H9c2 Cardiomyocytes Research Articles

Osthole protects H9c2 cardiomyocytes against trastuzumab-induced damage by enhancing autophagy through the p38MAPK/mTOR signaling pathway

Trastuzumab (TRZ) is a novel targeted anti-tumor agent that significantly improve the survival of patients with human epidermal growth factor receptor (HER2) positive breast cancer. However, its clinical application is limited due to the side effects of cardiotoxicity. Osthole (OST), a coumarin derivative isolated from Cnidium monnieri (L.) Cusson, has previously demonstrated cardioprotective effects. The aim of this study was to observe the protective effect of OST on TRZ-induced cardiomyocytes damage and to explore its potential mechanism. The results showed that OST pretreatment could significantly inhibit TRZ-induced cardiomyocytes damage, markedly increase the ratio of LC3II/I and Beclin-1 protein expression, and reduce the protein expression of p62. OST pretreatment significantly attenuated oxidative stress and apoptosis induced by TRZ, as evidenced by reducing intracellular ROS level, the Bax/Bcl-2 ratio, and Caspase-3 protein expression. Additionally, OST markedly increased the phosphorylation level of p38MAPK and decreased the mTOR phosphorylation level. However, the effects of OST on enhancing autophagy, reducing oxidative stress, apoptosis, and the phosphorylation level of mTOR were reversed after the addition of 3-MA or SB203580. Molecular docking results indicated that OST exerted a good binding ability with the p38MAPK protein. Our findings suggested that OST could protect TRZ-induced cardiomyocytes damage by enhancing autophagy via the p38MAPK/mTOR signaling pathway.

Stachydrine ameliorates hypoxia reoxygenation injury of cardiomyocyte via enhancing SIRT1-Nrf2 pathway

BackgroundHypoxia/reoxygenation (H/R)-induced cardiomyocyte cell apoptosis is critical in developing myocardial infarction. Stachydrine (STA), an active constituent of Leonurus heterophyllus sweet, could have a protective effect on myocardial H/R injury, which remains unexplored. Therefore, the study aimed to investigate the protective effects and mechanisms of STA on H/R injury of cardiomyocytes.MethodsRat cardiomyocyte H9c2 cells underwent H/R (hypoxia for 4 h and reoxygenation for 12 h). Cells were pretreated with STA (50 µM) 2 h before H/R. Cardiomyocyte injury was evaluated by CCK-8 assay and lactate dehydrogenase (LDH) release. Apoptosis was assessed by TUNEL staining and caspase-3 activity. Oxidative stress was assessed by lipid oxidation product MDA and a ROS-scavenging enzyme SOD in culture media. Western blot was performed to measure the protein expressions of SIRT1, Nrf2, and heme oxygenase-1 (HO-1).ResultsSTA reversed the decrease in cell viability and increased LDH release in H9c2 cells with the H/R insult. STA significantly suppressed oxidative stress, reduced MDA content, and increased SOD activity in H9c2 cells exposed to H/R. STA reduced apoptosis in H9c2 cells exposed to H/R, as evidenced by the reduced TUNEL positive cells and caspase-3 activity. In addition, STA enhanced SIRT1, Nrf2, and HO-1 protein expression in H/R-stimulated H9c2 cells. SIRT1 and Nrf2 involved the protective effect of STA in H/R-exposed H9c2 cells, as the changes in cell viability and caspase-3 activity by STA can be reversed by SIRT1 inhibitor EX-527 or Nrf2 siRNA.ConclusionsOur data speculated that STA protects H/R injury and inhibits oxidative stress and apoptosis in cardiomyocytes by activation of the SIRT1-Nrf2 pathway.

Everolimus prevents doxorubicin-induced apoptosis in H9c2 cardiomyocytes but not in MCF-7 cancer cells: Cardioprotective roles of autophagy, mitophagy, and AKT

Cardiotoxicity is a severe side effect of the chemotherapeutic agent doxorubicin (DOX). We recently showed that DOX-induced cardiomyocyte apoptosis and death were attenuated through autophagy pre-induction. Herein, we assessed how the autophagy/mitophagy-inducing antitumor drug everolimus (EVL) affected DOX-induced cytotoxicity in the rat cardiomyocyte cell line H9c2 and human breast cancer cell line MCF-7. Apoptosis was assessed using annexin V assay. Autophagy and mitophagy were assessed using fluorescence assays. Cellular protein levels were determined using western blotting. Pretreatment with EVL (1 nM) before DOX exposure inhibited mammalian target of rapamycin (mTOR) activity, induced autophagy and mitophagy, and activated protein kinase B (AKT) in H9c2 cells. In mitochondria, DOX (1 μM) induced structural damage (decreased membrane potential and release of cytochrome c), increased superoxide levels, decreased apoptosis inhibitor Bcl-2, and increased apoptosis inducer Bax, leading to apoptosis and reduced viability in H9c2 cells. EVL pretreatment suppressed DOX-induced changes. EVL anti-apoptotic effects were inhibited by treatment with MK-2206, a selective AKT inhibitor. Furthermore, EVL suppressed DOX-induced cardiotoxicity through autophagy/mitophagy and AKT activation but did not attenuate DOX-induced apoptosis or reduction in viability in MCF-7 cells. Altogether, EVL can protect cardiomyocytes from DOX-induced apoptosis and toxicity without reducing DOX antitumor effects, allowing safer chemotherapy.

CircMAP3K5 promotes cardiomyocyte apoptosis in diabetic cardiomyopathy by regulating miR-22-3p/DAPK2 Axis.

Diabetic cardiomyopathy (DCM) is one of the serious complications of the accumulated cardiovascular system in the long course of diabetes. To date, there is no effective treatment available for DCM. Circular RNA (circRNA) is a novel r2egulatory RNA that participates in a variety of cardiac pathological processes. However, the regulatory role of circular RNA MAP3K5 (circMAP3K5) in DCM is largely unclear. Microarray analysis of DCM rats' heart circular RNAs was performed and the highly species-conserved circRNA mitogen-activated protein kinase kinase kinase 5 (circMAP3K5) was identified, which participates in DCM processes. High glucose-provoked cardiotoxicity leads to the up-regulation of circMAP3K5, which mechanistically contributes to cardiomyocyte cell death. Also, in high glucose-induced H9c2 cardiomyocytes, the level of apoptosis was significantly increased, as well as the expression of circMAP3K5. In contrast, the depletion of circMAP3K5 could reduce high glucose-induced apoptosis in cardiomyocytes. In terms of mechanism, circMAP3K5 acts as a miR-22-3p sponge and miR-22-3p directly target death-associated protein kinase 2 (DAPK2) in H9c2 cardiomyocytes, where in circMAP3K5 upregulates DAPK2 expression by targeting miR-22-3p. Moreover, we also found that miR-22-3p inhibitor and pcDNA DAPK2 could antagonize the protective effects brought by the depletion of circMAP3K5. CircMAP3K5 is a highly conserved noncoding RNA that is upregulated during DCM process. We concluded that circMAP3K5 promotes high glucose-induced cardiomyocyte apoptosis by regulating the miR-22-3p/DAPK2 axis. The results of this study highlight a novel and translationally important circMAP3K5-based therapeutic approach for DCM.

Tanshinone IIA inhibits cardiomyocyte pyroptosis through TLR4/NF-κB p65 pathway after acute myocardial infarction.

Background: Tanshinone IIA, derived from Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bunge), constitutes a significant component of this traditional Chinese medicine. Numerous studies have reported positive outcomes regarding its influence on cardiac function. However, a comprehensive comprehension of the intricate mechanisms responsible for its cardioprotective effects is still lacking. Methods: A rat model of heart failure (HF) induced by acute myocardial infarction (AMI) was established via ligation of the left anterior descending coronary artery. Rats received oral administration of tanshinone IIA (1.5mg/kg) and captopril (10mg/kg) for 8weeks. Cardiac function was assessed through various evaluations. Histological changes in myocardial tissue were observed using staining techniques, including Hematoxylin and Eosin (HE), Masson, and transmission electron microscopy. Tunel staining was used to detect cell apoptosis. Serum levels of NT-pro-BNP, IL-1β, and IL-18 were quantified using enzyme-linked immunosorbent assay (ELISA). Expression levels of TLR4, NF-κB p65, and pyroptosis-related proteins were determined via western blotting (WB). H9C2 cardiomyocytes underwent hypoxia-reoxygenation (H/R) to simulate ischemia-reperfusion (I/R) injury, and cell viability and apoptosis were assessed post treatment with different tanshinone IIA concentrations (0.05μg/ml, 0.1μg/ml). ELISA measured IL-1β, IL-18, and LDH expression in the cell supernatant, while WB analysis evaluated TLR4, NF-κB p65, and pyroptosis-related protein levels. NF-κB p65 protein nuclear translocation was observed using laser confocal microscopy. Results: Tanshinone IIA treatment exhibited enhanced cardiac function, mitigated histological cardiac tissue damage, lowered serum levels of NT-pro-BNP, IL-1β, and IL-18, and suppressed myocardial cell apoptosis. Moreover, tanshinone IIA downregulated the expression of TLR4, NF-κB p65, IL-1β, pro-IL-1β, NLRP3, Caspase-1, and GSDMD-N pyroptosis-related proteins in myocardial tissue. Additionally, it bolstered H/R H9C2 cardiomyocyte viability, curbed cardiomyocyte apoptosis, and reduced the levels of TLR4, NF-κB p65, IL-1β, pro-IL-1β, NLRP3, Caspase-1, and GSDMD-N pyroptosis-related proteins in H/R H9C2 cells. Furthermore, it hindered NF-κB p65 protein nuclear translocation. Conclusion: These findings indicate that tanshinone IIA enhances cardiac function and alleviates myocardial injury in HF rats following AMI. Moreover, tanshinone IIA demonstrates potential suppression of cardiomyocyte pyroptosis. These effects likely arise from the inhibition of the TLR4/NF-κB p65 signaling pathway, presenting a promising therapeutic target.

Down-regulation of the mitochondrial fusion protein Opa1/Mfn2 promotes cardiomyocyte hypertrophy in Su5416/hypoxia-induced pulmonary hypertension rats

BackgroundMaladaptive right ventricular (RV) remodeling is the most important pathological feature of pulmonary hypertension (PH), involving processes such as myocardial hypertrophy and fibrosis. A growing number of studies have shown that mitochondria-associated endoplasmic reticulum membranes (MAMs) are involved in various physiological and pathological processes, such as calcium homeostasis, lipid metabolism, inflammatory response, mitochondrial dynamics, and autophagy/mitophagy. The abnormal expression of MAMs-related factors is closely related to the occurrence and development of heart-related diseases. However, the role of MAM-related factors in the maladaptive RV remodeling of PH rats remains unclear. Methods and resultsWe first obtained the transcriptome data of RV tissues from PH rats induced by Su5416 combined with hypoxia treatment (SuHx) from the Gene Expression Omnibus (GEO) database. The results showed that two MAMs-related genes (Opa1 and Mfn2) were significantly down-regulated in RV tissues of SuHx rats, accompanied by significant up-regulation of cardiac hypertrophy-related genes (such as Nppb and Myh7). Subsequently, using the SuHx-induced PH rat model, we found that the downregulation of mitochondrial fusion proteins Opa1 and Mfn2 may be involved in maladaptive RV remodeling by accelerating mitochondrial dysfunction. Finally, at the cellular level, we found that overexpression of Opa1 and Mfn2 could inhibit hypoxia-induced mitochondrial fission and reduce ROS production in H9c2 cardiomyocytes, thereby retarded the progression of cardiomyocyte hypertrophy. ConclusionsThe down-regulation of mitochondrial fusion protein Opa1/Mfn2 can accelerate cardiomyocyte hypertrophy and then participate in maladaptive RV remodeling in SuHx-induced PH rats, which may be potential targets for preventing maladaptive RV remodeling.

MiR-21 attenuates FAS-mediated cardiomyocyte apoptosis by regulating HIPK3 expression.

MicroRNA-21 (miR-21) plays an anti-apoptotic role following ischemia-reperfusion (I/R) injury (IRI) in vivo; however, its underlying mechanism remains unclear. This study explored the effects of miR-21 and homeodomain interacting protein kinase 3 (HIPK3) on cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R) in vitro. To this end, the rat cardiomyocyte H9C2 cell line was exposed to H/R and the roles of miR-21 and HIPK3 in regulating cell viability and apoptosis were evaluated by cell counting kit-8 assay, terminal-deoxynucleotidyl-transferase -mediated dUTP nick end labeling, and flow cytometry. Immunofluorescence and western blotting were performed to detect the expression/phosphorylation of apoptosis-related proteins. miR-21 expression was measured with quantitative real-time polymerase chain reaction. The putative interaction between miR-21 and HIPK3 was evaluated using the luciferase reporter assay. Our results showed that: (i) miR-21 overexpression or HIPK3 downregulation significantly attenuated H9C2 cells apoptosis after H/R; (ii) suppression of miR-21 expression promoted apoptosis; (iii) miR-21 overexpression inhibited HIPK3 expression; (iv) HIPK3 was the direct and main target of miR-21; (v) miR-21/HIPK3 formed part of a reciprocal, negative feedback loop; and (vi) HIPK3 downregulation decreased FAS-mediated apoptosis by inhibiting the phosphorylation of FADD, which subsequently inhibited the expression of BAX and cleaved caspase-3 and increased the expression of BCL2. Our study indicates that miR-21 attenuates FAS-mediated cardiomyocyte apoptosis by regulating HIPK3 expression, which could eventually have important clinical implications for patients with acute myocardial infarction.

Dapagliflozin attenuates high glucose-and hypoxia/reoxygenation-induced injury via activating AMPK/mTOR-OPA1-mediated mitochondrial autophagy in H9c2 cardiomyocytes

This study investigated the protective effect of dapagliflozin on H9c2 cardiomyocyte function under high glucose and hypoxia/reoxygenation (HG-H/R) conditions and identified the underlying molecular mechanisms. Dapagliflozin reduced the level of lactate dehydrogenase and reactive oxygen species in cardiomyocytes under HG-H/R conditions and was accompanied by a decrease in caspase-3/9 activity. In addition, Dapagliflozin significantly reduced mitochondrial permeability transition pore opening and increased ATP content, accompanied by upregulation of OPA1 with autophagy-related protein molecules and activation of the AMPK/mTOR signalling pathway in HG-H/R treated cardiomyocytes. OPA1 knockdown or compound C treatment attenuated the protective effects of dapagliflozin on the cardiomyocytes under HG-H/R conditions. Downregulation of OPA1 expression increased mitochondrial intolerance in cardiomyocytes during HG-H/R injury and the AMPK-mTOR-autophagy signalling is a key mechanism for protecting mitochondrial function and reducing cardiomyocyte apoptosis. Collectively, dapagliflozin exerted protective effects on the cardiomyocytes under HG-H/R conditions. Dapagliflozin attenuated myocardial HG-H/R injury by activating AMPK/mTOR-OPA1-mediated mitochondrial autophagy.

Licochalcone a improves cardiac functions after ischemia-reperfusion via reduction of ferroptosis in rats

Myocardial ischemia-reperfusion (I/R) injury triggers several cell death types, including apoptosis, autophagy, and ferroptosis. Licochalcone A (LCA), a natural flavonoid compound isolated from the root of Glycyrrhiza glabra, has been demonstrated to exert potential pharmacological benefits, such as antioxidant, antitumor, and anti-inflammatory activities. The present study aimed to investigate the involvement of ferroptosis in the pathogenesis of I/R and determine whether LCA can inhibit ferroptosis to prevent the myocardial I/R injury in rats. The effects of LCA on myocardial I/R injury were detected by examining the left ventricular-developed pressure and triphenyltetrazolium chloride staining. We conducted Western blotting analyses, ELISA assay, and quantitative real-time PCR to determine the levels of ferroptosis-related molecules. To demonstrate the cardioprotective effect of LCA in vitro, H9c2 and primary neonatal rat cardiomyocytes were co-treated with ferroptosis inducers (erastin, RSL3, or Fe-SP) and LCA for 16 and 24 h. Our ex vivo study showed that LCA increased the cardiac contractility, and reduced the infarct volume and ferroptosis-related biomarkers in rat hearts after I/R. Moreover, LCA reduced the levels of ferroptosis inducers-induced reactive oxygen species generation, lipid peroxidation, and ferroptosis-related biomarkers in cultured H9c2 cells and cardiomyocytes. LCA also reduced the Fe-SP-increased nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 protein levels in cultured cardiomyocytes. In the present study, we showed that the LCA-induced cardioprotective effects in attenuating the myocardial I/R injury were correlated with ferroptosis regulation, and provided a possible new therapeutic strategy for prevention or therapy of the myocardial I/R injury.

Network Pharmacology and Experimental Validation to Explore Mechanism of Tetrahydropalmatine on Acute Myocardial Ischemia.

To explore the potential molecular mechanism of tetrahydropalmatine (THP) on acute myocardial ischemia (AMI). First, the target genes of THP and AMI were collected from SymMap Database, Traditional Chinese Medicine Database and Analysis Platform, and Swiss Target Prediction, respectively. Then, the overlapping target genes between THP and AMI were evaluated for Grene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction network analysis. The binding affinity between the protein and THP was assessed by molecular docking. Finally, the protective effects of THP on AMI model and oxygen and glucose deprivation (OGD) model of H9C2 cardiomyocyte were explored and the expression levels of target genes were detected by RT-qPCR in vivo and in vitro. MMP9, PPARG, PTGS2, SLC6A4, ESR1, JAK2, GSK3B, NOS2 and AR were recognized as hub genes. The KEGG enrichment analysis results revealed that the potential target genes of THP were involved in the regulation of PPAR and hormone pathways. THP improved the cardiac function, as well as alleviated myocardial cell damage. Furthermore, THP significantly decreased the RNA expression levels of MMP9, PTGS2, SLC6A4, GSK3B and ESR1 (P<0.05, P<0.01) after AMI. In vitro, THP significantly increased H9C2 cardiomyocyte viability (P<0.05, P<0.01) and inhibited the RNA expression levels of PPARG, ESR1 and AR (P<0.05, P<0.01) in OGD model. THP could improve cardiac function and alleviate myocardial injury in AMI. The underlying mechanism may be inhibition of inflammation, the improvement of energy metabolism and the regulation of hormones.

The novel peptide athycaltide-1 attenuates Ang II-induced pathological myocardial hypertrophy by reducing ROS and inhibiting the activation of CaMKII and ERK1/2

Pathological myocardial hypertrophy initially develops as an adaptive response to cardiac stress, which can be induced by many diseases. It is accompanied by adverse cardiovascular events, including heart failure, arrhythmias, and death. The purpose of this research was to explore the molecular mechanism of a novel peptide Athycaltide-1 (ATH-1) in the treatment of Ang II-induced pathological myocardial hypertrophy. In this study, the mRNA of Control group, Ang II group, ATH-1 group and Losartan group mice were sequenced by high-throughput sequencing technology. The results showed that the differentially expressed genes (DEGs) were significantly enriched in cell response to oxidative stress, regulation of reactive oxygen species metabolism and calmodulin binding. Then, the oxidation level of mouse hearts and H9c2 cardiomyocytes in each group and the expression of key proteins of CaMKII/HDAC/MEF2C and ERK1/2 signaling pathways were detected to preliminarily verify the positive effect of ATH-1. At the same time, the effect of ATH-1 was further determined by adding reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) and CaMKII inhibitor AIP in vitro. The results showed that ATH-1 could significantly reduce the level of oxidative stress in hypertrophic cardiomyocytes and inhibiting the activation of CaMKII and ERK1/2.

Polymorphic variants at NDUFC2, encoding a mitochondrial complex I subunit, associate with cardiac hypertrophy in human hypertension

BackgroundA dysfunction of NADH dehydrogenase, the mitochondrial Complex I (CI), associated with the development of left ventricular hypertrophy (LVH) in previous experimental studies. A deficiency of Ndufc2 (subunit of CI) impairs CI activity causing severe mitochondrial dysfunction. The T allele at NDUFC2/rs11237379 variant associates with reduced gene expression and impaired mitochondrial function. The present study tested the association of both NDUFC2/rs11237379 and NDUFC2/rs641836 variants with LVH in hypertensive patients. In vitro studies explored the impact of reduced Ndufc2 expression in isolated cardiomyocytes.MethodsTwo-hundred-forty-six subjects (147 male, 59.7%), with a mean age of 59 ± 15 years, were included for the genetic association analysis. Ndufc2 silencing was performed in both H9c2 and rat primary cardiomyocytes to explore the hypertrophy development and the underlying signaling pathway.ResultsThe TT genotype at NDUFC2/rs11237379 associated with significantly reduced gene expression. Multivariate analysis revealed that patients carrying this genotype showed significant differences for septal thickness (p = 0.07), posterior wall thickness (p = 0.008), RWT (p = 0.021), LV mass/BSA (p = 0.03), compared to subjects carrying either CC or CT genotypes. Patients carrying the A allele at NDUFC2/rs641836 showed significant differences for septal thickness (p = 0.017), posterior wall thickness (p = 0.011), LV mass (p = 0.003), LV mass/BSA (p = 0.002) and LV mass/height2.7(p = 0.010) after adjustment for covariates. In-vitro, the Ndufc2 deficiency-dependent mitochondrial dysfunction caused cardiomyocyte hypertrophy, pointing to SIRT3-AMPK-AKT-MnSOD as a major underlying signaling pathway.ConclusionsWe demonstrated for the first time a significant association of NDUFC2 variants with LVH in human hypertension and highlight a key role of Ndufc2 deficiency-dependent CI mitochondrial dysfunction on increased susceptibility to cardiac hypertrophy development.

Riboflavin protects against heart failure via SCAD-dependent DJ-1-Keap1-Nrf2 signalling pathway.

Our recent studies have shown that flavin adenine dinucleotide (FAD) exerts cardiovascular protective effects by supplementing short-chain acyl-CoA dehydrogenase (SCAD). The current study aimed to elucidate whether riboflavin (the precursor of FAD) could improve heart failure via activating SCAD and the DJ-1-Keap1-Nrf2 signalling pathway. Riboflavin treatment was given to the mouse transverse aortic constriction (TAC)-induced heart failure model. Cardiac structure and function, energy metabolism and apoptosis index were assessed, and relevant signalling proteins were analysed. The mechanisms underlying the cardioprotection by riboflavin were analysed in the cell apoptosis model induced by tert-butyl hydroperoxide (tBHP). In vivo, riboflavin ameliorated myocardial fibrosis and energy metabolism, improved cardiac dysfunction and inhibited oxidative stress and cardiomyocyte apoptosis in TAC-induced heart failure. In vitro, riboflavin ameliorated cell apoptosis in H9C2 cardiomyocytes by decreasing reactive oxygen species (ROS). At the molecular level, riboflavin significantly restored FAD content, SCAD expression and enzymatic activity, activated DJ-1 and inhibited the Keap1-Nrf2/HO1 signalling pathway in vivo and in vitro. SCAD knockdown exaggerated the tBHP-induced DJ-1 decrease and Keap1-Nrf2/HO1 signalling pathway activation in H9C2 cardiomyocytes. The knockdown of SCAD abolished the anti-apoptotic effects of riboflavin on H9C2 cardiomyocytes. DJ-1 knockdown hindered SCAD overexpression anti-apoptotic effects and regulation on Keap1-Nrf2/HO1 signalling pathway in H9C2 cardiomyocytes. Riboflavin exerts cardioprotective effects on heart failure by improving oxidative stress and cardiomyocyte apoptosis via FAD to stimulate SCAD and then activates the DJ-1-Keap1-Nrf2 signalling pathway.

Mitochonic acid 5 rescues cardiomyocytes from doxorubicin-induced toxicity via repressing the TNF-α/NF-κB/NLRP3-mediated pyroptosis

AimsDoxorubicin (DOX) is an effective anti-tumor drug, but the cardiotoxicity severely limits its clinical use. Interestingly, a hypothesis has emerged suggesting an association between DOX-induced cardiotoxicity and mitochondrial disorders and oxidative stress. The mitochonic acid 5 (MA5) shows promise in alleviating mitochondrial dysfunction by promoting mitochondrial ATP synthesis and reducing reactive oxygen species (ROS) accumulation, though its potential in ameliorating DOX-induced cardiotoxicity remains elusive. MethodsNetwork pharmacology approach, molecular docking techniques, and molecular dynamics simulation (MDS) were used to reveal the specific drug targets and pharmaceutical mechanisms involved in the treatment of DOX-induced cardiotoxicity using MA5. For experimental verification, cardiomyocytes (H9c2) and mice were exposed to DOX in the presence or absence of MA5. Our investigation involved the assessment of echocardiographic parameters, cardiac enzymes, inflammatory factors, mitochondrial function, myocardial structure, and cardiomyocyte pyroptosis. ResultsAmong the 100 core targets identified in network pharmacology, MA5 was pharmacologically active against DOX-induced cardiotoxicity via pathways implicated in cancer, prostate cancer, lipids and atherosclerosis. Molecular docking analysis confirmed that MA5 docked well with TNF-α, interleukin-6 (IL-6), and caspase-3. Furthermore, MA5 exhibited a stronger affinity toward TNF-α than IL-6 and caspase-3. Subsequent MDS revealed the stability of binding between MA5 and TNF-α. The DOX-challenged mice also displayed abnormal myocardial enzymogram, disrupted systolic and diastolic function, and elevated inflammation and cardiomyocyte pyroptosis, which could be mitigated by the administration of MA5. Similarly, H9c2 cells exposed to DOX showed increased intracellular ROS production and impaired mitochondrial function, which were relieved by MA5 treatment. ConclusionOur findings suggest that MA5 attenuates DOX-induced cardiac anomalies through the TNF-α-mediated regulation of inflammation and pyroptosis. These insights offer a potential therapeutic strategy for managing DOX-induced cardiac complications, thereby improving the safety and efficacy of cancer treatments.

Sex-specific cytotoxicity of ostarine in cardiomyocytes

Ostarine is the most popular compound in the selective androgen receptor modulator group (SARMs). Ostarine is used as a physical performance-enhancing agent. The abuse of this agent in higher doses may lead to severe side effects. Here, we evaluate the effects of ostarine on the heart.We utilized a cardiomyocyte H9C2 cell line, isolated primary female and male cardiac fibroblast cells, as well as hearts obtained from rats.Ostarine increased the accumulation of two fibrosis protein markers, αSMA and fibronectin (p < 00.1) in male, but not in female fibroblast cells. Ostarine increased the expression of the cardiomyopathy marker βMhc in the H9C2 cell line (p < 0.05) and in the heart in rats (p < 0.01). The unfavorable changes were observed at high ostarine doses. Moreover, a decrease in viability and an increase in cytotoxicity marker LDH were observed already at lowest dose (1 nmoL/l). Taken together, our results suggest that ostarine is cardiotoxic which may be more relevant in males than in females.

Pharmacological potential of new metronidazole/eugenol/dihydroeugenol hybrids against Trypanosoma cruzi in vitro and in vivo.

From well-delimited immunomodulatory, redox and antimicrobial properties; metronidazole and eugenol were used as structural platforms to assembly two new molecular hybrids (AD06 and AD07), whose therapeutic relevance was analyzed on T. cruzi infection in vitro and in vivo. Non-infected, T. cruzi-infected H9c2 cardiomyocytes, and mice non-treated and treated with vehicle, benznidazole (Bz - reference drug), AD06 and AD07 were investigated. Parasitological, prooxidant, antioxidant, microstructural, immunological, and hepatic function markers were analyzed. Our findings indicated that in addition to having a direct antiparasitic effect on T. cruzi, metronidazole/eugenol hybrids (especially AD07) attenuated cellular parasitism, reactive species biosynthesis and oxidative stress in infected cardiomyocytes in vitro. Although AD06 and AD07 exerted no relevant impact on antioxidant enzymes activity (CAT, SOD, GR and GPx) in host cells, these drugs (especially AD07) attenuated trypanothione reductase activity in T. cruzi, which increased parasite's susceptibility to in vitro pro-oxidant challenge. AD06 and AD07 were well tolerated and do not determine humoral response suppression, mortality (100% survival) or hepatotoxicity in mice, as indicated by transaminases plasma levels. AD07 also induced relevant in vivo antiparasitic and cardioprotective effects, attenuating parasitemia, cardiac parasite load and myocarditis in T. cruzi-infected mice. Although this cardioprotective response is potentially related to AD07 antiparasitic effect, a direct anti-inflammatory potential of this molecular hybrid cannot be ruled out. Taken together, our findings indicated that the new molecular hybrid AD07 stood out as a potentially relevant candidate for the development of new, safe and more effective drug regimens for T. cruzi infection treatment.

Curcuminoids Attenuate Myocardial Ischemia-Reperfusion Injury by Regulating Total RNA M6a Levels: In Vitro Study.

Myocardial ischemia-reperfusion (IR) injury is an unresolved medical problem with a high incidence. This study aims to analyze the novel molecular mechanism by which curcuminoids protect cardiomyocytes from IR injury. A IR model In Vitro of rat cardiomyocytes H9c2 cells was structured. Curcumin (CUR) and its derivatives, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) treated H9c2 cells, and reactive oxygen species (ROS) production, viability, apoptosis, mitochondrial membrane potential (MMP), oxidative stress and total RNA m6A levels of H9c2 cells were detected by using DCFH-DA stain, CCK-8, flow cytometry, Hoechst 33342 stain, TMRM stain, ELISA and RTqPCR. FB23 was used in rescue experiments. IR significantly increased ROS production, decreased cell viability, and induced apoptosis, MMP loss, and oxidative stress. In addition, IR induced an increase in total RNA m6A levels and changes in m6A-related proteins expression. CUR (10 μM), DMC (10 μM) and BDMC (10 μM), significantly inhibited IR-induced ROS production, apoptosis, MMP loss and oxidative stress, and enhanced cell viability. Furthermore, CUR, DMC and BDMC altered the expression pattern of m6A-related proteins and reduced IR-induced total m6A levels. There was no significant difference in the effects of the three. CUR's protective effect was partially reduced by FB23. Curcuminoids attenuate myocardial IR injury by regulating total RNA m6A levels.

Hydroxysafflor Yellow A Alleviates Acute Myocardial Ischemia/Reperfusion Injury in Mice by Inhibiting Ferroptosis via the Activation of the HIF-1α/SLC7A11/GPX4 Signaling Pathway.

Ferroptosis is closely associated with the pathophysiology of myocardial ischemia. Hydroxysafflor yellow A (HSYA), the main active ingredient in the Chinese herbal medicine safflower, exerts significant protective effects against myocardial ischemia/reperfusion injury (MI/RI). The aim of this study was to investigate the protective effects of HSYA against MI/RI and identify the putative underlying mechanisms. An in vivo model of acute MI/RI was established in C57 mice. Subsequently, the effects of HSYA on myocardial tissue injury were evaluated by histology. Lipid peroxidation and myocardial injury marker contents in myocardial tissue and serum and iron contents in myocardial tissue were determined using biochemical assays. Mitochondrial damage was assessed using transmission electron microscopy. H9C2 cardiomyocytes were induced in vitro by oxygen-glucose deprivation/reoxygenation, and ferroptosis inducer erastin was administered to detect ferroptosis-related indicators, oxidative-stress-related indicators, and expressions of ferroptosis-related proteins and HIF-1α. In MI/RI model mice, HSYA reduced myocardial histopathological damage, ameliorated mitochondrial damage in myocardial cells, and decreased total cellular iron and ferrous ion contents in myocardial tissue. HSYA increased the protein levels of SLC7A11, HIF-1α, and GPX4 and mitigated erastin- or HIF-1α siRNA-induced damage in H9C2 cells. In summary, HSYA alleviated MI/RI by activating the HIF-1α/SLC7A11/GPX4 signaling pathway, thereby inhibiting ferroptosis.

Loganin alleviates myocardial ischemia-reperfusion injury through GLP-1R/NLRP3-mediated pyroptosis pathway.

Myocardial ischemia-reperfusion (I/R) injury is one of main pathological manifestations of cardiovascular outcomes related to NLRP3 inflammasome-mediated pyroptosis pathway. Loganin is an iridoid glycoside extracted from traditional Chinese medicines, which has multiple activities. However, the roles and mechanism of loganin in myocardial I/R injury remain largely unknown. The models of myocardial I/R injury were established using I/R-treated rats or OGD/R-treated H9C2 cardiomyocytes. Myocardial damage was assessed by TTC and hematoxylin-eosin staining. Pyroptosis-related marker levels were detected by immunohistochemistry, immunofluorescence and western blotting assays. Cell proliferation was examined via EdU assay. Cell apoptosis was investigated by LDH release and flow cytometry. The integrity of cell membrane was analyzed via Dil staining. GLP-1R and NLRP3 levels were detected by immunofluorescence and western blotting assays. Our results showed that loganin suppressed I/R-induced myocardial damage in rats by reducing myocardial infarct, injury and pyroptosis. In addition, loganin attenuated OGD/R-induced cardiomyocyte apoptosis through increasing cell proliferation and reducing LDH release and apoptotic rate. Loganin also mitigated OGD/R-induced cardiomyocyte pyroptosis by reducing cell membrane damage and levels of cleaved caspase-1, IL-1β and IL-18. Furthermore, loganin repressed GLP-1R/NLRP3 pathway activation in OGD/R-treated H9C2 cardiomyocytes by enhancing GLP-1R expression and decreasing NLRP3 level. GLP-1R/NLRP3 activation by GLP-1R inhibition or NLRP3 overexpression reversed the suppressive effects of loganin on OGD/R-induced cardiomyocyte pyroptosis. These data indicated that loganin prevented OGD/R-induced proliferation inhibition, apoptosis and pyroptosis in OGD/R-treated cardiomyocytes by inhibiting GLP-1R/NLRP3 activity, indicating the therapeutic potential of loganin in myocardial I/R injury.

Epicardial Adipose Tissue-Derived Leptin Promotes Myocardial Injury in Metabolic Syndrome Rats Through PKC/NADPH Oxidase/ROS Pathway.

Background The epicardial adipose tissue (EAT) of metabolic syndrome (MetS) is abnormally accumulated with dysfunctional secretion of adipokines, closely relating to cardiac dysfunction. The current study was designed to identify the effects of EAT-derived leptin on the myocardium of MetS rats and explore the potential molecular mechanisms. Methods and Results A MetS rat model was established in 8-week-old Wistar rats by a 12-week high-fat diet. MetS rats exhibited increased leptin secretion from EAT, cardiac hypertrophy, and diastolic dysfunction with preserved systolic function. The myocardium of MetS rats had abnormal structure, increased oxidative stress injury, and higher inflammatory factor levels, especially the subepicardial myocardium, which was correlated with the EAT-derived leptin level but not the serum leptin. The EAT was separated from each group of rats to prepare EAT-conditioned medium. H9C2 rat cardiomyoblasts were treated with EAT-conditioned medium or leptin, plus various inhibitors. EAT-derived leptin from MetS rats promoted mitochondrial oxidative stress and dysfunction, induced mitochondrial pathway apoptosis, and inhibited cell viability in H9C2 cardiomyoblasts via the protein kinase C/reduced nicotinamide adenine dinucleotide phosphate oxidase/reactive oxygen species (PKC/NADPH oxidase/ROS) pathway. EAT-derived leptin from MetS rats stimulated inflammation in H9C2 cardiomyocytes by promoting activator protein 1 nuclear translocation via the PKC/NADPH oxidase/ROS pathway. Leptin promoted the interaction between p-p47phox and gp91phox in H9C2 cardiomyocytes via protein kinase C, activating nicotinamide adenine dinucleotide phosphate oxidase, increasing reactive oxygen species generation, and inhibiting cell viability. Conclusions EAT-derived leptin induces MetS-related myocardial injury through the following 2 cooperative ways via PKC/NADPH oxidase/ROS pathway: (1) inducing mitochondrial pathway apoptosis by promoting mitochondrial oxidative stress and dysfunction; and (2) stimulating inflammation by promoting activator protein 1 nuclear translocation.