Rat Cardiomyocytes Research Articles

Virtual drug screen reveals context-dependent inhibition of cardiomyocyte hypertrophy.

Pathological cardiomyocyte hypertrophy is a response to cardiac stress that typically leads to heart failure. Despite being a primary contributor to pathological cardiac remodelling, the therapeutic space that targets hypertrophy is limited. Here, we apply a network model to virtually screen for FDA-approved drugs that induce or suppress cardiomyocyte hypertrophy. A logic-based differential equation model of cardiomyocyte signalling was used to predict drugs that modulate hypertrophy. These predictions were validated against curated experiments from the prior literature. The actions of midostaurin were validated in new experiments using TGFβ- and noradrenaline (NE)-induced hypertrophy in neonatal rat cardiomyocytes. Model predictions were validated in 60 out of 70 independent experiments from the literature and identify 38 inhibitors of hypertrophy. We additionally predict that the efficacy of drugs that inhibit cardiomyocyte hypertrophy is often context dependent. We predicted that midostaurin inhibits cardiomyocyte hypertrophy induced by TGFβ, but not noradrenaline, exhibiting context dependence. We further validated this prediction by cellular experiments. Network analysis predicted critical roles for the PI3K and RAS pathways in the activity of celecoxib and midostaurin, respectively. We further investigated the polypharmacology and combinatorial pharmacology of drugs. Brigatinib and irbesartan in combination were predicted to synergistically inhibit cardiomyocyte hypertrophy. This study provides a well-validated platform for investigating the efficacy of drugs on cardiomyocyte hypertrophy and identifies midostaurin for consideration as an antihypertrophic drug.

Multiple ingredients of a Chinese medicine formula Sheng-Mai-San coordinately attenuate doxorubicin-induced cardiotoxicity

BackgroundDoxorubicin (DOX) is one of the most widely used antitumor drugs. However, continuous DOX treatment would cause crucial cardiotoxicity side effect, accompanied with the accumulation of reactive oxygen species (ROS), impaired mitochondrial functions and increased cardiomyocyte death. Sheng-Mai-San (SMS), a traditional Chinese medicine widely used for cardiovascular diseases, has been clinically used to counteract cardiotoxicity of DOX, yet the underlying mechanism is largely unknown. MethodsWe utilized JC-1 assay to examine the effects of SMS and its components under DOX treatment in rat cardiomyocytes (H9c2 cells), and further investigated the functional mechanisms of active components in SMS formula via transcriptome sequencing, bioinformatics analysis and molecular experiments. ResultsWe identified three functional components of SMS in protecting cardiomyocytes from DOX-induced damages and revealed the coordinated regulatory mechanisms of these components, namely ginsenoside Rh2 (Rh2), Ophiopogonin D (OpD) and Schisandrin B (SchB). We found that Rh2 mainly acts to restore mitochondrial functions and to decrease ROS generation by enhancing fatty acid metabolism and antioxidant activity, OpD has assistive roles in mitochondrial function repair, and SchB mainly functions by inhibiting cell death, as well as activating cytoskeleton and ECM organization. ConclusionThis study provides supporting evidence for the clinical application of SMS to reduce the cardiotoxicity effects of DOX treatment, and also sheds light for the mechanistic studies of traditional Chinese medicine.

MFN2 deficiency promotes cardiac response to hypobaric hypoxia by reprogramming cardiomyocyte metabolism.

Under hypobaric hypoxia (HH), the heart triggers various defense mechanisms including metabolic remodeling against lack of oxygen. Mitofusin 2 (MFN2), located at the mitochondrial outer membrane, is closely involved in the regulation of mitochondrial fusion and cell metabolism. To date, however, the role of MFN2 in cardiac response to HH has not been explored. Loss- and gain-of-function approaches were used to investigate the role of MFN2 in cardiac response to HH. In vitro, the function of MFN2 in the contraction of primary neonatal rat cardiomyocytes under hypoxia was examined. Non-targeted metabolomics and mitochondrial respiration analyses, as well as functional experiments were performed to explore underlying molecular mechanisms. Our data demonstrated that, following 4 weeks of HH, cardiac-specific MFN2 knockout (MFN2 cKO) mice exhibited significantly better cardiac function than control mice. Moreover, restoring the expression of MFN2 clearly inhibited the cardiac response to HH in MFN2 cKO mice. Importantly, MFN2 knockout significantly improved cardiac metabolic reprogramming during HH, resulting in reduced capacity for fatty acid oxidation (FAO) and oxidative phosphorylation, and increased glycolysis and ATP production. In vitro data showed that down-regulation of MFN2 promoted cardiomyocyte contractility under hypoxia. Interestingly, increased FAO through palmitate treatment decreased contractility of cardiomyocyte with MFN2 knockdown under hypoxia. Furthermore, treatment with mdivi-1, an inhibitor of mitochondrial fission, disrupted HH-induced metabolic reprogramming and subsequently promoted cardiac dysfunction in MFN2-knockout hearts. Our findings provide the first evidence that down-regulation of MFN2 preserves cardiac function in chronic HH by promoting cardiac metabolic reprogramming.

Integrin beta-like 1 mediates fibroblast-cardiomyocyte crosstalk to promote cardiac fibrosis and hypertrophy.

Crosstalk between fibroblasts and cardiomyocytes (CMs) plays a critical role in cardiac remodelling during heart failure (HF); however, the underlying molecular mechanisms remain obscure. Recently, a secretory protein, Integrin beta-like 1 (ITGBL1) was revealed to have detrimental effects on several diseases, such as tumours, pulmonary fibrosis, and hepatic fibrosis; whereas the effect of ITGBL1 on HF is unclear. The purpose of this study was to evaluate its contribution to volume overload-induced remodelling. In this study, we identified ITGBL1 was highly expressed in varied heart diseases and validated in our TAC mice model, especially in fibroblasts. To investigate the role of ITGBL1 in in vitro cell experiments, neonatal rat fibroblasts (NRCFs) and cardiomyocytes (NRCMs) were performed for further study. We found that in comparison to NRCMs, NRCFs expressed high levels of ITGBL1. Meanwhile, ITGBL1 was upregulated in NRCFs, but not in NRCMs following angiotensin-II (AngII) or phenylephrine stimulation. Furthermore, ITGBL1 overexpression promoted NRCFs activation, whereas knockdown of ITGBL1 alleviated NRCFs activation under AngII treatment. Moreover, NRCFs-secreted ITGBL1 could induce NRCMs hypertrophy. Mechanically, ITGBL1-NME/NM23 nucleoside diphosphate kinase 1 (NME1)-TGF-β-Smad2/3 and Wnt signalling pathways were identified to mediate NRCFs activation and NRCMs hypertrophy, respectively. Finally, the knockdown of ITGBL1 in mice subjected to transverse aortic constriction (TAC) surgery recapitulated the in vitro findings, demonstrating blunted cardiac fibrosis, hypertrophy, and improved cardiac function. ITGBL1 is an important functional mediator between fibroblast-cardiomyocyte crosstalk and could be an effective target for cardiac remodelling in HF patients.

Experimental study on co-culture of DiI-labeled rat bone marrow mesenchymal stem cells and neonatal rat cardiomyocytes to induce differentiation into cardiomyocyte-like cells.

Myocardial infarction is a serious clinical disease with high mortality and poor prognosis. Cardiomyocytes (CMs) have limited regeneration abilities after ischemic injury. Their growth and differentiation can be enhanced by contact co-culture with stem cells. The aim was to study the contact co-culture of Dil-labeled bone marrow mesenchymal stem cells (BMSCs) and CMs for inducing differentiation of CMs from stem cells for treating myocardial infarction. After contact co-culture, the differentiation of BMSCs into CMs was analyzed qualitatively by detecting myocardial markers (cardiac troponin T and α-smooth muscle actin) using immunofluorescence and quantitatively using flow cytometry. To examine the mechanism, possible gap junctions between BMSCs and CMs were analyzed by detecting gap junction protein connexin 43 (C×43) expression in BMSCs using immunofluorescence. The functionality of gap junctions was analyzed using dye transfer experiments. The results revealed that BMSCs in contact with CMs exhibited myocardial markers and a significant increase in differentiation rate (P <0.05); they also proved the existence and function of gap junctions between BMSCs and CMs. It was shown that contact co-culture can induce Dil-labeled BMSCs to differentiate into CM-like cells and examined the principle of gap junction-mediated signaling pathways involved in inducing stem cells to differentiate into cardiomyocytes.

A novel onco-cardiological mouse model of lung cancer-induced cardiac dysfunction and its application in identifying potential roles of tRNA-derived small RNAs

An increasing body of research suggests cancer-induced cardiovascular diseases, leading to the appearance of an interdisciplinary study known as onco-cardiology. Lung cancer has the highest incidence and mortality. Cardiac dysfunction constitutes a major cause of death in lung cancer patients. However, its mechanism has not been elucidated because suitable animal models that adequately mimic clinical features are lacking. Here, we established a novel chemically induced lung cancer mouse model using benzo[a]pyrene and urethane to recapitulate the general characteristics of cardiac dysfunction caused by lung cancer, the cardiac disorders in the context of the progression of lung cancer were evaluated using echocardiographic and histological approaches. The pathological changes included myocardial ischaemia, pericarditis, cardiac pre-cachexia, and pulmonary artery hypertension. We performed sequencing to detect the tRNA-derived fragments and tRNA-derived stress-induced RNAs (tRFs/tiRNAs) expressions in mouse heart tissue. 22 upregulated and 16 downregulated tRFs/tiRNAs were identified. Subsequently, the top 10 significant results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were presented. The in vitro model was established by exposing neonatal rat cardiomyocytes and myocardial fibroblasts to lung tumour cell-conditioned medium, respectively. Western blotting revealed significant changes in cardiac failure markers (atrial natriuretic peptide and α-myosin heavy chain) and cardiac fibrosis markers (Collagen-1 and Collagen-3). Our model adequately reflects the pathological features of lung cancer-induced cardiac dysfunction. Furthermore, the altered tRF/tiRNA profiles showed great promise as novel targets for therapies. These results might pave the way for research on therapeutic targets in onco-cardiology.

Asiaticoside promotes heart autonomic nerve repair through receptor activator of nuclear factor kappa-B ligand pathway in coronary heart disease

This experiment intends to observe the effects of Asiaticoside on angiotensin II (AngII)-induced coronary heart disease (CHD) by regulating nuclear factor κ-B ligand receptor activator and RANK pathway (RANKL/RANK). Cardiomyocytes of rats with CHD were divided into AngII group (2 μmol/L), blank control group, and asiaticoside group (2 μmol/L and 10 μmol/L). After 24 hours of intervention, proliferation and apoptosis was assessed. At the same time, the expressions of RANKL, RANK and α-smooth muscle actin (α-SMA) were measured. Compared with blank group, the proliferation ability of cardiomyocytes with CHD in AngII group was significantly decreased and it increased after asiaticoside treatment (P &lt;0.05). The apoptosis of cardiomyocytes in AngII group was higher than blank group and asiaticoside group. Cardiomyocytes proliferation in asiaticoside group was the most obvious (P &lt;0.05). α-SMA expression decreased in AngII group and increased after asiaticoside treatment (P &lt;0.05). RANKL and RANK mRNA in CHD were significantly upregulated after administration of asiaticoside (P &lt;0.05). RANKL/RANK expression was high in normal CHD cardiomyocytes and reduced after Ang II stimulation. Asiaticoside promotes cardiomyocyte proliferation in CHD by promoting RANKL/RANK signaling, thereby reducing the possibility of heart failure in CHD.

MXene functionalized collagen biomaterials for cardiac tissue engineering driving iPSC-derived cardiomyocyte maturation

Electroconductive biomaterials are gaining significant consideration for regeneration in tissues where electrical functionality is of crucial importance, such as myocardium, neural, musculoskeletal, and bone tissue. In this work, conductive biohybrid platforms were engineered by blending collagen type I and 2D MXene (Ti3C2Tx) and afterwards covalently crosslinking; to harness the biofunctionality of the protein component and the increased stiffness and enhanced electrical conductivity (matching and even surpassing native tissues) that two-dimensional titanium carbide provides. These MXene platforms were highly biocompatible and resulted in increased proliferation and cell spreading when seeded with fibroblasts. Conversely, they limited bacterial attachment (Staphylococcus aureus) and proliferation. When neonatal rat cardiomyocytes (nrCMs) were cultured on the substrates increased spreading and viability up to day 7 were studied when compared to control collagen substrates. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were seeded and stimulated using electric-field generation in a custom-made bioreactor. The combination of an electroconductive substrate with an external electrical field enhanced cell growth, and significantly increased cx43 expression. This in vitro study convincingly demonstrates the potential of this engineered conductive biohybrid platform for cardiac tissue regeneration.

CaMKK2 alleviates myocardial ischemia/reperfusion injury by inhibiting oxidative stress and inflammation via the action on the AMPK-AKT-GSK-3β/Nrf2 signaling cascade.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) can regulate numerous biological processes and is implicated in diverse pathological processes. Yet its role in myocardial ischemia/reperfusion (MI/R) injury remains unknown. This project explored the possible functions and mechanisms of CaMKK2 in MI/R injury. A rat model of MI/R in vivo was established using the left anterior descending coronary artery ligation method. Rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) in vitro to establish a cell model. Overexpression of CaMKK2 was achieved by infecting recombinant adeno-associated virus or adenovirus expressing CaMKK2. Real-time quantitative PCR, immunoblotting, TTC staining, TUNEL assay, ELISA, oxidative stress detection assays, flow cytometry, and CCK-8 assay were carried out. A decline in CaMKK2 levels was induced by MI/R in vivo or H/R in vitro. Up-modulation of CaMKK2 in rats ameliorated the cardiac injury evoked by MI/R injury accompanied by suppression of cardiac apoptosis, oxidative stress, and proinflammatory response. Rat cardiomyocytes with CaMKK2 overexpression were also protected from H/R damage by inhibiting apoptosis, oxidative stress, and proinflammatory response. CaMKK2 overexpression led to increased phosphorylation of AMPK, AKT, and GSK-3β, and enhanced activation of Nrf2 under MI/R or H/R conditions. Inhibition of AMPK abolished CaMKK2-mediated Nrf2 activation and relevant cardioprotective effect. Restraint of Nrf2 also diminished CaMKK2-mediated relevant cardioprotective effect. Up-regulation of CaMKK2 provides a therapeutic benefit in the rat model of MI/R injury by boosting the Nrf2 pathway through regulation of AMPK/AKT/GSK-3β, which suggests CaMKK2 as a new molecular target for the treatment of MI/R injury.

Therapeutic potentials of terbium hydroxide nanorods for amelioration of hypoxia-reperfusion injury in cardiomyocytes

Myocardial hypoxia reperfusion (H/R) injury is the paradoxical exacerbation of myocardial damage, caused by the sudden restoration of blood flow to hypoxia affected myocardium. It is a critical contributor of acute myocardial infarction, which can lead to cardiac failure. Despite the current pharmacological advancements, clinical translation of cardioprotective therapies have proven challenging. As a result, researchers are looking for alternative approaches to counter the disease. In this regard, nanotechnology, with its versatile applications in biology and medicine, can confer broad prospects for treatment of myocardial H/R injury. Herein, we attempted to explore whether a well-established pro-angiogenic nanoparticle, terbium hydroxide nanorods (THNR) can ameliorate myocardial H/R injury. For this study, in vitro H/R-injury model was established in rat cardiomyocytes (H9c2 cells). Our investigations demonstrated that THNR enhance cardiomyocyte survival against H/R-induced cell death. This pro-survival effect of THNR is associated with reduction of oxidative stress, lipid peroxidation, calcium overload, restoration of cytoskeletal integrity and mitochondrial membrane potential as well as augmentation of cellular anti-oxidant enzymes such as glutathione-s-transferase (GST) and superoxide dismutase (SOD) to counter H/R injury. Molecular analysis revealed that the above observations are traceable to the predominant activation of PI3K-AKT-mTOR and ERK-MEK signalling pathways by THNR. Concurrently, THNR also exhibit apoptosis inhibitory effects mainly by suppression of pro-apoptotic proteins like Cytochrome C, Caspase 3, Bax and p53 with simultaneous restoration of anti-apoptotic protein, Bcl-2 and Survivin. Thus, considering the above attributes, we firmly believe that THNR have the potential to be developed as an alternative approach for amelioration of H/R injury in cardiomyocytes.

Activation of AMPKα2 attenuated doxorubicin-induced cardiotoxicity via inhibiting lipid peroxidation associated ferroptosis

Ferroptosis has been suggested to involve in doxorubicin (DOX)-induced cardiotoxicity. However, the underlying mechanisms and regulatory targets of cardiomyocyte ferroptosis remains to be understood. This study demonstrated that the up-regulation of ferroptosis associated proteins genes were accompanied with the down-regulation of AMPKα2 phosphorylation in DOX treated mouse heart or neonatal rat cardiomyocytes (NRCMs). AMPKα2 knockout (AMPKα2−/−) significantly exacerbated mouse cardiac dysfunction, increased mortality, promoting ferroptosis associated mitochondrial injuries, enhanced ferroptosis associated proteins and genes expression, and lead to accumulation of lactate dehydrogenase (LDH) and malondialdehyde (MDA) in mouse serum and hearts respectively. Ferrostatin-1 administration markedly improved cardiac function, decreased mortality, inhibited mitochondrial injuries and ferroptosis associated proteins and genes expression, and depressed accumulation of LDH and MDA in DOX treated AMPKα2−/− mouse. Moreover, Adeno-associated virus serotype 9 AMPKα2 (AAV9-AMPKα2) or AICAR treatment mediated AMPKα2 activation could significantly improve cardiac function and depress ferroptosis in mouse. AMPKα2 activation or silence could also inhibit or promote ferroptosis associated injuries in DOX treated NRCMs respecitively. Mechanistically, AMPKα2/ACC mediated lipid metabolism has been suggested to involve in regulating DOX-treatment induced ferroptosis other than mTORC1 or autophagy dependent pathway. The metabolomics analysis exhibited that AMPKα2−/− significantly enhanced accumulation of polyunsaturated fatty acids (PFAs), oxidized lipid, and phosphatidylethanolamine (PE). Finally, this study also demonstrated that metformin (MET) treatment could inhibit ferroptosis and improve cardiac function via activating AMPKα2 phosphorylation. The metabolomics analysis exhibited that MET treatment significantly depressed PFAs accumulation in DOX treated mouse hearts. Collectively, this study suggested that AMPKα2 activation might protect against anthracycline chemotherapeutic drugs mediated cardiotoxicity via inhibiting ferroptosis.

Trastuzumab potentiates doxorubicin-induced cardiotoxicity via activating the NLRP3 inflammasome in vivo and in vitro

Trastuzumab (Tra), the first humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (HER2), is commonly used alongside doxorubicin (Dox) as a combination therapy in HER2-positive breast cancer. Unfortunately, this leads to a more severe cardiotoxicity than Dox alone. NLRP3 inflammasome is known to be involved in Dox-induced cardiotoxicity and multiple cardiovascular diseases. However, whether the NLRP3 inflammasome contributes to the synergistic cardiotoxicity of Tra has not been elucidated. In this study, primary neonatal rat cardiomyocyte (PNRC), H9c2 cells and mice were treated with Dox (15 mg/kg in mice or 1 μM in cardiomyocyte) or Tra (15.75 mg/kg in mice or 1 μM in cardiomyocyte), or Dox combined Tra as cardiotoxicity models to investigate this question. Our results demonstrated that Tra significantly potentiated Dox-induced cardiomyocyte apoptosis and cardiac dysfunction. These were accompanied by the increased expressions of NLRP3 inflammasome components (NLRP3, ASC and cleaved caspase-1), the secretion of IL-β and the pronounced production of ROS. Inhibiting the activation of NLRP3 inflammasome by NLRP3 silencing significantly reduced cell apoptosis and ROS production in Dox combined Tra-treated PNRC. Compared with the wild type mice, the systolic dysfunction, myocardial hypertrophy, cardiomyocyte apoptosis and oxidative stress induced by Dox combined Tra were alleviated in NLRP3 gene knockout mice. Our data revealed that the co-activation of NLRP3 inflammasome by Tra promoted the inflammation, oxidative stress and cardiomyocytes apoptosis in Dox combined Tra-induced cardiotoxicity model both in vivo and in vitro. Our results suggest that NLRP3 inhibition is a promising cardioprotective strategy in Dox/Tra combination therapy.

Metabolic changes of glycerophospholipids during the reparative phase after myocardial infarction injury.

Myocardial infarction (MI) is a fatal manifestation of coronary heart disease, and its underlying mechanism is still largely unknown. Lipid levels and composition alterations predict the risk of MI complications. Glycerophospholipids (GPLs) are important bioactive lipids and play a crucial role in the development of cardiovascular diseases. However, the metabolic changes in the GPLs profile during post-MI injury remain unknown. In the current study, we constructed a classic MI model by ligating the left anterior descending branch and assessed the alterations in both plasma and myocardial GPLs profiles during the reparative phase post-MI by liquid chromatography-tandem mass spectrometry analysis. We found that myocardial GPLs, but not plasma GPLs, were markedly changed after MI injury. Importantly, MI injury is associated with decreased phosphatidylserine (PS) levels. Consistently, the expression of phosphatidylserine synthase 1 (PSS1), which catalyzes the formation of PS from its substrate phosphatidylcholine, was significantly reduced in heart tissues after MI injury. Furthermore, oxygen-glucose deprivation (OGD) inhibited PSS1 expression and reduced PS levels in primary neonatal rat cardiomyocytes, while overexpression of PSS1 restored the inhibition of PSS1 and the reduction in PS levels caused by OGD. Moreover, overexpression of PSS1 abrogated, whereas knockdown of PSS1 aggravated, OGD-induced cardiomyocyte apoptosis. Our findings revealed that GPLs metabolism was involved in the reparative phase post-MI, and cardiac decreased PS levels, resulting from inhibition of PSS1, are important contributor to the reparative phase post-MI. PSS1 overexpression represents a promising therapeutic strategy to attenuate MI injury.

CRISPR/Cas9 Knock-Out in Primary Neonatal and Adult Cardiomyocytes Reveals Distinct cAMP Dynamics Regulation by Various PDE2A and PDE3A Isoforms.

Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50-60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes.

Cardioprotective Effects of Dexmedetomidine in an Oxidative-Stress In Vitro Model of Neonatal Rat Cardiomyocytes.

Preterm birth is a risk factor for cardiometabolic disease. The preterm heart before terminal differentiation is in a phase that is crucial for the number and structure of cardiomyocytes in further development, with adverse effects of hypoxic and hyperoxic events. Pharmacological intervention could attenuate the negative effects of oxygen. Dexmedetomidine (DEX) is an α2-adrenoceptor agonist and has been mentioned in connection with cardio-protective benefits. In this study, H9c2 myocytes and primary fetal rat cardiomyocytes (NRCM) were cultured for 24 h under hypoxic condition (5% O2), corresponding to fetal physioxia (pO2 32-45 mmHg), ambient oxygen (21% O2, pO2 ~150 mmHg), or hyperoxic conditions (80% O2, pO2 ~300 mmHg). Subsequently, the effects of DEX preconditioning (0.1 µM, 1 µM, 10 µM) were analyzed. Modulated oxygen tension reduced both proliferating cardiomyocytes and transcripts (CycD2). High-oxygen tension induced hypertrophy in H9c2 cells. Cell-death-associated transcripts for caspase-dependent apoptosis (Casp3/8) increased, whereas caspase-independent transcripts (AIF) increased in H9c2 cells and decreased in NRCMs. Autophagy-related mediators (Atg5/12) were induced in H9c2 under both oxygen conditions, whereas they were downregulated in NRCMs. DEX preconditioning protected H9c2 and NRCMs from oxidative stress through inhibition of transcription of the oxidative stress marker GCLC, and inhibited the transcription of both the redox-sensitive transcription factors Nrf2 under hyperoxia and Hif1α under hypoxia. In addition, DEX normalized the gene expression of Hippo-pathway mediators (YAP1, Tead1, Lats2, Cul7) that exhibited abnormalities due to differential oxygen tensions compared with normoxia, suggesting that DEX modulates the activation of the Hippo pathway. This, in the context of the protective impact of redox-sensitive factors, may provide a possible rationale for the cardio-protective effects of DEX in oxygen-modulated requirements on survival-promoting transcripts of immortalized and fetal cardiomyocytes.

Pristimerin protects against pathological cardiac hypertrophy through improvement of PPARα pathway

Pristimerin (PM), serving as a biological component mainly obtained from Celastraceae and Hippocrateaceae families, has been extensively explored for its numerous pharmacological activities, especially anti-cancer activity. However, the function of PM on pathological cardiac hypertrophy is poorly understood. This work was intended to investigate the effects of PM on pressure-overload induced myocardial hypertrophy and its potential pathways. Mouse model of pathological cardiac hypertrophy was generated by transverse aortic constriction (TAC) or minipump administration of the β-adrenergic agonist ISO for 4 weeks, and PM (0.5 mg/Kg/d, i.p.) was treated for 2 weeks. PPARα−/− mice received TAC surgery were used for mechanism exploration. Moreover, neonatal rat cardiomyocytes (NRCMs) were utilized to explore the effect of PM following Angiotensin II (Ang II, 1.0 μM) administration. We found that PM attenuated pressure-overload induced cardiac dysfunction, myocardial hypertrophy and fibrosis in mice. Likewise, PM incubation dramatically reversed Ang II-mediated cardiomyocytes hypertrophy in NRCMs. RNA-Sequence showed that PM selectively contributed to improvement of PPARα/PGC1 signaling, while silencing PPARα abrogated the beneficial effects of PM on Ang II-treated NRCMs. Importantly, PM ameliorated Ang II-induced mitochondrial dysfunction and decrease in metabolic genes, whereas knockdown of PPARα eliminated these alterations in NRCMs. Similarly, PM presented limited protective effects on pressure-overload induced systolic dysfunction and myocardial hypertrophy in PPARα deficient mice. Overall, this study revealed that PM exerted protective activity against pathological cardiac hypertrophy through improvement of PPARα/PGC1 pathway.

Epigallocatechin-3-gallate restores mitochondrial homeostasis impairment by inhibiting HDAC1-mediated NRF1 histone deacetylation in cardiac hypertrophy.

Decompensated cardiac hypertrophy is accompanied by impaired mitochondrial homeostasis, whether histone acetylation is involved in this process is yet to be determined. The role of HDAC1-mediated NRF1 histone deacetylation was investigated in transverse aortic constriction (TAC)-induced hypertrophy in rats and phenylephrine (PE)-induced hypertrophic cardiomyocytes. Administration of epigallocatechin-3-gallate (EGCG), an inhibitor of HDAC1, restored cardiac function, decreased heart/body weight and fibrosis, increased the ratio of mtDNA/nDNA and the percentage of LysoTracker+ CMs in TAC, compared with TAC without receiving EGCG. In PE-treated hypertrophic H9C2 cells, EGCG attenuated cell hypertrophy and increased LC3B II+MitoTracker+ puncta, as well as the ratio of mtDNA/nDNA. Interestingly, NRF1 but not PGC-1α expression was decreased in TAC- or PE-induced hypertrophic hearts or cells, respectively, while EGCG upregulated both NRF1 and PGC-1α in vitro. EGCG treatment also increased the interaction between PGC-1α and NRF1. In addition to inhibiting HDAC1 expression, EGCG decreased the binding of HDAC1 and increased the binding of acH3K9 or acH3K14 in the promotor regions of PGC-1α and NRF1. In neonatal rat cardiomyocytes, restored NRF1, TFAM and FUNDC1 were abolished by the overexpression of HDAC1. Collectively, data suggest that NRF1 reduction was averted by EGCG via inhibiting HDAC1-mediated histone deacetylation. Acetylation of NRF1 histone may play a key role in maintaining mitochondrial homeostasis associated with cardiac hypertrophy.

ANGIOTENSIN II REDUCES THE EXPRESSION OF CX43 AND INHIBITS THE C-SRC /MAPK/ERK PATHWAY VIA CX43 IN CARDIOMYOCYTES

Objective: Hypertension is associated with abnormal renin angiotensin system (RAS). Angiotensin II (AngII), an important substance in RAS, is closely related to cardiomyocyte injury. Connexin43 (Cx43), a key structural protein in cardiomyocyte membrane, takes part in signal transduction and substance transport. Cx43 may cause extracellular interstitial fibrosis. It is known that Cx43 increases collagen I in heart tissue by regulating nuclear factor kappa B and Mechanistic metalloproteinase. However, the relationship between Ang II and Cx43 has been controversial,and how Ang II affects Cx43-related pathway is unclear. Otherwise, the activation and inhibition of the c-SRC /MAPK/ERK pathway may is related to Cx43, but the fact which is the forward is not sure. Objective:The purpose of this study is to explore whether Ang II affects the expression of Cx43 and inhibits c-Src /MAPK/ERK pathway via Cx43. Design and method: First, we given rat cardiomyocytes (H9C2 cells) different concentrations of Ang II (10-5M,10-6M and 10-7M,for 48h), and establish Ang II groups. There is a contral group, which was given normal culture without Ang II. Second,we given H9C2 cells the Gap26,which is the inhibition of Cx43. Proteins of Cx43 and c-SRC /MAPK/ERK pathway from all groups were detected by Western Blot, and mRNA of these proteins were detected by PCR. SPSS was used for statistical analysis to compare the difference between all the groups. Results: First, compared with the control group, protein of Cx43, c-SRC, ERK2 and ERK1 were significantly decreased in Ang II group, and when the concentration of Ang II was 10-5M, these proteins were the least. mRNA of Cx43, ERK2 and ERK1 also were cut down after giving Ang II (only 10-5M). Second, the Gap26-intervention group had the lower expression protein and mRNA of Cx43, c-SRC, ERK2 and ERK1. Conclusions: We proved that Angiotensin II can reduce the expression of Cx43 and inhibit c-SRC /MAPK/ERK pathway via Cx43 in cardiomyocytes. Based on the role of Cx43 and c-Src /MAPK/ERK pathway, hypertesion may injury the cardiomyocytes via RAS and these pathways.

Radiation fractionation effect on IDO1 through NF-kB/COX2 axis in non-small cell lung cancer.

e20613 Background: Lung cancer is the leading cancer mortality worldwide. Radiotherapy(RT) plays an important role in lung cancer treatment. Different does fractionation of RT has different effects on the prognosis of lung cancer. For example, stereotactic body radiotherapy can significantly improve the prognosis compared with conventionally Fractionated Radiotherapy. In non-small cell lung cancer, we have previously shown that Indoleamine 2,3-dioxygenase 1 (IDO1) metabolites and radiation induced changes had significant effects on long-term survival in patients treated with either Stereotactic body Radiation Therapy of 10Gy fraction size or conventional fractionated radiotherapy of 2Gy fraction size. However, the underlining molecular mechanism is unknown. In this study, we aimed to elaborate the molecular mechanism of such clinical differential effects in the laboratory. Methods: This study applied two dose/fractionation regimens with a similar BED10: 10Gyx1 and 2Gyx8. 10Gyx1 and 2Gyx8 were tested in cell lines of A549, H1299, Calu-1, TC-1 of human and murine NSCLC as well as normal rat cardiomyocyte cell line H9c2, and in vivo (TC-1 implanted C57Bl/6 mice). The mRNA and protein levels of IDO1, p-NF-kB, COX2 from cells and tumor tissues were determined by using qPCR, western blot and immunohistochemistry. Survival outcome was tested by colony formation assay in vitro and tumor growth curve in vivo. Results: In vitro, 10Gyx1 and 2Gyx8 induced IDO1 expression in all tested cell lines and the level of IDO1 expression induced by 10Gyx1 was lower than 2Gyx8. Both 10Gyx1 and 2Gyx8 increased p-NF-kB and COX2 expression and the level induced by 10Gyx1 was lower than 2Gyx8. In tumor bearing mice, two dose/fractionation regimens also induced intratumor IDO1 expression and increased the levels of p-NF-kB and COX2. Interestingly, the increased levels of IDO1, p-NF-kB, COX2 after 10Gyx1 were also significantly lower than 2Gyx8 in vivo. IDO inhibitor synergized with RT and enhanced cell killing efficacy of RT. Conclusions: The findings of this study suggest different does fractionation of RT induced up-regulation of IDO1 through NF-kB/COX2 axis in NSCLC and non-cancerous cell lines, what’s more, hypofractionated radiotherapy 10Gyx1 had less upregulation that of conventionally fractionated radiotherapy 2Gy x8 on IDO1 immune inhibitory pathway. The finding of hypofractionated radiotherapy less immune suppression differ from the traditional belief of more immune stimulation. Our current study provides a rationale in using IDO1 as a biomarker to design RT dose fractionation and/or immunotherapy in combination and possibilities for clinical trials to design IDO1 inhibitors in combination with radiotherapy.

Akt2 deficiency alleviates oxidative stress in the heart and liver via up-regulating SIRT6 during high-fat diet-induced obesity.

The present study aims to investigate the role of AKT2 in the pathogenesis of hepatic and cardiac lipotoxicity induced by lipid overload-induced obesity and identify its downstream targets. WT and Akt2 KO mice were fed either normal diet, or high-fat diet (HFD) to induce obesity model in vivo. Human hepatic cell line (L02 cells) and neonatal rat cardiomyocytes (NRCMs) were used as in vitro models. We observed that during HFD-induced obesity, Akt2 loss-of-function mitigated lipid accumulation and oxidative stress in the liver and heart tissue. Mechanistically, down-regulation of Akt2 promotes SIRT6 expression in L02 cells and NRCMs, the latter deacetylates SOD2, which promotes SOD2 activity and therefore alleviates oxidative stress-induced injury of hepatocytes and cardiomyocytes. Furthermore, we also proved that AKT2 inhibitor protects hepatocytes and cardiomyocytes from HFD-induced oxidative stress. Therefore, our work prove that AKT2 plays an important role in the regulation of obesity-induced lipid metabolic disorder in the liver and heart. Our study also indicates AKT2 inhibitor as a potential therapy for obesity-induced hepatic and cardiac injury.