Doxorubicin-induced Cardiomyocyte Injury Research Articles

The SPATA2/CYLD pathway contributes to doxorubicin-induced cardiomyocyte ferroptosis via enhancing ferritinophagy

Ferroptosis is an iron-dependent cell death and contributes to doxorubicin-induced cardiotoxicity, but the mechanisms behind intracellular iron overload in cardiomyocyte after administration of doxorubicin remain largely unknown. Ferritinophagy is a selective type of autophagy and could be a novel source for intracellular free iron. Spermatogenesis-associated protein 2 (SPATA2), a member of the TNF signaling pathway, can recruit cylindromatosis (CYLD, a deubiquitinating enzyme) to regulate cell death. This study aims to explore whether ferritinophagy is the source for intracellular iron overload in cardiomyocyte upon doxorubicin treatment and whether the SPATA2/CYLD pathway is involved in regulation of nuclear receptor coactivator 4 (NCOA4) level, the selective cargo receptor for ferritinophagy. The C57BL/6J mice were subjected to a single injection of doxorubicin, which showed the compromised cardiac functions, accompanied by the upregulation of SPATA2 and CYLD and the enhanced interaction between them, the increases in ferritinophagy (reflecting by increases in NCOA4 and ratio of LC3Ⅱ/LC3Ⅰ while decreases in NCOA4 ubiquitination and ferritin) and ferroptosis (reflecting by intracellular iron overload and increase of acyl-CoA synthetase long chain family member 4). Consistently, similar results were achieved in the cultured cardiomyocytes after incubation with doxorubicin. Knocked down of SPATA2 notably reduced doxorubicin-induced cardiomyocyte injury concomitant with the attenuated ferritinophagy and the decreased ferroptosis. Based on these observations, we conclude that a novel pathway of SPATA2/CYLD has been identified, which contributes to doxorubicin-induced cardiomyocyte ferroptosis via enhancing ferritinophagy through a mechanism involving the deubiquitination of NCOA4.

HMOX1 silencing prevents doxorubicin-induced cardiomyocyte injury, mitochondrial dysfunction, and ferroptosis by downregulating CTGF.

Doxorubicin is a type of effective antitumor drug but can contribute to cardiomyocyte injuries. We aimed to dissect the mechanism of the HMOX1/CTGF axis in DOX-induced cardiomyocyte injury, mitochondrial dysfunction, and ferroptosis. Bioinformatics analysis was conducted to retrieve differentially expressed genes in a DOX-induced mouse model. Mouse cardiomyocytes, HL-1 cells, were induced with l µM DOX, after which gain- or loss-of-function assays were applied. CCK-8, fluorescent probe assay, flow cytometry, and corresponding kits were employed to detect cell viability, ROS levels, mitochondrial membrane potential and cell apoptosis, and GSH and Fe2+ contents, respectively. qRT-PCR or Western blot assay was adopted to test HMOX1, CTGF, BCL-2, Caspase3, Cleaved-Caspase3, and GPX4 expression. Bioinformatics analysis showed that HMOX1 and CTGF were highly expressed in DOX-induced mice and correlated with each other. Also, HMOX1 and CTGF expression was high in HL-1 cells after DOX treatment, along with an obvious decrease in cell viability and GSH and GPX4 expression, an increase in ROS levels, apoptosis, and Fe2+ contents, and mitochondrial membrane potential dysfunction or loss. HMOX1 or CTGF silencing diminished cell apoptosis, Cleaved-Caspase3 expression, Fe2+ contents, and ROS levels, enhanced cell viability and the expression of GSH, GPX4, and BCL-2, and recovered mitochondrial membrane potential in DOX-induced HL-1 cells. Nevertheless, the effects of HMOX1 silencing on the viability, apoptosis, ferroptosis, and mitochondrial dysfunction of DOX-induced HL-1 cells were counteracted by CTGF overexpression. In conclusion, HMOX1 silencing decreased CTGF expression to alleviate DOX-induced injury, mitochondrial dysfunction, and ferroptosis of mouse cardiomyocytes.

Solid Lipid Nanoformulation of Berberine Attenuates Doxorubicin Triggered in vitro Inflammation in H9c2 Rat Cardiomyocytes.

The aim of this study was to evaluate the efficacy of solid lipid nanoparticles of berberine against doxorubicin-induced cardiotoxicity. Berberine (Ber) is cardioprotective, but its oral bioavailability is low, and its effect on chemotherapy-induced cardiotoxicity has not been studied. Solid lipid nanoparticles (SLNs) of berberine chloride were prepared, characterized and evaluated in vitro against doxorubicin-induced cardiomyocyte injury. Berberine-loaded SLNs (Ber-SLNs) were synthesized using the water-in-oil microemulsion technique with tripalmitin, Tween 80 and poloxamer 407. Ber-SLNs were evaluated for preventive effect against toxicity of doxorubicin in H9c2 cells. The culture was pre-treated (24 h) with Ber (10 μM) and Ber-SLNs (1 and 10 μM), and 1 μM of doxorubicin (Dox) was added for 3 h. The cell viability assay (MTT (3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) and LDH (Lactate dehydrogenase)), levels of Creatine kinase-MB (CK-MB), Nitrite, MDA (Malondialdehyde), ROS (Reactive oxygen species) generation, and apoptotic DNA (Deoxyribonucleic acid) content were assessed. Ber-SLNs had a mean particle size of 13.12±1.188 nm, the zeta potential of -1.05 ± 0.08 mV, poly-dispersity index (PDI) of 0.317 ± 0.05 and entrapment efficiency of 50 ± 4.8%. Cell viability was 81 ± 0.17% for Ber-SLNs (10 μM) and 73.22 ± 0.83% for Ber (10 μM) treated cells in the MTT assay. Percentage cytotoxicity calculated from LDH release was 58.91 ± 0.54% after Dox, 40.3 ± 1.3% with Ber (10 μM) and 40.7 ± 1.3% with Ber-SLNs (1 μM) (p<0.001). Inflammation and oxidative stress markers were lower with Ber and Ber-SLNs. Attenuation of ROS generation and apoptosis of cardiomyocytes were noted on fluorescence microscopy. Ber SLNs effectively prevented doxorubicin-induced inflammation and oxidative stress in rat cardiomyocytes. The results demonstrate that microemulsion is a simple and costeffective technique to prepare Ber-SLNs, and may be considered as a drug delivery vehicle for berberine.

MiR-488-3p Protects Cardiomyocytes against Doxorubicin-Induced Cardiotoxicity by Inhibiting CyclinG1.

Objective To investigate the protective effects and regulatory mechanism of miR-488-3p on doxorubicin-induced cardiotoxicity. Methods The C57BL/6 mice and primary cardiomyocytes were used to construct doxorubicin-induced cardiomyocyte injury models in vivo and in vitro. The levels of miR-488-3p and its downstream target genes were analyzed by quantitative real-time PCR. Mouse cardiac function, cell survival, cellular injury-related proteins, and the apoptosis level of cardiomyocytes were analyzed by echocardiography, MTT analysis, Western blotting, and DNA laddering separately. Results Cardiomyocyte injury caused by a variety of stimuli can lead to the reduction of miR-488-3p level, especially when stimulated with doxorubicin. Doxorubicin led to significant decrease in cardiac function, cell autophagic flux blockage, and apoptosis in vivo and in vitro. The expression of miR-488-3p's target gene, CyclinG1, increased remarkably in the doxorubicin-treated neonatal mouse cardiomyocytes. Overexpression of miR-488-3p inhibited CyclinG1 expression, increased cardiomyocyte viability, and attenuated doxorubicin-induced cardiomyocyte autophagic flux blockage and apoptosis. Conclusions miR-488-3p is one of the important protective miRNAs in doxorubicin-induced cardiotoxicity by inhibiting the expression of CyclinG1, which provides insight into the possible clinical application of miR-488-3p/CyclinG1 as therapeutic targets in doxorubicin-induced cardiovascular diseases.

The role of angiotensin signalling in anthracycline-induced cardiotoxicity

Abstract Anthracyclines (e.g. epirubicin, doxorubicin, daunorubicin) are widely used for the treatment of adult and paediatric cancers. Despite their therapeutic efficacy, anthracyclines are associated with both acute and late-onset cardiac toxicities. Meta-analyses report an overt cardiotoxicity incidence of 6.3%, whilst sub-clinical cardiotoxicity incidence is 17.9% (1). Angiotensin converting enzyme (ACE) inhibitors are used to treat anthracycline-induced cardiotoxicity (AIC) (2) and despite their efficacy being well studied for the treatment of heart failure, hypertension and post-acute coronary syndromes, their mechanism(s) for treating and preventing AIC remain unknown. Using in vitro cardiomyocytes, we evaluated the angiotensin signalling mechanisms stimulated by doxorubicin chemotherapy, applying quantitative PCR, immunofluorescence and real-time cell analysis technologies. In vitro adult human ventricular cardiomyocytes (AC10 cell line) treated with clinically relevant sub-toxic concentrations of doxorubicin, demonstrate a dose and time-dependent increase in angiotensin II type-1 receptor (AT1R) gene expression. Maximal AT1R expression was observed after 24 hours' exposure at 250 nanomolar (nM), with qPCR recording up to 13-fold increases in expression relative to control (figure 1). Consistent with gene expression studies, doxorubicin also induced expression of AT1R at the protein level, with immunofluorescence imaging displaying up-regulation of AT1R in association with doxorubicin concentrations up to 500nM (figure 2). Western blot results also support the induction of AT1R, however no relationship was observed between either doxorubicin concentration or drug exposure time. Cellular growth and morphological changes of cardiomyocytes in response to clinically relevant doses of doxorubicin treatment were evaluated with real-time cell analysis using impedance-based xCELLigence technology. During the early phases of doxorubicin exposure, an increase in cell size was observed, whilst experiments modelling the pharmacokinetics and serial half-lives of doxorubicin demonstrated reversibility of doxorubicin-induced cardiomyocyte injury following drug elimination. These data support the mechanistic hypothesis that a relationship exists between AIC and modulation of the angiotensin signalling pathway in cardiomyocytes. We demonstrate that cardiomyocyte exposure to doxorubicin induces AT1R gene and protein expression, whilst doxorubicin-induced cardiomyocyte injury displays reversibility following drug elimination. Genetic polymorphisms within the ACE gene have been associated with cardiomyopathy and left ventricular hypertrophy. Our research now provides the platform to ascertain whether the ACE genotype contributes to heart failure from AIC, and whether an elevation in pro-hypertrophic angiotensin II levels could exacerbate anthracycline-induced hypertrophy and promote the development of late-onset anthracycline-induced heart failure. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Cancer Research UK PhD research grant

MicroRNA miR-215-5p Regulates Doxorubicin-induced Cardiomyocyte Injury by Targeting ZEB2.

Doxorubicin (DOX) is a chemotherapeutic drug for treating various cancers. However, the DOX-induced cardiotoxicity greatly limits its clinical application. MicroRNAs are emerged as critical mediators of cardiomyocyte injury. This work explored the function of miR-215-5p in the regulation of DOX-induced mouse HL-1 cardiomyocyte injury. An in vitro model of DOX-treated cardiotoxicity was established in cardiac mouse cell line HL-1. Gene expression was measured by reverse transcription quantitative polymerase chain reaction. Cell viability was detected using CCK-8. Cell death and apoptosis were tested using transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), flow cytometry, and caspase-3/7 activity assays. Luciferase reporter assay was used to examine the target of miR-215-5p. We found that DOX induced cardiomyocyte injury and upregulated miR-215-5p in HL-1 cells. Inhibition of miR-215-5p attenuated DOX-induced cardiomyocyte death and apoptosis in vitro. Mechanistical experiments indicated that zinc finger E-box-binding homeobox (ZEB2) was targeted by miR-215-5p. In addition, ZEB2 expression was reduced in DOX-treated HL-1 cells. Rescue assays indicated that ZEB2 knockdown reversed the effects of miR-215-5p inhibition. In conclusion, miR-215-5p inhibition protects HL-1 cells against DOX-induced injury by upregulating ZEB2 expression.

Protective Effect of miR-204 on Doxorubicin-Induced Cardiomyocyte Injury via HMGB1.

The toxicity of doxorubicin (DOX) limits its clinical application. Nevertheless, at present, there is no effective drug to prevent DOX-induced cardiac injury. miR-204 is a newly discovered miRNA with many protective effects on cardiovascular diseases. However, little research has been done on the effects of miR-204 on DOX-induced cardiac injury. Our study is aimed at investigating the effect of miR-204 on DOX-induced myocardial injury. An adenoassociated virus system was used to achieve cardiac-specific overexpression of miR-204. Two weeks later, the mice were intraperitoneally injected with DOX (15 mg/kg) to induce cardiac injury. H9c2 myocardial cells were used to validate the role of miR-204 in vitro. Our study showed that miR-204 expression was decreased in DOX-treated hearts. miR-204 overexpression improved cardiac function and alleviated cardiac inflammation, apoptosis, and autophagy induced by DOX. In addition, our results showed that miR-204 prevented DOX-induced injury in cardiomyocytes by directly decreasing HMGB1 expression. Moreover, the overexpression of HMGB1 could offset the protective effects of miR-204 against DOX-induced cardiac injury. In summary, our study showed that miR-204 protected against DOX-induced cardiac injury via the inhibition of HMGB1, and increasing miR-204 expression may be a new treatment option for patients with DOX-induced cardiac injury.

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.

Dexrazoxane ameliorates doxorubicin-induced cardiotoxicity by inhibiting both apoptosis and necroptosis in cardiomyocytes

Doxorubicin, as a first line chemotherapeutic agent, its usage is limited owing to cardiotoxicity. Necroptosis is a new form of programmed cell death, and recent investigations indicated that necroptosis is vitally involved in serious cardiac pathological conditions. Dexrazoxane is the only cardiac protective drug approved by FDA for anthracycline. We aimed to explore whether and how dexrazoxane regulates doxorubicin-induced cardiomyocyte necroptosis. First, doxorubicin could cause heart failure and reduce cardiomyocyte viability by promoting cell apoptosis and necroptosis in vivo and in vitro. Second, necroptosis plays an important role in doxorubicin induced cardiomyocyte injury, which could be inhibited by Nec-1. Third, dexrazoxane increased cell viability and protect heart function by decreasing both cardiomyocyte apoptosis and necroptosis after doxorubicin treatment. Forth, dexrazoxane attenuated doxorubicin-induced inflammation and necroptosis by the inhibition of p38MAPK/NF-κB pathways. These results indicated that dexrazoxane ameliorates cardiotoxicity and protects heart function by attenuating both apoptosis and necroptosis in doxorubicin induced cardiomyocyte injury.

Protective effect of Shengxian decoction and each single herb component against adriamycin-induced cardiomyocyte injury

Objective To study the protective effect of Shengxian decoction (SXT) and its each single herb component against doxorubicin induced cardiomyocyte injury. Methods Myocardial cells isolated from the neonatal rats with hypoxia/reoxygenation injury of cardiomyocytes were divided into eight groups,control group (Con group),doxorubicin-induced injury group (M group),and treatment groups with Shengxian decoction and its each single herb decoction (SXT,A-E groups) following doxorubicin-induced injury.The indicators tested included cell viability,cell ROS,Ca2+ and LDH levels. Results Compared with the control group,doxorubicin induced obvious myocardial injury in model group.Compared with the model group,all tested drug groups increased the cell viability,reduced the levels of cell Ca2+ and LDH.Besides,SXT and the single herb decoction reduced the concentration of ROS except the decoction of Platycodon grandiflorum and Cimicifuage Rhizoma.The experimental results suggested that the application of SXT in the early episode could reduce the cardiac toxicity of adriamycin to a certain extent. Conclusion SXT and its each single herb decoction showed protective effect against doxorubicin-induced cardiomyocyte injury.

Downregulation of vitamin C transporter SVCT-2 in doxorubicin-induced cardiomyocyte injury

Vitamin C (Vit C) has been shown to be protective against doxorubicin (Dox)-induced cardiotoxicity. However, Vit C uptake into cardiomyocytes is poorly understood. Furthermore, whether the antioxidant enzyme reserve is enhanced by Vit C is also not known. The present study investigated an influence of Dox on Vit C transporters, expression of endogenous antioxidant reserve as well as enzymes, oxidative stress, and apoptosis in isolated cardiomyocytes. Cardiomyocytes isolated from adult Sprague-Dawley rats were exposed to control (culture medium 199 alone), Dox (10 μM), Vit C (25 μM), and Vit C + Dox for 24 h. Vit C transporter expression and localization, oxidative stress, antioxidant enzymes, and apoptosis were studied. Expression and localization of sodium-dependent vitamin C transporter-2 (SVCT-2) in the sarcolemma was reduced by Dox, but Vit C supplementation was able to blunt this change. There was a decrease in the expression of antioxidant enzymes glutathione peroxidase (GPx), catalase, and Cu/Zn superoxide dismutase (SOD) due to Dox, but only GPx expression was completely prevented and Cu/Zn SOD was partially rescued by Vit C. Dox-induced decrease in antioxidant reserve and increase in oxidative stress were partially mitigated by Vit C. Dox-induced apoptosis was ameliorated by Vit C. It is suggested that cardioprotection offered by Vit C in Dox-induced cardiomyopathy may involve an upregulation of SVCT-2 transporter followed by a reduction in oxidative stress as well as blunting of cardiomyocyte injury.

Cardioprotective effects of epigallocatechin-3-gallate against doxorubicin-induced cardiomyocyte injury

Doxorubicin-induced generation of reactive oxygen species is a leading cause of cardiomyopathy, the major side-effect limiting the clinical use of this anti-cancer drug. Epigallocatechin-3-gallate (EGCG), a main catechin in green tea that possesses a strong antioxidant property. This study aims to investigate whether EGCG can protect cardiac myocytes against doxorubicin-induced myocyte injury. Myocyte viability was measured with an MTT assay. Reactive oxygen species were measured with fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate. Myocyte shortening and intracellular Ca 2+ levels were determined with a spectrofluorometer connected to a video edge detection system. EGCG concentration-dependently increased cell viability and inhibited the generation of reactive oxygen species in doxorubicin-treated myocytes. Doxorubicin significantly decreased the amplitudes of cell shortening, the maximum velocity of cell contraction (+ dl/dt) and relaxation (− dl/dt) in electrically-stimulated myocytes in the presence or absence of isoprenaline, which was attenuated by EGCG. The present data suggest that EGCG may protect myocytes against oxidative stress-induced cellular injury in doxorubicin-treated cardiac myocytes. The effect of EGCG on Ca 2+ handling was also examined. EGCG increased the amplitudes of both electrically- and caffeine-induced Ca 2+ transients in doxorubicin-treated myocytes, suggesting that EGCG may reverse doxorubicin-induced intracellular Ca 2+ depletion in the sarcoplasmic reticulum. We found in the present study that EGCG may protect heart against doxorubicin-induced myocyte injury by improving Ca 2+ handling through scavenging reactive oxygen species. Our results imply that EGCG may be used together with doxorubicin to minimize its cardiac toxic effects.

Bicarbonate exacerbates oxidative injury induced by antitumor antibiotic doxorubicin in cardiomyocytes.

Doxorubicin, a broad-spectrum antitumor antibiotic, causes dose-dependent cardiomyopathy and heart failure. Although the exact molecular mechanisms of cardiotoxicity are not well established, oxidative mechanisms involving doxorubicin-induced superoxide anion production have been proposed. In this study, we show that bicarbonate, a physiologically relevant tissue component, greatly amplified doxorubicin-induced cardiomyocyte injury. Bicarbonate also enhanced inactivation of aconitase, a crucial tricarboxylic acid cycle enzyme, in cardiomyocytes exposed to doxorubicin. The cell-permeable superoxide dismutase mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin, reversed doxorubicin-induced cardiomyocyte injury. Bicarbonate enhanced the inactivation of purified mitochondrial aconitase in the xanthine/xanthine oxidase system, generating superoxide. The results suggest that bicarbonate amplifies the prooxidant effect of superoxide. Bicarbonate also caused an increased loading of cardiomyocytes with doxorubicin. We conclude that the bicarbonate-mediated increase in doxorubicin toxicity is due to increased intracellular loading of doxorubicin in cardiomyocytes and subsequent exacerbation of superoxide-mediated cardiomyocyte injury.