H9c2 Cells Research Articles

PI3K p85α/HIF-1α accelerates the development of pulmonary arterial hypertension by regulating fatty acid uptake and mitophagy

Abstract Background Pulmonary arterial hypertension (PAH) is characterized by lipid accumulation and mitochondrial dysfunction. This study was designed to investigate the effects of hypoxia-inducible factor-1α (HIF-1α) on fatty acid uptake and mitophagy in PAH. Methods Peripheral blood samples were obtained from PAH patients. Human pulmonary arterial smooth muscle cells and rat cardiac myoblasts H9c2 were subjected to hypoxia treatment. Male Sprague–Dawley rats were treated with monocrotaline (MCT). Right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), pulmonary artery remodeling, and lipid accumulation were measured. Cell proliferation and ROS accumulation were assessed. Mitochondrial damage and autophagosome formation were observed. Co-immunoprecipitation was performed to verify the interaction between HIF-1α and CD36/PI3K p85α. Results HIF-1α, CD36, Parkin, and PINK1 were upregulated in PAH samples. HIF-1α knockdown or PI3K p85α knockdown restricted the expression of HIF-1α, PI3K p85α, Parkin, PINK1, and CD36, inhibited hPASMC proliferation, promoted H9c2 cell proliferation, reduced ROS accumulation, and suppressed mitophagy. CD36 knockdown showed opposite effects to HIF-1α knockdown, which were reversed by palmitic acid. The HIF-1α activator dimethyloxalylglycine reversed the inhibitory effect of Parkin knockdown on mitophagy. In MCT-induced rats, the HIF-1α antagonist 2-methoxyestradiol (2ME) reduced RVSP, RVHI, pulmonary artery remodeling, lipid accumulation, and mitophagy. Recombinant CD36 abolished the therapeutic effect of 2ME but inhibited mitophagy. Activation of Parkin/PINK1 by salidroside (Sal) promoted mitophagy to ameliorate the pathological features of PAH-like rats, and 2ME further enhanced the therapeutic outcome of Sal. Conclusion PI3K p85α/HIF-1α induced CD36-mediated fatty acid uptake and Parkin/PINK1-dependent mitophagy to accelerate the progression of experimental PAH. Graphical Abstract

H2 protects H9c2 cells from hypoxia/reoxygenation injury by inhibiting the Wnt/CX3CR1 signaling pathway

H2 protects H9c2 cells from hypoxia/reoxygenation injury by inhibiting the Wnt/CX3CR1 signaling pathway

Berberine ameliorates septic cardiomyopathy through protecting mitochondria and upregulating Notch1 signaling in cardiomyocytes

IntroductionSeptic cardiomyopathy (SCM) arises as a consequence of sepsis-associated cardiovascular dysfunction, for which there is currently no specific targeted therapy available. Previous studies have demonstrated the beneficial therapeutic effect of berberine (BBR) on SCM; however, the underlying mechanisms of action remain unclear. The objective of this is to elucidate how BBR alleviates SCM.MethodsSeptic cardiomyopathy rat model was established by performing cecal ligation and puncture (CLP), while a cardiomyocyte injury model was provoked in H9C2 cells using lipopolysaccharide (LPS). Cardiac function was assessed through echocardiography, and myocardial histopathology was examined with hematoxylin-eosin (HE) staining. Cardiomyocyte viability was determined through Cell Counting Kit-8 (CCK8) assay, and measurement of ATP levels was done with an ATP assay kit. Mitochondrial ultrastructure was observed using transmission electron microscopy. Real-time polymerase chain reaction (RT-PCR) and Western blotting were employed to analyze the expression of Notch1 signaling pathway components and downstream molecules in myocardial tissues and cells.ResultIn vivo, BBR markedly improved symptoms and cardiac function in SCM rats, leading to enhanced ATP content, and ameliorated mitochondrial structure. Additionally, BBR increased Notch1 protein expression in myocardial tissue of the rats. In vitro, BBR elevated the survival rates of H9C2 cell, improved mitochondrial morphology, and raised ATP levels. The mRNA expression of Notch1, Hes1, and Hes2, and Notch1 protein expression was upregulated by BBR. While these effects were reversed upon inhibiting the Notch1 signaling pathway.ConclusionBBR improves septic cardiomyopathy by modulating Notch1 signaling to protect myocardial mitochondria.

Exploring the mechanism of sesamin for the treatment of PM2.5-induced cardiomyocyte damage based on transcriptomics, network pharmacology and experimental verification

IntroductionExposure to fine particulate matter (PM2.5) is known to be associated with cardiovascular diseases. Sesamin (Ses) is a natural phenolic compound found in sesame seeds and sesame oil. Ferroptosis is a novel mode of cell death characterised by iron-dependent lipid peroxidation. This study aims to explore whether PM2.5 can induce ferroptosis in H9C2 cells and to investigate the precise protective mechanism of Ses.MethodsBased on transcriptomic data, PM2.5 may induce ferroptosis in cardiomyocytes. The ferroptosis inducer erastin and ferroptosis inhibitor ferrostatin-1 (Fer-1) were used to illustrate the mechanisms involved in PM2.5-induced H9C2 cell injury. Using network pharmacology, the pharmacological mechanism and potential therapy targets of Ses were explored for the treatment of PM2.5-induced cardiomyocyte injury. H9C2 cells were cultured and pretreated with Fer-1 or different concentrations of Ses, and then cardiomyocyte injury model was established using erastin or PM2.5. Indicators of oxidative responses, including total superoxide dismutase, reduced glutathione, glutathione peroxidase and malondialdehyde, were measured. The expression levels of ferroptosis-related proteins were determined through Western blot analysis.ResultsResults demonstrate that PM2.5 induces ferroptosis in H9C2 cells and Ses exerts a protective effect by suppressing ACSL4-mediated ferroptosis.DiscussionOverall, these findings elucidate a novel mechanism by which Ses ameliorates the detrimental effects of PM2.5 on cardiomyocytes.

Resveratrol relieves myocardial ischemia–reperfusion injury through inhibiting AKT nitration modification

ABSTRACT Objective The aim of this study was to clarify whether Protein kinase B (PKB)/AKT is nitrated in myocardial ischemia and reperfusion injury (MIRI) resveratrol (RSV)’s protective effect during this process. Methods We blocked blood flow of the left coronary artery (LAD) of mice and used H9c2 cells under an oxygen-glucose deprivation (OGD) environment as animal and cell models of MIRI. N-methyl-D-aspartic acid receptor (NMDAR) inhibitor MK801, neuronal nitric oxide synthase (nNOS) inhibitor 7-NI and RSV were used as interventions. Nitration of proteins, infarction area, cardiomyocyte apoptosis and AKT nitration sites were detected during this study. Results During in-vivo study, AKT nitration was induced through the NMDAR/nNOS/peroxynitrite (ONOO–) pathway, leading to decreased phosphorylation of AKT and increased cardiomyocyte apoptosis. AKT nitration was decreased and phosphorylation was elevated when administrated with RSV, MK801 and 7-NI. In in-vitro study, AKT nitration and TUNEL positive cells was elevated when administrated with NO donor H9c2 cells after OGD/R, when administrated with RSV, MK801 and 7-NI, AKT nitration and apoptosis was deceased in H9c2 cells. Mass spectrometry revealed that nitration sites of AKT included 14 Tyrosine residues. Discussion RSV could inhibit AKT nitration and elevated phosphorylation through suppressing NMDAR/nNOS/ONOO– pathway and further reduce the apoptosis of cardiomyocytes in of myocardial I/R.

CMC/Gel/GO 3D-printed cardiac patches: GO and CMC improve flexibility and promote H9C2 cell proliferation, while EDC/NHS enhances stability.

In this research, carboxymethyl cellulose (CMC)/gelatin (Gel)/graphene oxide (GO)-based scaffolds were produced by using extrusion-based 3D printing for cardiac tissue regeneration. Rheological studies were conducted to evaluate the printability of CMC/Gel/GO inks, which revealed that CMC increased viscosity and enhanced printability. The 3D-printed cardiac patches were crosslinked with N-(3-dimethylaminopropyl)-n'-ethylcarbodiimide hydrochloride (EDC)/ N-hydroxysuccinimide (NHS) (100:20 mM, 50:10 mM, 25:5 mM) and then characterized by mechanical analysis, electrical conductivity testing, contact angle measurements and degradation studies. Subsequently, cell culture studies were conducted to evaluate the viability of H9C2 cardiomyoblast cells by using the Alamar Blue assay and fluorescence imaging. A high concentration of EDC/NHS (100:20 mM) led to the stability of the patches; however, it drastically reduced the flexibility of the scaffolds. Conversely, a concentration of 25:5 mM resulted in flexible but unstable scaffolds in PBS solution. The suitable EDC/NHS concentration was found to be 50:10 mM, as it produced flexible, stable, and stiff cardiac scaffolds with high ultimate tensile strength (UTS). Mechanical characterization revealed that % the strain at break of C15/G7.5/GO1 exhibited a remarkable increase of 61.03% compared to C15/G7.5 samples. The improvement of flexibility was attributed to the hydrogen bonding between CMC, Gel and GO. The electrical conductivity of 3D printed CMC/Gel/GO cardiac patches was 7.0×10-3 S/cm, demonstrating suitability for mimicking the desired electrical conductivity of human myocardium. The incorporation of 1 wt% of GO and addition of CMC concentration from 7.5 wt % to 15 wt % significantly enhanced relative % cell viability. Overall, although this research is at its infancy, CMC/Gel/GO cardiac patches have potential to improve the physiological function of cardiac tissue.

Role of the ROS/NLRP3/caspase-1 pathway in NiSO4-induced cellular pyroptosis and apoptosis in H9c2 cells

According to comprehensive research, the cardiovascular system is damaged by nickel exposure. The present study selected rat cardiomyocytes (H9c2 cells) and subjected them to varying doses of sodium nickel sulfate (NiSO4) for 24hours to better understand the mechanism of cardiovascular damage caused by NiSO4 exposure. The relevant indicators were detected employing biochemical analysis, real-time quantitative polymerase chain reaction (RT-qPCR), and western blot, and flow cytometry was used to detect the cell apoptosis rate. The study revealed that the survival rate of H9c2 cells fell significantly when the concentration of NiSO4 exposure rose. Moreover, it caused oxidative stress in H9c2 cells by raising the expression of reactive oxygen species and the concentration of LDH in the cell supernatants. After NiSO4 exposure, the levels of ASC, NLRP3, gasdermin D, and caspase-1 in H9c2 cells increased, suggesting that H9c2 cells underwent pyroptosis induced by NiSO4. In addition, NiSO4 exposure also led to inflammation, with increased levels of interleukin [IL]-18, IL-1β. After adding the antioxidant N-Acetyl-L-cysteine (NAC), the level of ROS indicated that the oxidative stress level in H9c2 cells was reduced, western blot inhibited inflammation, the level of pyroptosis was reduced, and the activity of the NLRP3/caspase1 signaling pathway was reduced. To examine the connection between pyroptosis and apoptosis, the cells were treated with the caspase1 inhibitor Z-YVAD-Fluoromethyl Ketone (Z-YVAD-FMK, YVAD), which resulted in a significant decrease in the rate of cell apoptosis as well as a reduction in the activity of the related protein in the signaling pathway, which in turn decreased the level of pyroptosis. NiSO4 could induce pyroptosis in H9c2 cells through the ROS/NLRP3/caspase-1 axis. Furthermore, NiSO4-induced apoptosis and pyroptosis were found to be reduced by the addition of the caspase-1 inhibitor.

The Decreased Proliferation Capacity of Cardiomyocytes Induced By Androsterone Is Mediated By the Interactions Between Androgen Receptor and Retinoblastoma Protein.

Our previous study has demonstrated that the decline in cardiomyocytes proliferation capacity induced by maternal androgen excess was mainly attributed to the accumulation of androsterone in the heart. However, the underlying mechanism by which androsterone inhibits cardiomyocytes proliferation remains unknown. In this study, pregnant mice were injected subcutaneously daily with dihydrotestosterone (DHT) from gestational day (GD) 16.5 to GD18.5. On GD18.5, fetal heart tissue was dissected and used for analyzing androgen receptor (AR) levels. H9c2 cells and primary cardiomyocytes, isolated from fetal hearts, were applied to investigate the mechanism. H9c2 cells under androsterone treatment were subjected to RNA sequencing analysis and the results showed that genes were primarily enriched in cell cycle and DNA replication pathways. Elevated AR levels were observed in fetal cardiac tissue in the maternal DHT-treated group. Androsterone treatment increased the ratio of nuclear AR and cytoplasmic AR both in H9c2 cells and primary cardiomyocytes. The ablation and overexpression of AR can mildly reverse and aggravate cell cycle arrest induced by androsterone, respectively. ChIP-qPCR analysis suggested that AR can directly repress cell cycle and DNA replication-related gene expression, which was mediated by the recruitment of retinoblastoma protein (Rb). The repression of cell proliferation in response to androsterone was alleviated partly through the downregulation of Rb by siRNA transfection. In conclusion, AR repression to cell cycle and DNA replication-related gene expression, mediated by recruitment of Rb, may be one of the potential mechanisms of cell cycle arrest in cardiomyocytes induced by androsterone.

Comparative cytotoxicity and mitochondrial disruption in H9c2 cardiomyocytes induced by common pesticides

Chronic exposure to pesticides is believed to be associated with various human diseases, including cardiovascular diseases. However, the mechanisms by which pesticides lead to cardiovascular diseases remain unclear. In our study, we selected the following commonly used pesticides as typical examples: the herbicides glyphosate (GLY) and glufosinate ammonium (GLA); the insecticides imidacloprid (IMI) and thiamethoxam (THM); and the fungicides pyraclostrobin (PYR) and azoxystrobin (AZO). We employed H9c2 cells as a model to investigate the cytotoxic effects of these pesticides on myocardial cells at concentrations of 1, 10, 100, and 1000 mg/L. The results indicate that these pesticides can affect cell viability, alter the cell cycle, and significantly impact ATP content and mitochondrial complex levels, ultimately triggering oxidative stress responses in the cells. However, compared to herbicides GLY and GLA, insecticides IMI and THM, and fungicides PYR and AZO pesticides are more toxic to H9c2 cells. Additionally, GLY, GLA, IMI, THM, PYR, and AZO were found to cause structural changes in the mitochondria of H9c2 cells. Molecular docking results suggest that these pesticides can bind to proteins related to mitochondrial dynamics. Furthermore, IMI, THM, PYR, and AZO exhibit stronger binding abilities to mitochondrial dynamics-related proteins. These findings indicate that these pesticides significantly adverse effects on myocardial cells, mainly by causing mitochondrial dysfunction and inducing oxidative stress. Our findings highlight the importance of considering the differential toxicity of various classes of pesticides when assessing their risks to human health, particularly concerning cardiovascular diseases.

EMP1 knockdown mitigated high glucose-induced pyroptosis and oxidative stress in rat H9c2 cardiomyocytes by inhibiting the RAS/RAF/MAPK signaling pathway.

The purpose of this study was to investigate the mechanism of EMP1 action in high glucose (HG)-induced H9c2 cardiac cell pyroptosis and oxidative injury. Rat cardiomyocytes H9c2 were exposed to 33 mM glucose for 24, 48, or 72 h to induce cytotoxicity. EMP1-siRNA, NLRP3 agonist Nigericin, and pcNDA-RAS were used to treat H9c2 cells under HG conditions. Cell Counting Kit (CCK)-8 assay showed that cell proliferation was decreased following HG induction, which was rescued by EMP1 knockdown. Our results also suggested that EMP1 siRNA transfection significantly decreased the apoptosis and pyroptosis of HG-induced cells, as indicated by the reduction of NLRP3 IL-1β, ASC, GSDMD, cleaved-caspase1 and cleaved-caspase3 levels in HG-induced H9c2 cells. In addition, EMP1 knockdown alleviated HG-induced mitochondrial damage and oxidative stress in H9c2 cells. NLRP3 activation reversed the inhibitory effects of EMP1 knockdown on pyroptosis and oxidative stress in HG-induced H9c2 cells. Mechanistically, we found that EMP1 knockdown suppressed the RAS/RAF/MAPK signaling pathway in HG-induced H9c2 cells. RAS overexpression blocked the protective effect of EMP1 knockdown on HG-induced H9c2 cell apoptosis, pyroptosis, and oxidative injury. Our findings suggest that EMP1 knockdown treatment might provide a novel therapy for diabetic cardiomyopathy.

Icariin ameliorates Coxsackievirus B3-induced viral myocarditis by modulating the S100 calcium binding protein A6/β-catenin/c-Myc signaling pathway

BackgroundCoxsackievirus B3 (CVB3) is a leading cause of viral myocarditis and is currently lacking specific pharmacological treatments, highlighting the critical need for therapeutic development. Icariin (ICA), a prenylated flavonol glycoside, was previously found to exhibit several pharmacological effects, but its potential to combat CVB3 remains uninvestigated. PurposeThis study aimed to elucidate the anti-CVB3 efficacy of ICA and elucidate its molecular mechanisms. MethodsCVB3-infected HeLa cells, H9C2 cells and neonate rat ventricular cardiomyocytes (NRVCs) were selected as in vitro models, and were treated with ICA at 1 and 10 μM. Additionally, BALB/c mice that were infected with CVB3 via intraperitoneal injection were chosen as in vivo model and were treated with ICA or ribavirin over 3 days. The effect of ICA against CVB3 was determined by Cell Counting Kit-8 (CCK-8) assay, western blot, real-time fluorescence quantitative PCR (RT–qPCR), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and flow cytometry. ResultsIn this study, it was found that ICA is capable of reducing CVB3 viral load both in vitro and in vivo. Mechanistic studies suggested that ICA prevents cardiomyocyte apoptosis by attenuating the S100 calcium binding protein A6 (S100A6)/β-catenin/c-Myc signaling pathway. Additionally, ICA inhibits the secretion of proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β) and CXC motif chemokine ligand 2 (CXCL2) in heart tissue, thereby mitigating CVB3-induced myocarditis. Moreover, ICA also regulates the immune response of CD4+ T, CD8+ T and Treg cells by changing the cells numbers in spleen tissue. Lastly, ICA can reduce the load of other enteroviruses (such as CVA6, CVA16 and EV71) in rhabdomyosarcoma (RD) cells as well. ConclusionOur findings indicate that ICA provides significant protection against CVB3 infection by modulating the S100A6/β-catenin/c-Myc signaling pathway, suggesting its potential use as a novel drug against CVB3 infection in clinical application.

CaMKII Exacerbates Doxorubicin‐Induced Cardiotoxicity by Promoting Ubiquitination Through USP10 Inhibition

ABSTRACTBackgroundDoxorubicin (DOX) is an effective anticancer drug, but it has a problem of cardiotoxicity that cannot be ignored. Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is tightly associated with the pathological progression of DOX‐induced cardiotoxicity. Ubiquitin‐specific protease 10 (USP10) plays an important role in many biological processes and cancers. However, its association with DOX‐induced cardiotoxicity and CaMKII remains unclear.MethodsH9C2 cells, HL‐1 cells and C57BL/6 mice were used to establish the DOX‐induced cardiotoxicity model, and the CaMKII‐specific inhibitor KN‐93 and USP10 specific inhibitor Spautin‐1 were used to observe the CaMKII and USP10 effect. In cell experiments, CCK‐8 method was used to assess cell viability, LDH kit was used to assess lactate dehydrogenase expression, DCFH‐DA staining was used to observe changes in active oxygen content, TUNEL staining was used to observe cell apoptosis, and Western blotting method was used to detect relevant protein markers. The expression of p‐CaMKII and USP10 was assessed by immunofluorescence staining. In animal experiments, mouse echocardiograph was used were used to evaluate cardiac function, and HE staining and Masson staining were used to evaluate myocardial injury. Cardiomyocyte apoptosis was detected by TUNEL staining. Western blotting method was used to detect relevant protein markers.ResultsOur results demonstrated that activation of CaMKII and inhibition of USP10 pathway related to DOX‐induced cardiotoxicity. Inhibition of CaMKII with KN‐93 ameliorated DOX‐induced cardiac dysfunction and cytotoxicity. In addition, CaMKII inhibition prevented DOX‐induced apoptosis and ubiquitination. Furthermore, CaMKII inhibition increased USP10 expression in DOX‐treated mouse hearts, H9C2 cells and HL‐1 cells. At last, the USP10 inhibitor, Spautin‐1, blocked the regulatory effect of CaMKII inhibition on apoptosis and ubiquitination in DOX‐induced cardiotoxicity.ConclusionOur findings revealed that DOX‐induced myocardial apoptosis and activated CaMKII through cellular and animal levels, while providing a novel probe into the mechanism of CaMKII action: promoting ubiquitination by inhibiting USP10 aggravated apoptosis.

DanShen Decoction targets miR-93-5p to provide protection against MI/RI by regulating the TXNIP/NLRP3/Caspase-1 signaling pathway

BackgroundBone marrow mesenchymal stem cells (BMSCs) derived exosomes have demonstrated potential therapeutic efficacy on myocardial ischemia/reperfusion injury (MI/RI). This study has explored the underlying mechanisms of Danshen decoction (DSD) pretreated BMSCs-exosomes to treat MI/RI in vivo and in vitro. MethodsExtracellular vesicles extracted from BMSCs were identified, miRNA sequencing was performed to screen the effects of DSD, and verified to target TXNIP in vivo. After MI/RI modeling, rats were treated with BMSCs-exosomes pretreated with DSD or miRNA inhibitor. BMSCs-exosomes, DSD-pretreated BMSCs-exosomes, and miRNA inhibitor/anti-miRNA-pretreated BMSCs-exosomes were used to treat H9c2 cells or MI/RI rats. CCK-8, Tunnel staining, and flow cytometry were performed to measure cell viability. LDH, CK, CK-MB were detected to evaluate cell injury. MDA, SOD, and ROS were used to confirm oxidative stress. Furthermore, IL-1β, IL-18, cleaved-caspase-1, pro-caspase-1, NLRP3, TXNIP, and GSDMD were quantified for the TXNIP/NLRP3/Caspase-1 signaling activation. In addition, echocardiography was used to observe the heart function, and H&E stain was performed to detect pathological injury. ResultsFollowing DSD pretreatment, there was a marked elevation in the expression levels of miR-93-5p, miR-16-5p, and miR-15b-5p, with miR-93-5p exhibiting the highest baseMean value. The administration of a miR-93-5p inhibitor yielded effects counteractive to those observed with DSD treatment, leading to reduced cell proliferation, heightened oxidative stress (as indicated by increased levels of SOD and ROS, alongside a decrease in MDA), and enhanced cell apoptosis. Furthermore, DSD effectively mitigated the miR-93-5p-induced upregulation of key inflammatory and apoptotic markers, including IL-1β, IL-18, caspase-1, NLRP3, TXNIP, and GSDMD. Notably, exosomes derived from DSD-pretreated BMSCs demonstrated a capacity to alleviate cardiac damage. ConclusionDSD may target miR-93-5p within BMSC-derived exosomes to confer protection against cardiac damage by inhibiting the activation of the TXNIP/NLRP3/Caspase-1 signaling pathway, thereby mitigating cardiomyocyte pyroptosis. This study provides a theoretical foundation for the application of DSD in the treatment of MI/RI.

Detoxification and benefits on acute heart failure in mice- of fuziline using glycyrrhetinic acid: an integrated biochemical analysis

IntroductionAconiti Lateralis Radix Praeparata (lateral roots of Aconitum carmichaelii Debeaux, Fuzi), is commonly used to treat various cardiovascular diseases, particularly heart failure. However, its strong cardiotoxicity limits its clinical applicability. Glycyrrhizae radix et rhizoma, (the root of Glycyrrhiza uralensis Fisch., Gancao), is known to synergistically increase the cardiotonic effects of Fuzi and alleviate the myocardial injury caused by Fuzi to some extent. However, the detailed mechanism via which the combination of Fuzi and Gancao reduces toxicity and increases or preserves the efficacy of Fuzi requires further investigation.MethodsOxidative stress injury models in H9C2 cells and mice with acute heart failure were established to evaluate the optimal synergistic protective concentration of Fuziline and Glycyrrhetinic acid (GA). A GA probe was then synthesized and used for target fishing using chemical and biological methods. Finally, the target and its function were verified using fluorescence co-localization, Western blotting, protein interaction analysis, molecular docking, and calcium ion imaging.ResultsThe best pharmacodynamic potential was achieved with a 1:1 or 2:1 ratio of Fuziline and GA concentrations. At these ratios, they regulated the protein levels of the downstream players of the Ca2+ signaling pathway via MDH2 and CALR, thereby balancing Ca2+ homeostasis in the myocardial tissue and mitigating the effects of heart failure.ConclusionThis study aimed to investigate the compatibility of Fuziline and GA, the active metabolites of a traditional Chinese medicine (TCM) pair, in exerting their cardiac effects, identify the direct biological targets and verify the mechanism of compatibility.

Cardioprotective effects of cinnamoyl imidazole on apoptosis and oxidative stress in hypoxia/reoxygenation-induced H9C2 cell lines

BackgroundThis study explored the effects of cinnamoyl imidazole on alleviating oxidative stress and apoptosis in hypoxia/reoxygenation (H/R)-induced H9C2 cells, using computational analysis with in-vitro validation. MethodsComputational techniques, including SwissADME and Swiss Target Prediction, were employed to predict the ADME properties and to identify targets of cinnamoyl imidazole. Differential gene expression (DEG) analysis was conducted on myocardial infarction (MI) datasets obtained from the Gene Expression Omnibus. Gene enrichment and molecular simulation studies were done to focus on apoptotic pathways. The computational findings were validated through In vitro experiments on H9C2 cardiomyocytes subjected to 8 h of hypoxia followed by 24 h of reoxygenation. Antioxidant enzyme levels (catalase, GST, GSH-Px, and SOD), mitochondrial membrane potential (ΔΨm), caspase-3 activity, and the expression of CASP3, MAPK8, JAK2, and BCL2L1 were assessed. ResultsCinnamoyl imidazole has demonstrated favourable pharmacokinetic properties, characterized by high gastrointestinal absorption and low toxicity with negative toxicity for organ endpoints. Molecular docking studies revealed the strong binding affinities for CASP3, MAPK8, and JAK2. In vitro results showed a significant increase in cell viability (94.7 % at 10 μM, p < 0.001) and antioxidant enzyme activity, along with a 64.3 % reduction in caspase-3 activity at 1000 μM (p < 0.01). Cinnamoyl imidazole treatment preserved mitochondrial membrane potential, downregulated pro-apoptotic genes CASP3 and MAPK8, and upregulated the anti-apoptotic gene BCL2L1. ConclusionCinnamoyl imidazole effectively mitigates oxidative stress and apoptosis in H/R-induced H9C2 cells, enhancing cell viability and antioxidant defenses while maintaining mitochondrial integrity.

Myocardial ischemia-reperfusion injury upregulates nucleostemin expression via HIF-1α and c-Jun pathways and alleviates apoptosis by promoting autophagy

Myocardial ischemia-reperfusion (I/R) injury, often arising from interventional therapy for acute myocardial infarction, leads to irreversible myocardial cell death. While previous studies indicate that nucleostemin (NS) is induced by myocardial I/R injury and mitigates myocardial cell apoptosis, the underlying mechanisms are poorly understood. Here, our study reveals that NS upregulation is critical for preventing cardiomyocyte death following myocardial I/R injury. Elevated NS protein levels were observed in myocardial I/R injury mouse and rat models, as well as Hypoxia/reoxygenation (H/R) cardiac cell lines (H9C2 cells). We identified binding sites for c-Jun and HIF-1α in the NS promoter region. Inhibition of JNK and HIF-1α led to a significant decrease in NS transcription and protein expression. Furthermore, inhibition of autophagy and NS expression promoted myocardial cell apoptosis in H/R. Notably, the cell model showed reduced LC3I transformation to LC3II, downregulated Beclin1, upregulated p62, and altered expression of autophagy-related proteins upon NS interference in H/R cells. These findings suggest that NS expression, driven by c-Jun and HIF-1α pathways, facilitates autophagy, providing protection against both myocardial I/R injury and H/R-induced cardiomyocyte apoptosis.

SNORD3A: a new possible circulating biomarker of human heart failure

Abstract Background Heart failure (HF) is a complex clinical syndrome and the leading cause of mortality, morbidity and hospitalization in industrialized countries. Human cardiac biopsies are invaluable resources for identifying cardiac signaling pathways, however blood fractions and peripheral blood mononuclear cells (PBMCs) could be used as a source of surrogate biomarkers of signaling pathway activation in human heart failure. Purpose In the present study we aimed to identify novel circulating biomarkers mirroring human myocardial signaling in HF and to study the role played by SNORD3A in cardiomyocyte survival and death. Methods Cardiac left ventricle samples and PBLs from 8 HF patients undergoing heart transplantation and 2 control patients (CTRL) were analyzed by RNA sequencing (RNASeq) to identify transcripts that were regulated in both hearts and PBLs. Five identified transcripts, KCNQOT1, MIAT, SCRN1, MALAT1 and SNORD3A, were then validated by quantitative real-time PCR in PBMCs and in LVs. Next, we analyzed the expression of the Small Nucleolar RNA (snoRNA) SNORD3A in LVs and PBMCs from 8-week-old wild type C57BL/6 mice with pressure overload-induced HF by transverse aortic constriction (TAC). Sham-operated mice (sham) were used as controls. To further investigate the role of SNORD3A in cardiomyocyte function and survival, SNORD3A antisense oligonucleotides (SNOaso) and scramble control sequences (GFPaso) were synthesized and tested in cardiomyoblasts H9C2 cells under normoxic or hypoxic conditions to evaluate SNORD3A transcription levels, cell death and protein synthesis. Results SNORD3A expression was significantly increased in both LVs and PBMCs from HF patients compared to control subjects. Consistent with these results, SNORD3A levels were increased both in PBMCs and LVs from TAC mice compared to sham. In vitro hypoxia significantly increased SNORD3A expression levels in H9C2 cells, compared to normoxia. After SNOaso transfection, SNORD3A transcripts were downregulated, and they were further reduced following 3-hour-hypoxia. Depletion of SNORD3A levels increased cardiomyocyte death and reduced protein synthesis in H9C2 cells. Conclusions Our data suggest that SNORD3A might be involved in the regulation of cardiomyocyte survival in HF and could be useful for diagnostic and prognostic applications in HF.

Alarin regulates RyR2 and SERCA2 to improve cardiac function in heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF), a complex disease that is increasingly prevalent due to population aging, pose significant challenges in its treatment. The present study utilized the HFpEF rat model and H9C2 cells as research subjects to thoroughly investigate the potential mechanisms of alarin in protecting cardiac function in HFpEF. The study shows that under HFpEF conditions, oxidative stress significantly increases, leading to myocardial structural damage and dysfunction of calcium ion channels, which ultimately impairs diastolic function. Alarin, through its interaction with NADPH oxidase 1 (NOX1), effectively alleviates oxidative stress and modulates the activities of type 2 ryanodine receptor (RyR2) and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2), thereby facilitating the restoration of Ca2+ homeostasis and significantly improving cardiac function in the HFpEF model. This research not only uncovers the cardioprotective effects of alarin and its underlying molecular mechanisms but also provides new insights and potential therapeutic targets for HFpEF treatment strategies, suggesting a promising future for alarin and related therapies in the management of this debilitating condition.

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

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

Double Encapsulation of Resveratrol and Doxorubicin in Composite Nanogel—An Opportunity to Reduce Cardio- and Neurotoxicity of Doxorubicin

The simultaneous encapsulation of drugs into nanosized delivery systems could be beneficial for cancer therapies since it could alleviate adverse reactions as well as provide synergistic effects. However, the encapsulation of hydrophobic drugs into hydrophilic nanoparticles, such as nanogels, could be challenging. Therefore, innovative technological approaches are needed. In this research, a composite nanogel system was prepared from chitosan, albumin, and hydroxypropyl-β-cyclodextrin for co-delivery of the hydrophilic anticancer drug doxorubicin and hydrophobic antioxidant resveratrol. The nanoparticles were characterized using dynamic light scattering and found to have a hydrodynamic diameter of approx. 31 nm, narrow size distribution (PDI = 0.188), positive ƺ-potential (+51.23 mV), and pH-dependent release of the loaded drugs. FTIR and X-ray analyses proved the successful development of the composite nanogel. Moreover, the double-loaded system showed that the loading of resveratrol exerted protection against doxorubicin-induced toxicity in cardioblast H9c2 and neuroblast SH-SY5Y cells. The simultaneous loading did not influence the cytostatic effect of the antitumor agent in lymphoma L5178Y and L5178MDR cell lines.