heart weight and body weight ratio. These effects were confirmed biochemically by a substantially reduced serum creatine kinase-MB (CK-MB) and aspartate aminotransferase (AST) levels. AMF effectively restored nuclear respiratory factor-1(NRF-1), mitochondrial transcription factor A (TFAM), and normalized heat shock protein − 27(HSP-27) expression levels compared to the DOX group. Moreover, AMF mitigated oxidative stress conditions and significantly suppressed NADPH oxidase (NOX) expression levels. It also showed significant anti-inflammatory effects via suppressing interleukin-6 (IL-6) expression and decreasing nuclear factor Kabba B (NF-κb) immune-staining. In addition, AMF markedly reduced FAS ligand (FASL) expression and p53 immune staining in cardiac tissue. This study is the first for the in vivo potential beneficial effects of AMF against acute DOX cardiotoxicity, possibly via exerting antioxidant, anti-inflammatory, and anti-apoptotic effects and restoring mitochondrial function.

SIRT1 protects the heart against doxorubicin-induced cardiotoxicity by mediating the DNA damage response via deacetylation of histone H2AX

Abstract Background Doxorubicin induces DNA damage not only in tumor cells but also in the cardiomyocyte, and accumulation of damaged DNA has been implicated in doxorubicin-induced cardiotoxicity. We previously found that cardiomyocyte-specific deletion of SIRT1, a NAD+-dependent histone/protein deacetylase, worsens doxorubicin-induced cardiotoxicity in mice. However, its molecular mechanism remains unclear. Phosphorylation of histone H2AX at Ser139 catalyzed by ATM (mutated in ataxia-telangiectasia) at the sites of DNA damage is a critical mediator for DNA repair. Purpose Here, we tested the hypothesis that deacetylation of H2AX by SIRT1 mediates DNA damage response to counteract doxorubicin-induced cardiotoxicity. Methods and results Wild-type (WT) mice and tamoxifen-inducible cardiomyocyte-specific SIRT1 knockout (SIRT1-cKO) mice at 3 month of age received doxorubicin (4 IP injections of 5 mg/kg/week) or a vehicle. Immunoblotting of myocardial lysates from mice 1 week after final doxorubicin showed that doxorubicin increased phospho-Ser139-H2AX level by 1.6-fold in WT, but such a response was blunted in SIRT1-cKO. Ser1981-phosphorylations of ATM induced by doxorubicin were similar in WT and SIRT1-cKO. DNA fragmentation evaluated by TUNEL staining revealed that the increase in TUNEL-positive nuclei by doxorubicin was more in SIRT1-cKO (0.13% to 0.38%) than those in WT (0.07% to 0.19%), suggesting higher DNA damage in SIRT1-cKO. In H9c2 cardiomyocytes, knockdown of SIRT1 also abolished the doxorubicin-induced Ser139-phosphorylation of H2AX without changing phospho-ATM levels. Increases in DNA damage evaluated by comet assay and cleavage of caspase-3 by doxorubicin were also enhanced in SIRT1-knockdown cells. Immunostaining for acetyl-Lys5-H2AX in the heart sections revealed that acetyl-Lys5-H2AX levels were increased in SIRT1-cKO by 58% compared with those in WT. In H9c2 cells, acetyl-Lys5-H2AX level was also increased by SIRT1 knockdown and reduced by expression of wild-type SIRT1. To test the role of the increased acetyl-Lys5-H2AX level under SIRT1 inhibition, we generated a mutant in which Lys5 was substituted to glutamine (K5Q H2AX) as a mimic of acetylated Lys5. In COS7 cells expressing WT or K5Q H2AX, Ser139-phosphorylation induced by doxorubicin was suppressed in K5Q mutant. In addition, doxorubicin-induced cleavage of caspase-3 was enhanced in H9c2 cells expressing K5Q H2AX as well as S139A H2AX, that cannot be phosphorylated at Ser139, compared with cells expressing WT H2AX. Conclusions These findings suggest that the increased Lys5 acetylation of H2AX via SIRT1 inhibition interferes Ser139 phosphorylation, leading to accumulation of damaged DNA and promotion of the apoptotic response. Such regulation of the DNA damage response contributes to protection by SIRT1 against doxorubicin-induced cardiotoxicity. Funding Acknowledgement Type of funding sources: None.

Activation of invariant natural killer T cells by alpha-galactosylceramide ameliorates doxorubicin-induced cardiotoxicity in mice.

Activation of invariant natural killer T cells by alpha-galactosylceramide ameliorates doxorubicin-induced cardiotoxicity in mice.

Activation of invariant natural killer T cells ameliorates doxorubicin-induced cardiotoxicity in mice

Abstract Objective Doxorubicin (DOX) is one of the most important anticancer agents and widely used to treat cancers but clinical utility of DOX is limited for its dose-dependent cardiotoxicity. The precise mechanism of DOX-induced cardiotoxicity is still not fully understood but it has been reported that cardiac inflammation is involved in the cardiotoxicity. Invariant natural killer T (iNKT) cells, a unique subset of T lymphocytes that recognize glycolipid antigens and secrete a large amount of both Th1 and Th2 cytokines on activation, have been shown to play crucial roles in the regulation of immune responses. However, it remains unclear whether iNKT cells are involved in DOX-induced cardiotoxicity. Methods and results Male C57BL/6J mice were administered DOX (20mg/kg body weight; n=28) or vehicle (Vehicle; n=6). DOX-administered mice were further divided into 2 groups; those treated with α-galactosylceramide (αGC, 0.1μg/g body weight; DOX-αGC; n=14), which specifically activates iNKT cells, or those treated with PBS (DOX-PBS; n=14) by intraperitoneal injections (twice; 4 days before and 3 days after DOX administration).An echocardiography conducted at 14 days after DOX/Vehicle administration revealed that LV fractional shortening was significantly reduced in the DOX-PBS compared to the Vehicle (49.3±0.8% vs. 59.2±1.7%, P&amp;lt;0.05), and this decrease was completely attenuated in the DOX-αGC (57.7±1.3%, P&amp;lt;0.05 vs. DOX-PBS)without affecting LV end-diastolic diameter. Flow cytometric analysis revealed that the ratio of iNKT cells to mononuclear cells infiltrated into the heart tissue was significantly increased in the DOX+αGC compared to the Vehicle and the DOX+PBS (1.00±0.09% vs. 0.54±0.09% and 0.71±0.07%, P&amp;lt;0.05). Immuno-histochemistry revealed that the infiltration number of Iba1+macrophages in the heart tissue was significantly elevated in the DOX+αGC compared to the Vehicle and the DOX+PBS (55.4±3.2 cells/mm2 vs. 21.7±2.0 cells/mm2 and 37.5±5.9 cells/mm2, P&amp;lt;0.05) The ratio of fibrosis area to the heart tissue was markedly higher in the DOX-PBS than in Vehicle (4.3±0.5% vs. 2.2±0.1%, P&amp;lt;0.05), and this increase was completely attenuated in the DOX-αGC (2.8±0.1%, P&amp;lt;0.05 vs.DOX-PBS).Real-time PCR analysis revealed that mRNA expressions of M2 macrophage markers (Arginase 1 and Retnla) and IL-4 were significantly enhanced in the DOX+αGC compared to the DOX+PBS (Arginase 1: 2.5±0.4 vs. 1.6±0.3 [relative ratio to the Vehicle], P=0.08; Retnla: 2.4±0.5 vs. 1.1±0.2 [relative ratio to the Vehicle], P&amp;lt;0.05; IL-4: 1.0±0.3 vs. 8.94±2.8 [relative ratio to the DOX+PBS], P&amp;lt;0.05), while those of M1 macrophage markers (iNOS and MCP-1) did not change among all groups. Conclusions Activation of iNKT cells ameliorates DOX-induced cardiotoxicity in mice via enhanced M2 macrophage polarization with the upregulation of IL-4 and reducing cardiac fibrosis. iNKT cell activation can be a novel preventive strategy against DOX-induced cardiotoxicity. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Japan Agency for Medical Research and Development (18lm0203001j0002) and JSPS KAKENHI (18K15834).

Xanthine oxidase inhibition attenuates doxorubicin-induced cardiotoxicity in mice

Abstract Background Accumulating evidence suggests that high serum uric acid (UA) is associated with left ventricular (LV) dysfunction. Although xanthine oxidase (XO) activation is a critical regulatory mechanism of the terminal step in ATP and purine degradation, the pathophysiological role of cardiac tissue XO in LV dysfunction remains unclear. Objectives We hypothesized that cardiac XO is activated in doxorubicin-induced LV dysfunction, and XO inhibitors ameliorate LV function by inhibiting cell death signals as well as by modifying cardiac purine metabolism. Methods Either doxorubicin (10 mg/kg) or vehicle was intraperitonially administered in a single injection to ICR mice. Mice were treated with or without oral XO inhibitors (febuxostat 3 mg/kg/day or topiroxostat 5 mg/kg/day) for 8 days starting 24 hours before doxorubicin-injection. The LV function was assessed by echocardiography at day 6 and by ex vivo heart perfusion at day 7. Results Cardiac tissue XO activity measured by a highly sensitive assay with liquid chromatography/mass spectrometry (n=8 each) and cardiac UA content (n=3–6) were significantly increased in doxorubicin-treated mice at day 7 and dramatically reduced by XO inhibitors. Accordingly, XO inhibitors substantially improved LV ejection fraction (n=8 each) and LV developed pressure (n=9 each) that had been impaired by doxorubicin administration. Intriguingly, the expression of GPX4, a negative regulator of ferroptosis, was decreased in doxorubicin-treated hearts but improved by XO inhibitors (n=6 each). Furthermore, metabolome analyses revealed an enhanced purine metabolism in doxorubicin-treated hearts, and XO inhibitors suppressed the serial metabolic reaction of hypoxanthine–xanthine–UA. Conclusions Doxorubicin administration induces cardiac tissue XO activation associated with an impaired LV function. XO inhibition attenuates the doxorubicin-induced cardiotoxicity partly through an anti-ferroptotic effect and the conservation of tissue ATP levels by modulating purine metabolism. The present study suggests that pharmacological XO inhibition represents a potential therapeutic strategy for the treatment of doxorubicin-induced cardiotoxicity. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): This study was supported in part by grants-in-aid for Ministry of Education Culture, Sports, Science and Technology.

Xanthine oxidase inhibition attenuates doxorubicin-induced cardiotoxicity in mice

Accumulating evidence suggests that high serum uric acid (UA) is associated with left ventricular (LV) dysfunction. Although xanthine oxidase (XO) activation is a critical regulatory mechanism of the terminal step in ATP and purine degradation, the pathophysiological role of cardiac tissue XO in LV dysfunction remains unclear. We herein investigated the role and functional significance of tissue XO activity in doxorubicin-induced cardiotoxicity. Either doxorubicin (10 mg/kg) or vehicle was intraperitonially administered in a single injection to mice. Mice were treated with or without oral XO-inhibitors (febuxostat 3 mg/kg/day or topiroxostat 5 mg/kg/day) for 8 days starting 24 h before doxorubicin injection. Cardiac tissue XO activity measured by a highly sensitive assay with liquid chromatography/mass spectrometry and cardiac UA content were significantly increased in doxorubicin-treated mice at day 7 and dramatically reduced by XO-inhibitors. Accordingly, XO-inhibitors substantially improved LV ejection fraction (assessed by echocardiography) and LV developed pressure (assessed by ex vivo Langendorff heart perfusion) impaired by doxorubicin administration. This was associated with an increase in XO-derived hydrogen peroxide production with concomitant upregulation of apoptotic and ferroptotic pathways, all of which were reduced by XO-inhibitors. Furthermore, metabolome analyses revealed enhanced purine metabolism in doxorubicin-treated hearts, and XO-inhibitors suppressed the serial metabolic reaction of hypoxanthine–xanthine–UA, the paths of ATP and purine degradation. In summary, doxorubicin administration induces cardiac tissue XO activation associated with impaired LV function. XO-inhibitors attenuate doxorubicin-induced cardiotoxicity through inhibition of XO-derived oxidative stress and cell death signals as well as the maintenance of cardiac energy metabolism associated with modulation of the purine metabolic pathway.

Asiatic Acid Protects against Doxorubicin-Induced Cardiotoxicity in Mice.

The use of doxorubicin (DOX) can result in depression of cardiac function and refractory cardiomyopathy. Currently, there are no effective approaches to prevent DOX-related cardiac complications. Asiatic acid (AA) has been reported to provide cardioprotection against several cardiovascular diseases. However, whether AA could attenuate DOX-related cardiac injury remains unclear. DOX (15 mg/kg) was injected intraperitoneally into the mice to mimic acute cardiac injury, and the mice were given AA (10 mg/kg or 30 mg/kg) for 2 weeks for protection. The data in our study found that AA-treated mice exhibited attenuated cardiac injury and improved cardiac function in response to DOX injection. AA also suppressed myocardial oxidative damage and apoptosis without affecting cardiac inflammation in DOX-treated mice. AA also provided protection in DOX-challenged cardiomyocytes, improved cell viability, and suppressed intracellular reactive oxygen species (ROS) in vitro. Detection of signaling pathways showed that AA activated protein kinase B (AKT) signaling pathway in vivo and in vitro. Furthermore, we found that AA lost its protective effects in the heart with AKT inactivation. In conclusion, our results found that AA could attenuate DOX-induced myocardial oxidative stress and apoptosis via activation of the AKT signaling pathway.

The Tumor-Suppressive Human Circular RNA CircITCH Sponges miR-330-5p to Ameliorate Doxorubicin-Induced Cardiotoxicity Through Upregulating SIRT6, Survivin, and SERCA2a.

Doxorubicin is one of the most potent antitumor agents available; however, its clinical use is restricted because it poses a risk of severe cardiotoxicity. Previous work has established that CircITCH (circular RNA ITCH [E3 ubiquitin-protein ligase]) is a broad-spectrum tumor-suppressive circular RNA and that its host gene, ITCH (E3 ubiquitin protein ligase), is involved in doxorubicin-induced cardiotoxicity (DOXIC). Whether CircITCH plays a role in DOXIC remains unknown. We aimed to dissect the role of CircITCH in DOXIC and further decipher its potential mechanisms. Circular RNA sequencing was performed to screen the potentially involved circRNAs in DOXI pathogenesis. Quantitative polymerase chain reaction and RNA in situ hybridization revealed that CircITCH was downregulated in doxorubicin-treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in the autopsy specimens from cancer patients who suffered from doxorubicin-induced cardiomyopathy. Cell death/viability assays, detection of cardiomyocyte necrosis markers, microelectrode array, and cardiomyocyte functional assays revealed that CircITCH ameliorated doxorubicin-induced cardiomyocyte injury and dysfunction. Detection of cellular/mitochondrial oxidative stress and DNA damage markers verified that CircITCH alleviated cellular/mitochondrial oxidative stress and DNA damage induced by doxorubicin. RNA pull-down assays, Ago2 immunoprecipitation and double fluorescent in situ hybridization identified miR-330-5p as a direct target of CircITCH. Moreover, CircITCH was found to function by acting as an endogenous sponge that sequestered miR-330-5p. Bioinformatic analysis, luciferase reporter assays, and quantitative polymerase chain reaction showed that SIRT6 (sirtuin 6), BIRC5 (baculoviral IAP repeat containing 5, Survivin), and ATP2A2 (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2, SERCA2a [SR Ca2+-ATPase 2]) were direct targets of miR-330-5p and that they were regulated by the CircITCH/miR-330-5p axis in DOXIC. Further experiments demonstrated that CircITCH-mediated alleviation of DOXIC was dependent on the interactions between miR-330-5p and the 3'-UTRs of SIRT6, BIRC5, and ATP2A2 mRNA. Finally, AAV9 (adeno-associated virus serotype 9) vector-based overexpression of the well-conserved CircITCH partly prevented DOXIC in mice. CircITCH represents a novel therapeutic target for DOXIC because it acts as a natural sponge of miR-330-5p, thereby upregulating SIRT6, Survivin and SERCA2a to alleviate doxorubicin-induced cardiomyocyte injury and dysfunction.

Repeated Remote Ischemic Conditioning Reduces Doxorubicin-Induced Cardiotoxicity

ObjectivesThis study investigated the cardioprotective effect of repeated remote ischemic preconditioning (rRIC) on doxorubicin-induced cardiotoxicity in mice. BackgroundDoxorubicin is an effective chemotherapeutic agent for a wide range of tumor types but its use and dosing are limited by acute and chronic cardiotoxicity. Remote ischemic conditioning (RIC) is cardioprotective in multiple cardiovascular injury models, but the effectiveness of rRIC in doxorubicin-induced cardiotoxicity has not been fully elucidated. MethodsrRIC was performed on mice before and after doxorubicin administration. Cardiac function was assessed by echocardiography and myocardial biology was tested by molecular approaches. ResultsDoxorubicin administration induced acute cardiotoxicity, as indicated by reduced cardiac function, reduced myocyte cross-section area and increased extracellular collagen deposition, increased circulating cardiac muscle damage markers, and decreased heart weight. Doxorubicin also adversely affected other organs, including the kidney, liver, and spleen, as evaluated by circulating markers or organ weight loss. rRIC not only abrogated doxorubicin-induced cardiotoxicity (left ventricular ejection fraction, doxorubicin 47.5 ± 1.1%, doxorubicin + rRIC 51.6 ± 0.7%, p = 0.017), but also was associated with multiorgan protection. Within the myocardium, rRIC attenuated doxorubicin-induced cardiomyocyte apoptosis, reduced inflammation, and increased autophagy signaling. ConclusionsrRIC may be a promising approach to reduce doxorubicin-induced cardiotoxicity.

Cardamonin protects against doxorubicin-induced cardiotoxicity in mice by restraining oxidative stress and inflammation associated with Nrf2 signaling

The clinical application of doxorubicin (DOX) for cancer treatment is limited due to its cardiotoxicity. However, the basic pathophysiological molecular mechanisms underlying DOX-induced cardiomyopathy have not yet been completely clarified, and the disease-specific therapeutic strategies are lacking. The aim of the present study was to investigate the potential cardioprotective effect of cardamonin (CAR), a flavone found in Alpinia plant, on DOX-induced cardiotoxicity in a mouse model. At first, in DOX-treated mouse cardiomyocytes, CAR showed significantly cytoprotective effects through elevating nuclear factor erythroid-2 related factor 2 (Nrf2) signaling, and reducing the degradation of Nrf2. This process then improved the anti-oxidant system, as evidenced by the up-regulated expression levels of haem oxygenase-1 (HO1), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase modifier subunit (GCLM), superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT). In contrast, DOX-induced increases in malondialdehyde (MDA) and reactive oxygen species (ROS) were highly inhibited by CAR treatments. Additionally, DOX-induced apoptosis and inflammatory response in cardiomyocytes were diminished by CAR through reducing the Caspase-3 and nuclear factor-κB (NF-κB) signaling pathways, respectively. Then, in the DOX-induced animal model with cardiotoxicity, we confirmed that through improving Nrf2 signaling, CAR markedly suppressed oxidative stress, apoptosis and inflammatory response in hearts of mice, improving cardiac function eventually. Together, our findings demonstrated that CAR activated Nrf2-related cytoprotective system, and protected the heart from oxidative damage, apoptosis and inflammatory injury, suggesting that CAR might be a potential therapeutic strategy in the prevention of DOX-associated myocardiopathy.