Neonatal Mouse Cardiomyocytes Research Articles

Isoliquiritigenin alleviates myocardial ischemia-reperfusion injury by regulating the Nrf2/HO-1/SLC7a11/GPX4 axis in mice

Ischemia-reperfusion (I/R) injury, a multifaceted pathological process, occurs when the prolongation of reperfusion duration triggers ferroptosis-mediated myocardial damage. Isoliquiritigenin (ISL), a single flavonoid from licorice, exhibits a wide range of pharmacological impacts, but its function in ferroptosis caused by myocardial I/R injury remains unclear. This study delved into the protective effect of ISL on myocardial I/R injury-induced ferroptosis and its mechanism. Neonatal mouse cardiomyocytes (NMCM) underwent hypoxia/reoxygenation (H/R) to simulate the pathological process of myocardial I/R. ISL significantly attenuated H/R-triggered production of reactive oxygen species in NMCM, reduced the expression of malondialdehyde and the activity of lactate dehydrogenase, enhanced superoxide dismutase and catalase activity, and increased the expression of nuclear factor E2-related factor 2 (Nrf2) and its downstream heme oxygenase 1 (HO-1), thereby mitigating oxidative stress damage. CCK8 experiment revealed that the ferroptosis inhibitor Ferrostatin-1 significantly improved myocardial cell viability after 24 h of reoxygenation, and ISL treatment showed a similar effect. ISL reduced intracellular free iron accumulation, up-regulated glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression, and inhibited lipid peroxidation accumulation, thereby alleviating ferroptosis. The Nrf2-specific inhibitor ML385 counteracted ISL's defensive role against H/R-triggered oxidative stress damage and ferroptosis. In vivo experiments further confirmed that by regulating the translocation of Nrf2 into the nucleus, ISL treatment increased the levels of HO-1, GPX4, and SLC7A11, inhibited the expression of ACSL4, Drp1 to exert the antioxidant role, alleviated mitochondrial damage, and ferroptosis, ultimately reducing myocardial infarction area and injury induced by I/R. ML385 nearly abolished ISL's protective impact on the I/R model by inhibiting Nrf2 function. In summary, ISL is capable of mitigating oxidative stress, mitochondrial damage, and cardiomyocyte ferroptosis caused by I/R, thereby reducing myocardial injury. A key mechanism includes triggering the Nrf2/HO-1/SLC7A11/GPX4 pathway to prevent oxidative stress damage and cardiomyocyte ferroptosis caused by I/R.

BMP7 promotes cardiomyocyte regeneration in zebrafish and adult mice

Zebrafish have a lifelong cardiac regenerative ability after damage, whereas mammals lose this capacity during early postnatal development. This study investigated whether the declining expression of growth factors during postnatal mammalian development contributes to the decrease of cardiomyocyte regenerative potential. Besides confirming the proliferative ability of neuregulin 1 (NRG1), interleukin (IL)1b, receptor activator of nuclear factor kappa-Β ligand (RANKL), insulin growth factor (IGF)2, and IL6, we identified other potential pro-regenerative factors, with BMP7 exhibiting the most pronounced efficacy. Bmp7 knockdown in neonatal mouse cardiomyocytes and loss-of-function in adult zebrafish during cardiac regeneration reduced cardiomyocyte proliferation, indicating that Bmp7 is crucial in the regenerative stages of mouse and zebrafish hearts. Conversely, bmp7 overexpression in regenerating zebrafish or administration at post-mitotic juvenile and adult mouse stages, invitro and invivo following myocardial infarction, enhanced cardiomyocyte cycling. Mechanistically, BMP7 stimulated proliferation through BMPR1A/ACVR1 and ACVR2A/BMPR2 receptors and downstream SMAD5, ERK, and AKT signaling. Overall, BMP7 administration is a promising strategy for heart regeneration.

Mitochonic Acid 5 Improves Mitochondrial Respiration Following Alcohol-Induced Mitochondrial Dysfunction

Chronic alcohol exposure can lead to the development of Alcohol Induced Cardiomyopathy (ACM). The pathological changes in ACM have been suggested to be largely caused by mitochondrial damage, reduction of ATP, oxidative stress, and apoptosis of the cardiomyocytes. Cardiomyocytes, as cells with high energy demand, rely greatly on mitochondrial homeostasis and ATP production. Thus, therapeutic intervention to specifically target the mitochondria and ATP production can potentially alleviate ACM. Mitochonic acid 5 (MA-5) has been recently identified as a potential therapeutic compound that increases cellular ATP and improves mitochondrial function in several cardiac disorders. Given the potential role of MA-5 in improving mitochondrial function, I speculate that MA-5 could be an effective treatment for alcohol-induced cardiotoxicity. In the present study, I test the hypothesis that MA-5 can mitigate mitochondrial energetics in cultured cardiomyocytes exposed to alcohol. To investigate this, neonatal mouse cardiomyocytes (NMCMs) were isolated from mice at day 1-2 of age and cultured for 6 days with alcohol (50mM). The cells were treated with MA-5 (1μM, 5μM, or 10μM) for 4 hours with serum starvation and then cellular energetic changes were measured using the Mito Stress assay with the Seahorse Bioanalyzer (XF96pro, Agilent). To investigate the impacts of chronic alcohol exposure, rat embryonic myoblasts, H9C2 cells, were treated with 50mM alcohol for 3 weeks. Subsequently, cells were treated with MA-5 (1μM, 5μM, or 10μM) for 24 hours and then attached to the Resipher system (Lucid Lab) for continuous oxygen consumption monitoring and recording. NMCMs treated with 50mM alcohol showed a substantial reduction in basal respiration, maximal respiration, and spare respiratory capacity compared to controls. NMCMs treated with 10μM MA-5 showed a significant improvement in these parameters. Chronically exposed H9C2 cells showed a prolonged decline in OCR. MA-5 treatment improved and maintained oxygen consumption in these cells. In summary, these results demonstrated that alcohol exposed cardiomyocytes experience a decline in cellular energetics that could be corrected by MA-5 treatment. Future in vivo studies on preclinical ACM animal models are warranted to yield further insights into a potential novel therapy for ACM in patients. This research was supported by grant #T32AA007577 from the NIAAA at the National Institutes of Health. 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.

IF1 Knockout Protects Against Doxorubicin-Induced Cardiac Dysfunction by Improving Cardiomyocyte Energetics

Doxorubicin (DOX), a member of the Anthracycline antibiotics class, has been used as a potent chemotherapy agent against solid tumors. However, its use is limited by its cardiotoxicity. It is well-documented that mitochondrial dysfunction is one of the main mechanisms of DOX-induced cardiotoxicity. ATP synthase inhibitory subunit 1 (ATPIF1, or IF1) is an endogenous inhibitor of mitochondrial ATP synthase. Recent studies indicate that IF1 may suppress mitochondrial respiration via inhibiting ATP synthase. Others and our previous studies demonstrated that IF1 inactivation exerts hepatic and cardiac protective effects. We hypothesize that the absence of IF1 protects the heart from DOX-induced cardiotoxicity by improving mitochondrial energy metabolism. We first investigate the impact of IF1 KO on mitochondrial respiration using the high-resolution respirometer (OROBOROS) on freshly isolated cardiac mitochondria from IF1 KO and WT mice. IF1 KO mitochondria did show substantially increased state 2, state 3, and state 4 respiration rates compared with those of WT. We then investigated if WT and IF1 KO mice respond differently to DOX treatment. WT and IF1 KO mice at the ages of 12-16 weeks either received DOX 3mg/kg/every other day or vehicle intraperitoneally for two weeks. In vivo, cardiac function was evaluated using the VEVO F2 Echocardiographic system (Visual Sonic) 4 and 6 weeks after initiation of DOX and vehicle administration. Compared with the vehicle-treated WT mice, left ventricular ejection fraction (LVEF) was substantially decreased in DOX-treated WT mice compared with vehicle-treated WT mice. In contrast, LVEF in DOX-IF1 KO mice was substantially less reduced compared with vehicle-treated IF1 KO mice. We next investigated if improving cellular energetics is the critical mechanism underlying the protective effects of IF1 KO. Mouse neonatal cardiomyocyte (NMCM) isolated from WT and IF1 KO mice were cultured and treated with 0.5 and 1mM Dox for 24 hours. Cellular energetics were measured with the Mito stress assay using the Seahorse Bioanalyzer (96Xpro, Agilent). Basal, maximal, and ATP production-linked oxygen consumption rates were markedly decreased in DOX-treated-NMCM but not in DOX-treated IF1 KO NMCM compared with Vehicle-treated NMCM. In summary, the above in vivo and in vitro investigations support that IF1 inactivation is protective against DOX cardiac toxicity, at least partly, via improving cellular energetics; therefore, inactivating IF1 may be a potential therapeutic target to prevent and mitigate DOX-induced cardiomyopathy in patients. This research was supported by project number 5R01HL160969 from the National Heart Lung and Blood Institute at the NIH. 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.

YBX1 inhibits mitochondrial-mediated apoptosis in ischemic heart through the PI3K/AKT signaling pathway

Abstract Background Myocardial infarction (MI) is associated with higher morbidity and mortality in the world, especially in cold weather. YBX1 is an RNA-binding protein that is required for pathological growth of cardiomyocyte by regulating cell growth and protein synthesis. But YBX1, as an individual RNA-binding protein, regulates cardiomyocytes through signaling cascades during myocardial infarction remain largely unexplored. Methods In vivo, the mouse MI model was induced by ligating the left anterior descending coronary artery (LAD), and randomly divided into sham operation group, MI group, MI+ YBX1 knockdown/overexpression group and MI+ negative control (NC) group. The protective effect of YBX1 was verified by echocardiography and triphenyltetrazolium chloride staining. In vitro, mitochondrial-dependent apoptosis was investigated by using CCK8, TUNEL staining, reactive oxygen species (ROS) staining and JC-1 staining in hypoxic neonatal mouse cardiomyocytes (NMCMs). Results YBX1 expression of cardiomyocytes was downregulated in a mouse model and a cellular model on the ischemic condition. Compared to mice induced by MI, YBX1 overexpression mediated by adeno-associated virus serotype 9 (AAV9) vector reduced the infarcted size and improved cardiac function. Knockdown of endogenous YBX1 by shRNA partially aggravated ischemia-induced cardiac dysfunction. In hypoxic cardiomyocytes, YBX1 overexpression decreased lactic dehydrogenase (LDH) release, increased cell viability, and inhibited apoptosis by affecting the expression of apoptosis related proteins, while knockdown of endogenous YBX1 by siRNA had the opposite effect. Overexpression of YBX1 restored mitochondrial dysfunction in hypoxic NMCMs by increasing mitochondrial membrane potential and ATP content and decreasing ROS. In hypoxic NMCMs, YBX1 overexpression increased the expression of phosphorylated phosphatidylinositol 3 kinase (PI3K)/AKT, and the antiapoptosis effect of YBX1 was eliminated t by LY294002, PI3K/AKT inhibitor. Conclusion YBX1 protected the heart from ischemic damage by inhibiting the mitochondrial-dependent apoptosis through PI3K/AKT pathway. It is anticipated that YBX1 may serve as a novel therapeutic target for MI.

Exosomal miR-9-5p derived from iPSC-MSCs ameliorates doxorubicin-induced cardiomyopathy by inhibiting cardiomyocyte senescence

Doxorubicin (DOX) is a chemotherapeutic agent widely used for tumor treatment. Nonetheless its clinical application is heavily limited by its cardiotoxicity. There is accumulated evidence that transplantation of mesenchymal stem cell-derived exosomes (MSC-EXOs) can protect against Dox-induced cardiomyopathy (DIC). This study aimed to examine the cardioprotective effects of EXOs isolated from human induced pluripotent stem cell-derived MSCs (iPSC-MSCs) against DIC and explore the potential mechanisms. EXOs were isolated from the cultural supernatant of human BM-MSCs (BM-MSC-EXOs) and iPSC-MSCs (iPSC-MSC-EXOs) by ultracentrifugation. A mouse model of DIC was induced by intraperitoneal injection of Dox followed by tail vein injection of PBS, BM-MSC-EXOs, or iPSC-MSC-EXOs. Cardiac function, cardiomyocyte senescence and mitochondrial dynamics in each group were assessed. In vitro, neonatal mouse cardiomyocytes (NMCMs) were subjected to Dox and treated with BM-MSC-EXOs or iPSC-MSC-EXOs. The mitochondrial morphology and cellular senescence of NMCMs were examined by Mitotracker staining and senescence-associated-β-galactosidase assay, respectively. Compared with BM-MSC-EXOs, mice treated with iPSC-MSC-EXOs displayed improved cardiac function and decreased cardiomyocyte mitochondrial fragmentation and senescence. In vitro, iPSC-MSC-EXOs were superior to BM-MSC-EXOs in attenuation of cardiomyocyte mitochondrial fragmentation and senescence caused by DOX. MicroRNA sequencing revealed a higher level of miR-9-5p in iPSC-MSC-EXOs than BM-MSC-EXOs. Mechanistically, iPSC-MSC-EXOs transported miR-9-5p into DOX-treated cardiomyocytes, thereby suppressing cardiomyocyte mitochondrial fragmentation and senescence via regulation of the VPO1/ERK signal pathway. These protective effects and cardioprotection against DIC were largely reversed by knockdown of miR-9-5p in iPSC-MSC-EXOs. Our results showed that miR-9-5p transferred by iPSC-MSC-EXOs protected against DIC by alleviating cardiomyocyte senescence via inhibition of the VPO1/ERK pathway. This study offers new insight into the application of iPSC-MSC-EXOs as a novel therapeutic strategy for DIC treatment.Graphical

Xinyang tablet alleviated cardiac dysfunction in a cardiac pressure overload model by regulating the receptor-interacting serum/three-protein kinase 3/FUN14 domain containing 1-mediated mitochondrial unfolded protein response and mitophagy

Ethnopharmacological relevanceXinyang tablet (XYT) has been used for heart failure (HF) for over twenty years in clinical practice, but the underlying molecular mechanism remains poorly understood. Aims of the studyIn the present study, we aimed to explore the protective effects of XYT in HF in vivo and in vitro. Materials and methodsTransverse aortic constriction was performed in vivo to establish a mouse model of cardiac pressure overload. Echocardiography, tissue staining, and real-time quantitative PCR (qPCR) were examined to evaluate the protective effects of XYT on cardiac function and structure. Adenosine 5′-triphosphate production, reactive oxygen species staining, and measurement of malondialdehyde and superoxide dismutase was used to detect mitochondrial damage. Mitochondrial ultrastructure was observed by transmission electron microscope. Immunofluorescence staining, qPCR, and Western blotting were performed to evaluate the effect of XYT on the mitochondrial unfolded protein response and mitophagy, and to identify its potential pharmacological mechanism. In vitro, HL-1 cells and neonatal mouse cardiomyocytes were stimulated with Angiotensin II to establish the cell model. Western blotting, qPCR, immunofluorescence staining, and flow cytometry were utilized to determine the effects of XYT on cardiomyocytes. HL-1 cells overexpressing receptor-interacting serum/three-protein kinase 3 (RIPK3) were generated by transfection of RIPK3-overexpressing lentiviral vectors. Cells were then co-treated with XYT to determine the molecular mechanisms. ResultsIn the present study, XYT was found to exerta protective effect on cardiac function and structure in the pressure overload mice. And it was also found XYT reduced mitochondrial damage by enhancing mitochondrial unfolded protein response and restoring mitophagy. Further studies showed that XYT achieved its cardioprotective role through regulating the RIPK3/FUN14 domain containing 1 (FUNDC1) signaling. Moreover, the overexpression of RIPK3 successfully reversed the XYT-induced protective effects and significantly attenuated the positive effects on the mitochondrial unfolded protein response and mitophagy. ConclusionsOur findings indicated that XYT prevented pressure overload-induced HF through regulating the RIPK3/FUNDC1-mediated mitochondrial unfolded protein response and mitophagy. The information gained from this study provides a potential strategy for attenuating mitochondrial damage in the context of pressure overload-induced heart failure using XYT.

Cardiomyocyte maturation alters molecular stress response capacities and determines cell survival upon mitochondrial dysfunction

Cardiomyocyte maturation during pre- and postnatal development requires multiple intertwined processes, including a switch in energy generation from glucose utilization in the embryonic heart towards fatty acid oxidation after birth. This is accompanied by a boost in mitochondrial mass to increase capacities for oxidative phosphorylation and ATP generation required for efficient contraction. Whether cardiomyocyte differentiation is paralleled by augmented capacities to deal with reactive oxygen species (ROS), physiological byproducts of the mitochondrial electron transport chain (ETC), is less clear. Here we show that expression of genes and proteins involved in redox homeostasis and protein quality control within mitochondria increases after birth in the mouse and human heart. Using primary embryonic, neonatal and adult mouse cardiomyocytes in vitro we investigated how excessive ROS production induced by mitochondrial dysfunction affects cell survival and stress response at different stages of maturation. Embryonic and neonatal cardiomyocytes largely tolerate inhibition of ETC complex III by antimycin A (AMA) as well as ATP synthase (complex V) by oligomycin but are susceptible to complex I inhibition by rotenone. All three inhibitors alter the intracellular distribution and ultrastructure of mitochondria in neonatal cardiomyocytes. In contrast, adult cardiomyocytes treated with AMA undergo rapid morphological changes and cellular disintegration. At the molecular level embryonic cardiomyocytes activate antioxidative defense mechanisms, the integrated stress response (ISR) and ER stress but not the mitochondrial unfolded protein response upon complex III inhibition. In contrast, adult cardiomyocytes fail to activate the ISR and antioxidative proteins following AMA treatment. In conclusion, our results identified fundamental differences in cell survival and stress response in differentiated compared to immature cardiomyocytes subjected to mitochondrial dysfunction. The high stress tolerance of immature cardiomyocytes might allow outlasting unfavorable intrauterine conditions thereby preventing fetal or perinatal heart disease and may contribute to the regenerative capacity of the embryonic and neonatal mammalian heart.

Deubiquitinase OTUB1 regulates doxorubicin-induced cardiotoxicity via deubiquitinating c-MYC.

Doxorubicin (DOX), an anthracycline drug widely used in antitumor therapies, has dose-dependent toxicity that can cause cardiomyocyte apoptosis and oxidative stress, thus limiting its clinical application. OTUB1 (ovarian tumor associated proteinase B1) is an OTU superfamily deubiquitinase that effectively regulates cell proliferation, inflammatory responses, apoptosis, and oxidative stress by specifically removing K48- and K63-linked ubiquitination; however, its role in DOX-induced cardiotoxicity remains unknown. A DOX-induced subacute cardiotoxicity mouse model was established by intraperitoneal injection, and cardiac injury was assessed by echocardiography, serum cardiac markers, and histopathological staining. Western blotting, qRT-PCR, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) immunohistochemistry were used to analyze cell apoptosis, tissue oxidative stress was assessed by superoxide dismutase (SOD) activity, malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) activity. Cell counting kit-8 (CCK-8) assay, TUNEL staining, Western blotting, qRT-PCR, and reactive oxygen species (ROS) flow cytometry were applied on isolated neonatal mice cardiomyocytes to assess apoptosis and oxidative stress. Differentially expressed genes were analyzed using RNA sequencing and clustering analyses. c-MYC inhibitor 10,058-F4 and siRNA targeting c-Myc were used to investigate the roles of c-MYC in OTUB1's regulations of DOX-induced cardiotoxicity. Immunoprecipitation and Western blotting were performed to reveal the deubiquitinating effects of OTUB1 on c-MYC expression. We found that global Otub1-knockdown in vivo alleviated the subacute DOX treatment-induced cardiac dysfunction, fibrosis, and cardiomyocyte atrophy. Mechanistically, unbiased RNA sequencing and molecular biology experiments revealed that cardiomyocyte apoptosis, inflammation, and oxidative stress in DOX-induced cardiotoxicity were significantly compromised in the Otub1-knockdown group. Further in vitro studies have shown that c-MYC, a critical regulator of apoptosis, is indispensable in OTUB1's regulations of DOX-induced cardiotoxicity. Deubiquitinating effects of OTUB1 on K48- and K63-linked ubiquitination of c-MYC protein are essential for promoting cardiomyocyte apoptosis and oxidative responses. OTUB1-c-MYC inhibition protected cardiomyocytes against DOX-induced apoptosis and oxidative stress, suggesting that OTUB1 is a potential translational therapeutic target for preventing DOX-induced cardiotoxicity.

Differential sensitivity to longitudinal and transverse stretch mediates transcriptional responses in mouse neonatal ventricular myocytes.

To identify how cardiomyocyte mechanosensitive signaling pathways are regulated by anisotropic stretch, micropatterned mouse neonatal cardiomyocytes were stretched primarily longitudinally or transversely to the myofiber axis. Four hours of static, longitudinal stretch induced differential expression of 557 genes, compared with 30 induced by transverse stretch, measured using RNA-seq. A logic-based ordinary differential equation model of the cardiac myocyte mechanosignaling network, extended to include the transcriptional regulation and expression of 784 genes, correctly predicted measured expression changes due to anisotropic stretch with 69% accuracy. The model also predicted published transcriptional responses to mechanical load in vitro or in vivo with 63-91% accuracy. The observed differences between transverse and longitudinal stretch responses were not explained by differential activation of specific pathways but rather by an approximately twofold greater sensitivity to longitudinal stretch than transverse stretch. In vitro experiments confirmed model predictions that stretch-induced gene expression is more sensitive to angiotensin II and endothelin-1, via RhoA and MAP kinases, than to the three membrane ion channels upstream of calcium signaling in the network. Quantitative cardiomyocyte gene expression differs substantially with the axis of maximum principal stretch relative to the myofilament axis, but this difference is due primarily to differences in stretch sensitivity rather than to selective activation of mechanosignaling pathways.NEW & NOTEWORTHY Anisotropic stretch applied to micropatterned neonatal mouse ventricular myocytes induced markedly greater acute transcriptional responses when the major axis of stretch was parallel to the myofilament axis than when it was transverse. Analysis with a novel quantitative network model of mechanoregulated cardiomyocyte gene expression suggests that this difference is explained by higher cell sensitivity to longitudinal loading than transverse loading than by the activation of differential signaling pathways.

Cucurbitacin B protects against myocardial ischemia-reperfusion injury through activating JAK2/STAT3 signaling pathway.

Cucurbitacin B, a tetracyclic triterpenoid compound extracted from various plants, has been proven to exert a vital role in various diseases. However, the effect of cucurbitacin B on myocardial infarction (MI) and ischemia-reperfusion (I/R) injury is still relatively unclear. The main purpose of the present study was to investigate the effect of cucurbitacin B on cell apoptosis and oxidative damage after myocardial I/R injury in vitro and in vivo and elucidate the molecular mechanisms underlying its role. The 56-day-old adult mice and 1-day-old neonatal mice cardiomyocytes were used to construct I/R or oxygen-glucose deprivation/reoxygenation (OGD/R) injury models. The oxidative injury, western blot and TUNEL assay were performed to evaluate cardiomyocyte damage in the present study. In vitro， we confirmed that cucurbitacin B could attenuate LDH release, oxidative stress and cell apoptosis in cardiomyocytes exposed to OGD/R. Besides, we confirmed in an adult I/R mouse model that cucurbitacin B can improve cardiac repair and block cell apoptosis in the acute phase (24 h) post-myocardial I/R injury, as well as promote long-term cardiac function and fiber scar area after 28 days of I/R. Mechanically, we clarify that cucurbitacin B exerts cardiomyocyte protective effects through activating the JAK2/STAT3 signaling pathway. In conclusion, our study elucidates for the first time the protective role of cucurbitacin B in cardiac I/R injury, which provides a novel perspective for better prevention of I/R injury through the JAK2/STAT3 signaling pathway.

FOXO1 reduces STAT3 activation and causes impaired mitochondrial quality control in diabetic cardiomyopathy.

To investigate the role of FOXO1 in STAT3 activation and mitochondrial quality control in the diabetic heart. Type 1 diabetes mellitus (T1DM) was induced in rats by a single intraperitoneal injection of 60 mg · kg-1 streptozotocin (STZ), while type 2 diabetes mellitus (T2DM) was induced in rats with a high-fat diet through intraperitoneal injection of 35 mg · kg-1 STZ. Primary neonatal mouse cardiomyocytes and H9c2 cells were exposed to low glucose (5.5 mM) or high glucose (HG; 30 mM) with or without treatment with the FOXO1 inhibitor AS1842856 (1 μM) for 24 hours. In addition, the diabetic db/db mice (aged 8 weeks) and sex- and age-matched non-diabetic db/+ mice were treated with vehicle or AS1842856 by oral gavage for 15 days at a dose of 5 mg · kg-1 · d-1 . Rats with T1DM or T2DM had excessive cardiac FOXO1 activation, accompanied by decreased STAT3 activation. Immunofluorescence and immunoprecipitation analysis showed colocalization and association of FOXO1 and STAT3 under basal conditions in isolated cardiomyocytes. Selective inhibition of FOXO1 activation by AS1842856 or FOXO1 siRNA transfection improved STAT3 activation, mitophagy and mitochondrial fusion, and decreased mitochondrial fission in isolated cardiomyocytes exposed to HG. Transfection with STAT3 siRNA further reduced mitophagy, mitochondrial fusion and increased mitochondrial fission in HG-treated cardiomyocytes. AS1842856 alleviated cardiac dysfunction, pathological damage and improved STAT3 activation, mitophagy and mitochondrial dynamics in diabetic db/db mice. Additionally, AS1842856 improved mitochondrial function indicated by increased mitochondrial membrane potential and adenosine triphosphate production and decreased mitochondrial reactive oxygen species production in isolated cardiomyocytes exposed to HG. Excessive FOXO1 activation during diabetes reduces STAT3 activation, with subsequent impairment of mitochondrial quality, ultimately promoting the development of diabetic cardiomyopathy.

Cardiometabolic rewiring in the context of doxorubicin-induced cardiotoxicity: fuel preference changes from fatty acids to glucose oxidation

Abstract Background Doxorubicin (DOX) is a chemotherapeutic drug administered to treat a wide spectrum of tumours. However, its clinical use is hampered by the onset of cardiotoxic effects, with a reduction of LVEF that occurs within the first year from the treatment initiation. Recently it has been demonstrated that DOX accumulates into mitochondria, with metabolic perturbation and energetic imbalance. We previously described that phosphoinositide 3-kinase γ (PI3Kγ) contributes to DOX-induced cardiotoxicity, with the inhibition of autophagy and accumulation of damaged mitochondria into cardiac cells. Objective Here we intend to better understand the maladaptive metabolic rewiring occurring in DOX-treated hearts and the contribution of PI3Kg signalling to this process. Methods 10 weeks-old wild-type (WT) and knock-in mice expressing a kinase-inactive PI3Kγ (kinase-dead; KD) were treated with DOX (4 mg/Kg) at day 0, 7 and 14 (cumulative dose 12 mg/Kg). To explore the contribution of PI3Kγ on the early cardiac metabolic rewiring, metabolomic analysis were performed on animals sacrificed at day 3. Mitochondria respiration capacity and glycolytic enzymes activity were evaluated at day 3 and 42. To investigate the role of autophagy, 8 weeks-old mice were infected with an adeno-associated virus 9 (AAV9) carrying a vector expressing a short-hairpin RNA against ATG7 (ATG7sh). To test the role of PI3Kγ in glycolysis regulation, glucose uptake and plasma membrane exposure of GLUT-4 were measured in neonatal mouse cardiomyocytes (NMCs) with DOX acute treatment (1µM for 3h). To evaluate oxygen consumption with the administration of different substrates OROBOROS analysis were performed on myocardial slices from mice sacrificed at day 3. Results Metabolomic analysis of DOX-treated hearts revealed an accumulation of TCA cycle metabolites, with reduced levels of pyruvate, unchanged abundance of lactate but increased quantity of Acetyl-CoA, indicative of increased glucose oxidation and accelerated TCA cycle flux. Moreover, the activity of glycolytic enzymes was upregulated and fatty acid oxidation downregulated after DOX. In agreement, oxygen consumption was significantly increased in DOX-treated WT after pyruvate supplementation, with the formation of cytotoxic ROS rather than energy production. These metabolic changes were fully prevented in KD hearts. Interestingly, they failed to increase glucose oxidation in response to DOX even with autophagy inhibition, indicating that PI3Kγ likely controls the fuel preference after DOX through an autophagy-independent mechanism. In vitro experiments showed that genetic and pharmacological inhibition of PI3Kγ significantly prevented DOX-induced mobilization of GLUT-4-carrying vesicles to the plasma membrane and reduced glucose uptake. Conclusion These results demonstrate that PI3Kγ promotes a maladaptive metabolic rewiring in the heart in response to DOX, with an increased glucose utilization via a GLUT4-dependent mechanism.

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.

Abstract 12312: UBC9 Protects Against Cardiac Hypertrophy and Heart Failure via Regulating FOXO3-LMCD1 Pathway

Introduction: Pathological cardiac hypertrophy is a major risk factor for heart failure (HF) and a leading cause of morbidity and mortality worldwide. However, the molecular mechanisms underlying pathological cardiac hypertrophy remain unclear. Hypothesis: In the present study, we investigated the possible underlying effects of UBC9 on cardiac hypertrophy and heart failure. Methods: UBC9 was upregulated in samples of human hypertrophic hearts compared to normal controls using RNA sequencing analysis from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo). Real-time polymerase chain reaction (RT-PCR) and western blotting were used to analyze the expression of UBC9 in samples of human hypertrophic hearts and normal controls. Mice with cardiac-specific over-expression and low-expression of UBC9 by intravenous injection of adeno-associated virus 9 (AAV9)-encoding UBC9 under the control of cardiac troponin T (cTnT) promoter were subjected to transverse aortic construction (TAC). Co-immunoprecipitation assays were performed to determine the mechanism by which UBC9 regulates the expression of forkhead box O3 (FOXO3). Results: UBC9 expression increased in the hearts of patients with hypertrophic cardiomyopathy (HCM) and pressure overload-induced mice, as well as in the Neonatal Mouse Cardiomyocytes (NMCMs) treated with phenylephrine (PE). Cardiac cell-specific UBC9 over-expression alleviates cardiac hypertrophy and dysfunction. Cardiac cell-specific UBC9 knockout aggravates cardiac hypertrophy and heart failure. Mechanistically, UBC9 inhibits LIM and cysteine-rich domain 1 (LMCD1) expression by directly binding to forkhead box O3 (FOXO3), a transcription factor of LMCD1, and promoting FOXO3 dephosphorylation and nuclear translocation, independent of SUMOylation. Conclusions: This study provides a new insight that UBC9, as a potential intervention target in cardiac hypertrophy and HF, may contribute to exploring effective therapeutic strategies for pathological cardiac hypertrophy treatment.

Abstract 11344: Targeted Deletion of Arrdc4 Mitigates Cardiac Injury in Insulin-Deficient Diabetes

Introduction: Diabetic cardiomyopathy is an important contributor to mortality in the diabetic population. However, the pathogenesis of diabetic cardiomyopathy is unclear. Hypothesis: We recently reported that the interaction between glucose transporter 1 (GLUT1) and its adaptor protein Arrdc4 mediates metabolic stress in the ischemic heart. Here, we tested whether this mechanism plays a role in diabetic cardiomyopathy. Methods: To determine glucose responsiveness, mouse neonatal cardiomyocytes were treated with high glucose (25 mM). To investigate cardiac phenotype in vivo, we engineered Arrdc4-knockout (KO) mice by CRISPR/Cas9 technology and created an insulin-deficient diabetic model by intraperitoneal injection of streptozotocin. Results: Arrdc4 expression was robustly stimulated by D-glucose (324±10%, p<0.01) in cardiomyocytes. The glucose responsiveness was mediated by MondoA, a nutrient-sensing transcriptional factor, through a cytoplasmic-nuclear shuttling mechanism. Confocal live imaging showed that Arrdc4 triggered endocytic trafficking of GLUT1 into lysosomes. In the animal model, levels of insulin depletion were comparable between the genotypes. Nevertheless, Arrdc4-KO diabetic mice had enhanced glucose uptake in multiple organs including the heart (139±13%, p<0.01) compared to wild-type (WT) diabetic mice. Arrdc4-KO diabetic hearts had lower induction of oxidative stress, ER stress, and autophagy by hyperglycemia, leading to better mitochondrial function and less cardiomyocyte apoptosis (70±5%, p<0.01) than WT diabetic hearts. Although resting echocardiography could not detect early signs of left ventricular dysfunction, stress hemodynamic analysis uncovered the masked mechanical dysfunction by diabetes and found that Arrdc4-KO diabetic mice retained a greater cardiac response to beta-adrenergic stimulation than wild-type mice (149±5%, p<0.01). Conclusion: The metabolic rearrangements by Arrdc4 deletion improve glucose transport due to the absence of its inhibition towards GLUT1 function, protecting the heart against diabetic damage. These results provide a novel link between GLUT1 and Arrdc4 as a fundamental mechanism of the regulation of glucose homeostasis in diabetic cardiomyopathy.

Abstract 16405: Perm1 Protects the Heart From Pressure Overload-Induced Dysfunction by Promoting Oxidative Metabolism

Introduction: Defects in ATP production lead to cardiac dysfunction and heart failure (HF) while restoring reduced mitochondrial (Mito) function mitigates HF. Previously we showed that a novel Mito protein, Perm1 (PGC-1 and ERR-induced regulator, muscle 1), promotes Mito biogenesis in cardiomyocytes (CM). Here we studied the role of myocardial Perm1 in vivo . Hypothesis: Perm1 protects the heart from pressure overload-induced dysfunction. Methods: First, we generated cardiac-specific Perm1 transgenic mice (Perm1cTG). Next, we induced pressure overload by transverse aortic constriction (TAC) or performed Sham operations on 10-week-old control (CTL) and Perm1cTG male mice. Heart function, histology, and biochemical analyses were performed eight weeks postoperatively. To understand the mechanism of Perm1, the heart protein samples were subjected to quantitative mass spectrometry (MS) analysis, and we also identified Perm1 binding proteins in the heart by MS. Finally, mouse neonatal CM were infected with adenoviruses expressing shMic60 (knock down) or shControl in combination with Perm1 or LacZ, followed by oxygen consumption rate (OCR) analysis. Results: Sham-operated Perm1cTG and CTL mice showed no differences in function and morphology. Following TAC, CTL developed severely reduced heart function, while Perm1cTG had only minimal change. Perm1cTG TAC mice also showed reduced cardiac hypertrophy, Anf and Bnp mRNA expression, and fibrosis, compared to CTL TAC, showing Perm1 protects the heart from hemodynamic stress. Perm1cTG TAC hearts showed significantly increased OCR compared to CTL TAC, suggesting Perm1 preserves TAC-induced CM Mito dysfunction. MS analysis identified MICOS (mitochondrial contact site and cristae organizing system) and oxidative phosphorylation proteins were upregulated in Perm1cTG TAC hearts compared to CTL TAC. Moreover, we found that Perm1 binds to Mic60, which is a core component of the MICOS. Finally, Mic60 was required for Perm1-induced maximal oxidative capacity, suggesting Perm1 acts through Mic60. Conclusion: Perm1 protects the heart against pressure overload by restoring oxidative metabolism. Future studies will reveal the role of Perm1/Mic60 in the heart. This work is supported by NIH R01HL151239.

Polo-like kinase 1 promotes sepsis-induced myocardial dysfunction

Sepsis-induced myocardial dysfunction (SIMD) is the main cause of mortality in sepsis. In this study, we identified Polo-like kinase 1 (Plk-1) is a promoter of SIMD. Plk-1 expression was increased in lipopolysaccharide (LPS)-treated mouse hearts and neonatal rat cardiomyocytes (NRCMs). Inhibition of Plk-1 either by heterozygous deletion of Plk-1 or Plk-1 inhibitor BI 6727 alleviated LPS-induced myocardial injury, inflammation, cardiac dysfunction, and thereby improved the survival of LPS-treated mice. Plk-1 was identified as a kinase of inhibitor of kappa B kinase alpha (IKKα). Plk-1 inhibition impeded NF-κB signal pathway activation in LPS-treated mouse hearts and NRCMs. Augmented Plk-1 is thus essential for the development of SIMD and is a druggable target for SIMD.

Sphingosylphosphorylcholine alleviates pressure overload-induced myocardial remodeling in mice via inhibiting CaM-JNK/p38 signaling pathway.

Apoptosis plays a critical role in the development of heart failure, and sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid naturally occurring in blood plasma. Some studies have shown that SPC inhibits hypoxia-induced apoptosis in myofibroblasts, the crucial non-muscle cells in the heart. Calmodulin (CaM) is a known SPC receptor. In this study we investigated the role of CaM in cardiomyocyte apoptosis in heart failure and the associated signaling pathways. Pressure overload was induced in mice by trans-aortic constriction (TAC) surgery. TAC mice were administered SPC (10 μM·kg-1·d-1) for 4 weeks post-surgery. We showed that SPC administration significantly improved survival rate and cardiac hypertrophy, and inhibited cardiac fibrosis in TAC mice. In neonatal mouse cardiomyocytes, treatment with SPC (10 μM) significantly inhibited Ang II-induced cardiomyocyte hypertrophy, fibroblast-to-myofibroblast transition and cell apoptosis accompanied by reduced Bax andphosphorylation levels of CaM, JNK and p38, as well as upregulated Bcl-2, a cardiomyocyte-protective protein. Thapsigargin (TG) could enhance CaM functions by increasing Ca2+ levels in cytoplasm. TG (3 μM) annulled the protective effect of SPC against Ang II-induced cardiomyocyte apoptosis. Furthermore, we demonstrated that SPC-mediated inhibition of cardiomyocyte apoptosis involved the regulation of p38 and JNK phosphorylation, which was downstream of CaM. These results offer new evidence for SPC regulation of cardiomyocyte apoptosis, potentially providing a new therapeutic target for cardiac remodeling following stress overload.

Tetrahydrocurcumin ameliorates postinfarction cardiac dysfunction and remodeling by inhibiting oxidative stress and preserving mitochondrial function via SIRT3 signaling pathway

BackgroundMyocardial infarction (MI) often leads to sudden cardiac death. Persistent myocardial ischemia increases oxidative stress and impairs mitochondrial function, contributing significantly to postinfarction cardiac dysfunction and remodeling, and the subsequent progression to heart failure (HF). Tetrahydrocurcumin (THC), isolated from the rhizome of turmeric, has antioxidant properties and has been shown to protect against cardiovascular diseases. However, its effects on HF after MI are poorly understood. PurposeThe objective was the investigation of the pharmacological effects of THC and its associated mechanisms in the pathogenesis of HF after MI. MethodsA total of 120 mice (C57BL/6, male) were used for the in vivo experiments. An MI mouse model was created by permanent ligation of the left anterior descending coronary artery. The mice received oral dose of THC at 120 mg/kg/d and the effects on MI-induced myocardial injury were evaluated by assessment of cardiac function, histopathology, myocardial oxidative levels, and mitochondrial function. Molecular mechanisms were investigated by intraperitoneal injection of 50 mg/kg of the SIRT3 selective inhibitor 3-TYP. Meanwhile, mouse neonatal cardiomyocytes were isolated and cultured in a hypoxic incubator to verify the effects of THC in vitro. Lastly, SIRT3 and Nrf2 were silenced using siRNAs to further explore the regulatory mechanism of key molecules in this process. ResultsThe mouse hearts showed significant impairment in systolic function after MI, together with enlarged infarct size, increased myocardial fibrosis, cardiac hypertrophy, and apoptosis of cardiomyocytes. A significant reversal of these changes was seen after treatment with THC. Moreover, THC markedly reduced reactive oxygen species generation and protected mitochondrial function, thus mitigating oxidative stress in the post-MI myocardium. Mechanistically, THC counteracted reduced Nrf2 nuclear accumulation and SIRT3 signaling in the MI mice while inhibition of Nrf2 or SIRT3 reversed the effects of THC. Cell experiments showed that Nrf2 silencing markedly reduced SIRT3 levels and deacetylation activity while inhibition of SIRT3 signaling had little impact on Nrf2 expression. ConclusionThis is the first demonstration that THC protects against the effects of MI. THC reduced both oxidative stress and mitochondrial damage by regulating Nrf2-SIRT3 signaling. The results suggest the potential of THC in treating myocardial ischemic diseases.