Cultures Of Neonatal Rat Cardiomyocytes Research Articles

Ophthalmic acid is a bloodborne metabolite that contributes to age-induced cardiomyocyte hypertrophy.

Cardiac aging involves the development of left ventricular hypertrophy alongside a decline in functional capacity. Here, we use neutral blood exchange to demonstrate that the acute removal of age-accumulated blood factors significantly regresses cardiac hypertrophy in aged mice. The reversal of hypertrophy was not attributed to age-associated hemodynamic effects, implicating a role of blood-derived factors. In addition, the overarching paradigm of systemic aging maintains that the age-related overabundance of plasma proteins are largely responsible for causing pathological phenotypes in tissues. Our results suggest that blood metabolites, not proteins, drive cardiac hypertrophy instead. Upon analyzing serum metabolomics data sets, we identified ophthalmic acid as a circulating metabolite whose levels increase with advanced age. Treatment of adult mouse and neonatal rat cardiomyocytes in culture with ophthalmic acid increased their relative surface areas. This study uncovers a non-protein metabolite that may contribute to cardiomyocyte hypertrophy during aging. Identifying a method to counteract ophthalmic acid's hypertrophic effects may reveal novel therapeutic opportunities for cardiac rejuvenation.

Roles of aldosterone on ventricular arrhythmias, and associated gap junction and ion channel remodeling in hypertrophic heart

Abstract Background Ventricular arrhythmias are critical causes of death in patients with chronic heart failure (CHF). Aldosterone (Ald) is an exacerbating factor of CHF and mineral corticoid receptor antagonist (MRA) is usual drug for the management of CHF. MRA also suppressed sudden cardiac death, however, mechanisms of these effects remain unknown. We investigated the effects of MRA on ventricular arrhythmias and arrhythmogenic factors using Dahl Salt sensitive (DS) rats. Methods (in vivo) DS rats were divided into three groups; low salt group (LS), high salt group (HS), and eplerenone-, an MRA, treated high salt group (HS+EPL). Low salt (0.3% NaCl) or high salt (8% NaCl) were administrated for 8 weeks and EPL was administrated in the last 4 weeks. Cardiac geometry and function were assessed by echocardiography. Susceptibility of ventricular arrhythmias were assessed using programmed stimulation. Myocyte hypertrophy and cardiac fibrosis were examined by Masson’s trichrome staining. Expression and distribution of connexin43 (Cx43) were examined by immunostaining and western blotting. mRNA expression of Ca transient-related factors, such as phospholamban, SERCA2a, and ryanosin 2, and potassium ion channel-related factors, such as KCNE1 and KCNQ1, was examined by RT-PCR. (in vitro) Ald (1-100 nM) was administrated to cultured rat neonatal cardiomyocytes. Expression of Cx43, Ca transient-related, and potassium ion channel-related factors are also examined as mentioned above. Effects of Src tyrosin kinase inhibitor, PP2, and ERK inhibitor, PD98059, on Ald-induced Cx43 reduction were examined by RT-PCR and immunostaining. Results Systolic blood pressure and Ald concentrations in cardiac tissue were higher in HS group than LS group, and EPL did not affect them. In HS group left ventricular hypertrophy, dilatation and systolic dysfunction were observed in association with myocyte hypertrophy and interstitial fibrosis. EPL significantly recovered cardiac function and histological impairments. In addition, elongation of QTc in ECG and susceptibility of ventricular tachycardia were remarkable in HS group rather than LS and EPL attenuated them. Distribution of Cx43 was scattered in HS group, and EPL attenuated these changes. Decrease in SERCA2a and increase in KCNE1/KCNQ1 mRNA expression were found in HS group, and these changes were attenuated by EPL. In cultured myocytes, Ald decreased Cx43 mRNA and protein expression in a dose dependent fashion. Ald-induced Cx43 reduction was inhibited by PP2 and PD98059. In addition, Ald (100 nM) also decreased SERCA2a and increased KCNE1/KCNQ1 mRNA expression. Conclusion These results indicate that Ald reduced and dispersed the Cx43 expression reduction and dispersion through Src tyrosin kinase and ERK signaling pathways, and also mediated SRECA2A and KCNE1/KCNQ1 expression, which induced QTc prolongation and susceptibility of lethal ventricular arrhythmias in hypertensive cardiac hypertrophy.

Inhibition of miR-214-3p attenuates ferroptosis in myocardial infarction via regulating ME2

Myocardial infarction (MI) contributes to an increased risk of incident heart failure and sudden death, but there is still a lack of effective treatment in clinic. Recently, growing evidence has indicated that abnormal expression of microRNAs (miRNAs) plays a crucial role in cardiovascular diseases. In this research, the involvement of miRNA-214-3p in MI was explored. A mouse model of MI was established by ligation of the left anterior descending coronary artery, and primary cultures of neonatal rat cardiomyocytes (NRCMs) were submitted to hypoxic treatment to stimulate cellular injury in vitro. Our results showed that miR-214-3p level was significantly upregulated in the infarcted region of mouse hearts and in NRCMs exposed to hypoxia, accompanying with an obvious elevation of ferroptosis. Inhibition of miR-214-3p by antagomir injection improved cardiac function, decreased infarct size, and attenuated iron accumulation and oxidant stress in myocardial tissues. MiR-214-3p could also promote ferroptosis and cellular impairments in NRCMs, while miR-214-3p inhibitor effectively protected cells from hypoxia. Furthermore, dual luciferase reporter gene assay revealed that malic enzyme 2 (ME2) is a direct target of miR-214-3p. In cardiomyocytes, overexpression of ME2 ameliorated the detrimental effects and excessive ferroptosis induced by miR-214-3p mimic, whereas ME2 depletion compromised the protective role of miR-214-3p inhibitor against hypoxic injury and ferroptosis. These findings suggest that miR-214-3p contributes to enhanced ferroptosis during MI at least partially via suppressing ME2. Inhibition of miR-214-3p may be a new approach for tackling MI.

Interrelation between α-Cardiac Actin Treadmilling and Myocardin-Related Transcription Factor-A Nuclear Shuttling in Cardiomyocytes.

Myocardin-related transcription factors (MRTFs) play a central role in the regulation of actin expression and cytoskeletal dynamics that are controlled by Rho GTPases. SRF is a ubiquitous transcription factor strongly expressed in muscular tissues. The depletion of SRF in the adult mouse heart leads to severe dilated cardiomyopathy associated with the down-regulation of target genes encoding sarcomeric proteins including α-cardiac actin. The regulatory triad, composed of SRF, its cofactor MRTFA and actin, plays a major role in the coordination of the nuclear transcriptional response to adapt actin filament dynamics associated with changes in cell shape, and contractile and migratory activities. Most of the knowledge on the regulation of the SRF–MRTF–Actin axis has been obtained in non-muscle cells with α-actin and smooth muscle cells with α-smooth actin. Here, we visualized for the first time by a time-lapse video, the nucleocytoplasmic shuttling of MRTFA induced by serum or pro-hypertrophic agonists such as angiotensin II, phenylephrine and endothelin-1, using an MRTFA-GFP adenovirus in cultures of neonatal rat cardiomyocytes. We showed that an inhibitor of the RhoA/ROCK signaling pathway leads to an α-cardiac actin polymerization disruption and inhibition of MRTFA nucleocytoplasmic shuttling. Moreover, inhibition of the PI3K/Akt signaling pathway also prevents the entry of MRTFA into the nuclei. Our findings point out a central role of the SRF–MRTFA–actin axis in cardiac remodeling.

PEGylated Gold Nanoparticle Toxicity in Cardiomyocytes: Assessment of Size, Concentration, and Time Dependency.

Gold Nanoparticles (GNPs) have shown promising capabilities for use in many in-vivo applications such as gene and drug delivery, photothermal ablation of tumors, and tracking in many imaging modalities. Yet GNPs have thus far had limited use in cardiovascular medicine. Polyethylene glycol functionalized (PEGylated) GNPs have been extensively studied in a wide array of in vitro and in vivo models with results showing no apparent toxicity, but to our knowledge an investigation has never been performed to determine direct cardiomyocyte toxicity. In this study, we assessed if PEGylated GNPs exhibited direct toxicity to a primary culture of neonatal rat cardiomyocytes in order to establish PEGylated GNPs for potential future use in cardiovascular medicine applications. We present novel results that demonstrate both a particle size and concentration dependent relationship on cell viability. Cell viability was found to be significantly enhanced for many concentrations and sizes as compared to the control and increased linearly as a function of particle diameter. Additionally, viability increased in a parabolically dependent manner as a function of decreasing particle concentration. These new results could advance understanding of nanoparticle-cell interactions and lead to the development of new applications involving the use of gold nanoparticles in cardiovascular medicine.

MultiFRET: A Detailed Protocol for High-Throughput Multiplexed Ratiometric FRET.

The newly generated software plugin MultiFRET allows for real-time measurements of multiplexed fluorescent biosensors in a near high-throughput fashion. Here we describe a detailed protocol for setup and use of this software for any purpose requiring instant feedback during fluorescence measurement experiments. We further describe its non-primary features including beam splitter misalignment correction, custom calculations through input of simple equations typed in a .txt format, customizable Excel output, and offline bulk analysis of image stacks. Finally, we supply a usage example of a cAMP measurement in cultured rat neonatal cardiomyocytes.

MicroRNA-34c-5p provokes isoprenaline-induced cardiac hypertrophy by modulating autophagy via targeting ATG4B

Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs (miRNAs) play multiple roles in biological processes and participate in cardiovascular diseases. In the present research, we investigate the impact of miRNA-34c-5p on cardiac hypertrophy and the mechanism involved. The expression of miR-34c-5p was proved to be elevated in heart tissues from isoprenaline (ISO)-infused mice. ISO also promoted miR-34c-5p level in primary cultures of neonatal rat cardiomyocytes (NRCMs). Transfection with miR-34c-5p mimic enhanced cell surface area and expression levels of foetal-type genes atrial natriuretic factor (Anf) and β-myosin heavy chain (β-Mhc) in NRCMs. In contrast, treatment with miR-34c-5p inhibitor attenuated ISO-induced hypertrophic responses. Enforced expression of miR-34c-5p by tail intravenous injection of its agomir led to cardiac dysfunction and hypertrophy in mice, whereas inhibiting miR-34c-5p by specific antagomir could protect the animals against ISO-triggered hypertrophic abnormalities. Mechanistically, miR-34c-5p suppressed autophagic flux in cardiomyocytes, which contributed to the development of hypertrophy. Furthermore, the autophagy-related gene 4B (ATG4B) was identified as a direct target of miR-34c-5p, and miR-34c-5p was certified to interact with 3′ untranslated region of Atg4b mRNA by dual-luciferase reporter assay. miR-34c-5p reduced the expression of ATG4B, thereby resulting in decreased autophagy activity and induction of hypertrophy. Inhibition of miR-34c-5p abolished the detrimental effects of ISO by restoring ATG4B and increasing autophagy. In conclusion, our findings illuminate that miR-34c-5p participates in ISO-induced cardiac hypertrophy, at least partly through suppressing ATG4B and autophagy. It suggests that regulation of miR-34c-5p may offer a new way for handling hypertrophy-related cardiac dysfunction.

Platelet-rich plasma alleviates myocardial ischemia-reperfusion injury in rats

To investigate the protective effect of platelet-rich plasma (PRP) against acute myocardial ischemiareperfusion (IR) injury and the possible mechanism. Aortic blood samples were collected from 10 SD rats to prepare PRP, in which the concentrations of platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) were measured. Cell models of IR injury were established in primary cultures of neonatal SD rat cardiomyocytes by exposing the cells to 3 h of hypoxia. The cells were then reoxygenated and co-cultured with 1%, 5%, 10%, and 20% volume of PRP for 12 h, and the changes in cell viability was assessed. Immunofluorescence staining of the cardiomyocytes was performed, and the cellular expression of AMPK and its phosphorylation level were detected. The effects of PRP on the proliferation and migration of rat aortic endothelial cells (RAOECs) were examined. In a SD rat model of myocardial IR injury, 100 μL of PRP (n= 20) or normal saline (n=20) was injected at 4 sites around the ligation site immediately after cardiac reperfusion. One day after the injection, 6 rats were selected from each group for TTC staining of the myocardial tissues and measurement of troponin Ⅰ content. One week later, the cardiac function of the remaining rats was assessed by echocardiography, and HE staining of the myocardial tissues was performed. The effect of PRP treatment for 24 h on polarization of M1 and M2 macrophages was also examined by flow cytometry in RAW264.7 cells after hypoxic exposure for 3 h. The concentrations of PDGF-BB and TGF-β1 were significantly higher in PRP than in whole blood. Addition of 1% volume of PRP significantly reduced death of the cardiomyocytes following reoxygenation, and this effect was closely related with the activation of AMPK. Treatment with PRP obviously promoted the proliferation and migration of RAOECs. In rat models of acute myocardial IR injury, injections of PRP significantly reduced the infarct size and troponin Ⅰ concentration as compared with saline injection (P < 0.001). One week after PRP injection, the rats showed significantly improved cardiac function with a lowered level of inflammatory response in comparison with the rats with saline injection. In RAW264.7 cells with hypoxic exposure, treatment with PRP obviously decreased the number of M1 macrophages and increase the number of M2 macrophages. PRP can improve acute myocardial IR injury in rats by phosphorylating AMPK and regulating macrophage polarization, which produces a protective immunomodulatory effect on the ischemic myocardial tissues.

EPAC1 inhibition as a new therapeutic target in anthracyclines induced cardiotoxicity

Doxorubicin (Dox) is an Anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent often induces side effects such as cardiotoxicity leading to cardiomyocyte death and dilated cardiomyopathy (DCM). This cardiotoxicity has been related to reactive oxygen species generation, DNA intercalation, topoisomerase II inhibition and bioenergetics alterations resulting in DNA damages leading to cardiomyocyte death. There is a need for new therapeutic agents and strategies aiming at reducing Dox side effects in the heart. Among these mechanisms, the role of Epac (exchange protein directly activated by cAMP) could be worth investigating. Therefore we have investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57BL/6 vs. Epac1 KO mice, iv injections, 12 mg/kg) and in vitro (primary culture of neonatal rat cardiomyocytes (NRVM), Dox 1 μM). In vivo, Dox-treated mice developed a DCM associated with Ca 2+ homeostasis dysfunction. In vitro, as measured by flow cytometry and western blot, Dox induced DNA damage and cell death. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation, cell size reduction and relative plasma membrane integrity. We also observed that Dox led to a modification of the protein level of Epac1 and Epac2 isoforms. The inhibition of Epac1 (ESI09, CE3F4), but not of Epac2 (ESI05), prevented DNA/TopIIβ complexes, decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. These results were confirmed in vivo since Dox-induced cardiotoxicity was prevented in Epac1 KO mice as evidenced by unaltered cardiac function (no DCM) 15 weeks post-treatment. Inhibition of Epac1 could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy.

Protective action of the microbial metabolite butyrate against cardiomyocyte hypertrophy

Abstract Introduction Short-chain fatty acids are one of the gut microbial metabolites that may influence host physiology. We previously reported that gut dysbiosis was associated with heart failure, and that the proportions of butyrate-producing bacteria diminished prominently in the gut of patients with heart failure. Purpose We investigated the molecular mechanism of butyrate and investigated the protective mechanism against heart failure. Methods We searched for G protein-coupled receptors for short-chain fatty acids using single-cell transcriptome analysis of cardiomyocytes and non-cardiomyocytes isolated from murine hearts. In addition, we examined the effects of butyrate on endothelin-1 (ET1) or isoproterenol-induced hypertrophic responses and histone deacetylase (HDAC) activities in cultured neonatal rat cardiomyocytes. Results Single-cell transcriptome analysis and co-expression network analysis revealed that G protein-coupled receptors for short-chain fatty acid receptors were not expressed in cardiomyocytes and that Olfr78 was expressed in vascular smooth muscle cells in the heart. Treatment with butyrate inhibited ET1-induced hypertrophic growth and up-regulation of the genes such as Nppa, Acta1, and Myh7 in cultured rat neonatal cardiomyocytes. Moreover, butyrate increased the acetylation levels of histone H3, indicating that butyrate has an inhibitory effect on HDAC in cardiomyocytes. In addition, treatment with butyrate caused up-regulation of Inpp5f, encoding inositol polyphosphate-5-phosphatase f, which was associated with a significant decrease in the phosphorylation levels of Akt. These results suggest that butyrate may act as HDAC inhibitor to increase Inpp5f gene expression, leading to the activation of Akt-glycogen synthase kinase 3beta (Gsk3beta) pathway, and thereby protect against hypertrophic responses. Conclusion There was no known GPCR for short-chain fatty acid expressed in cardiomyocytes. However, butyrate suppressed cardiomyocyte hypertrophy through epigenetic modification of gene expression. Our results may uncover a potential role of the dysbiosis of intestinal microbiota in the pathogenesis of cardiac hypertrophy and failure. Funding Acknowledgement Type of funding source: None

Autoantibodies directed against \u03b11-adrenergic receptor and endothelin receptor A in patients with prostate cancer

BackgroundFor prostate cancer, signaling pathways induced by over-boarding stimulation of G-protein coupled receptors (GPCR) such as the endothelin, α1- and β-adrenergic, muscarinic and angiotensin 1 receptors were accused to support the carcinogenesis. However, excessive receptor stimulation by physiological receptor ligands is minimized by a control system that induces receptor sensitization and down-regulation. This system is missing when so-called “functional autoantibodies” bind to the GPCR (GPCR-AAB). If GPCR-AAB were found in patients with prostate cancer, uncontrolled GPCR stimulation could make these autoantibodies an additional supporter in prostate cancer.MethodsUsing the bioassay of spontaneously beating cultured rat neonatal cardiomyocytes, GPCR-AAB were identified, quantified and characterized in the serum of 25 patients (aged 56–78 years, median 70 years) with prostate cancer compared to 10 male patients (aged 48–82 years, median 64) with urinary stone disorders (controls).ResultsOf the cancer patients, 24 (96%) and 17 (68%), respectively, carried autoantibodies directed against the α1-adrenergic receptor (α1-AAB) and endothelin receptor A (ETA-AAB). No patient was negative for both GPCR-AAB. In contrast, ETA-AAB and α1-AAB were absent in all (100%) and 9 (90%) of the 10 control patients, respectively. While α1-AAB targeted a specific epitope of the first extracellular loop of the α1-adrenergic receptor subtype A, an epitope of the second extracellular loop of the ETA receptor was identified as a target of ETA-AAB. As demonstrated in vitro, the functional activity of both autoantibodies found in prostate cancer can be neutralized by the aptamer BC007.ConclusionsWe hypothesized that α1-AAB and ETA-AAB, which are highly present in prostate cancer patients, could by their functional activity support carcinogenesis by excessive receptor stimulation. The in vitro demonstrated neutralization of α1- and ETA-AAB by the aptamer BC007 could open the door to complement the treatments already available for prostate cancer.

EPAC1 inhibition prevents adverse cardiotoxicity induced by anticancer treatment

Anticancer treatments (chemo- and radiation therapies), such as Doxorubicin (Dox), used to treat many types of cancer are associated with many side effects such as cardiotoxicity mainly leading to a dilated cardiomyopathy (DCM) in chronic. There is a need for new treatment options and strategies aiming at reducing Doxorubicin side effects in the heart. Among these mechanisms Epac (exchange protein directly activated by cAMP) signaling could be worth investigating. We have investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57Bl63/Knock-out Epac1 mice, iv injections, 12 mg/kg cumulative dose) and in vitro using primary culture of neonatal rat cardiomyocytes (NRVM, 24 h, Dox 1 μM). Then we investigated the effect of Epac inhibition on human cancer cell lines (24 h, Dox 1-10 μM). Dox-treated mice developed a DCM associated with Ca2+ homeostasis dysfunction. In vitro, as measured by flow cytometry and western blot, Dox induced DNA damages and cell death. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation, cell size reduction and relative plasma membrane integrity. The inhibition of Epac1 (ESI09, CE3F4), but not Epac2 (ESI05), prevented DNA/TopIIβ complexes, decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. These results were confirmed in vivo since Dox-induced cardiotoxicity was prevented in Epac1 KO mice as evidenced by unaltered cardiac function (no DCM) 15 weeks post-treatment. Interestingly, the protection conferred by Epac1 inhibition was not transferred to human cancer cell lines treated by Dox. Inhibition of Epac1 may thus represents a novel strategy to prevent Anthracyclines-induced cardiotoxicity. Moreover, Epac1 inhibition seems to be a valuable therapeutic strategy to treat cancer by potentiating Dox toxicity.

Solvent-Free Fabrication of Carbon Nanotube/Silk Fibroin Electrospun Matrices for Enhancing Cardiomyocyte Functionalities.

Cardiac tissue engineering holds great potential in regenerating functional cardiac tissues for various applications. The major strategy is to design scaffolds recapitulating the native cardiac microenvironment to enhance cell and tissue functionalities. Among various biomaterial systems, nanofibrous matrices with aligned morphologies and enhanced conductivity incline to induce the formation of oriented engineered cardiac tissues with enhanced functionalities. The challenge is to functionalize the scaffolds with conductive additives without influencing their biocompatibility. In this study, we developed a fully aqueous process for the fabrication of conductive carbon nanotube/silk fibroin (CNT/silk) electrospun scaffolds. The carbon nanotubes are well dispersed within the nanofibers, providing the scaffolds with enhanced conductivity and excellent biocompatibility for the culture of neonatal rat cardiomyocytes with improved cell spreading and enhanced expression of cardiac-specific proteins. Moreover, the aligned CNT/silk fibroin composite scaffolds exhibit abilities to guide the oriented organization of cardiac tissues and the biomimicking distribution of sarcomeres and gap junctions. The findings demonstrate the great potential of the CNT/silk scaffolds prepared through this aqueous processing method in supporting the formation of cardiac tissues with enhanced functionalities.

High content screening identifies licoisoflavone A as a bioactive compound of Tongmaiyangxin Pills to restrain cardiomyocyte hypertrophy via activating Sirt3

Background: Cardiac hypertrophy is a prominent feature of heart remodeling, which may eventually lead to heart failure. Tongmaiyangxin (TMYX) pills are a clinically used botanical drug for treating multiple cardiovascular diseases including chronic heart failure. The aim of the current study was to identify the bioactive compounds in Tongmaiyangxin pills that attenuate cardiomyocytes hypertrophy, and to investigate the underlying mechanism of action. Methods and Results: The anti-hypertrophy effect of TMYX was validated in isoproterenol-induced cardiac hypertrophy model in C57BL/6 mice. After TMYX treatment for 2 weeks, the heart ejection fraction and fractional shortening of the mice model was increased by approximately 20% and 15%, respectively, (p < 0.05). Besides, TMYX dose-dependently reduced the cross section area of cardiomyocytes in the angiotensin-II induced hypertrophy H9c2 model (p < 0.01). Combining high content screening and liquid chromatography mass spectrometry, four compounds with anti-cardiac hypertrophy effects were identified from TMYX, which includes emodin, licoisoflavone A, licoricone and glyasperin A. Licoisoflavone A is one of the compounds with most significant protective effect and we continued to investigate the mechanism. Primary cultures of neonatal rat cardiomyocytes were treated with a hypertrophic agonist phenylephrine (PE) in the presence or absence of licoisoflavone A. After 48 h of treatment, cells were harvested and mitochondrial acetylation was analyzed by western blotting and Image analysis. Interestingly, the results suggested that the anti-hypertrophic effects of licoisoflavone A depend on the activation of the deacetylase Sirt3 (p < 0.01). Finally, we showed that licoisoflavone A-treatment was able to decrease relative ANF and BNP levels in the hypertrophic cardiac cells (p < 0.01), but not in cells co-treated with Sirt3 inhibitors (3-TYP) (p > 0.05). Conclusion: TMYX exerts its anti-hypertrophy effect possibly through upregulating Sirt3 expression. Four compounds were identified from TMYX which may be responsible for the anti-hypertrophy effect. Among these compounds, licoisoflavone A was demonstrated to block the hypertrophic response of cardiomyocytes, which required its positive regulation on the expression of Sirt3. These results suggested that licoisoflavone A is a potential Sirt3 activator with therapeutic effect on cardiac hypertrophy.

P3116New role of EPAC1 in Anthracycline-induced cardiotoxicity and anticancer therapy

Abstract Introduction Doxorubicin (Dox) is an anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent induces many side effects such as cardiotoxicity leading to dilated cardiomyopathy (DCM). The cardiotoxicity of Dox has been related to reactive oxygen species generation, DNA intercalation, topoisomerase II inhibition and bioenergetics alterations leading to cardiomyocyte death. Objective Nowadays the challenge is to find new treatment options aiming at reducing Dox cardiotoxicity. Epac (exchange protein directly activated by cAMP) signaling could be worth investigating as Epac activates small G proteins which are known to be involved in Dox-induced cardiotoxicity. Methods We investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57Bl63/ Knock-out Epac1 mice, iv injections, 12mg/kg cumulative dose) and in vitro (primary culture of neonatal rat cardiomyocytes (NRVM, 24h, Dox 1μM). Results In vivo, Dox-treated mice developed a DCM associated with Ca2+ homeostasis dysfunction (increase of Ca2+ waves and Ca2+ leaks). In vitro, as measured by flow cytometry and western blot, Dox (1μM) induced DNA damages and cell death in NRVM. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation and cell size reduction. The inhibition of Epac1 (ESI09, CE3F4) decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. Moreover, in vivo, Epac1 KO mice were protected against Dox-induced cardiotoxicity by unaltered cardiac function (no DCM) and calcium homeostasis at 15 weeks post-treatment. Conclusion Inhibition of Epac1 could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy. Indeed, preliminary data show also that preventing Dox-induced cardiotoxicity, the inhibition of Epac1 can also potentiate cancerous cells death. Acknowledgement/Funding Labex Lermit (ANR 0033), Torino and Inserm

1,25-Vitamin D3 protects against cooking oil fumes-derived PM2.5-induced cell damage through its anti-inflammatory effects in cardiomyocytes

The functional role of 1,25-vitamin D3 in cooking oil fumes (COFs)-derived PM2.5-induced cell damage is largely unexplored. The present study investigated the protective role of 1,25-vitamin D3 against cell injury by possible involvement of JAK/STAT and NF-κB signaling pathways in cardiomyocytes. Cell viability was measured using CCK-8 assay, and cell apoptosis was analyzed by flow cytometry, qRT-PCR and Western blot in cultured rat neonatal cardiomyocytes treated with 1,25-vitamin D3 and COFs-derived PM2.5. Expressions of JAK/STAT and NF-κB signaling pathway were measured by Western blot. The results suggested that treatment with COFs-derived PM2.5 significantly decreased cell viability and increased apoptosis and oxidative stress in cultured rat neonatal cardiomyocytes. 1,25-vitamin D3 pretreatment alleviated the cell injury by increasing cell viability and decreasing apoptosis in the cardiomyocytes. 1,25-vitamin D3 pretreatment also decreased the ROS level and inflammation in the cardiomyocytes. Furthermore, 1,25-vitamin D3 pretreatment alleviated COFs-derived PM2.5-evoked elevation of JAK/STAT and NF-κB signaling pathways. Our study showed that 1,25-vitamin D3 pretreatment protected cardiomyocytes from COFs-derived PM2.5-induced injury by decreasing ROS, apoptosis and inflammation level via activations of the JAK/STAT and NF-κB signaling pathways.

Anticancer therapy induced cardiotoxicity: Involvement of EPAC1 in cardioprotection and anticancer therapy

Doxorubicin (Dox) is an anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent induces many side effects such as cardiotoxicity leading to dilated cardiomyopathy (DCM). The cardiotoxicity of Dox has been related to reactive oxygen species generation, DNA intercalation and topoisomerase II inhibition, bioenergetics alterations leading to cardiomyocyte death. Nowadays the challenge is to find new treatment options aiming at reducing Dox cardiotoxicity. Epac (exchange protein directly activated by cAMP) signaling could be worth investigating as Epac activates small G proteins which are known to be involved in Dox-induced cardiotoxicity. We investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57Bl63/Knock-out Epac1 mice, iv injections, 12mg/kg cumulative dose) and in vitro (primary culture of neonatal rat cardiomyocytes (NRVM, 24h, Dox 1μM). In vivo, Dox-treated mice developed a DCM associated with Ca2 + homeostasis dysfunction (increase of Ca2 + waves and Ca2 + leaks). In vitro, as measured by flow cytometry and western blot, Dox (1μM) induced DNA damage and cell death in NRVM. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation and cell size reduction. The inhibition of Epac1 (ESI09, CE3F4) decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. Moreover, in vivo, Epac1 KO mice were protected against Dox-induced cardiotoxicity by unaltered cardiac function (no DCM) and calcium homeostasis at 15 weeks post-treatment. Inhibition of Epac1 could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy. Indeed, preliminary data show also that preventing Dox-induced cardiotoxicity, the inhibition of Epac1 can also potentiate cancerous cells death.

Active uptake of hydrophilic copper complex Cu(ii)-TETA in primary cultures of neonatal rat cardiomyocytes.

Myocardial ischemia leads to copper efflux from the heart. The ischemic tissue with a low copper content fails to take up copper from the circulation even under the conditions of serum copper elevation. Cardiac copper repletion thus requires other available forms of this element than those currently known to bind to copper transport proteins. The copper complex of triethylenetetramine (TETA) is a metabolite of TETA, which has the potential to increase cardiac copper content in vivo. In the present study, we synthesized Cu(ii)-TETA, analyzed its crystal structure, and demonstrated the role of this compound in facilitating copper accumulation in primary cultures of neonatal rat cardiomyocytes. The Cu(ii)-TETA compound formed a square pyramidal chloride salt [Cu(TETA)Cl]Cl structure, which dissociates from chloride in aqueous solution to yield the four-coordinate dication Cu(ii)-TETA. Cu(ii)-TETA was accumulated as an intact compound in cardiomyocytes. Analysis from time-dependent copper accumulation in cardiomyocytes defined a different dynamic process in copper uptake between Cu(ii)-TETA and CuCl2 exposure. An additive copper accumulation in cardiomyocytes was found when the cells were exposed to both CuCl2 and Cu(ii)-TETA. Gene silencing of copper transport 1 (CTR1) did not affect cross-membrane transportation of Cu(ii)-TETA, but inhibited copper cellular accumulation from CuCl2. Furthermore, the uptake of Cu(ii)-TETA by cardiomyocytes was ATP-dependent. It is thus concluded that the formation of Cu(ii)-TETA facilitates copper accumulation in cardiomyocytes through an active CTR1-independent transportation process.

Detection and quantitation of iron in ferritin, transferrin and labile iron pool (LIP) in cardiomyocytes using 55Fe and storage phosphorimaging

Dysregulated iron metabolism has a detrimental effect on cardiac function. The importance of iron homeostasis in cardiac health and disease warrants detailed studies of cardiomyocyte iron uptake, utilization and recycling at the molecular level. In this study, we have performed metabolic labeling of primary cultures of neonatal rat cardiomyocytes with radioactive iron coupled with separation of labeled iron-containing molecules by native electrophoresis followed by detection and quantification of incorporated radioiron by storage phosphorimaging. For the radiolabeling we used a safe and convenient beta emitter 55Fe which enabled sensitive and simultaneous detection and quantitation of iron in cardiomyocyte ferritin, transferrin and the labile iron pool (LIP). The LIP is believed to represent potentially dangerous redox–active iron bound to uncharacterized molecules. Using size-exclusion chromatography spin micro columns, we demonstrate that iron in the LIP is bound to high molecular weight molecule(s) (≥5000 Da) in the neonatal cardiomyocytes.

The Involvement of Cytochrome c Oxidase in Mitochondrial Fusion in Primary Cultures of Neonatal Rat Cardiomyocytes.

Cytochrome c oxidase (CCO) is a copper-dependent enzyme of mitochondrial respiratory chain. In pressure overload-induced cardiac hypertrophy, copper level and CCO activity are both depressed, along with disturbance in mitochondrial fusion and fission dynamics. Copper repletion leads to recovery of CCO activity and normalized mitochondrial dynamics. The present study was undertaken to define the link between CCO activity and mitochondrial dynamic changes. Primary cultures of neonatal rat cardiomyocytes were treated with phenylephrine to induce cell hypertrophy. Hypertrophic cardiomyocytes were then treated with copper to reverse hypertrophy. In the hypertrophic cardiomyocytes, CCO activity was depressed and mitochondrial fusion was suppressed. Upon copper repletion, CCO activity was recovered and mitochondrial fusion was reestablished. Depression of CCO activity by siRNA targeting CCO assembly homolog 17 (COX17), a copper chaperone for CCO, led to fragmentation of mitochondria, which was not recoverable by copper supplementation. This study thus demonstrates that copper-dependent CCO is critical for mitochondrial fusion in the regression of cardiomyocyte hypertrophy.