Model Of DOX-induced Cardiotoxicity Research Articles

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.

Preventive effect of imperatorin against doxorubicin-induced cardiotoxicity through suppression of NLRP3 inflammasome activation.

Cardiotoxicity is one of the major obstacles to anthracycline chemotherapy. Anthracycline cardiotoxicity is closely associated with inflammation. Imperatorin (IMP), a furocoumarin ingredient extracted from Angelica dahurica, might have potential activity in preventing anthracycline cardiotoxicity due to its anti-cancer, anti-inflammatory, anti-oxidant, cardioprotective properties. This study aims to reveal the effect of IMP on doxorubicin (DOX)-induced cardiotoxicity and its underlying mechanism. We established a rat model of DOX-induced cardiotoxicity by intraperitoneal injection with DOX (1.25mg/kg twice weekly for 6weeks), and found that both IMP (25mg/kg and 12.5mg/kg) and dexrazoxane 12.5mg/kg relieved DOX-induced reductions in heart weight, change in cardiac histopathology, and elevated serum levels of LDH, AST and CK-MB. Moreover, DOX upregulated mRNA levels of NLRP3, CASP1, GSDMD, ASC, IL-1β and IL-18, elevated protein expressions of NLRP3, ASC, GSDMD-FL, GSDMD-N, pro‑caspase‑1, caspase‑1 p20, pro‑IL‑1β and IL‑1β in heart tissues, as well as increased serum levels of pro-inflammatory cytokines including IL-1β and IL-18, however both of IMP and dexrazoxane suppressed these alterations. In addition, we carried out neonatal rat cardiomyocytes experiments to confirm the results of the in vivo study. Consistently, pretreatment with IMP 25µg/mL relieved DOX (1μg/mL)-induced cardiomyocytes injury, including decreased cell viability and reduced supernatant LDH. IMP inhibited DOX-induced activation of NLRP3 inflammasome in cardiomyocytes. In conclusion, IMP had a protective effect against DOX-induced cardiotoxicity via repressing the activation of NLRP3 inflammasome. These findings suggest that IMP may be a promising alternative or adjunctive drug for the prevention of anthracycline 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.

Metformin-mediated protection against doxorubicin-induced cardiotoxicity

BackgroundA phase II clinical trial of metformin (MET) for the treatment of doxorubicin (DOX)-induced cardiotoxicity (NCT02472353) failed. ObjectivesThe aims of this study were to confirm MET-mediated protection against DOX-induced cardiotoxicity and its mechanism using H9C2 cells, and to establish a Wistar rat model of DOX-induced cardiotoxicity. Subsequently, Wistar rats were utilized to identify clinically relevant indicators for evaluating MET-mediated protection against DOX-induced cardiotoxicity, thereby facilitating early transition towards successful clinical trials. MethodsMET-mediated protection was assessed using cell viability and cytotoxicity experiments. Additionally, intramitochondrial reactive oxygen species (ROS) levels were measured using an ROS fluorescent probe (dihydroethidium) to confirm the oxidative stress mechanism. Eighteen Wistar rats were randomly allocated to the control, DOX, and DOX+MET groups; and the body weight, adverse drug reactions (ADRs), myocardial injury, cardiac function, oxidative stress, and histopathology of heart tissues were compared between groups. ResultsH9C2 cells treated with MET/Dexrazoxane demonstrated dose-dependent protection against DOX-induced cardiotoxicity. The fluorescence intensity of H9C2 cells suggested DOX-induced cardiomyocyte toxicity and MET-mediated protection against DOX-induced cardiotoxicity. In vivo experiments confirmed that a rat model of DOX-induced cardiotoxicity was successfully established, but MET-mediated protection against DOX-induced cardiotoxicity was not demonstrated. This was attributed to insufficient energy intake because of ADRs, such as vomiting. ConclusionsWe confirmed the MET-mediated protection against DOX-induced cardiomyocyte toxicity and its mechanism involving the inhibition of oxidative stress in vitro experiments. It is imperative to investigate the optimal conditions for MET-mediated protection against DOX-induced cardiotoxicity in vivo or clinical trials.

Asiatic acid ameliorates doxorubicin-induced cardiotoxicity by promoting FPN-mediated iron export and inhibiting ferroptosis.

Doxorubicin (DOX), a common chemotherapeutic agent in cancer therapy, is accompanied by pronounced cardiotoxicity. Ferroptosis has been implicated in the pathogenesis and therapeutics of DOX-induced cardiotoxicity (DIC). Asiatic acid (AA), a pentacyclic triterpene from the Chinese medicinal herb Centella asiatica, displays antioxidant, anti-inflammatory, and antiapoptotic activities. In this study, we investigated the beneficial effects of AA against DOX-induced ferroptosis and cardiotoxicity and the underlying mechanisms. A chronic DIC model was established by challenging mice with DOX (5 mg/kg, i.p.) once per week for 4 weeks. Concurrent with DOX insult, the mice were administered AA (25 mg·kg-1·d-1, i.g.). Cardiac function and mechanical properties of isolated cardiomyocytes were evaluated at the end of treatment. We showed that AA administration preserved cardiac function, significantly reduced cardiac injury, and improved cardiomyocyte contractile function in DIC mice. The beneficial effects of AA were causally linked to the inhibition of DOX-induced ferroptosis both in vivo and in vitro. We revealed that AA attenuated DOX-induced iron accumulation in HL-1 cells by increasing FPN-mediated iron export, in a Nrf2-dependent manner. AA upregulated Nrf2 expression and promoted Nrf2 nuclear translocation in DOX-treated HL-1 cells. Moreover, AA-offered benefits against DOX-induced cardiac dysfunction and ferroptosis were abolished by Nrf2 inhibitor ML385 (30 mg·kg-1·d-1, i.p.) administrated 30 min before AA in DIC mice. Our data favor that AA promotes FPN-mediated iron export to inhibit iron overload and ferroptosis in DIC, suggesting its therapeutic potential in the treatment of DIC.

Abstract Mo001: Inhibition of TAOK1-mediated Cardiomyocyte Death Attenuates Doxorubicin-Induced Cardiotoxicity

Background and Objective: Doxorubicin (Dox)-induced cardiotoxicity (DIC) is one of the most serious side effects of cancer treatments. However, no effective treatment is currently available. In this study, we aimed to identify novel therapeutic targets using CRISPR/Cas9-based screening of human cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). Methods and Results: We performed a kinase-focused CRISPR gene knockout screen in hPSC-CMs and identified thousand and one amino acid protein kinase 1 (TAOK1 ) as a potential regulator of DOX-induced cardiomyocyte death. We generated TAOK1-knockout (KO) hPSC-CMs using CRISPR/Cas9 and found that TAOK1-KO hPSC-CMs showed higher cell viability than control KO cells after 1μM DOX treatment (49.3% vs. 30.5%, P = 0.03). TAOK1-KO decreased DOX-induced ROS production (-58.8%, P = 0.01) and DNA damage (-28.8%, P < 0.01) in hPSC-CMs. Transcriptome analysis using RNA-seq of TAOK1-KO hPSC-CMs identified 483 differentially expressed genes, and Hallmark pathway analysis revealed upregulation of the oxidative phosphorylation pathway (FDR < 0.01) in TAOK1-KO hPSC-CMs compared to the control. Extracellular flux analysis demonstrated higher basal and maximal respiratory capacity in TAOK1-KO hPSC-CMs compared to control after DOX treatment (1.2 vs. 0.6, and 3.8 vs. 2.0 pmol/min/μg, respectively, P < 0.01 for all). Next, we overexpressed TAOK1 in hPSC-CMs using adenovirus vector and found that TAOK1 overexpression (OE) augmented DOX-induced cell viability reduction in hPSC-CMs compared to control after 0.5μM DOX treatment (-63.4 % vs. -19.2%, P < 0.01). Western blot analysis showed that DOX-induced p38 phosphorylation was suppressed by TAOK1-KO (P < 0.01) and enhanced (P = 0.01) by TAOK1-OE in hPSC-CMs. Augmented DOX-induced cardiomyocyte death in TAOK1-OE hPSC-CMs was partially rescued by inhibitors of p38 MAPK or apoptosis. Lastly, we demonstrated that TAOK1 suppression using AAV-mediated gene silencing mitigated the decline in the left ventricular ejection fraction in a mouse model of DIC (-30.0% vs. -17.7%, P < 0.01). Histological analysis of heart tissue revealed that TAOK1 suppression reduced myocardial fibrosis and apoptosis of cardiac cells in a mouse model of DIC (6.6% vs. 4.3%, P = 0.02, and 0.31% vs. 0.18%, P< 0.01, respectively). Conclusion: Our results suggest that TAOK1 regulates DOX-induced cardiomyocyte death; thus, its inhibition could be a potential therapeutic approach for DIC.

Micafungin protects mouse heart against doxorubicin-induced oxidative injury via suppressing MALT1-dependent k48-linked ubiquitination of Nrf2

Oxidative stress contributes greatly to doxorubicin (DOX)-induced cardiotoxicity. Down-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) is a key factor in DOX-induced myocardial oxidative injury. Recently, we found that mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1)-dependent k48-linked ubiquitination was responsible for down-regulation of myocardial Nrf2 in DOX-treated mice. Micafungin, an antifungal drug, was identified as a potential MALT1 inhibitor. This study aims to explore whether micafungin can reduce DOX-induced myocardial oxidative injury and if its anti-oxidative effect involves a suppression of MALT1-dependent k48-linked ubiquitination of Nrf2. To establish the cardiotoxicity models in vivo and in vitro, mice were treated with a single dose of DOX (15 mg/kg, i.p.) and cardiomyocytes were incubated with DOX (1 μM) for 24 h, respectively. Using mouse model of DOX-induced cardiotoxicity, micafungin (10 or 20 mg/kg) was shown to improve cardiac function, concomitant with suppression of oxidative stress, mitochondrial dysfunction, and cell death in a dose-dependent manner. Similar protective roles of micafungin (1 or 5 μM) were observed in DOX-treated cardiomyocytes. Mechanistically, micafungin weakened the interaction between MALT1 and Nrf2, decreased the k48-linked ubiquitination of Nrf2 while elevated the protein levels of Nrf2 in both DOX-treated mice and cardiomyocytes. Furthermore, MALT1 overexpression counteracted the cardioprotective effects of micafungin. In conclusion, micafungin reduces DOX-induced myocardial oxidative injury via suppression of MALT1, which decreases the k48-linked ubiquitination of Nrf2 and elevates Nrf2 protein levels. Thus, micafungin may be repurposed for treating DOX-induced cardiotoxicity.

CREG1 attenuates doxorubicin-induced cardiotoxicity by inhibiting the ferroptosis of cardiomyocytes

ObjectiveDoxorubicin (DOX)-induced cardiotoxicity limits the application of DOX in cancer patients. Currently, there is no effective prevention or treatment for DOX-induced cardiotoxicity. The cellular repressor of E1A-stimulated genes (CREG1) is a cardioprotective factor that plays an important role in the maintenance of cardiomyocytes differentiation and homeostasis. However, the role and mechanism of CREG1 in DOX-induced cardiotoxicity has not yet been elucidated. MethodsIn vivo, C57BL/6J mice, CREG1 transgenic and cardiac-specific CREG1 knockout mice were used to establish a DOX-induced cardiotoxicity model. H&E staining, Masson’s trichrome, WGA staining, real-time PCR, and western blotting were performed to examine fibrosis and ferroptosis in the myocardium. In vitro, neonatal mouse cardiomyocytes (NMCMs) were cultured and stimulated with DOX, CREG1-overexpressed adenovirus, and small interfering RNA was used to establish CREG1 overexpression or knockdown cardiomyocytes. Transcriptomics, real-time PCR, western blotting, and immunoprecipitation were used to examine the roles and mechanisms of CREG1 in cardiomyocytes ferroptosis. ResultsThe mRNA and protein levels of CREG1 were reduced in the hearts and NMCMs after DOX treatment. CREG1 overexpression alleviated myocardial damage and inhibited DOX-induced ferroptosis in the myocardium. CREG1 deficiency in the heart aggravated DOX-induced cardiotoxicity and ferroptosis. In vitro, CREG1 overexpression inhibited cardiomyocytes ferroptosis induced by DOX, and CREG1 knockdown aggravated DOX-induced cardiotoxicity. Mechanistically, CREG1 inhibited the mRNA and protein expression of pyruvate dehydrogenase kinase 4 (PDK4) by regulating the F-box and WD repeat domain containing 7 (FBXW7)-forkhead box O1 (FOXO1) pathway. PDK4 deficiency reversed the effects of CREG1 knockdown on cardiomyocytes ferroptosis following DOX treatment. ConclusionCREG1 alleviated DOX-induced cardiotoxicity by inhibiting ferroptosis in cardiomyocytes. Our findings may help clarify the new roles of CREG1 in the development of DOX-induced cardiotoxicity.

Inhibition of cytochrome P450 epoxygenase promotes endothelium-to-mesenchymal transition and exacerbates doxorubicin-induced cardiovascular toxicity

BackgroundDoxorubicin (DOX) is a potent chemotherapy widely used in treating various neoplastic diseases. However, the clinical use of DOX is limited due to its potential toxic effect on the cardiovascular system. Thus, identifying the pathway involved in this toxicity may help minimize chemotherapy risk and improve cancer patients’ quality of life. Recent studies suggest that Endothelial-to-Mesenchymal transition (EndMT) and endothelial toxicity contribute to the pathogenesis of DOX-induced cardiovascular toxicity. However, the molecular mechanism is yet unknown. Given that arachidonic acid and associated cytochrome P450 (CYP) epoxygenase have been involved in endothelial and cardiovascular function, we aimed to examine the effect of suppressing CYP epoxygenases on DOX-induced EndMT and cardiovascular toxicity in vitro and in vivo.Methods and ResultsTo test this, human endothelial cells were treated with DOX, with or without CYP epoxygenase inhibitor, MSPPOH. We also investigated the effect of MSPPOH on the cardiovascular system in our zebrafish model of DOX-induced cardiotoxicity. Our results showed that MSPPOH exacerbated DOX-induced EndMT, inflammation, oxidative stress, and apoptosis in our endothelial cells. Furthermore, we also show that MSPPOH increased cardiac edema, lowered vascular blood flow velocity, and worsened the expression of EndMT and cardiac injury markers in our zebrafish model of DOX-induced cardiotoxicity.ConclusionOur data indicate that a selective CYP epoxygenase inhibitor, MSPPOH, induces EndMT and endothelial toxicity to contribute to DOX-induced cardiovascular toxicity.

Vericiguat attenuates doxorubicin-induced cardiotoxicity through the PRKG1/PINK1/STING axis

Doxorubicin (DOX) is restricted due to its severe cardiotoxicity. There is still a lack of viable and effective drugs to prevent or treat DOX-induced cardiotoxicity(DIC). Vericiguat is widely used to treat heart failure with reduced ejection fraction. However, it is not clear whether vericiguat can improve DIC. In the present study, we constructed a DIC model using mice and neonatal rat cardiomyocytes and found that vericiguat ameliorated DOX-induced cardiac insufficiency in mice, restored DOX-induced mitochondrial dysfunction in neonatal rat cardiomyocytes, and inhibited the expression of inflammatory factors. Further studies showed that vericiguat improved mitochondrial dysfunction and reduced mtDNA leakage into the cytoplasm by up-regulating PRKG1, which activated PINK1 and then inhibited the STING/IRF3 pathway to alleviate DIC. These findings demonstrate for the first time that vericiguat has therapeutic potential for the treatment of DIC.

Apigenin alleviates doxorubicin-induced myocardial pyroptosis by inhibiting glycogen synthase kinase-3β in vitro and in vivo.

Apigenin, a natural flavonoid compound found in chamomile (Matricaia chamomilla L.) from the Asteraceae family, has been shown in our previous study to possess antimyocardial hypertrophy and anti-cardiac fibrosis effects. However, its effects and mechanisms on the pyroptosis of cardiomyocytes induced by doxorubicin (DOX) are poorly understood. The objective of this study was to investigate the role of GSK-3β and the effects of apigenin in DOX-induced cardiotoxicity. H9c2 cells stimulated with DOX were treated with SB216763 and apigenin. Additionally, a mouse model of DOX-induced cardiotoxicity was prepared and further treated with apigenin and SB216763 for 30 days. The findings revealed that treatment with SB216763 or apigenin resulted in a significant reduction in the levels of pyroptosis-related factors. Furthermore, the phosphorylation of GSK-3β was enhanced while the phosphorylation of nuclear factor-kB (NF-κB) p65 was reduced following treatment with either SB216763 or apigenin. Conversely, the effects of apigenin treatment were nullified in siRNA-GSK-3β-transfected cells. Results from computer simulation and molecular docking analysis supported that apigenin could directly target the regulation of GSK-3β. Therefore, our study confirmed that the inhibition of GSK-3β and treatment with apigenin effectively suppressed the pyroptosis of cardiomyocytes in both DOX-stimulated H9c2 cells and mice. These benefits may be attributed in part to the decrease in GSK-3β expression and subsequent reduction in NF-κB p65 activation. Overall, our findings revealed that the pharmacological targeting of GSK-3β may offer a promising therapeutic approach for alleviating DOX-induced cardiotoxicity.

Doxorubicin treatment reduces expression of cardiac iron transporters

Doxorubicin (DOX) is an effective anticancer therapy. Unfortunately, the toxicity of DOX is not limited to cancerous cells, with the heart particularly prone to DOX-induced damage. While the mechanisms of DOX cardiotoxicity remain unclear, accumulation of iron within the heart may exacerbate the detrimental effects of DOX. Thus, understanding the effects of DOX on iron transport and storage protein expression would provide further insight on the mechanisms of disrupted iron handling and may lead to potential therapeutic targets for cardioprotection. Our goal was to investigate changes in gene expression of iron transporters in a preclinical model of DOX-induced cardiotoxicity. Female Sprague Dawley rats (2-3 month-old) were randomly divided to receive DOX or saline consistent with standard clinical treatment (i.e., intravenous infusion once every 3 weeks for 4 total cycles and a cumulative dose of ~160 mg/m2). One week following the fourth cycle measures of cardiac function, assessed by echocardiography, and cardiorespiratory capacity, determined by a graded exercise tolerance test, were completed. Cardiac tissue was also collected and cardiac mitochondria were isolated for measurement of reactive oxygen species production. At endpoint, DOX treatment resulted in a lower heart weight to tibia length ratio, reduced cardiorespiratory capacity, impaired ventricular fractional shortening and greater cardiac mitochondrial hydrogen peroxide emission. We found significant reductions in expression of transmembrane/cytoplasmic iron transporters (transferrin receptor 1 and divalent metal transporter 1) and the inner mitochondrial membrane transporter mitoferrin-2 with DOX treatment compared to saline. Expression of genes encoding for ferroportin-1, mitoferrin-1, ABCB8 and ferrochelatase were also downregulated in the DOX-treated group compared to saline, but these did not reach statistical significance. These findings are the first to describe changes in gene expression of proteins involved in iron homeostasis in a clinically relevant model of DOX-induced cardiotoxicity. Further studies are needed to investigate interventional approaches that improve cardiac iron handling to determine if they provide cardioprotection during DOX treatment. This research was funded by the National Institute of Health (NIH), R01 HL144858. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exercise Rehabilitation Preserves Cardiorespiratory Muscle Function Following Doxorubicin Treatment

Doxorubicin (DOX) is a highly effective chemotherapeutic agent. Unfortunately, cancer patients who have completed DOX treatment often experience exercise intolerance, reduced cardiac function, dyspnea and unfavorable changes to body composition, ultimately, leading to increased incidence of heart failure. Exercise has been shown to reduce the risk of developing chemotherapy-induced cardiorespiratory dysfunction; however, the ability of cancer patients to exercise during treatment can be limited by cancer type, stage and fatigue. Therefore, these experiments utilized a chronic DOX-induced cardiotoxicity model to determine the effects of rehabilitative exercise on cardiorespiratory muscle function. Female Sprague-Dawley rats were treated with DOX once every three weeks for four total cycles via intravenous infusion (i.e., cumulative dose of ~160mg/m2). One-week following the completion of DOX treatment, rats remained sedentary (Sed-DOX) or were prescribed 10-weeks of moderate intensity treadmill running at 30m/min, 0% grade, 1-hour/day, and 3x/week (Mod-DOX). Body composition, exercise tolerance and echocardiography were assessed at three timepoints: 1) pre-treatment; 2) post-treatment and; 3) post-rehabilitation. Diaphragm muscle specific force production was measured post-rehabilitation. From pre- to post-treatment each group gained weight and no differences existed between groups. However, from post-treatment to post-rehabilitation body weight of the DOX-treated groups did not change and was significantly reduced compared to sedentary saline-treated (Sed-Sal) rats from week 15 onwards. Although mean body weight did not differ between the DOX-treated groups, lean mass increased in Mod-DOX rats between each timepoint with no change in fat mass, while fat mass increased in Sed-DOX rats and an initial increase in lean mass was attenuated from post-treatment to post-rehabilitation. Exercise time to fatigue did not change between timepoints within each group; however, exercise run time from post-treatment to post-rehabilitation was significantly longer in Mod-DOX rats compared to Sed-DOX and Sed-Sal. Cardiac fractional shortening percentage (FS%) was reduced in Sed-DOX rats at post-treatment and post-rehabilitation compared to pre-treatment. Additionally, posterior wall shortening velocity (PWSV) was reduced in Sed-DOX rats at post-treatment compared to pre-treatment. No differences in FS% or PWSV were seen between any timepoint in the Mod-DOX group. Finally, ex vivo diaphragm muscle function showed significant diaphragm muscle weakness in Sed-DOX rats at 15-60Hz stimulations compared to Sed-Sal. Diaphragm force production did not differ in Mod-DOX rats compared to Sed-Sal at any stimulation frequency. These results demonstrate a beneficial effect of moderate intensity rehabilitation exercise for the maintenance of body composition, cardiac function and diaphragm muscle function following DOX treatment compared to sedentary rats. NIH R01 HL144858, NIH R01 HL146443, NIH HL134621. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of METTL3 ameliorates doxorubicin-induced cardiotoxicity through suppression of TFRC-mediated ferroptosis

BackgroundDoxorubicin (DOX) is a chemotherapeutic drug, while its clinical use is greatly limited by the life-threatening cardiotoxicity. N6-methyladenosine (m6A) RNA modification participates in varieties of cellular processes. Nonetheless, it remains elusive whether m6A modification and its methyltransferase METTL3 are involved in the progression of DOX-induced cardiotoxicity (DIC). MethodsMice were administrated with DOX (accumulative dosage of 20 mg/kg) repeatedly to establish a chronic DIC model. Cardiomyocyte-specific conditional METTL3 knockout mice were employed to evaluate the effects of altered m6A RNA modification on DIC. The effects of METTL3 on cardiomyocyte ferroptosis were also examined in response to DOX stimulation. ResultsDOX led to increased levels in m6A modification and METTL3 expression in cardiomyocytes in a c-Jun-dependent manner. METTL3-knockout mice exhibited improved cardiac function, remodeling and injury following DOX insult. Besides, inhibition of METTL3 alleviated DOX-induced iron accumulation and ferroptosis in cardiomyocytes, whereas METTL3 overexpression exerted the opposite effects. Mechanistically, METTL3 promoted m6A modification of TFRC mRNA, a critical gene governing iron uptake, and enhanced its stability through recognition of the m6A reader protein, IGF2BP2. Moreover, pharmacological administration of a highly selective METTL3 inhibitor STM2457 effectively ameliorated DIC in mice. ConclusionMETTL3 plays a cardinal role in the etiology of DIC by regulating cardiac iron metabolism and ferroptosis through TFRC m6A modification. Inhibition of METTL3 might be a potential therapeutic avenue for DIC.

Pyridoxamine Limits Cardiac Dysfunction in a Rat Model of Doxorubicin-Induced Cardiotoxicity.

The use of doxorubicin (DOX) chemotherapy is restricted due to dose-dependent cardiotoxicity. Pyridoxamine (PM) is a vitamin B6 derivative with favorable effects on diverse cardiovascular diseases, suggesting a cardioprotective effect on DOX-induced cardiotoxicity. The cardioprotective nature of PM was investigated in a rat model of DOX-induced cardiotoxicity. Six-week-old female Sprague Dawley rats were treated intravenously with 2 mg/kg DOX or saline (CTRL) weekly for eight weeks. Two other groups received PM via the drinking water next to DOX (DOX+PM) or saline (CTRL+PM). Echocardiography, strain analysis, and hemodynamic measurements were performed to evaluate cardiac function. Fibrotic remodeling, myocardial inflammation, oxidative stress, apoptosis, and ferroptosis were evaluated by various in vitro techniques. PM significantly attenuated DOX-induced left ventricular (LV) dilated cardiomyopathy and limited TGF-β1-related LV fibrotic remodeling and macrophage-driven myocardial inflammation. PM protected against DOX-induced ferroptosis, as evidenced by restored DOX-induced disturbance of redox balance, improved cytosolic and mitochondrial iron regulation, and reduced mitochondrial damage at the gene level. In conclusion, PM attenuated the development of cardiac damage after DOX treatment by reducing myocardial fibrosis, inflammation, and mitochondrial damage and by restoring redox and iron regulation at the gene level, suggesting that PM may be a novel cardioprotective strategy for DOX-induced cardiomyopathy.

Mesenchymal Stem Cell-Derived Exosomes Ameliorate Doxorubicin-Induced Cardiotoxicity.

Doxorubicin (DOX) is an incessantly used chemotherapeutic drug that can cause detrimental dose-dependent effects such as cardiotoxicity and congestive heart failure. Hence, there is a need to discover innovative therapeutic approaches to counteract DOX-induced cardiotoxicity (DIC). MSC-Exos have shown to reduce apoptosis and cardiac fibrosis and promote cardiomyocyte proliferation in myocardial infracted mice. However, the effect of MSC-Exos on ameliorating DOX-induced pyroptosis has not been investigated. In this current study, H9c2 were first exposed to DOX to stimulate pyroptosis, followed by subsequent treatment with MSC-Exos, with further analysis performed through immunocytochemistry, western blotting, and RT-PCR. Our data depicted that post-treatment with MSC-Exos significantly (p < 0.05) reduced the HMGB1/TLR4 axis, inflammasome formation (NLRP3), pyroptotic markers (caspase-1, IL-1β, and IL-18), and the pyroptotic executioner (GSDMD) in DOX-treated H9c2 cells. In conclusion, our data show that MSC-Exos attenuates inflammation-induced pyroptosis in our in vitro DIC model. Our findings indicate that MSC-Exos may serve as a promising therapeutic intervention for mitigating DIC, as they maintain the therapeutic capabilities of MSCs while circumventing the drawbacks associated with traditional stem cell therapy.

Poncirus trifoliata Aqueous Extract Protects Cardiomyocytes against Doxorubicin-Induced Toxicity through Upregulation of NAD(P)H Dehydrogenase Quinone Acceptor Oxidoreductase 1.

Doxorubicin (DOX), an anthracycline-based chemotherapeutic agent, is widely used to treat various types of cancer; however, prolonged treatment induces cardiomyotoxicity. Although studies have been performed to overcome DOX-induced cardiotoxicity (DICT), no effective method is currently available. This study investigated the effects and potential mechanisms of Poncirus trifoliata aqueous extract (PTA) in DICT. Changes in cell survival were assessed in H9c2 rat cardiomyocytes and MDA-MB-231 human breast cancer cells. The C57BL/6 mice were treated with DOX to induce DICT in vivo, and alterations in electrophysiological characteristics, serum biomarkers, and histological features were examined. The PTA treatment inhibited DOX-induced decrease in H9c2 cell viability but did not affect the MDA-MB-231 cell viability. Additionally, the PTA restored the abnormal heart rate, R-R interval, QT interval, and ST segment and inhibited the decrease in serum cardiac and hepatic toxicity indicators in the DICT model. Moreover, the PTA administration protected against myocardial fibrosis and apoptosis in the heart tissue of mice with DICT. PTA treatment restored DOX-induced decrease in the expression of NAD(P)H dehydrogenase quinone acceptor oxidoreductase 1 in a PTA concentration-dependent manner. In conclusion, the PTA inhibitory effect on DICT is attributable to its antioxidant properties, suggesting the potential of PTA as a phytotherapeutic agent for DICT.

FUDNC1 ameliorates doxorubicin-induced cardiotoxicity by restoring MERCs structure and blocked autophagic flux

Abstract Background Doxorubicin (DOX)-induced cardiotoxicity (DIC) is frequently observed in clinical practice. Autophagy dysregulation is known to be a mechanism of DIC. Mitochondrial-Endoplasmic Reticulum Contact sites (MERCs) is where autophagy initiates and autophagosomes form. However, the role of MERCs in autophagy dysregulation in DIC remains elusive. FUNDC1 is a mitochondria outer membrane protein that serves as a mitophagy receptor as well as a tethering protein of MERCs. Purpose The aim of our study was to investigate the effect of DOX on MERCs and explore whether it is involved in the dysregulation of autophagy. Our hypothesis is that FUNDC1 overexpression ameliorates DIC by restoring MERCs structure and dysregulated autophagic flux. Method We use confocal microscopy and transmission electron microscopy to assess MERCs structure. Overall autophagic flux was analyzed by RFP-EGFP-LC3B fluorescence assay and western blotting for LC3BII. A total dose of 15 mg/kg of doxorubicin was administrated in C57BL/6J mice to construct a DIC model in vivo. Adeno-associated virus (AAV) was used to cardiac-specifically overexpress FUNDC1. Cardiac remodeling was evaluated by Masson’s trichrome staining. Cardiac function (left ventricle ejection fraction, LVEF; left ventricle fraction shortening, LVFS) was measured by echocardiography. Results DOX treatment led to a significant downregulation of multiple tethering proteins (including FUNDC1) of MERCs and inhibition of MERCs formation, as demonstrated by decreased colocalization coefficients of mitochondria and endoplasmic reticulum in cardiomyocytes. The overexpression of FUNDC1 abolished DOX-induced inhibition on MERCs formation. In addition, DOX treatment blocked the normal autophagic flux, as demonstrated by the decreased autophagosome synthesis and impaired autophagosome turnover. Maintaining the MERCs structure by FUNDC1 overexpression was able to restore the blocked autophagic flux mentioned above. Moreover, FUNDC1 overexpression restored the blocked autophagic flux in vivo, as demonstrated by the increased LC3B turnover compared to the DOX treatment group. Importantly, global heart function of FUNDC1 overexpressed mice was significantly improved post DOX treatment compared to the Wildtype DOX treatment group, as demonstrated by the increased LVEF and LVFS. In addition, LV fibrosis level was found to be significantly decreased in FUNDC1 overexpressed mice treated with DOX compared to the Wildtype DOX treatment group. Conclusion We reported for the first time that the impaired MERCs formation in DOX-treated cardiomyocytes and revealed that restoring the disrupted MERCs structure by FUNDC1 overexpression was able to revert the blocked autophagic flux induced by DOX treatment. Furthermore, cardiac-specific overexpression of FUNDC1 protected DOX-treated mice against adverse cardiac remodeling and improved cardiac function. These findings may shed light on the prevention and treatment of DIC.Figure

SGK1 is involved in doxorubicin-induced chronic cardiotoxicity and dysfunction through activation of the NFκB pathway

Breast cancer is the predominant cancer among women worldwide, and chemotherapeutic agents, such as doxorubicin (DOX), have the potential to significantly prolong survival, albeit at the cost of inducing severe cardiovascular toxicity. Inflammation has emerged as a crucial biological process contributing to the remodeling of cardiovascular toxicity. The role of serum glucocorticoid kinase 1 (SGK1) in various inflammatory diseases has been extensively investigated. Here, we studied the molecular mechanisms underlying the function of SGK1 in DOX-induced cardiotoxicity in HL-1 cardiomyocyte cell lines and in a tumor-bearing mouse model. SGK1 was upregulated in the DOX-induced cardiotoxicity model, accompanied by increased levels of inflammatory factors. Furthermore, inhibition of SGK1 suppresses the phosphorylation of nuclear factor-kappa B (NFκB) in cardiomyocytes, which inhibits the production of inflammatory factors and apoptosis of cardiomyocytes, and has cardioprotective effects. Simultaneously, small interfering RNA targeting SGK1 inhibited the proliferation of breast cancer cells. Conversely, overexpression of SGK1 increases the phosphorylation of NFκB and aggravates myocardial injury. In conclusion, our study demonstrates that SGK1 promotes DOX-induced cardiac inflammation and apoptosis by promoting NFκB activity. Our results indicate that inhibiting SGK1 might be an effective treatment strategy that can provide both tumor-killing and cardioprotective functions. Further in vivo research is needed to fully elucidate the effects and mechanisms of combination therapy with SGK1 inhibitors and DOX in breast cancer treatment.

Abstract 12306: CREG1 Attenuates Doxorubicin-Induced Cardiotoxicity via Inhibiting the Ferroptosis of Cardiomyocytes

Introduction: Doxorubicin (DOX) is currently one of the most used anthracycline anticancer drugs. However, the cardiotoxicity of DOX limits its application in cancer patients. Cellular repressor of E1A-stimulated genes (CREG1) is an important cardioprotective factor. Hypothesis: We investigated the underlying effects and mechanism of of CREG1 in DOX-induced cardiotoxicity have not been reported. Methods: In vivo, a mouse DOX-induced cardiotoxicity model was established by DOX intraperitoneal injection, the mRNA and protein expression of CREG1 in the myocardium was examined using real-time PCR and western blot. CREG1 transgenic mice and cardiac-specific CREG1 knockout mice were used to establish DOX-induced cardiotoxicity model. HE staining, Masson staining, WGA staining and western blot were applied to examine fibrosis, myocardial hypertrophy and ferroptosis. In vitro, neonatal mouse cardiomyocytes (NMCMs) were stimulated with DOX, CREG1 overexpression adenovirus and small interfering RNA was used to examine the role of CREG1 on the ferroptosis of NMCMs. Results: In vivo, the mRNA and protein expression of CREG1 were significantly reduced in DOX-treated myocardium. CREG1 overexpression alleviated the myocardial damage induced by DOX, and CREG1 deficiency aggravated the DOX-induced cardiotoxicity. The abnormal increase in ferroptosis was shown in the DOX-treated heart tissues. CREG1 overexpression inhibited the ferroptosis, and CREG1 deficiency aggravated the ferroptosis induced by DOX. In vitro, the mRNA and protein of CREG1 was reduced in DOX-treated NMCMs. CREG1 overexpression reduced the ferroptosis of cardiomyocytes, CREG1 knockdown aggravated the ferroptosis of cardiomyocytes induced by DOX. Mechanically, CREG1 inhibited the mRNA and protein expression of pyruvate dehydrogenase kinase 4 (PDK4). The effect of CREG1 overexpression on ferroptosis of cardiomyocytes was reversed by PDK4 overexpression. Conclusion: CREG1 alleviated DOX-induced cardiotoxicity by inhibiting ferroptosis of cardiomyocytes. Our findings might help clarify new roles of CREG1 in the development of DOX-induced cardiotoxicity.