H9c2 Myocardial Cells Research Articles

Apelin-13's Actions in Controlling Hypertension-Related Cardiac Hypertrophy and the Expressions of Inflammatory Cytokines.

As a key molecule for improving cardiovascular diseases, Apelin-13 was surveyed in this work to explain its actions in controlling inflammation, pyroptosis, and myocardial hypertrophy. First, mouse models with myocardial hypertrophy were established. Then, assessments were made on the pathological variation in the heart of mouse, on the cardiac functions, as well as on the expressions of cardiac hypertrophy markers (β-MHC, ANP, and BNP), inflammatory factors (TNF-α, COX2, IL-6, ICAM-1, and VCAM-1), myocardial cell pyroptosis markers (NLRP3, ASC, c-caspase-1, and GSDMD-N), and Hippo pathway proteins (p-YAP, YAP, LATS1, and p-LATS1) by HE staining, echocardiography scanning, and western blot tests separately. The expressions of such inflammatory factors as in myocardial tissue were acquired by ELISA. After inducing the phenotype of H9c2 cell hypertrophy by noradrenaline, we used CCK-8 kits to know about the activity of H9c2 cells treated with Apelin-13, and performed ɑ-actinin staining to measure the changes in volumes of such cells. As unraveled through this work, Apelin-13 refrained the activation of the Hippo pathway, which in turn attenuated the hypertrophy, inflammation, and pyroptosis of myocardial tissue and H9c2 cells. Hence, Apelin-13 can be considered as a target for hypertension treatment.

TRPC6 regulates necroptosis in myocardial ischemia/reperfusion injury via Ca2+/CaMKII signaling pathway

Myocardial ischemia-reperfusion injury (MIRI) frequently complicates postoperative cardiovascular disease treatment. Necroptosis, a cell death mechanism similar to apoptosis, is regulated by specific signaling pathways and plays an important role in MIRI. Receptor-interacting protein 3 (RIP3), a key protein regulating necroptosis during MIRI, directly phosphorylates calmodulin-dependent protein kinase II (CaMKII). Leading to mitochondrial permeablity transition pore (mPTP) opening and inducing necroptosis. Transient receptor potential canonical channel 6 (TRPC6) regulats Ca2+ entry, is linked to CaMKII as an important upstream effector. However, the connection between TRPC6 and MIRI necroptosis remains unclear. The study aimed to investigate the relationship between TRPC6 and MIRI necroptosis, with a specific focus on elucidating the role of TRPC6 in regulating CaMKII phosphorylation during cardiac necroptosis via Ca2+ modulation. Methods and resultsThe experiment used wild-type (WT) and TRPC6 knockout (TRPC6−/−) mice for I/R model construction, and H9c2 myocardial cell line for H/R model. After ischemia-reperfusion (I/R), TRPC6 protein levels in mice significantly increased, exacerbating myocardial injury, infarct size (IS), and cardiac function in WT mice. In contrast, TRPC6 knockout attenuated myocardial injury, IS, and improved cardiac function. The results showed a significant correlation between changes in CaMKII and TRPC6. TRPC6 knockout led to decreased intracellular calcium levels, CaMKII phosphorylation, reactive oxygen species levels, mPTP opening, and improve mitochondrial structure. ConclusionI/R upregulates TRPC6, which mediates Ca2+ entry and CaMKII phosphorylation, exacerbates oxidative stress, and induces necroptosis. These findings suggest a potential therapeutic avenue for mitigating MIRI by targeting TRPC6.

Effect of extracellular matrix stiffness on efficacy of Dapagliflozin for diabetic cardiomyopathy

BackgroundExtracellular matrix (ECM) stiffness is closely related to the progress of diabetic cardiomyopathy (DCM) and the response of treatment of DCM to anti-diabetic drugs. Dapagliflozin (Dapa) has been proven to have cardio-protective efficacy for diabetes and listed as the first-line drug to treat heart failure. But the regulatory relationship between ECM stiffness and treatment efficacy of Dapa remains elusive.Materials and methodsThis work investigated the effect of ECM stiffness on DCM progression and Dapa efficacy using both in vivo DCM rat model and in vitro myocardial cell model with high glucose injury. First, through DCM rat models with various levels of myocardial injury and administration with Dapa treatment for four weeks, the levels of myocardial injury, myocardial oxidative stress, expressions of AT1R (a mechanical signal protein) and the stiffness of myocardial tissues were obtained. Then for mimicking the stiffness of myocardial tissues at early and late stages of DCM, we constructed cell models through culturing H9c2 myocardial cells on the polyacrylamide gels with two stiffness and exposed to a high glucose level and without/with Dapa intervention. The cell viability, reactive oxygen species (ROS) levels and expressions of mechanical signal sensitive proteins were obtained.ResultsThe DCM progression is accompanied by the increased myocardial tissue stiffness, which can synergistically exacerbate myocardial cell injury with high glucose. Dapa can improve the ECM stiffness-induced DCM progression and its efficacy on DCM is more pronounced on the soft ECM, which is related to the regulation pathway of AT1R-FAK-NOX2. Besides, Dapa can inhibit the expression of the ECM-induced integrin β1, but without significant impact on piezo 1.ConclusionsOur study found the regulation and effect of biomechanics in the DCM progression and on the Dapa efficacy on DCM, providing the new insights for the DCM treatment. Additionally, our work showed the better clinical prognosis of DCM under early Dapa intervention.

Mesenchymal stem cells ameliorate inflammation and pyroptosis in diabetic cardiomyopathy via the miRNA-223-3p/NLRP3 pathway

BackgroundDiabetic cardiomyopathy (DCM) stands as the primary cause of heart failure and mortality among patients with diabetes. Nevertheless, conventional treatment approaches are limited in their ability to effectively prevent myocardial tissue damage itself. Mesenchymal stem cell (MSC) therapy exhibits immense potential for treating DCM; however, the precise mechanisms involved in regulating inflammatory responses and pyroptosis processes, an emerging form of cellular death, within myocardial cells remain elusive. Hence, it is imperative to further elucidate the precise underlying mechanisms to facilitate the clinical implementation of MSC therapy.MethodsIn vivo, we established a DCM mouse model by administering streptozotocin and fed the mice a high-glucose and high-fat diet, followed by MSC therapy. Cardiac function and myocardial injury were evaluated through echocardiography and histological analysis. Furthermore, the levels of inflammation and pyroptosis were assessed using ELISA, Western blotting, and qRT-PCR. In vitro experiments involved inducing H9C2 myocardial cell damage with high glucose treatment, followed by coculture with MSCs to investigate their role in modulating inflammation and pyroptosis mechanisms.ResultsMSCs can maintain cardiac function and alleviate myocardial injury in mice with DCM. Moreover, they effectively suppress the activation of NLRP3 and reduce the release of inflammatory factors (such as IL-1β and ROS), thereby further downregulating the expression of pyroptosis-related proteins including NLRP3, Caspase-1, and GSDMD. Additionally, we experimentally validated that MSCs exert their therapeutic effects by promoting the expression of miR-223-3p in cardiac myocytes; however, this effect can be reversed by an miR-223-3p inhibitor.ConclusionMSCs effectively mitigate the release of inflammatory factors and cell lysis caused by pyroptosis through the regulation of the miR-223-3p/NLRP3 pathway, thereby safeguarding cardiomyocytes against damage in DCM. This mechanism establishes a novel theoretical foundation for the clinical treatment of cardiac conditions utilizing MSCs.

Overexpression of NDNF Improves the Cytoprotective Effects of Aged Human Bone Marrow Mesenchymal Stem Cells by Modulating Oxidative Stress and Apoptosis.

The therapeutic potential of autologous stem cell transplantation for heart repair diminishes in the elderly due to stem cell aging. Rejuvenating aged stem cells to enhance their protective effects on injured cardiomyocytes is crucial for aging patients with heart failure. In this study, we aimed to investigate whether neuron-derived neurotrophic factor (NDNF) over-expression improves the protective effect of aged stem cells for injured cardiomyocytes and explore the underlying mechanism. Human bone marrow was collected from both young and old patients, and bone marrow mesenchymal stem cells (BMSCs) were cultured. Lentivirus expression vectors carrying NDNF genes were used to transfect aged BMSCs. Fatal hypoxia-induced injury in H9C2 cells served as an in vitro ischemia model. The conditioned medium from different BMSC groups was applied to assess the beneficial effects on hypoxia-induced damage in myocardial H9C2 cells. Results revealed that the conditioned medium of NDNF over-expressed old BMSCs increased H9C2 cell viability and reduced oxidative stress and apoptosis levels under fatal hypoxia. NDNF over-expressed old BMSCs exhibited an antiapoptotic role by upregulating the antiapoptotic gene Bcl-2 and downregulating the proapoptotic genes Bax. Additionally, the protective effects were mediated through the elevation of phosphorylated AKT. Our data support the promise of NDNF as a potential target to enhance the protective effects of autologous aged BMSCs on ischemic cardiomyocytes and then improve the curative effects of stem cell for ischemic heart injury in aged patients.

MiR-92a-3p regulates ethanol-induced apoptosis in H9c2 cardiomyocytes.

The role of miR-92a-3p in the ethanol-induced apoptosis of H9c2 cardiomyocytes remains unclear. In this study, we explored the role of miR-92a-3p in the ethanol-induced apoptosis of H9c2 cardiomyocytes and identified its target genes and signaling pathways. H9c2 cells were cultured with or without 100mM ethanol for 24h. The differential expression of miR-92a-3p was verified in H9c2 cells through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To manipulate the expression of miR-92a-3p, both a mimic and an inhibitor were transfected into H9c2 cells. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit and apoptosis-related antibodies were used for apoptosis detection through flow cytometry and Western blotting, respectively. Target genes were verified through RT-qPCR, Western blotting, and double luciferase reporter gene assays. miR-92a-3p was significantly overexpressed in ethanol-stimulated H9c2 cardiomyocytes (P<0.001). After ethanol stimulation, H9c2 myocardial cells exhibited increased apoptosis. The apoptosis rate was higher in the miR-92a-3p mimic group than in the control group. However, the apoptosis rate was lower in the miR-92a-3p inhibitor group than in the control group, indicating that miR-92a-3p promotes the ethanol-induced apoptosis of H9c2 myocardial cells. RT-qPCR and Western blotting revealed that the miR-92a-3p mimic and inhibitor significantly regulated the mRNA and protein expression levels of mitogen- and stress-activated protein kinase 2 and cyclic AMP-responsive element-binding protein 3-like protein 2 (CREB3L2), suggesting that miR-92a-3p promotes the apoptosis of H9c2 cardiomyocytes by inhibiting the MSK2/CREB/Bcl-2 pathway. Therefore, the apoptosis of H9c2 cardiomyocytes increases after ethanol stimulation, and miR-92a-3p can directly target MSK2 and CREB3L2, thereby promoting the ethanol-induced apoptosis of H9c2 myocardial cells.

WSB1, as an E3 ligase, restrains myocardial ischemia–reperfusion injury by activating β-catenin signaling via promoting GSK3β ubiquitination

BackgroundReperfusion is the most effective strategy for myocardial infarct, but induces additional injury. WD repeat and SOCS box containing protein 1 (WSB1) plays a protective role in ischemic cells. This study aims to investigate the effects of WSB1 on myocardial ischemia–reperfusion (IR) injury.MethodsThe myocardial IR was induced by left anterior descending (LAD) ligation for 45 min and subsequent reperfusion. The overexpression of WSB1 was mediated by tail vein injection of AAV9 loaded with WSB1 encoding sequence two weeks before IR surgery. H9c2 myocardial cells underwent oxygen-sugar deprivation/reperfusion (OGD/R) to mimic IR, and transfected with WSB1 overexpression or silencing plasmid to alter the expression of WSB1.ResultsWSB1 was found highly expressed in penumbra of myocardial IR rats, and the WSB1 overexpression relieved IR-induced cardio dysfunction, myocardial infarct and pathological damage, and cardiomyocyte death in penumbra. The ectopic expression of WSB1 in H9c2 myocardial cells mitigated OGD/R-caused apoptosis, and silencing of WSB1 exacerbated the apoptosis. In addition, WSB1 activated β-catenin signaling, which was deactivated under the ischemic condition. The co-immunoprecipitation results revealed that WSB1 mediated ubiquitination and degradation of glycogen synthase kinase 3 beta (GSK3β) as an E3 ligase in myocardial cells. The effects of WSB1 on myocardial cells under ischemic conditions were abolished by an inhibitor of β-catenin signaling.ConclusionWSB1 activated β-catenin pathway by promoting the ubiquitination of GSK3β, and restrained IR-induced myocardial injury. These findings might provide novel insights for clinical treatment of myocardial ischemic patients.

Potential Effects of Orally Ingesting Polyethylene Terephthalate Microplastics on the Mouse Heart.

Polyethylene terephthalate microplastics (PET MPs) are widespread in natural environment, and can enter organisms and accumulate in the body, but its toxicity has not been well studied. Therefore, in order to investigate the toxic effects of PET microplastics on mammals, this study investigated the toxic effects of PET MPs on ICR mice and H9C2 cells by different treatment groups. The results indicated the cardiac tissue of mice in the PET-H (50µg/mL) group showed significant capillary congestion, myocardial fiber breakage, and even significant fibrosis compared to the PET-C (control) group (P < 0.01). Results of the TUNEL assay demonstrated significant apoptosis in myocardial tissue in the PET-H and PET-M (5µg/mL) groups (P < 0.01). Meanwhile, Western blotting showed increased expression of the apoptosis-related protein Bax and decreased expression of PARP, caspase-3, and Bcl-2 proteins in both myocardial tissues and H9C2 cells. In addition, flow cytometry confirmed that PET MPs decreased the mitochondrial membrane potential and apoptosis in H9C2 cells; however, this trend was reversed by N-acetylcysteamine application. Moreover, PET MP treatment induced the accumulation of reactive oxygen species (ROS) in H9C2 cells, while the MDA level in the myocardial tissue was elevated, and the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were decreased (P < 0.01), indicating a change in the redox environment. In conclusion, PET MPs promoted cardiomyocyte apoptosis by inducing oxidative stress and activating mitochondria-mediated apoptotic processes, ultimately leading to myocardial fibrosis. This study provides ideas for the prevention of PET MP toxicity and promotes thinking about enhancing plastic pollution control.

Songorine ameliorates LPS-induced sepsis cardiomyopathy by Wnt/β-catenin signaling pathway-mediated mitochondrial biosynthesis.

Septic cardiomyopathy (SCM) is manifested by impairment of cardiac contractile function with myocardial mitochondrial dysregulation. Natural product, songorine (SGR), a diterpenoid alkaloid derived from the lateral root of Aconitum carmichaeli, has been reported for the treatment of heart failure. Here, the protective role of SGR in heart injury of SCM was investigated and its underlying action of mechanism was explored. Firstly, the mouse and cardiomyocytes (H9C2 cell) SCM model induced by LPS were established to evaluate the therapeutic effect of SGR. The in vivo results exhibited that SGR rescued the survival rate of SCM mice, restored the loss of ejection fraction (EF) and fractional shortening (FS), and reduced left ventricular systolic diameter and left ventricular diastole diameter (LVIDs, LVIDd) by echocardiography. SGR improved the mitochondrial biosynthesis and myocardial fiber structure and arranged them neatly by transmission electron microscope (TEM). Further, SGR inhibited inflammatory targets myeloperoxidase (MPO) and tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and plasminogen activator inhibitor-1 (PAI-1). And SGR activated the mitochondrial biosynthesis-related peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), β-catenin, and matrix metallopeptidase 2 (MMP2) proteins. Meanwhile, the in vitro results showed that SGR promoted the increased the myocardial H9C2 cell viability, and mitochondrial biosynthesis and structure. SGR also blocked the inflammatory factors and reversed PGC-1α, β-catenin, and MMP2 in vitro, while SGR alleviated the myocardial cell apoptosis via flow cytometry. The findings indicate that SGR mitigates sepsis-caused myocardial damage by Wnt/β-catenin signaling pathway-mediated mitochondrial biosynthesis. SGR may be a promising candidate for treatment of SCM.

Therapeutic Potential of Ginsenoside Rb1-PLGA Nanoparticles for Heart Failure Treatment via the ROS/PPARα/PGC1α Pathway.

(1) Background: Ginsenoside Rb1-PLGA nanoparticles (GRb1@PLGA@NPs) represent a novel nanotherapeutic system, yet their therapeutic efficacy and underlying mechanisms for treating heart failure (HF) remain unexplored. This study aims to investigate the potential mechanisms underlying the therapeutic effects of GRb1@PLGA@NPs in HF treatment; (2) Methods: The left anterior descending coronary artery ligation was employed to establish a HF model in Sprague-Dawley rats, along with an in vitro oxidative stress model using H9c2 myocardial cells. Following treatment with GRb1@PLGA@NPs, cardiac tissue pathological changes and cell proliferation were observed. Additionally, the serum levels of biomarkers such as NT-proBNP, TNF-α, and IL-1β were measured, along with the expression of the ROS/PPARα/PGC1α pathway; (3) Results: GRb1@PLGA@NPs effectively ameliorated the pathological status of cardiac tissues in HF rats, mitigated oxidative stress-induced myocardial cell damage, elevated SOD and MMP levels, and reduced LDH, MDA, ROS, NT-proBNP, TNF-α, and IL-1β levels. Furthermore, the expression of PPARα and PGC1α proteins was upregulated; (4) Conclusions: GRb1@PLGA@NPs may attenuate myocardial cell injury and treat HF through the ROS/PPARα/PGC1α pathway.

Shengmai injection inhibits palmitic acid-induced myocardial cell inflammatory death via regulating NLRP3 inflammasome activation

ObjectiveTo determine the protective effect of Shengmai injection (SMI) on myocardial injury in diabetic rats and its mechanism based on NLRP3/Caspase1 signaling pathway. Materials and methodsRat H9c2 cardiomyocytes were cultured in vitro, and the cell survival rate of different concentrations of palmitate acid (PA) and different concentrations of SMI were detected by CCK-8. The myocardial injury cell model was induced with PA, treated with SMI, and combined with NLRP3 specific inhibitor (MCC950) to interfere with the high-fat-induced rat H9c2 myocardial cell injury model. The cell changes were observed by Hoechst/PI staining and the expression levels of MDA, SOD, and ROS in each group were detected. The protein and gene changes of the NLRP3/Caspase-1 signaling pathway were detected by Western blot and RT-qPCR, respectively. Results200 μmol/L of PA were selected to induce the myocardial injury cell model and 25 μL/mL of SMI was selected for intervention concentration. SMI could significantly reduce MDA expression, increase SOD level, and decrease ROS production. SMI could decrease the gene expression levels of NLRP3, ASC, Caspase-1, and GSDMD, and the protein expressions of NLRP3, ASC, Cleaved Caspase-1, GSDMD, and GSDMD-N. ConclusionSMI can inhibit the high-fat-induced activation of the NLRP3/Caspase-1 signaling pathway, intervene in cardiomyocyte pyroptosis, and prevent diabetic cardiomyopathy.

The protective effects of uric acid against myocardial ischemia via the Nrf2 pathway

Uric acid (UA) possesses both pro- and anti-oxidative properties in ischemic heart disease, but the underlying mechanism remains unclear. We aimed to investigate UA’s protective effect on myocardial ischemia by examining its effects on ECG Ischemic Alterations (EIA) and H2O2-induced oxidative stress in H9C2 myocardial cells. The incidence of EIA decreased over time and was more prevalent among women than men. A U-shaped relationship was observed between UA levels and EIA incidence, with the third quartile exhibiting a protective association. Addition of 237.9 μmol/L UA improved cellular damage and oxidative stress in H2O2-treated H9C2 cells, as determined by cell viability, LDH release, ROS levels, and total antioxidant capacity assays. UA activated the Nrf2 pathway, evidenced by increased expression of Nrf2, GCLC, and HO-1 proteins. By reversing cell cycle blockage, promoting wound healing ability, improving colony-forming capacity, and increasing angiogenesis in H2O2-treated cells, UA exhibited positive effects on cardiomyocyte growth characteristics. Additionally, use of Nrf2 inhibitor ML385 confirmed the involvement of the Nrf2 pathway by negating UA’s effects on oxidatively damaged cardiomyocytes. Our findings suggest that UA induces downstream antioxidant factors to ameliorate oxidative stress by activating the Nrf2 pathway, which could be one of the targets responsible for UA’s beneficial effects in myocardial ischemia.

AP39 inhibits ferroptosis by inhibiting mitochondrial autophagy through the PINK1/parkin pathway to improve myocardial fibrosis with myocardial infarction

Background and purposeResearch has revealed the involvement of mitochondrial autophagy and iron death in the pathogenesis of myocardial fibrosis. The objective of this study is to investigate whether the mitochondrial-targeted H2S donor AP39 inhibits mitochondrial autophagy and antagonizes myocardial cell iron death through the PINK1/Parkin pathway, thereby improving myocardial fibrosis in rats with myocardial infarction. Experimental approachA rat model of myocardial infarction was created by intraperitoneal injection of a high dose of isoproterenol, and H9c2 myocardial cells were subjected to hypoxic injury induced by CoCl2. Western blot, RT-PCR, transmission electron microscopy, immunohistochemistry, as well as echocardiography, and studies on isolated hearts were employed. Key resultsIn the hearts of rats with myocardial infarction, there was a significant accumulation of interstitial collagen fibers, accompanied by downregulation of CSE protein expression, activation of the PINK1/Parkin signaling pathway, and activation of mitochondrial autophagy. Intervention with AP39 resulted in a significant improvement of the aforementioned changes, which could be reversed by the addition of PAG. Similar results were observed in vitro experiments. Furthermore, the addition of CCCP reversed the antagonistic effect of AP39 on myocardial cell iron death, while the addition of RSL3 reversed the inhibitory effect of AP39 on collagen production in myocardial cells. Conclusion and implicationsThe mitochondrial-targeted H2S donor AP39 can inhibit mitochondrial autophagy through the PINK1/Parkin pathway, antagonize myocardial cell iron death, and improve myocardial fibrosis in rats with myocardial infarction.

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy.

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 µM) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.

PFKFB2 inhibits ferroptosis in myocardial ischemia/reperfusion injury through AMPK activation.

6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 (PFKFB2) is a key regulator of glycolytic enzyme. This study identified if PFKFB2 can regulate myocardial ferroptosis in ischemia/reperfusion (I/R) injury. Mice myocardial (I/R) injury and H9c2 cells oxygen-glucose deprivation/reperfusion (OGD/R) models were established. PFKFB2 expression was enhanced in I/R mice and OGD/R H9c2 cells. Overexpression of PFKFB2 improves heart function in I/R mice. Overexpression of PFKFB2 inhibits I/R and OGD/R-induced ferroptosis in mice and H9c2 cells. Mechanistically, overexpression of PFKFB2 activating the adenosine monophosphate activated protein kinase (AMPK). AMPK inhibitor compound C reverses effect of PFKFB2 overexpression in reducing ferroptosis under OGD/R treatment. In conclusion, PFKFB2 protects hearts against I/R-induced ferroptosis through activation of AMPK signaling pathway.

Propofol reduces lipopolysaccharide‑induced cardiomyocyte injury in sepsis by activating SIRT1‑mediated autophagy.

Myocardial injury is an indicator of poor prognosis in sepsis, whereas propofol has been reported to protect the myocardium. Therefore, the present study investigated the effect of propofol on myocardial injury in sepsis and its mechanism. An in vitro model of myocardial cell injury was established in myocardial H9C2 cells using lipopolysaccharide (LPS). The Cell Counting Kit 8 (CCK8) assay was used to investigate the effect of propofol pretreatment on the viability of normal and LPS-challenged H9C2 cells, whereas the lactate dehydrogenase (LDH) detection kit was used to measure the levels of LDH. The expression levels of LC3 were analyzed using an immunofluorescence assay. Western blotting was performed to analyze the expression levels of autophagy-related proteins. Following treatment with the autophagy inhibitor 3-methyladenine, CCK8 assay, TUNEL assay, western blotting, 2,7-dichlorohydrofluorescein diacetate assay and ELISA were performed to investigate whether propofol exerted its effects on cell viability, apoptosis, oxidative stress and inflammation via autophagy. Moreover, to further explore the regulatory mechanism of propofol in myocardial injury, sirtuin 1 (SIRT1) was knocked down via transfection with small interfering RNA, and SIRT1 protein was inhibited via the addition of the SIRT1 inhibitor EX527. The present study demonstrated that propofol activated autophagy in LPS-induced cardiomyocytes, and reversed the effects of LPS on viability, apoptosis, oxidative stress and the inflammatory response. Moreover, SIRT1 knockdown and inhibition decreased the activation of autophagy and the protective effect of propofol on LPS-induced cardiomyocytes. In conclusion, propofol reduced LPS-induced cardiomyocyte injury by activating SIRT1-mediated autophagy.

Two new phenolic amides from Allium chinense

ObjectiveTo isolate the phenolic amides from the dried bulbs of Allium chinense and investigate their myocardium protective activities. MethodsThe chemical constituents were isolated and purified by combining with silica gel column, Sephadex LH-20 column, HPLC and other chromatography techniques. Their structures were elucidated by NMR techniques and mass spectrometry. The isolated compounds were evaluated to determine their protective effect for myocardium cells in vitro. ResultsTwo new phenolic amides, namely, alichinemide I (1) and alichinemide II (2), and six konwn amides were isolated from the dried bulbs of A. chinense. The structures of compounds 3–8 were identified as 3-indolcarbaldehyde (3), 1-(2-aminophenyl)urea (4), 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid (5), N-trans-feruloyltyramine (6), N-trans-p-coumaroyltyramine (7), and N-(3,4-dimethoxyphenethyl) acetamide (8). Compound 3 (50 μmol/L) showed significant inhibitory effect on the damage of H9c2 myocardial cells induced by H2O2in vitro. ConclusionCompounds 1 and 2 were new phenolic amides. Compound 3 could be one of the potential myocardium protective constituents of A. chinense.

Plantamajoside alleviates hypoxia-reoxygenation injury through integrin-linked kinase/c-Src/Akt and the mitochondrial apoptosis signaling pathways in H9c2 myocardial cells

Myocardial ischemia–reperfusion injury(MIRI) is one of the common complications after myocardial infarction surgery, Oxidative stress is among the main mechanisms of myocardial ischemia–reperfusion injury. Plantamajoside (PMS), the main effective ingredient in the genus Plantain, has been reported to possess an antioxidation, anti-inflammatory and anti-apoptosis role. However, whether PMS can attenuate myocardial ischemia–reperfusion injury is not yet known. Herein, we explored the effects of PMS on hypoxia-reoxygenation (H/R) injury in H9c2 cardiomyocytes and the underling molecular mechanisms of the treatment. Network pharmacological analysis screened the top 31 key genes in the treatment of MIRI disease treated with PMS, and the result of molecular docking further illustrated the roles that the PMS play in the treatment of MIRI through its interference with integrin-linked kinase (ILK) target protein. PMS was not cytotoxic in the concentration range of 5–40 μM and increased cell survival after H/R injury in a concentration-dependent manner without affecting proliferation or growth. PMS significantly reduced the levels of lactate dehydrogenase, malonic dialdehyde, reactive oxygen species and cell apoptosis, and increased soperoxide dismutase activity compared with those of the H/R injury group. PMS promoted the protein and mRNA expression of ILK and Bcl-2, the protein expression of p-Akt, and reduced the protein and mRNA expression of Bax, Caspase-3, and Cytochrome c, the protein expression of p–c-Src. PMS has protective effects against H/R injury in H9c2 cells, and its protective mechanism may be related to reactive oxygen species clearance, activation of the ILK/c-Src/Akt pathway and inhibition of the mitochondrial apoptosis.

Ring finger protein 10 improves pirarubicin-induced cardiac inflammation by regulating the AP-1/Meox2 signaling pathway

ObjectivesPirarubicin (THP) is widely used in clinical antitumor therapy, but its cardiotoxicity seriously affects the therapeutic effect in patients. In the study, we investigated the role of ring finger protein 10 (RNF10) in cardiotoxicity induced by THP. Materials and methodsA cardiac toxicity model in Sprague-Dawley (SD) rats induced by THP was established. Changes in diet, weight, electrocardiogram (ECG), and echocardiography were observed. Serum levels of brain natriuretic peptide (BNP), creatine kinase MB (CK-MB), cardiac troponin T (cTnT), and lactate dehydrogenase (LDH) were measured. The expression of RNF10 in myocardium was observed by immunohistochemistry. The expressions of RNF10, activator protein-1 (AP-1), mesenchyme homeobox 2 (Meox2), total nuclear factor (NF)-κB p65 (T-P65), phosphorylated NF-κB p65 (PP65), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and mature IL-1β were detected by Western blot. A THP-induced H9c2 myocardial cell injury model was established. RNF10 was downregulated or overexpressed by RNF10 siRNA and a RNF10 lentiviral vector, respectively. Then, cell viability was measured. The expression of RNF10 in H9c2 cells was observed by immunofluorescence. All of the above signaling pathways were verified by Western blots. FindingsTHP caused a series of cardiotoxic manifestations in SD rats. Our studies suggested that THP caused cardiac inflammation by inhibiting the expression of RNF10, while overexpression of RNF10 antagonized the cardiotoxicity induced by THP. SignificanceOur study showed RNF10 improved THP-induced cardiac inflammation by regulating the AP-1/Meox2 signaling pathway. RNF10 may be a new target to treat THP-induced cardiotoxicity.

DUSP12 ameliorates myocardial ischemia-reperfusion injury through HSPB8-induced mitophagy.

This study aimed to explore the role of dual specificity phosphatase 12 (DUSP12) in regulating myocardial ischemia-reperfusion (I/R) injury and the underlying mechanism. The expression of DUSP12 in myocardial tissuesand heat-shock protein beta-8(HSPB8) and mitophagy-related proteins in myocardial tissues and H9c2 cells were detected by western blot analysis. The serum creatine kinase isoenzymes (CK-MB)and lactate dehydrogenase (LDH), levels of reactive oxygen speciesand malondialdehyde,superoxide dismutase activity in myocardial tissuesand H9c2 cells, and caspase-3 activity in H9c2 cells were analyzed by corresponding assay kits. The infarct area in therat's heart was observed by triphenyl tetrazolium chloridestaining. The apoptosis of myocardial cells in myocardial tissues and H9c2 cells was detected by terminal-deoxynucleotidyl transferase dUTP-biotin nick-end labelingassay. The interaction between DUSP12 and HSPB8 was clarified by the coimmunoprecipitation assay. The transfection efficacy of si-HSPB8#1 and si-HSPB8#2 in H9c2 cells was confirmed by real-time quantitative-polymerasechain reactionand western blot analysis. As a result, DUSP12 expression was downregulated in I/R rats, which was promoted by lentivirus-expressing DUSP12. DUSP12 overexpression reduced the serumcreatine kinase isoenzymes (CK-MB) and LDH, decreased the infarct area in the rat's heart, and suppressed the apoptosis and oxidative stress in myocardial tissues. DUSP12 overexpression also upregulated the expression of HSPB8 to promote mitophagy. The coimmunoprecipitation assay indicated that DUSP12 could be combined with HSPB8. In addition, DUSP12 overexpression could inhibit hypoxia/reoxygenation-elicited apoptosis as well as oxidative stress in H9c2 cells by upregulating HSPB8 expression to promote mitophagy, which was countervailed by HSPB8 deficiency. In conclusion, DUSP12 overexpression decreased the apoptosis and oxidative stress in myocardial I/R injury through HSPB8-induced mitophagy.