Doxorubicin-induced Cardiotoxicity Research Articles

Deubiquitinase MYSM1 promotes doxorubicin-induced cardiotoxicity by mediating TRIM21-ferroptosis axis in cardiomyocytes

Anthracycline antitumor drug doxorubicin (DOX) induces severe cardiotoxicity. Deubiquitinating enzymes (DUBs) are crucial for protein stability and function and play a significant role in cardiac pathophysiology. By comparing RNA sequencing datasets and conducting functional screening, we determined that Myb-like, SWIRM, and MPN domains 1 (MYSM1) is a key regulator of DOX-induced cardiotoxicity. In this study, we aimed to explore the function and regulatory mechanisms of MYSM1 in DOX-induced cardiotoxicity. Genetic knockdown of MYSM1 significantly mitigated DOX-induced cardiomyopathy. Correspondingly, cardiomyocyte-specific knockdown of MYSM1 by AAV9 protected the heart from DOX-induced cardiotoxicity. Gain- and loss-of-function analysis verified that MYSM1 mediated DOX-induced cardiomyocyte injury in vitro. Through a Co-IP combined with LC-MS/MS analysis, we discovered that MYSM1 directly interacted with tripartite motif-containing protein 21 (TRIM21). Mechanistic investigations revealed that MYSM1 regulates the deubiquitination and the stability of TRIM21 via its MPN domain. Furthermore, MYSM1 exacerbated DOX-induced cardiotoxicity by enhancing ferroptosis. This study identified MYSM1 as a potential therapeutic target for DOX-induced cardiotoxicity and illustrated a MYSM1-TRIM21-ferroptosis axis in regulating DOX-induced cardiotoxicity.

Bag2 protects against doxorubicin-induced cardiotoxicity by maintaining Pink1-mediated mitophagy

The clinical application of Doxorubicin (DOX) is limited due to its cardiotoxicity. Mitophagy dysfunction is the primary cause of DOX-induced cardiotoxicity (DIC). However, the precise mechanism by which DOX regulates mitophagy remains elusive. Bag2 (BCL2-associated athanogene 2) is a cochaperone implicated in multiple pathological states. The aim of this study was to investigate the potential cardio-protective effects of Bag2 in DIC. C57BL/6 mice and AC16 cells were used to establish DIC model. The expression of Bag2 were measured by western blotting and immunohistochemical. The effects of Bag2 on DIC were assessed through functional gain and loss experiments. Through in vitro and in vivo experiments, we found that Bag2 expression was significantly reduced after DOX treatment. Both Bag2 knockdown and DOX administration resulted in apoptosis, mitochondrial dysfunction, and impaired mitophagy. Conversely, Bag2 overexpression exerted protective effects against these phenotypes induced by DOX stimulation. Mechanistically, Bag2 maintained mitophagy activation by binding to Pink1 and protecting it from proteasome-dependent degradation, thereby preserving mitochondrial function and protecting against myocardial lesions. Our findings suggest that Bag2 may serve as a promising therapeutic target for the treatment of DIC.

Isoliquiritin targeting m5C RNA methylation improves mitophagy in doxorubicin-induced myocardial cardiotoxicity

BackgroundDoxorubicin (DOX)-induced myocardial cardiotoxicity (DIC) severely limits its clinical application, and there is no optimal therapeutic agent available. Recent studies revealed that activation of BNIP3-mediated mitophagy and the inhibition of m5C RNA methylation played a crucial role in DIC. Isoliquiritin (ISL) has remarkable cardiac protective effect. But its potential mechanisms against DIC still remains unknown. PurposeTo investigate the therapeutic effect and potential mechanism of Isoliquiritin(ISL) on doxorubicin(DOX)-induced myocardial cardiotoxicity(DIC). MethodsBioinformatics analyses and network pharmacology were carried out to identify effective target of ISL against DIC. Molecular docking and surface-plasmon resonance (SPR) were used to confirm the predict. The mechanism of ISL regulating mitophagy through M5C methylation to improve DIC was demonstrated in vitro and in vivo experiments. The methylation modification was verified by MeRIP-qPCR. Cell model of DIC was constructed to evaluate mitochondrial function by measuring cell viability, myocardial enzyme level, mitochondrial quality, mCherry-EGFP analysis and TEM morphometry with the application of mitophagy inhibitor (Baf A1) and inducer (CCCP). Myocardial injury in mice with DIC was assessed by survival rate, myocardial enzyme level, HE staining, echocardiography and detection of mitophagy markers. ResultsThe decreased level of m5C writer TRDMT1 and mitochondrial marker (BNIP3) were chosen for the research. After the docking and SPR verification between ISL and TRDMT1, the improvement of ISL on TRDMT1 and TRDMT1-associated m5C level of BNIP3 was identified. In vitro and in vivo experiments showed that the cardiac markers, heart function, and mitochondrial function were recovered after ISL application. Meanwhile, the results manifested that there was blocked autophagy flow indicated by mCherry-EGFP analysis, then the suppressed mitophagy caused the mitochondria damage associated cell death. ISL could alleviate the autophagy block, and Baf A1 couldn't influnce the ISL effect. Compared to CCCP group, Mitochondrial maker TOMM20 significantly elevated treated with both CCCP and DOX, indicating that DOX impaired mitophagy for clearing damaged mitochondrial proteins. After ISL treated, a higher level of co-localization between mitochondrial and BNIP3 was observed, inducing restoration of mitochondrial function. ConclusionThis study showed that ISL may exert cardioprotective role restoring BNIP3-mediated mitophagy by targeting TRDMT1 to alleviate DOX-induced macro-autophagy-dependent protein homeostasis and acquired blocking of mitophagy, providing a new idea for the clinical treatment of DIC.

The Protective Effects of Carvacrol Against Doxorubicin-Induced Cardiotoxicity In Vitro and In Vivo.

Doxorubicin (DOX) is a remarkable chemotherapeutic agent, however, its adverse effect on DOX-induced cardiotoxicity (DIC) is a rising concern. Recent research has identified carvacrol (CAR), an antioxidant and anti-inflammatory agent, as a promising natural compound for protecting against DIC. This study aims to investigate the potential cardioprotective effects properties of CAR in vitro and in vivo. The cardioprotective effect of CAR was assessed by pretreating H9c2 cells with non-toxic CAR for 24h, followed by co-treatment with DOX (10μM) for an additional 24h. The cell viability was determined using an MTT assay. For the in vivo study, male Sprague-Dawley rats (200-250g) were randomly divided into three groups: control, cardiotoxicity (DOX), and treatment (CAR + DOX) groups. CAR (50mg/kg, BW) was administered orally to the CAR + DOX groups for 14days. Then, a single dose of DOX (15mg/kg/i.p, BW) was administered on day 15 for DOX and CAR + DOX groups. The rats were allowed to recover for 3days before being sacrificed. Our results demonstrated that DOX (10µM) significantly reduced H9c2 cell viability by 50% (p < 0.0001), and CAR (0.067µM) protected H9c2 cells from DIC (p = 0.0045). In the rat model, CAR pretreatment effectively mitigated DOX-induced reductions in systolic pressure (p = 0.0007), pulse pressure (p = 0.0213), hypertrophy (p = 0.0049), and cardiac fibrosis (p = 0.0006). However, the pretreatment did not alter the heart function, oxidative stress, and antioxidant enzymes. In conclusion, our results indicate that CAR could potentially serve as an adjuvant to reduce cardiotoxicity by ameliorating myocardial fibrosis and hypertrophy.

Vanillic acid attenuates doxorubicin-induced cardiotoxicity by regulating PINK1/Parkin/Mfn2 signaling pathway

ObjectivesThe aim of this study was to explore the potential protective effects of vanillic acid (VA) against doxorubicin (DOX)-induced cardiotoxicity and unravel the underlying molecular mechanisms involved. MethodsThe DOX -induced myocardial toxicity model was established in Sprague-Dawley (SD) rats by intraperitoneal injection of doxorubicin. Simultaneously, different concentrations of VA were administered for intervention. After modeling, rat cardiac function was evaluated using an ultrasound machine. The detection of creatine kinase isoenzyme MB (CK-MB) levels in rat serum using a biochemical analyzer. HE staining method was used to observe myocardial pathological changes, Masson's trichrome staining was performed to assess the degree of myocardial fibrosis. The observation of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) levels in myocardial tissue using assay kits. Western blotting and immunohistochemistry was conducted to detect protein expression levels of PINK1, Parkin, P62, LC3I/II, Mfn2, Drp1, OPA, and Fis1. ResultsCompared to the DOX group, intervention with VA demonstrates ameliorative effects on doxorubicin-induced cardiotoxicity, as evidenced by improvements in left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), and a reduction in left ventricular end-diastolic diameter (LVEDd) in rats. Additionally, the application of VA reduced the levels of CK-MB in rat serum. However, there were no statistically significant differences observed in heart rate. Histological examination reveals that VA administration positively impacts the arrangement of myocardial fibers and mitigates myocardial fibrosis in the rat hearts subjected to doxorubicin-induced injury. Moreover, relative to the DOX group, the use of VA alleviates DOX-induced myocardial cell apoptosis. VA treatment also upregulates the expression levels of PINK1 and Parkin, leading to the activation of mitochondrial autophagy. Furthermore, VA enhances the expression levels of mitochondrial dynamics proteins Mfn2, Drp1, and Fis1 while downregulating the expression of OPA. Among them, the high-dose group of VA (60 mg/kg·d) showed the most pronounced improvement in DOX-induced cardiotoxicity. ConclusionVA attenuated DOX-induced myocardial injury by up regulating the PINK1/Parkin/Mfn2 signaling pathway.

Doxorubicin-Induced Cardiotoxicity Through SIRT1 Loss Potentiates Overproduction of Exosomes in Cardiomyocytes.

Mutual interaction between doxorubicin (DOX) and cardiomyocytes is crucial for cardiotoxicity progression. Cardiomyocyte injury is an important pathological feature of DOX-induced cardiomyopathy, and its molecular pathogenesis is multifaceted. In addition to the direct toxic effects of DOX on cardiomyocytes, DOX-induced exosomes in the extracellular microenvironment also regulate the pathophysiological states of cardiomyocytes. However, the mechanisms by which DOX regulates exosome secretion and subsequent pathogenesis remain incompletely understood. Here, we found that DOX significantly increased exosome secretion from cardiomyocytes, and inhibiting this release could alleviate cardiomyocyte injury. DOX promoted exosome secretion by reducing cardiomyocyte silencing information regulator 1 (SIRT1) expression, exacerbating cardiotoxicity. DOX impaired lysosomal acidification in cardiomyocytes, reducing the degradation of intracellular multivesicular bodies (MVBs), resulting in an increase in MVB volume before fusing with the plasma membrane to release their contents. Mechanistically, SIRT1 loss inhibited lysosomal acidification by reducing the expression of the ATP6V1A subunit of the lysosomal vacuolar-type H+ ATPase (V-ATPase) proton pump. Overexpressing SIRT1 increased ATP6V1A expression, improved lysosomal acidification, inhibited exosome secretion, and thereby alleviated DOX-induced cardiotoxicity. Interestingly, DOX also induced mitochondrial-derived vesicle formation in cardiomyocytes, which may further increase the abundance of MVBs and promote exosome release. Collectively, this study identified SIRT1-mediated impairment of lysosomal acidification as a key mechanism underlying the increased exosome secretion from cardiomyocytes induced by DOX, providing new insights into DOX-induced cardiotoxicity pathogenesis.

Abstract 4137609: Lentiviral shRNA Library Screen Using Human Pluripotent Stem Cell-derived Cardiomyocytes Identified Mediators of Protection via MAP4K4 Inhibition against Doxorubicin-induced Cardiotoxicity

Background: Doxorubicin (Dox) is a common component in chemotherapy for a wide range of tumors, but the cardiotoxicity has restricted its use. There is an unmet need for novel therapeutics of Dox-induced cardiotoxicity (DIC). We previously identified the protein kinase MAP4K4 as a mediator of DIC. Notably, we showed that blocking MAP4K4 activity via DMX-5804 protected against Dox-induced cell death and dysfunction in human iPSCs derived cardiomyocytes (hiPSC-CMs). As a powerful tool to study the protective mechanism, pooled library screen performs high-throughput phenotypic examination, but its potential on hiPSC-CMs is poorly explored. Hypothesis: A pooled lentiviral shRNA library screen in hiPSC-CMs treated with Dox ± DMX-5804 could identify mediators of protection against DIC. Methods: To maximize the chances to uncover key factors mediating DMX-5804 protection, we performed a high-throughput loss-of-function screen. A pooled library of 1731 lentiviral shRNAs was generated. hiPSC-CMs were transduced, then treated with Dox ± DMX-5804. Cardiac troponin I ELISA was used to examine myocyte injury. Genomic DNA was isolated, followed by amplification and sequencing of the integrated shRNAs. Log2 fold change was calculated and transformed into Z score to identify significantly shifted shRNAs. Results: To build the shRNA library we included 1) genes identified via transcriptomic analysis of hiPSC-CMs treated with Dox ± DMX-5804; 2) genes in the MAP4K4 signaling cascade; 3) previously defined pro-death or pro-survival genes in DIC. To ensure single gene silenced per cell, we used a low MOI of 0.5. After transduction, cells were treated with Dox ± DMX-5804. Confirming the cardiotoxicity and protection, Dox induced a 2-fold increase of cardiac troponin I release and DMX-5804 significantly reversed it. Next, we recovered and sequenced the integrated shRNAs, and quantified the frequency of each. We identified 146 significantly enriched or depleted shRNAs (|Z score| &gt; 2). When comparing Dox treated group to vehicle or DMX-5804 protected group, shRNA enrichment indicates pro-death function of the targeted gene. Conversely, pro-survival genes can be inferred from shRNA depletion. Amongst the 5 most consistently targeted genes, we identified SIRT6 and GATA4 which are known pro-survival in DIC, suggesting the screen is potent to define key genes. Conclusion: We performed a proof-of-concept pooled library screen in human cardiomyocytes and identified mediators of protection against DIC.

Abstract 4126333: TRPC6 A404V is a gain-of-function variant associated with doxorubicin-induced cardiotoxicity in patients

Background: Gain-of-function mutations in the canonical transient receptor potential 6 (TRPC6) channel contribute to heart failure (HF) and focal segmental glomerulosclerosis in humans. Our GWAS results indicated that the TRPC6 A404V variant, having a minor allele frequency of 12% in the population, is associated with doxorubicin-induced cardiotoxicity. However, the underlying ionic mechanisms of this variant have not been fully examined. Methods: Combining patch clamp recording, site-directed mutagenesis, and in-silico analysis, we evaluated the TRPC6 A404V variant function. Results: Whole-cell TRPC6 currents were elicited in HEK293 cells using a voltage ramp protocol from -100 mV to +100 mV with a holding potential of -60 mV, 48 hours after transfection with TRPC6 WT or A404V cDNAs. The inward-current densities at -100 mV and the outward-current densities at +100 mV for TRPC6 WT and A404V variant were similar at baseline. However, after exposure to 50 μM 1-oleoyl acetyl-sn-glycerol (OAG, a TRPC6 activator), these current densities were significantly increased by 2.6-fold and 4.5-fold respectively in the WT (n=20 cells), and by 8.4-fold and 8.9-fold respectively in the A404V variant (n=12 cells, p&lt;0.05 vs. WT). The OAG effects were abolished by superfusion with 1 μM BI-749327 (a specific TRPC6 inhibitor). Moreover, a 24-hour treatment with 0.5 μM doxorubicin (DOX) further amplified the effects of OAG, increasing the total inward- and outward-current densities by 7.2-fold and 9.5-fold respectively in TRPC6 WT (n=11 cells), and by 13.9-fold and 14.7-fold respectively in the A404V variant (n=13 cells, p&lt;0.05 vs. WT). In comparison, the OAG effects on TRPC6 WT and A404V channels were not potentiated by treatment with 0.5 μM doxorubicinol (DOXol, a metabolite of DOX) for 24 h (n=10-12 cells, p=N.S.). Molecular docking studies showed that OAG had a lower binding energy (-5.60 kcal/mol) and a smaller apparent equilibrium constant (Kd) of 0.079 mM with the A404V variant, compared to those of -4.49 kcal/mol and 0.511 mM respectively with TTRPC6 WT. Conclusion: TRPC6 A404V is a gain-of-function variant with a higher binding affinity to OAG. Treatment with DOX but not DOXol greatly enhances TRPC6 WT and A404V channel activation by OAG. Thus, cancer patients carrying the TRPC6 A404V variant may be more vulnerable to develop HF upon treatment with anthracycline.

Abstract 4137349: Cardiomyocyte resilience to Doxorubicin with NAD + supplementation in cardiotoxicity

Introduction: Doxorubicin (DOX) is a potent chemotherapy drug that carries a significant risk of cardiotoxicity leading tso heart failure by presenting a notable challenge in cancer treatment. Despite being characterized by oxidative stress and inflammation, fully understanding Doxorubicin-induced cardiotoxicity (DIC) mechanisms remains elusive. Ongoing research emphasizes the promising role of nicotinamide adenine dinucleotide (NAD + ) and its precursors in cardiovascular health. Therefore, we sought to evaluate the effectiveness of NAD + in preventing or reducing DIC. Methods: H9c2 rat cardiomyocytes were pre-treated with NAD + 100uM for 48 hours, subsequently exposed to DOX 500nM for 24 hours, and received a follow-up NAD + 100uM treatment for an additional 48 hours. Cell viability was evaluated via the MTT assay. Mitochondrial reactive oxygen species (ROS) production was measured using the MitoSOX assay. The expression of genes crucial for cardiac cell development and function was analyzed through quantitative polymerase chain reaction (qPCR). Furthermore, Western blotting (WB) was conducted to detect changes in the expression of proteins associated with oxidative stress and apoptosis, thus providing an in-depth assessment of NAD + 's potential therapeutic effects against anthracycline-induced cardiac complications. Results: NAD + treatment maintained myocardial cell viability in the presence of DOX and modulated gene expression beneficial for cardiac function, including downregulation of pro-inflammatory cytokines such as IL1b and TNF-a. Furthermore, it reduced oxidative stress markers, decreased levels of pro-apoptotic Caspase 3, and increased levels of the antioxidant SOD2 via Sirt1 pathway regulation. Conclusion: Our findings highlight the critical importance of a multidisciplinary research approach to elucidate the cardioprotective capacity of NAD + against DIC, potentially paving the way for new targeted strategies in oncological therapies.

Hypolipidemic and Cardioprotective Efficacy of Peltophorum pterocarpum Linn in Doxorubicin Induced Cardiotoxicity in Rats

Background: Cardiovascular disease (CVD) refers to a common group of illnesses affecting the heart and blood arteries. It encompasses heart valve problems, atherosclerosis, angina, cardiomyopathy, coronary artery disease (CAD), and infections of the heart which are considered the main causes of death. Modern medications have some success but they typically come with unfavorable side effects. Researchers are paying attention to herbal remedies because of their limited or no negative effects. This study was conducted to find out the hypolipidemic and cardioprotective potential of the ethanolic extract of Peltophorum pterocarpum (P. pterocarpum) Linn leaves in doxorubicin-induced cardiac damage in rats. Methods: The ethanolic extract of P. pterocarpum leaves was subjected to compound identification by GC-MS analysis. For in vivo analyses, the rats were divided into six groups. Cardiac damage was induced by doxorubicin at a dosage of 1.5 ml/kg b.wt. various dosages of extract (200 and 300 mg/kg b.wt.) were injected and propranolol (25 mg/kg b.wt.) is used as a standard drug. After the treatment of the extract, biochemical parameters were evaluated followed by in silico analysis. Result: The levels of cardiac marker and lysosomal enzymes significantly increased in toxin-treated rats, and the mitochondrial enzyme levels were significantly reduced. After treatment with P. pterocarpum leaf extract, the levels significantly (P less than 0.05) returned to normal. These results may prove the cardiac protective effect of the ethanolic extract of P. pterocarpum leaves in DOX-induced cardiotoxicity rats.

New targets and biomarkers for doxorubicin-induced cardiotoxicity in humans: implications drawn from toxicogenomic data and molecular modelling

The doxorubicin-induced cardiotoxicity continues to be a life-threatening adverse effect in the clinic. Doxorubicin-induced acute cardiotoxicity is reversible, whereas chronic cardiotoxicity is irreversible, leading to dilated cardiomyopathy and heart failure. The aim of this study was to identify the molecular mechanisms associated with doxorubicin metabolites in doxorubicin-induced chronic cardiotoxicity. For this purpose, literature searches and in silico toxicogenomic analyses were conducted using various tools, including the Comparative Toxicogenomic Database, GeneMANIA, Metascape, MIENTURNET, ChEA3, and AutoDock. Additionally, molecular dynamics simulations were performed for 500 ns using Schrödinger software to assess the stability and dynamics of the representative docked complexes. We observed that doxorubicin biotransformed into five metabolites in the human heart and identified 11 common genes related to doxorubicin, its metabolites, dilated cardiomyopathy, and heart failure. Our findings revealed that doxorubicin and its metabolites primarily exhibited binding affinity to the beta-1 adrenergic receptor and fatty acid synthase. Furthermore, we identified several key transcription factors, especially the Homeobox protein Nkx-2.6, and hsa-miR-183-3p associated with this cardiotoxicity. Finally, we observed that, in addition to doxorubicinol, 7-deoxidoxorubicinone, another metabolite of doxorubicin, may also contribute to this cardiotoxicity. These findings contribute to our understanding of the processes underlying doxorubicin-induced chronic cardiotoxicity.

Small Molecules Targeting Mitochondria: A Mechanistic Approach to Combating Doxorubicin-Induced Cardiotoxicity.

Doxorubicin (Dox) is a commonly used chemotherapy drug effective against a range of cancers, but its clinical application is greatly limited by dose-dependent and cumulative cardiotoxicity. Mitochondrial dysfunction is recognized as a key factor in Dox-induced cardiotoxicity, leading to oxidative stress, disrupted calcium balance, and activation of apoptotic pathways. Recent research has emphasized the potential of small molecules that specifically target mitochondria to alleviate these harmful effects. This review provides a comprehensive analysis of small molecules that offer cardioprotection by preserving mitochondrial function in the context of doxorubicin-induced cardiotoxicity (DIC). The mechanisms of action include the reduction of reactive oxygen species (ROS) production, stabilization of mitochondrial membrane potential, enhancement of mitochondrial biogenesis, and modulation of key signaling pathways involved in cell survival and apoptosis. By targeting mitochondria, these small molecules present a promising therapeutic strategy to prevent or reduce the cardiotoxic effects associated with Dox treatment. This review not only discusses the mechanistic actions of these agents but also emphasizes their potential in improving cardiovascular outcomes for cancer patients. Gaining insight into these mechanisms can help in creating more effective strategies to safeguard the heart during chemotherapy, allowing for the ongoing use of Dox with a lower risk to the patient's cardiovascular health. This review highlights the critical role of mitochondria-targeted therapies as a promising approach in addressing DIC.

CaMKII Exacerbates Doxorubicin-Induced Cardiotoxicity by Promoting Ubiquitination Through USP10 Inhibition.

Doxorubicin (DOX) is an effective anticancer drug, but it has a problem of cardiotoxicity that cannot be ignored. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is tightly associated with the pathological progression of DOX-induced cardiotoxicity. Ubiquitin-specific protease 10 (USP10) plays an important role in many biological processes and cancers. However, its association with DOX-induced cardiotoxicity and CaMKII remains unclear. H9C2 cells, HL-1 cells and C57BL/6 mice were used to establish the DOX-induced cardiotoxicity model, and the CaMKII-specific inhibitor KN-93 and USP10 specific inhibitor Spautin-1 were used to observe the CaMKII and USP10 effect. In cell experiments, CCK-8 method was used to assess cell viability, LDH kit was used to assess lactate dehydrogenase expression, DCFH-DA staining was used to observe changes in active oxygen content, TUNEL staining was used to observe cell apoptosis, and Western blotting method was used to detect relevant protein markers. The expression of p-CaMKII and USP10 was assessed by immunofluorescence staining. In animal experiments, mouse echocardiograph was used were used to evaluate cardiac function, and HE staining and Masson staining were used to evaluate myocardial injury. Cardiomyocyte apoptosis was detected by TUNEL staining. Western blotting method was used to detect relevant protein markers. Our results demonstrated that activation of CaMKII and inhibition of USP10 pathway related to DOX-induced cardiotoxicity. Inhibition of CaMKII with KN-93 ameliorated DOX-induced cardiac dysfunction and cytotoxicity. In addition, CaMKII inhibition prevented DOX-induced apoptosis and ubiquitination. Furthermore, CaMKII inhibition increased USP10 expression in DOX-treated mouse hearts, H9C2 cells and HL-1 cells. At last, the USP10 inhibitor, Spautin-1, blocked the regulatory effect of CaMKII inhibition on apoptosis and ubiquitination in DOX-induced cardiotoxicity. Our findings revealed that DOX-induced myocardial apoptosis and activated CaMKII through cellular and animal levels, while providing a novel probe into the mechanism of CaMKII action: promoting ubiquitination by inhibiting USP10 aggravated apoptosis.

CRISPRi/a screens in human iPSC-cardiomyocytes identify glycolytic activation as a druggable target for doxorubicin-induced cardiotoxicity

Doxorubicin is limited in its therapeutic utility due to its life-threatening cardiovascular side effects. Here, we present an integrated drug discovery pipeline combining human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs), CRISPR interference and activation (CRISPRi/a) bidirectional pooled screens, and a small-molecule screening to identify therapeutic targets mitigating doxorubicin-induced cardiotoxicity (DIC) without compromising its oncological effects. The screens revealed several previously unreported candidate genes contributing to DIC, including carbonic anhydrase 12 (CA12). Genetic inhibition of CA12 protected iCMs against DIC by improving cell survival, sarcomere structural integrity, contractile function, and calcium handling. Indisulam, a CA12 antagonist, can effectively attenuate DIC in iCMs, engineered heart tissue, and animal models. Mechanistically, doxorubicin-induced CA12 potentiated a glycolytic activation in cardiomyocytes, contributing to DIC by interfering with cellular metabolism and functions. Collectively, our study provides a roadmap for future drug discovery efforts, potentially leading to more targeted therapies with minimal off-target toxicity.

Machine Learning-Based ECG Signal Classification for Enhanced Early Detection of Doxorubicin-Induced Cardiotoxicity in Rats

Cardiotoxicity, which leads to irreversible myocardial damage, is a major adverse effect associated with chemotherapy. Electrocardiogram (ECG) is an inexpensive, rapid, and simple tool that may provide valuable diagnostic information pertinent to cardiotoxicity. An automatic interpretation and classification of the ECG signals by machine learning algorithms is considered superior to human interpretation of the ECG which may not be able to early detect subtle alterations in the ECG and vary according to the experience of the specialist. The present work aimed at using different machine learning algorithms to classify ECG signals recorded from doxorubicin-injected rats. Rats were divided into four groups and each group was intraperitoneally injected with different cumulative doses of doxorubicin (0, 6, 12, and 18 mg/kg). ECG signal classification depended on multiple features that were extracted from the recorded signals under different conditions. K nearest-neighbors’ algorithm achieved higher classification accuracy (99.83%) than random forest (99.56%), decision tree (99.54%), artificial neural network (99.50%), and support vector machine (99.38%). Furthermore, the dose-dependent cardiotoxicity was validated via a histopathological examination of the left ventricle that indicated significant pathological alterations in the cardiac tissue. The present findings emphasized the potential of the machine learning-based enhanced detection of cardiotoxicity and validated the dose-dependent toxicity of doxorubicin in the cardiac left ventricle. This approach might be applicable clinically to avoid cardiotoxicity in chemotherapy-treated patients.

RBM20 promotes doxorubicin-induced cardiotoxicity by Atp6v0a1 alternative splicing

Abstract Background &amp; Objective: Doxorubicin (dox), an effective chemotherapy drug, is extremely limited in clinical practice by its lethal cardiotoxicity. Whereas dysregulation of splice-regulatory networks has been reported in cardiomyopathies, the role of RNA binding motif protein 20 (RBM20), an adaptive splice regulator, in doxorubicin-induced cardiotoxicity (DIC) remained to be elucidated. Purpose Here we aimed to show the role of RBM20 in DIC and elucidate the relationship between alternative splicing and lysosome acidification impairment. Method Mice were treated with intraperitoneal injection of cumulative 20mg/kg doxorubicin with four equal injections over a period of 2 weeks to induce DIC. The contribution of RBM20 in cardiotoxicity was evaluated in conditional cardiomyocyte-specific RBM20 overexpression (RBM20tg/+/Myh6-CreERT) mice. The regulatory factor of RBM20 was determined through luciferase assay and chromatin immunoprecipitation in vitro. Autophagic flux was evaluated by transmission electron microscope in RBM20tg/+/Myh6-CreERT mice and by lysotracker and a tandem fluorescence RFP-GFP-LC3 reporter system in RBM20 overexpressed or knockdown cardiomyocyte. RBM20-dependent splicing events were identified by rMATS 3.2.5 on the RNA-sequencing data. Results In the DIC human myocardium and murine model, we observed significant elevation of RBM20 in cardiomyocytes. Cardiomyocyte-specific RBM20 overexpression resulted in severe cardiac dysfunction reflected by dilated ventricles, declined left ventricular ejection fraction, increased chamber stiffness and lower survival rate. After screening for potential upstream transcription factors, c-Jun was identified to bind and upregulate RBM20 directly. The inhibition of c-Jun by SR 11302 could partially mitigate DIC in vitro. Mechanistically, RBM20 was positively related with autophagic flux blockade and impaired lysosome acidification by shifting Atp6v0a1, the a1-subunit of the V0 domain of vacuolar H+-ATPases, toward an exon 6-7 exclusion isoform without altering total transcript levels. By restoring exon 6-7-containing ATP6V0a1 level, we were able to reduce DIC by restoring normal autophagic flux. Conclusion Overall, our study reveals that elevation of RBM20 disturbed lysosome acidification in doxorubicin induced cardiotoxicity via the alternative splicing of Atp6v0a1, leading to heart failure.JUN-Rbm20 upregulattion promoted DICRBM20 alternative spliced atp6v0a1

Ivabradine prevents doxorubicin-induced cardiotoxicity via attenuating mitochondrial dysfunction and mitochondrial dynamic imbalance in H9C2 cells and rats

Abstract Background Doxorubicin (DOX) is a widely used chemotherapeutic agent, and cardiotoxicity is one of its well-known side effects, which is mediated by cardiac mitochondrial dysfunction. Ivabradine (IVA), a heart rate reduction drug, exerts benefits in various cardiac diseases as shown in both preclinical and clinical studies. However, its roles on DOX-induced cardiotoxicity have never been investigated. Purpose We determined whether IVA reduces DOX-induced cardiotoxicity by directly promoting mitochondrial function and maintaining mitochondrial dynamic balance. Methods H9C2 cells were divided into 4 groups: 1) Control, 2) DOX (10 μM, 25 h), 3) IVA (3 μM, 25 h), 4) Pretreatment with IVA (3 μM, 1 h), followed by DOX treatment (10 μM, 24 h). Then, cell viability and mitochondrial function were determined. In animal study, male Wistar rats were divided into 4 groups: 1) Control, 2) IVA (10 mg/kg/d, PO), 3) DOX (3 mg/kg/d, 6 doses, IP), 4) Co-treatment with IVA (10 mg/kg/d, PO) and DOX (3 mg/kg/d, 6 doses, IP) for 30 days. Then, heart rate, cardiac function, and cardiac mitochondrial dynamic proteins were determined. Result DOX reduced %cell viability of H9C2 cells via increasing cellular and mitochondrial oxidative stress, and reducing mitochondrial respiration (Figure 1A, B). Under DOX-induced cellular injury condition, pretreatment with IVA resulted in increasing %cell viability by 11%, decreasing cellular and mitochondrial oxidative stress; however, IVA did not affect mitochondrial respiration. Consistent with in vitro study, DOX induced cardiac dysfunction as indicated by reduced heart rate and ejection fraction, impaired mitochondrial function and mitochondrial dynamics balance (Figure 1C, D). Co-treatment with DOX and IVA had no further impact on heart rate. DOX-IVA attenuated cardiac mitochondrial dysfunction and mitochondrial dynamics imbalance, thus prevented cardiac dysfunction. Conclusion IVA effectively protected against DOX-induced cardiotoxicity through improvement of mitochondrial function, mitochondrial dynamics, and reduction of oxidative stress. These findings suggest the therapeutic potential of IVA in preventing Dox-induced cardiotoxicity.

Role of liraglutide on cardiotoxicity and gut microbiota changes induced by doxorubicin in rats

Abstract Background Cancer is one of the main causes of morbidity and mortality worldwide. Despite doxorubicin (DOX) being an effective chemotherapy drug, DOX-induced cardiotoxicity is the most severe side effect, limiting its use. There is no effective treatment for preventing DOX-induced cardiotoxicity. Gut microbiota seems to have a role in cardiovascular disease. Glucagon like peptide-1 (GLP-1) analogs such as liraglutide have shown benefits in cardiovascular diseases and seem to affect gut microbiota. Purpose To evaluate the role of liraglutide in modulating acute DOX-induced cardiotoxicity and gut microbiota. Methods Sixty male Wistar rats were allocated into four groups: Control (C), DOX (D), Liraglutide (L), and DOX + Liraglutide (DL). Animals in L and DL groups received a subcutaneous injection of 0.6 mg/kg liraglutide daily for 2 weeks. After 12 days, D and DL groups received an intraperitoneal injection of DOX (20 mg/kg). Rats were subjected to echocardiogram and isolated heart functional study 48 hours after DOX injection. At the end of the experiment, feces were collected to evaluate microbiota and short chain fatty acid concentration. Statistical analyses: Generalized linear model (GLM), ANCOVA and PERMANOVA (p&amp;lt;0.05). Results DOX-treated rats had worse cardiac function than rats that did not receive DOX in both echocardiography and isolated heart study; liraglutide did not change any functional parameters (Table). In feces, the phylum Bacteroidota was reduced in D and L groups compared to C and the phylum Pseudomonadota was increased in D and DL compared to C and L. Alpha-diversity did not differ between groups. Beta-diversity differed between C, D and L groups, and was similar between D and DL (Figure). Feces short chain fatty acid concentration was reduced in DOX-treated rats, with no effect from liraglutide (Figure). Conclusion DOX altered the function and composition of gut microbiota and caused acute cardiotoxicity; liraglutide changed gut microbiota and did not improve cardiac function.Table.Left ventricular functional dataFigure.Microbiota

USP14 modulates cell pyroptosis and ameliorates doxorubicin-induced cardiotoxicity by deubiquitinating and stabilizing SIRT3

This study investigates the role of the deubiquitinating enzyme USP14 in alleviating doxorubicin (DOX)-induced cardiotoxicity (DIC), particularly concerning its mechanism of regulating pyroptosis through the stabilization of the mitochondrial protein SIRT3. Using in vivo and in vitro models, the research demonstrated that USP14 overexpression protects against DOX-induced cardiac damage by modulating pyroptosis. Silencing SIRT3 via siRNA revealed that SIRT3 is a key intermediary molecule in USP14-mediated regulation of pyroptosis. Notably, DOX exposure resulted in decreased USP14 expression, while its overexpression preserved mitochondrial function and reduced oxidative stress by stabilizing SIRT3. Immunoprecipitation confirmed that USP14 stabilizes SIRT3 through deubiquitination. These findings position USP14 as a promising therapeutic target for mitigating DOX-induced cardiotoxicity by stabilizing SIRT3 and maintaining mitochondrial integrity, suggesting potential novel strategies for cardio-protection in chemotherapy.

A Cardiac-Targeting and Anchoring Bimetallic Cluster Nanozyme Alleviates Chemotherapy-Induced Cardiac Ferroptosis and PANoptosis.

Doxorubicin (DOX), a potent antineoplastic agent, is commonly associated with cardiotoxicity, necessitating the development of strategies to reduce its adverse effects on cardiac function. Previous research has demonstrated a strong correlation between DOX-induced cardiotoxicity and the activation of oxidative stress pathways. This work introduces a novel antioxidant therapeutic approach, utilizing libraries of tannic acid and N-acetyl-L-cysteine-protected bimetallic cluster nanozymes. Through extensive screening for antioxidative enzyme-like activity, an optimal bimetallic nanozyme (AuRu) is identified that possess remarkable antioxidant characteristics, mimicking catalase-like enzymes. Theoretical calculations reveal the surface interactions of the prepared nanozymes that simulate the hydrogen peroxide decomposition process, showing that these bimetallic nanozymes readily undergo OH⁻ adsorption and O₂ desorption. To enhance cardiac targeting, the atrial natriuretic peptide is conjugated to the AuRu nanozyme. These cardiac-targeted bimetallic cluster nanozymes, with their anchoring capability, effectively reduce DOX-induced cardiomyocyte ferroptosis and PANoptosis without compromising tumor treatment efficacy. Thus, the therapeutic approach demonstrates significant reductions in chemotherapy-induced cardiac cell death and improvements in cardiac function, accompanied by exceptional in vivo biocompatibility and stability. This study presents a promising avenue for preventing chemotherapy-induced cardiotoxicity, offering potential clinical benefits for cancer patients.