Apoptosis In Rat Cardiomyocytes Research Articles

Annexin-A1 short peptide alleviates septic myocardial injury by upregulating SIRT3 and inhibiting myocardial cell apoptosis.

Septic myocardial injury is a common complication of severe sepsis, which occurs in about 50% of cases. Patients with this disease may experience varying degrees of myocardial damage. Annexin-A1 short peptide (ANXA1sp), with a molecular structure of Ac-Gln-Ala-Tyr, has been reported to exert an organ protective effect in the perioperative period by modulating sirtuin-3 (SIRT3). Whether it possesses protective activity against sepsis-induced cardiomyopathy is worthy of study. This study aimed to investigate whether ANXA1sp exerts its anti-apoptotic effect in septic myocardial injury in vitro and in vivo via regulating SIRT3. In this study, we established in vivo and in vivo models of septic myocardial injury based on C57BL/6 mice and primary cardiomyocytes by lipopolysaccharide (LPS) induction. Results showed that ANXA1sp pretreatment enhanced the seven-day survival rate, improved left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), and cardiac output (CO), and reduced the levels of creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and lactate dehydrogenase (LDH). Western blotting results revealed that ANXA1sp significantly increased the expression of SIRT3, Bcl-2, and downregulated Bax expression. TUNEL staining and flow cytometry results showed that ANXA1sp could attenuate the apoptosis rate of cardiomyocytes, whereas this anti-apoptotic effect was significantly attenuated after SIRT3 knockout. To sum up, ANXA1sp can alleviate LPS-induced myocardial injury by reducing myocardial apoptosis via SIRT3 upregulation.

Regulation of optimized new Shengmai powder on cardiomyocyte apoptosis and ferroptosis in ischemic heart failure rats: The mediating role of phosphatidylinositol-3-kinase/protein kinase B/tumor protein 53 signaling pathway

Ethnopharmacological relevanceOptimized New Shengmai Powder (ONSMP) is a sophisticated traditional Chinese medicinal formula renowned for bolstering vital energy, optimizing blood circulation, and mitigating fluid retention. After years of clinical application, ONSMP has shown a significant impact in improving myocardial injury and cardiac function and has a positive effect on treating heart failure. However, many unknowns exist about the molecular biological mechanisms of how ONSMP exerts its therapeutic effects, which require further research and exploration. Aim of the studyExploring the potential molecular biological mechanisms by which ONSMP ameliorates cardiomyocyte apoptosis and ferroptosis in ischemic heart failure (IHF). Materials and methodsFirst, we constructed a rat model of IHF by inducing acute myocardial infarction through surgery and using echocardiography, organ coefficients, markers of heart failure, antioxidant markers, and histopathological examination to assess the effects of ONSMP on cardiomyocyte apoptosis and ferroptosis in IHF rats. Next, we used bioinformatics analysis techniques to analyze the active components, signaling pathways, and core targets of ONSMP and calculated the interactions between core targets and corresponding elements. Finally, we detected the positive expression of apoptosis and ferroptosis markers and core indicators of signaling pathways by immunohistochemistry; detected the mean fluorescence intensity of core indicators of signaling pathways by immunofluorescence; detected the protein expression of signaling pathways and downstream effector molecules by western blotting; and detected the mRNA levels of p53 and downstream effector molecules by quantitative polymerase chain reaction. ResultsONSMP can activate the Ser83 site of ASK by promoting the phosphorylation of the PI3K/AKT axis, thereby inhibiting the MKK3/6-p38 axis and the MKK4/7-JNK axis signaling to reduce p53 expression, and can also directly target and inhibit the activity of p53, ultimately inhibiting p53-mediated mRNA and protein increases in PUMA, SAT1, PIG3, and TFR1, as well as mRNA and protein decreases in SLC7A11, thereby inhibiting cardiomyocyte apoptosis and ferroptosis, effectively improving cardiac function and ventricular remodeling in IHF rat models. ConclusionONSMP can inhibit cardiomyocyte apoptosis and ferroptosis through the PI3K/AKT/p53 signaling pathway, delaying the development of IHF.

Ulinastatin protects against myocardial ischemia/reperfusion injury in rats via Rho/ROCK signaling pathway.

We aimed to study the influences of ulinastatin on diseased myocardial tissues, cardiomyocyte apoptosis and inflammatory reaction in rats with myocardial ischemia/reperfusion injury (IRI) via the Ras homolog (Rho)/Rho-associated kinase (ROCK) signaling pathway and its mechanism. The rats were randomly divided into three groups: control group (C group), IR model group (IR group) and IR model + ulinastatin treatment group (UR group). The pathological changes in myocardial tissues were detected via HE staining, the markers of myocardial injury were examined using kits, and apoptosis was determined through TUNEL assay. Moreover, ELISA was applied to measure the expressions of TNF-α, interleukin-6 (IL-6) and IL-8 in cardiac tissues, and Western blotting was performed to detect the protein expression levels of RhoA, ROCK2 and MLCP. The myocardial infarction area in the IR group was markedly larger than that in the C group (P<0.01) but was significantly reduced after ulinastatin treatment (P<0.05), and the IR group had higher levels of AST, cTnI, CK-MB and LDH than C group, but the levels of those indexes were significantly reduced after ulinastatin treatment.The cardiomyocyte apoptosis was increased in the IR group compared with that in the C group, while it was decreased in the UR group in comparison with that in the IR group. Besides, the UR group exhibited lowered expression levels of the Rho/ROCK signaling pathway-related proteins compared with the IR group. Ulinastatin may ameliorate the prognosis of rats with myocardial IRI via the Rho/ROCK signaling pathway.

Shuxuening Injection Inhibits Apoptosis and Reduces Myocardial Ischemia-Reperfusion Injury in Rats through PI3K/AKT Pathway.

To investigate the main components and potential mechanism of Shuxuening Injection (SXNI) in the treatment of myocardial ischemia-reperfusion injury (MIRI) through network pharmacology and in vivo research. The Traditional Chinese Medicine Systems Pharmacology (TCMSP) and PharmMapper databases were used to extract and evaluate the effective components of Ginkgo biloba leaves, the main component of SXNI. The Online Mendelian Inheritance in Man (OMIM) and GeneCards databases were searched for disease targets and obtain the drug target and disease target intersections. The active ingredient-target network was built using Cytoscape 3.9.1 software. The STRING database, Metascape online platform, and R language were used to obtain the key targets and signaling pathways of the anti-MIRI effects of SXNI. In order to verify the therapeutic effect of different concentrations of SXNI on MIRI in rats, 60 rats were first divided into 5 groups according to random number table method: the sham operation group, the model group, SXNI low-dose (3.68 mg/kg), medium-dose (7.35 mg/kg), and high-dose (14.7 mg/kg) groups, with 12 rats in each group. Then, another 60 rats were randomly divided into 5 groups: the sham operation group, the model group, SXNI group (14.7 mg/kg), SXNI+LY294002 group, and LY294002 group, with 12 rats in each group. The drug was then administered intraperitoneally at body weight for 14 days. The main biological processes were validated using in vivo testing. Evans blue/triphenyltetrazolium chloride (TTC) double staining, hematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis were used to investigate the efficacy and mechanism of SXNI in MIRI rats. Eleven core targets and 30 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were selected. Among these, the phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT) pathway was closely related to SXNI treatment of MIRI. In vivo experiments showed that SXNI reduced the myocardial infarction area in the model group, improved rat heart pathological damage, and reduced the cardiomyocyte apoptosis rate (all P<0.01). After SXNI treatment, the p-PI3K/PI3K and p-AKT/AKT ratios as well as B-cell lymphoma-2 (Bcl-2) protein expression in cardiomyocytes were increased, while the Bax and cleaved caspase 3 protein expression levels were decreased (all P<0.05). LY294002 partially reversed the protective effect of SXNI on MIRI. SXNI protects against MIRI by activating the PI3K/AKT signaling pathway.

Protein PDK4 Interacts with HMGCS2 to Facilitate High Glucoseinduced Myocardial Injuries.

As a distinct type of cardiomyopathy, diabetic cardiomyopathy (DCM) is featured as diastolic or systolic cardiac dysfunction in diabetic patients. In order to broaden the understanding of molecular mechanisms in DCM, we intended to explore the mechanism of the interaction between PDK4 protein and Hmgcs2 in high glucose (HG)-induced myocardial damage. PDK4 and Hmgcs2 expression in the myocardium of diabetes mellitus (DM) model rats and HG-incubated cardiomyocyte line H9C2 was analyzed by western blot analysis. Echocardiography and TUNEL assay were utilized for respective assessment of cardiac structure and function and cardiomyocyte apoptosis in DM rats after silencing PDK4 or/and Hmgcs2. In vitro, the impact of PDK4 and Hmgcs2 on HG-induced cardiomyocyte injuries was identified with cell counting kit-8 and flow cytometry assays, along with detection of LDH release, caspase-3/7 activities, and reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Moreover, a coimmunoprecipitation assay was utilized to test the interaction between PDK4 and Hmgcs2. Both PDK4 and Hmgcs2 were highly expressed in the myocardial tissues of DM rats. Mechanistically, PDK4 interacted with Hmgcs2 to upregulate Hmgcs2 expression in HG-induced H9C2 cells. Silencing PDK4 improved cardiac function and reduced cardiomyocyte apoptosis in DM rats. In HG-induced H9C2 cells, PDK4 or Hmgcs2 silencing enhanced cell viability and reduced LDH release, caspase-3/7 activities, cell apoptosis, and ROS and MDA levels, and these trends were further promoted by the simultaneous silencing of PDK4 and Hmgcs2. In summary, the silencing of PDK4 and Hmgcs2 alleviated HG-induced myocardial injuries through their interaction.

Cardioprotective Role of Tinospora cordifolia against Trimethylamine-NOxide and Glucose Induced Stress in Rat Cardiomyocytes.

Type 2 diabetes has become a concern issue that affects the quality of life and can increase the risk of cardiac insufficiency elevating the threat to the life safety of patients. A recognized cause of cardiac insufficiency is diabetic cardiomyopathy, chronic hyperglycemia, and myocardial lipotoxicity which can reduce the myocardial contractile performance, and enhance the cardiomyocyte hypertrophy and interstitial fibrosis. The cause of diabetic cardiomyopathy is multi-factorial which includes oxidative stress, insulin resistance, inflammation, apoptosis, and autophagy. Recent clinical studies have suggested the dysbiosis of gut microbiota, secretion of metabolites, and their diffusion in to the host as to have direct detrimental effects on the cardiac contractility. In the present paper, we have done in silico studies including molecular interaction of phytoconstituents of Tinospora cordifolia against reactive oxygen species producing proteins. Whereas, in vitro studies were conducted on H9C2 cardiac cells including cell morphological examination, level of reactive oxygen species, cell count-viability, apoptotic status, in the presence of high glucose, trimethylamine-n-oxide, and plant extracts which were determined through cell analyzer and microscopic assays. The treatment of high glucose and trimethylamine-n-oxide was found to be increase the cardiac stress approximately two fold by attenuating hypertrophic conditions, oxidative stress, and apoptosis in rat cardiomyocytes, and Tinospora cordifolia was found to be a cardioprotective agent. Conclusively, our study has reported that the Indian medicinal plant Tinospora cordifolia has the ability to treat diabetic cardiomyopathy. Our study can open up a new herbal therapeutic strategy against diabetic cardiomyopathy.

Saponins of Panax japonicus ameliorates cardiac aging phenotype in aging rats by enhancing basal autophagy through AMPK/mTOR/ULK1 pathway

Heart disease is a significant health concern for elderly individuals, with heart aging being the primary cause. Recent studies have shown that autophagy can play a protective role in preventing cardiac aging. Our previous research confirmed that Chikusetsu saponin IVa, a fundamental component of Saponins of Panax japonics (SPJ), can enhance basic autophagy levels in cardiomyocyte of isoproterenol induced cardiac fibrosis mice. However, it remains unclear whether SPJ possesses a protective effect on cardiac dysfunction during the natural aging process. Rats were randomly divided into four groups: adult control group (6 months old), aging group (24 months old), aging group treated with 10 mg/kg SPJ, and aging group treated with 30 mg/kg SPJ. The heart function, blood pressure, and heart mass index (HMI) were measured. Hematoxylin and eosin staining (H&E) and Wheat Germ Agglutinin (WGA) staining were used to observe the changes in morphology, while Masson staining was used to examine collagen deposition in the rat hearts and CD45 immunohistochemistry was conducted to examine the macrophage infiltration in heart tissues. TUNEL kit was used to detect apoptosis level of cardiomyocyte, and western blot was used to evaluate autophagy-related proteins as well as AMPK/mTOR/ULK1 pathway-related markers. SPJ treatment improved the cardiac function, reduced HMI, attenuated myocardial fiber disorder, inhibited inflammatory cell infiltration, and decreased collagen deposition and cardiomyocyte apoptosis in aging rats. Additionally, SPJ treatment decreased the expression of aging-related proteins and restored the expression of autophagy-related markers. SPJ activated autophagy through the activation of AMPK, which in turn increased the phosphorylation of ULK1(Ser555), while inhibited the phosphorylation of mTOR and ULK1(Ser757). Our study demonstrates that SPJ improves the cardiac function of aging rats by enhancing basal autophagy through the AMPK/mTOR/ULK1 pathway. These results offer a theoretical foundation and empirical evidence to support the clinical advancement of SPJ in enhancing age-related cardiac dysfunction.

Alcohol Intake Provoked Cardiomyocyte Apoptosis Via Activating Calcium-Sensing Receptor and Increasing Endoplasmic Reticulum Stress and Cytosolic [Ca2+]i.

Cardiomyocyte apoptosis plays an important role in alcoholic cardiac injury. However, the association between calcium-sensing receptor (CaSR) and alcohol-induced cardiomyocyte apoptosis remain unclear. Therefore, we investigated the role and its moleculer mechanism of CaSR in rat cardiomyocyte apoptosis induced by alcohol. Alcohol-induced cardiomyocyte apoptosis in vivo and in vitro model of rats were applied in this study. The expression of CaSR, endoplasmic reticulum stress markers and apoptosis were tested by immunohistological staining, western blot, TUNEL and flow cytometry, respectively. [Ca2+]i were detected by confocal laser scanning microscopy. Compared with the control group, alcohol intake (AI) led to abnormal arrangements of cardiomyocytes and obvious increase of myocardial apoptosis. Moreover, AI also significantly upregulated protein expression of CaSR, GRP94, caspase-12 and CHOP. Alcohol induced apoptosis of cultured cardiomyocytes of rats in a dose-dependent way. Activation of CaSR markedly enhanced cardiomyocyte apoptosis and ERS induced by alcohol, ERS inducer also significantly increased cardiomyocyte apoptosis without activating CaSR. Furthermore, GdCl3 augmented alcohol-induced increase of [Ca2+]i in cardiomyocytes, which was attenuated by NPS2390 but not 4-PBA pre-treatment. Alcohol could induce cardiomyocyte apoptosis in rats in vivo and in vitro, which was mediated probably via activating CaSR, and then ERS and the increase of the cytosolic [Ca2+]i. This provides a potential target for preventing cardiomyocyte apoptosis and cardiomyopathy induced by alochol.

Dexmedetomidine attenuates myocardial ischemia-reperfusion injury in hyperlipidemic rats by inhibiting inflammation, oxidative stress and NF-κB.

The present study was conducted to determine the protective effect of Dexmedetomidine (DEX) in myocardial ischemia-reperfusion injury in hyperlipidemic rats. Towards this, the effect of DEX was first evaluated on the infarct size and the histopathology of cardiac tissues using TTC and H and E staining, and it was found that DEX significantly improved the infarct size and architecture of the myocardial tissues following the I/R injury. DEX also showed significant improvement in various examined hemodynamic parameters (e.g., LVSP, and ± dp/dtmax ) in a dose-dependent manner. The lipid profile (LDL, VLDL, TC, TG, and HDL level) of the rats were also found significantly improved in DEX-treated rats. The level of various pro-inflammatory cytokines (IL-1β, IL-6, IL-10, IL-17, and TNF-α), cardiac injury (CK, CK-MB, Troponin I AST, ALT, and LDH), and oxidative stress (MDA, SOD, and GSH) biomarkers were also found to be restored near to the normal in DEX-treated group. It has been found that DEX also significantly reduces apoptosis of rat cardiomyocytes. In western blot analysis, DEX showed a significant reduction in the activation of NF-κB. In conclusion, our study demonstrated the protective effect of Dexmedetomidine in myocardial ischemia-reperfusion injury in hyperlipidemic rats possibly via amelioration of oxidative stress, and inflammation apoptosis.

Cardiomyocyte Exosomal Nanovesicles Mediating p53 to Regulate Autophagy and Protect Myocardial Infarction

This study explores the protective mechanism of cardiomyocyte exosome nanovesicles on myocardial infarction. Enzyme activity was measured and apoptosis of rat cardiomyocytes was assessed by TdT-mediated dUTP Nick-End Labeling (TUNEL) along with analysis of myocardial function by color Doppler echocardiogram. In addition, triphenyl tetrazolium chloride (TTC) staining evaluated the myocardial ischemia. BMSCs were co-cultured with primary cardiomyocytes followed by measuring autophagy and exosome morphology of myocardial tissue by transmission electron microscope and protein expression by Western blot. Exosomes secreted by Bone marrow mesenchymal stem cells (BMSCs) were successfully isolated. LDH, CK, CK-MB activity, cardiomyocyte apoptosis rate, LVESD, LVEDD, LVEDP, myocardial infarction area, cell area occupied by autophagic vesicles and p53 expression in model group were higher than control group (P &lt;0.05) and they were lower in exosome group than model group. In addition, LVEF, LVFS, and LVSP were lower in model group and higher in exosome group (P &lt;0.05). Model group showed significantly promoted autophagy activation and autophagosome formation, which were inhibited by cardiomyocyte exosomes. Moreover, exosome-like vesicles were found with a diameter of 60–90 nm. Furthermore, PKH-26 staining showed that cardiomyocytes could uptake exosomes well. Myocardial injury significantly promotes autophagy activation. Moreover, the apoptosis rate of cardiomyocytes and the area of myocardial ischemia were reduced and the cardiac function of rats with myocardial infarction was improved.

HDAC3 Impairs Cardiac Function in Chronic Heart Failure Rats via Mediating MicroRNA-26b-3p to Target High Mobility Group AT-Hook 2.

Histone deacetylase 3 (HDAC3) has been studied in chronic heart failure (CHF), while the regulatory mechanism of HDAC3 on the development of CHF in regulating microRNA (miR)-26b-3p/high mobility group AT-hook 2 (HMGA2) axis has not been extensively investigated. This study aimed to probe the effects of HDAC3, miR-26b-3p and HMGA2 on CHF. CHF rat models were established using aortic coarctation. HDAC3, miR-26b-3p and HMGA2 levels in CHF rats were examined. Thereafter, the CHF rats were injected with relative oligonucleotides and plasmids of HDAC3, miR-26b-3p and HMGA2 to detect the cardiac function, inflammatory reaction, myocardial tissue pathological changes, and cardiomyocyte apoptosis. The binding relationship between miR-26b-3p and HMGA2 and the interaction between HDAC3 and miR-26b-3p were validated. HDAC3 and HMGA2 were elevated, while miR-26b-3p was decreased in CHF rats. The reduced HDAC3 or HMGA2 or enriched miR-26b-3p attenuated cardiac dysfunction, inflammatory reaction, myocardial tissue pathological changes and cardiomyocyte apoptosis in CHF rats, while the reduction of miR-26b-3p exerted the opposite effects. Furthermore, the inhibition of the miR-26b-3p or elevation of HMGA2 reversed the effect of reduced HDAC3 on mitigating CHF progression. Mechanically, miR-26b-3p targeted HMGA2 and HDAC3 bound to miR-26-3p. Downregulation of HDAC3 relieves cardiac function in CHF rats via mediating miR-26b-3p/HMGA2 axis. This study provides novel theory references and a distinct direction for the therapy strategies of CHF.

Tanshinone IIA inhibits hypoxia/reoxygenation-induced cardiomyocyte apoptosis and autophagy by regulating ABCE1

To investigate the effects of tanshinone IIA on apoptosis and autophagy induced by hypoxia/reoxygenation in H9C2 cardiomyocytes and its mechanism. H9C2 cardiomyocytes in logarithmic growth phase were divided into control group, hypoxia/reoxygenation model group and tanshinone IIA low-dose, medium-dose and high-dose groups (50, 100, 200 mg/L tanshinone IIA were treated after hypoxia/reoxygenation respectively). The dose with good therapeutic effect was selected for follow-up study. The cells were divided into control group, hypoxia/reoxygenation model group, tanshinone IIA+pcDNA3.1-NC group and tanshinone IIA+pcDNA3.1-ABCE1 group. The cells were transfected with the overexpressed plasmids pcDNA3.1-ABCE1 and pcDNA3.1-NC and then treated accordingly. Cell counting kit-8 (CCK-8) was used to detect H9C2 cell activity in each group. The apoptosis rate of cardiomyocytes was detected by flow cytometry. The ATP-binding cassette transporter E1 (ABCE1), apoptosis-related proteins Bcl-2 and Bax, caspase-3, autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3II/I) and p62 mRNA expression level of H9C2 cells in each group were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The protein expression levels of the above indexes in H9C2 cells were detected by Western blotting. (1) Cell activity and ABCE1 expression: tanshinone IIA inhibited the activity of H9C2 cells induced by hypoxia/reoxygenation, and the effect was significant at medium-dose [(0.95±0.05)% vs. (0.37±0.10)%, P < 0.01], mRNA and protein expression of ABCE1 were significantly reduced [ABCE1 mRNA (2-ΔΔCt): 2.02±0.13 vs. 3.74±0.17, ABCE1 protein (ABCE1/GAPDH): 0.46±0.04 vs. 0.68±0.07, both P < 0.05]. (2) Expression of apoptosis-related proteins: medium-dose of tanshinone IIA inhibited the apoptosis of H9C2 cells induced by hypoxia/reoxygenation [apoptosis rate: (28.26±2.52)% vs. (45.27±3.07)%, P < 0.05]. Compared with the hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated the protein expression of Bax and caspase-3 in H9C2 cells induced by hypoxia/reoxygenation, and significantly up-regulated the protein expression of Bcl-2 [Bax (Bax/GAPDH): 0.28±0.03 vs. 0.47±0.03, caspase-3 (caspase-3/GAPDH): 0.31±0.02 vs. 0.44±0.03, Bcl-2 (Bcl-2/GAPDH): 0.53±0.02 vs. 0.37±0.05, all P < 0.05]. (3) Expression of autophagy-related proteins: compared with the control group, the positive rate of LC3 in the hypoxia/reoxygenation model group was significantly increased, while the positive rate of LC3 in the medium-dose of tanshinone IIA group was significantly decreased [(20.67±3.09)% vs. (42.67±3.86)%, P < 0.01]. Compared with hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated Beclin-1, LC3II/I and p62 protein expressions [Beclin-1 (Beclin-1/GAPDH): 0.27±0.05 vs. 0.47±0.03, LC3II/I ratio: 0.24±0.05 vs. 0.47±0.04, p62 (p62/GAPDH): 0.21±0.03 vs. 0.48±0.02, all P < 0.05]. (4) Expression of apoptosis and autophagy related proteins after transfection with overexpressed ABCE1 plasmid: compared with tanshinone IIA+pcDNA3.1-NC group, the protein expression levels of Bax, caspase-3, Beclin-1, LC3II/I and p62 in tanshinone IIA+pcDNA3.1-ABCE1 group were significantly up-regulated, while the protein expression level of Bcl-2 was significantly down-regulated. 100 mg/L tanshinone IIA could inhibit autophagy and apoptosis of cardiomyocytes by regulating the expression level of ABCE1. So, it protects H9C2 cardiomyocytes injury induced by hypoxia/reoxygenation.

Mechanism of new optimized Sheng-Mai-San Formula to regulate cardiomyocyte apoptosis through NMDAR pathway

Background and objectivesIschemic heart failure (HF) has become a disease that seriously endangers people's life and health. As a herbal formula widely used in clinical practice, new optimized Sheng-Mai-San (NO-SMS) has been shown to be significantly effective in improving cardiac function, increasing exercise tolerance, and slowing the progression of myocardial fibrosis in heart failure patients in multi-center clinical studies in various regions of China. In our previous pharmacodynamic and toxicological studies, we found that a medium-dose formulation (8.1 g of raw drug/kg) was the most effective in the treatment of heart failure, but its mechanism of action is still being investigated. The present study is exploring its relationship with cardiomyocyte apoptosis. Materials and methodsWe investigated and verified this through two parts of experiments, in vivo and in vitro. Firstly, we prepared male SD rats with heart failure models by ligating the left anterior descending branch of the coronary artery (EF ≤ 50%), which were treated with NO-SMS Formula (8.1 g of raw drug/kg/d), Ifenprodil (5.4 mg/kg/d) or Enalapril (0.9 mg/kg/d) prepared suspensions by gavage for 4 weeks. The cardiac and structural changes were evaluated by echocardiography, H&E, and MASSON staining. The apoptosis of cardiomyocytes in each group was detected by Western blot, qRT-PCR, and ELISA. In vitro cell experiments include H9c2 cardiomyocyte injury induced by H2O2 and NMDA respectively, and the groups were incubated with NO-SMS and Ifenprodil-containing serum for 24 h. Apoptosis was detected by Annexin V-FITC/PI double-staining method, and the rest of the assays were consistent with the in vivo experiments. ResultsCompared with the model group, the NO-SMS formula group and the Ifenprodil group could significantly improve cardiac function, delay myocardial fibrosis, reduce the expression of pro-apoptotic proteins, mRNA, and the concentration levels of Ca2+ and ROS in heart failure rats and H9c2 cardiomyocytes with H2O2 and NMDA-induced injury, which could significantly reduce the apoptosis rate of damaged cardiomyocytes and effectively inhibit the apoptosis of cardiomyocytes. ConclusionNO-SMS Formula improved cardiac function, inhibited ventricular remodeling and cardiomyocyte apoptosis in HF rats, and its mechanism may be related to the regulation of the NMDAR signaling pathway, inhibition of large intracellular Ca2+ inward flow, and ROS production in cardiomyocytes.

Highlighting the novel effects of high-intensity interval training on some histopathological and molecular indices in the heart of type 2 diabetic rats.

Type 2 diabetes is one of the most common metabolic diseases in recent years and has become an important risk factor for cardiovascular disorders. The first goal is to reduce type 2 diabetes, and in the case of cardiovascular disease, the second goal is to reduce and manage that disorder. The rats were divided into 4 groups: Healthy Control (n=8), Diabetes Control (n=8), Diabetes Training (n=8), and Healthy Training (n=8). The protocol consisted of 8 weeks of High-intensity interval (5 sessions per week), where the training started with 80% of the peak speed in the first week, and 10% was added to this speed every week. To measure the level of B-catenin, c-MYC, GSK3B, and Bcl-2 proteins using the western blot method, cardiac pathological changes were measured using hematoxylin and eosin staining, Masson's trichrome and PAS staining and apoptosis using the TUNEL method. Histological results showed that diabetes causes significant pathological hypertrophy, fibrosis, and severe apoptosis in heart tissue. HIIT training significantly reduced pathological hypertrophy and fibrosis in heart tissue, and the rate of cardiomyocyte apoptosis was greatly reduced. This research showed that diabetes disorder increases the levels of B-catenin and c-Myc proteins and causes a decrease in the expression of GSK3B and Bcl-2 proteins. After eight weeks of HIIT training, the levels of B-catenin and c-Myc proteins decreased significantly, and the levels of GSK3B and Bcl-2 proteins increased. This study showed that HIIT could be a suitable strategy to reduce cardiomyopathy in type 2 diabetic rats. However, it is suggested that in future studies, researchers should perform different intensities and exercises to promote exercise goals in type 2 diabetic cardiomyopathy.

Effect of miR-499-5p/SOX6 axis on atrial fibrosis in rats with atrial fibrillation.

Atrial fibrosis is involved in the progression of atrial fibrillation (AF). miR-499-5p is the most downregulated microRNA in arrhythmogenic cardiomyopathy hearts. Sry-related high-mobility-group box 6 (SOX6) is associated with apoptosis, inflammatory responses, and fibrosis. This study investigated the mechanism of miR-499-5p in ameliorating AF rats by regulating SOX6. AF rat models were established by injecting Ach-CaCl2 mixture, and the rats were treated with Lv-miR-499-5p/oe-SOX6/si-SOX6 before modeling. AF duration was recorded using electrocardiogram. miR-499-5p and SOX6 expression levels in the myocardium were determined by reverse transcription-quantitative polymerase chain reaction. The binding of miR-499-5p and SOX6 was validated. The atrial fibrosis degree and cardiomyocyte apoptosis were assessed using the Masson and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining methods. Levels of SOX6, atrial fibrosis markers (collage I/α-SMA/TGFβ1), cell cycle-related proteins (p21/CDC25/Cyclin B1), and cell senescence markers (SA-β-gal/γ-H2AX) were measured using Western blotting and immunohistochemistry. miR-499-5p was downregulated and SOX6 was upregulated in AF rats. miR-499-5p overexpression shortened the AF duration, alleviated atrial fibrosis, and decreased collage I/α-SMA/TGFβ1. miR-499-5p targeted SOX6 to ameliorate atrial fibrosis. AF rats exhibited increased p21/CDC25/Cyclin B1/SA-β-gal/γ-H2AX levels and raised cardiomyocyte apoptosis. SOX6 silencing downregulated p21 and alleviated cardiomyocyte cycle arrest, cell senescence, and apoptosis in AF rats. Shortly, miR-499-5p suppresses atrial fibrosis and cardiomyocyte senescence by targeting SOX6 and downregulating p21, thus mitigating AF in rats.

Platelet Membrane-Fused Circulating Extracellular Vesicles Protect the Heart from Ischemia/Reperfusion Injury.

Myocardial ischemia/reperfusion injury (I/RI) may potentiate cardiac remodeling and heart failure, while effective therapies for I/RI remain lacking. Circulating human plasma-derived extracellular vesicles (hEV) have great potential to protect against I/RI. However, the effective delivery of hEV in vivo remains a limiting factor for clinical application. The present study constructs a biomimetic delivery system of platelet membrane-fused hEV (P-hEV), utilizing the natural affinity of platelets for hEV delivery to the injured vascular and myocardial sites. The results show that platelet membrane and hEV membrane fusion can be achieved through repeated extrusion. Compared to non-modified hEV, P-hEV uptake is greatly enhanced in human umbilical vein endothelial cells (HUVECs) stressed by oxygen-glucose deprivation/reperfusion (OGD/R). Functionally, P-hEV inhibits HUVECand neonatal rat cardiomyocyte(NRCM) apoptosis and promotes HUVECsmigration and tube formation under OGD/R stress in vitro. Intravenous delivery of P-hEV more effectively targets and accumulates at injury sites in the heart. Furthermore, P-hEV significantly enhances protection against acute I/RI and attenuates cardiac remodeling at three weeks post-I/RI. In conclusion, the platelet membrane-fused hEV delivery system enhances the target delivery of EV to protect against myocardial I/RI, presenting a novel drug delivery system for ischemic heart diseases.

Peptidase Inhibitor 16 Attenuates Left Ventricular Injury and Remodeling After Myocardial Infarction by Inhibiting the HDAC1-Wnt3a-β-Catenin Signaling Axis.

Background Myocardial infarction (MI) is a cardiovascular disease with high morbidity and mortality. PI16 (peptidase inhibitor 16), as a secreted protein, is highly expressed in heart diseases such as heart failure. However, the functional role of PI16 in MI is unknown. This study aimed to investigate the role of PI16 after MI and its underlying mechanisms. Methods and Results PI16 levels after MI were measured by enzyme-linked immunosorbent assay and immunofluorescence staining, which showed that PI16 was upregulated in the plasma of patients with acute MI and in the infarct zone of murine hearts. PI16 gain- and loss-of-function experiments were used to investigate the potential role of PI16 after MI. In vitro, PI16 overexpression inhibited oxygen-glucose deprivation-induced apoptosis in neonatal rat cardiomyocytes, whereas knockdown of PI16 exacerbated neonatal rat cardiomyocyte apoptosis. In vivo, left anterior descending coronary artery ligation was performed on PI16 transgenic mice, PI16 knockout mice, and their littermates. PI16 transgenic mice showed decreased cardiomyocyte apoptosis at 24 hours after MI and improved left ventricular remodeling at 28 days after MI. Conversely, PI16 knockout mice showed aggravated infract size and remodeling. Mechanistically, PI16 downregulated Wnt3a (wingless-type MMTV integration site family, member 3a)/β-catenin pathways, and the antiapoptotic role of PI16 was reversed by recombinant Wnt3a in oxygen-glucose deprivation-induced neonatal rat cardiomyocytes. PI16 also inhibited HDAC1 (class I histone deacetylase) expression, and overexpression HDAC1 abolished the inhibition of apoptosis and Wnt signaling of PI16. Conclusions In summary, PI16 protects against cardiomyocyte apoptosis and left ventricular remodeling after MI through the HDAC1-Wnt3a-β-catenin axis.

Dexmedetomidine postconditioning attenuates myocardial ischemia/reperfusion injury by activating the Nrf2/Sirt3/SOD2 signaling pathway in the rats

ABSTRACTObjectivesTo observe the protective effects of dexmedetomidine (Dex) postconditioning on myocardial ischemia/reperfusion injury (IRI) and to explore its potential molecular mechanisms.MethodsOne-hundred forty-seven male Sprague-Dawley rats were randomly divided into five groups receiving the different treatments: Sham, ischemia/reperfusion (I/R), Dex, Brusatol, Dex + Brusatol. By the in vivo rat model of myocardial IRI, cardioprotective effects of Dex postconditioning were evaluated by assessing serum CK-MB and cTnI levels, myocardial HE and Tunel staining and infarct size. Furthermore, the oxidative stress-related markers including intracellular ROS level, myocardial tissue MDA level, SOD and GSH-PX activities were determined.ResultsDex postconditioning significantly alleviated myocardial IRI, decreased intracellular ROS and myocardial tissue MDA level, increased SOD and GSH-PX activities. Dex postconditioning significantly up-regulated myocardial expression of Bcl-2, down-regulated Bax and cleaved caspase-3 and decreased cardiomyocyte apoptosis rate. furthermores, Dex postconditioning promoted Nrf2 nuclear translocation, increased myocardial expression of Sirt3 and SOD2 and decreased Ac-SOD2. However, brusatol reversed cardioprotective benefits of Dex postconditioning, significantly decreased Dex-induced Nrf2 nuclear translocation and reduced myocardial expression of Sirt3 and SOD2.ConclusionsDex postconditioning can alleviate myocardial IRI by suppressing oxidative stress and apoptosis, and these beneficial effects are at least partly mediated by activating the Nrf2/Sirt3/SOD2 signaling pathway.

Compound Kushen injection attenuates angiotensin II‑mediated heart failure by inhibiting the PI3K/Akt pathway.

Compound Kushen injection (CKI) is a type of traditional Chinese medicine that has previously been studied for the treatment of various types of cancer. Previous studies have reported that CKI regulates cell apoptosis by downregulating the PI3K/Akt pathway. The present study aimed to determine whether CKI alleviates heart failure (HF) by attenuating cardiomyocyte apoptosis via the inhibition of the PI3K/Akt pathway. Angiotensin II (Ang II) was used to elicit HF, and osmotic minipumps with either Ang II (2µg/kg/day) or phosphate‑buffered saline (PBS; 200µl) were subcutaneously implanted into 6‑week‑old male C57BL/6 mice for 3weeks. In addition, PBS or CKI (25mg/kg/day) were subcutaneously infused once a day for 3weeks. Echocardiography was used to examine hemodynamics. The myocardial injury biomarkers, cardiac troponin I and N‑terminal (NT)‑pro hormone B‑type natriuretic peptide, were assessed using enzyme‑linked immunosorbent assay. Transmission electron microscopy was used to determine the morphology of the myocardium. The rate of apoptosis was detected using TUNEL staining and flow cytometry (FCM), and the expression levels of apoptosis‑related proteins were measured using western blot (WB) analysis. Moreover, H9C2 cells were treated with CKI (2mg/ml) or LY294002 (an inhibitor of the PI3K/Akt pathway; 25µmol/l) in combination with Ang II (1µmol/l) for 48h. Cell Counting Kit‑8 assay, FCM and WB analysis were performed in the H9C2 cells to examine cell viability, cell cycle distribution and representative signaling proteins. It was found that CKI promoted healthy cardiac function, reduced myocardial structural damage and reduced the rate of cardiomyocyte apoptosis. CKI markedly attenuated the expression of apoptosis‑related proteins in the PI3K/Akt pathway. The results of the invitro experiments indicated that CKI promoted cardiomyocyte proliferation and inhibited apoptosis, similar to LY294002. On the whole, the present study demonstrates that CKI reduces cardiomyocyte apoptosis, promotes healthy cardiac function and attenuates Ang II‑mediated HF. These ameliorative effects may be associated with the inhibition of the PI3K/Akt pathway.

The Protective Effect of Sheng Mai Yin on Diabetic Cardiomyopathy via NLRP3/Caspase-1 Pathway.

Sheng Mai Yin (SMY) has therapeutic effects on myocardial infarction (MI), heart failure (HF), diabetic cardiomyopathy (DCM), and myocarditis. To study whether SMY can relieve pyroptosis and play a protective role in diabetic cardiomyopathy, a molecular docking technique was used to predict the possible mechanism of SMY against DCM. Then, a DCM rat model was induced by intraperitoneal injection of streptozotocin (STZ), divided into 5 groups: the DM group (model), SMY-L group (2.7 mL/kg SMY), SMY-M group (5.4 mL/kg SMY), SMY-H group (10.8 mL/kg SMY), and Met group (120 mg/kg metformin). Rats in the CTL group (control) and DM group were given normal saline. After 8 weeks, the levels of blood glucose, lipids, and myocardial enzymes were detected according to the kit instructions. Cardiac function was detected by echocardiography. HE and Masson were used to observing the pathological changes, collagen deposition, and collagen volume fraction (CVF). The apoptosis rate of cardiomyocytes was determined by Tunel. The IL-1β level was determined by ELISA and RT-PCR. The expressions of NLRP3, caspase-1, and GSDMD were measured using RT-PCR and Western blotting. The docking results suggested that SMY may act on NLRP3 and its downstream signal pathway. The in vivo results showed that SMY could reduce blood glucose and lipid levels, improve heart function, improve histopathological changes and myocardial enzymes, and alleviate cardiomyocyte apoptosis and myocardial fibrosis. SMY inhibited the mRNA and protein expressions of NLRP3, ASC, Caspase-1, and GSDMD and IL-1β production. SMY can reduce DCM by regulating the NLRP3/caspase-1 signaling pathway, providing a new research direction for the treatment of DCM.