Cardiomyocyte Apoptosis In Mice Research Articles

Cyanidin prevents cardiomyocyte apoptosis in mice after myocardial infarction.

Myocardial infarction is a worldwide disease with high morbidity and mortality and a major cause of chronic heart failure, seriously affecting patients' quality of life. Natural medicine has been used to cure or prevent cardiovascular disease for decades. As a natural flavonoid, anthocyanidin has been used to treat many diseases due to its antioxidative, anti-inflammatory, and other properties. A mouse model (C57BL/6) weighing 30-40g was utilized to induce myocardial infarction by ligating the left anterior descending coronary artery. Cyanidin (30mg/kg) was administered orally to mice for four weeks. A variety of assessments were used to evaluate cardiac function. The gene expression was measured using RNAseq and Western blot. Histological changes in myocardial tissue were assessed using staining techniques, including Masson, Hematoxylin Eosin (HE), and transmission electron microscopy. Tunnel staining was implemented as a method to detect cellular apoptosis. For the quantification of B-type natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) in the serum, an enzyme-linked immunosorbent assay (ELISA) was employed. Furthermore, autodock simulation was executed in order to assess the interaction between cyanidin and a subset of genes. Cyanidin treatment inhibited myocardial cell apoptosis, improved cardiac function, and reduced serum concentrations of BNP and atrial natriuretic peptide ANP, as well as mitigated histological cardiac tissue damage. Cyanidin also inhibited the activity of matrix metalloproteinases (MMP2/9) and Fibronectin 1 (Fn1). Cyanidin improves heart function and reduces myocardial damage in mice after MI. Furthermore, cyanidin can prevent cardiomyocyte apoptosis. These effects are most likely caused by suppression of MMP9/2 and control of the Akt signaling pathway, suggesting an appropriate therapeutic target.

Mycn ameliorates cardiac hypertrophy-induced heart failure in mice by mediating the USP2/JUP/Akt/β-catenin cascade

BackgroundPathological cardiac hypertrophy is associated with cardiac dysfunction and is a key risk factor for heart failure and even sudden death. This study investigates the function of Mycn in cardiac hypertrophy and explores the interacting molecules.MethodsA mouse model of cardiac hypertrophy was induced by isoproterenol (ISO). The cardiac dysfunction was assessed by the heart weight-to-body weight ratio (HW/BW), echocardiography assessment, pathological staining, biomarker detection, and cell apoptosis. Transcriptome alteration in cardiac hypertrophy was analyzed by bioinformatics analysis. Gain- or loss-of-function studies of MYCN proto-oncogene (Mycn), ubiquitin specific peptidase 2 (USP2), and junction plakoglobin (JUP) were performed. The biological functions of Mycn were further examined in ISO-treated cardiomyocytes. The molecular interactions were verified by luciferase assay or immunoprecipitation assays.ResultsMycn was poorly expressed in ISO-treated mice, and its upregulation reduced HW/BW, cell surface area, oxidative stress, and inflammation while improving cardiac function of mice. It also reduced apoptosis of cardiomyocytes in mice and those in vitro induced by ISO. Mycn bound to the USP2 promoter to activate its transcription. USP2 overexpression exerted similar myocardial protective functions. It stabilized JUP protein by deubiquitination modification, which blocked the Akt/β-catenin pathway. Knockdown of JUP restored phosphorylation of Akt and β-catenin protein level, which negated the protective effects of USP2.ConclusionThis study demonstrates that Mycn activates USP2 transcription, which mediates ubiquitination and protein stabilization of JUP, thus inactivating the Akt/β-catenin axis and alleviating cardiac hypertrophy-induced heart failure.

MALAT1/miR-185-5p mediated high glucose-induced oxidative stress, mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway.

To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR-185-5p with MALAT1 and RhoA were validated by dual-luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D-glucose (HG) in the presence or absence of MALAT1-shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR-185-5p expression were determined by real-time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1S616 , mitofusin 2 and apoptosis-related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC-1. MALAT1 was significantly up-regulated while miR-185-5p was down-regulated in myocardium of db/db mice and HG-induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG-induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p and mediated HG-induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.

The Action and Mechanism of Trehalose on GATA4 Autophagy Degradation and Ventricular Remodeling.

To probe the effect of trehalose on myocardial hypertrophy and its specific molecular mechanism. C57BL/6J male mice were divided into four subgroups: Sham operation subgroup (Sham), negative sham subgroup (Sham+Trehalose), transverse aortic constriction (TAC), and trehalose treatment subgroup (TAC+Trehalose). Immediately after the TAC operation, trehalose at a dose of 10 mg/kg was given daily via gavage. After four weeks, changes in cardiac function were evaluated using ultrasound to measure EF (ejection fraction), FS (fractional shortening), IVRT (isovolumic relaxation time), MPI (myocardial performance index), Tau (isovolumic relaxation time constant), LVESP (left ventricular end-systolic pressure), and EDPVR (end-diastolic pressure-volume relationship). The profiles of autophagy-associated proteins (p62, LC3II/I, and Beclin-1) and GATA4 protein in mice myocardial tissues were assessed by Western blotting (WB). Myocardial cells were classified from TAC mice into five groups: Control, Trehalose, Phenylephrine (PE), PE+Trehalose, and PE+Trehalose+autophagy inhibitor chloroquine groups. In the PE group, cardiomyocytes were treated with 50 μmol/L PE. Then, the cells were treated with trehalose (100 μmol/L), trehalose (100 μmol/L)+autophagy (20 μmol/L) for 24 hours respectively. The Control group was treated with the same amount of normal saline. Flow cytometry was utilized to detect myocardial cell apoptosis in each subgroup. The alterations in apoptosis and autophagy-correlated proteins (p62, LC3II/I, and Beclin-1) were assessed by WB. Additionally, the level of GATA4 protein upstream of autophagy was estimated. Furthermore, the expression levels of pro-apoptotic proteins Bad, BAX, Cleaved-caspase-3, and anti-apoptotic protein Bcl-2 were examined by WB. The TAC operation significantly augmented myocardial hypertrophy, heart weight-to-body weight ratio, and myocardial cell apoptosis in mice (p < 0.05). Trehalose significantly improved cardiac hypertrophy, cardiomyocyte apoptosis, and cardiac function decline in mice. Additionally, it also significantly enhanced autophagy in mouse cardiac tissues (p < 0.05). At the cellular level, trehalose significantly decreased PE-elicited apoptosis and promoted the protein expressions of Beclin-1 and LC3 II/I in cardiomyocytes while significantly dampening the profiles of p62 and GATA4 in cells. The effect of trehalose and chloroquine treatment was significantly greater than that of the trehalose group. Trehalose significantly abates myocardial hypertrophy and pressure overload-induced cardiomyocyte apoptosis in mice. The cardioprotective effect of trehalose on enhanced autophagy is attributed, at least in part, to the promotion of autophagic degradation of GATA4.

Bilobalide Prevents Apoptosis andImproves Cardiac Function in Myocardial Infarction.

Myocardial infarction (MI) is an extremely severe cardiovascular disease, which ranks as the leading cause of sudden death worldwide. Studies have proved that cardiac injury following MI can cause cardiomyocyte apoptosis and myocardial fibrosis. Bilobalide (Bilo) from Ginkgo biloba leaves have been widely reported to possess excellent cardioprotective effects. However, concrete roles of Bilo in MI have not been investigated yet. We here designed both in vitro and in vivo experiments to explore the effects of Bilo on MI-induced cardiac injury and the underlying mechanisms of its action. We conducted in vitro experiments using oxygen-glucose deprivation (OGD)-treated H9c2 cells. Cell apoptosis in H9c2 cells was assessed by conducting flow cytometry assay and evaluating apoptosis-related proteins with western blotting. MI mouse model was established by performing left anterior descending artery (LAD) ligation. Cardiac function of MI mice was determined by assessing ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD). Histological changes were analyzed, infarct size and myocardial fibrosis were measured by hematoxylin and eosin (H&E) and Masson staining in cardiac tissues from the mice. The apoptosis of cardiomyocytes in MI mice was assessed by TUNEL staining. Western blotting was applied to detect the effect of Bilo on c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinases (p38 MAPK) signaling both in vitro and in vivo. Bilo inhibited OGD-induced cell apoptosis and lactate dehydrogenase (LDH) release in H9c2 cells. The protein levels of p-JNK and p-p38 were significantly downregulated by Bilo treatment. SB20358 (inhibitor of p38) and SP600125 (inhibitor of JNK) suppressed OGD-induced cell apoptosis as Bilo did. In MI mouse model, Bilo improved the cardiac function and significantly reduced the infarct size and myocardial fibrosis. Bilo inhibited MI-induced cardiomyocytes apoptosis in mice. Bilo suppressed the protein levels of p-JNK and p-p38 in cardiac tissues from MI mice. Bilo alleviated OGD-induced cell apoptosis in H9c2 cells and suppressed MI-induced cardiomyocyte apoptosis and myocardial fibrosis in mice via the inactivation of JNK/p38 MAPK signaling pathways. Thus, Bilo may be an effective anti-MI agent.

Inhibition of MicroRNA-122-5p Relieves Myocardial Ischemia-Reperfusion Injury via SOCS1.

Evidence has shown that microRNA (miR)-122-5p is a diagnostic biomarker of acute myocardial infarction. Here, we aimed to uncover the functions of miR-122-5p in the pathological process of myocardial ischemia-reperfusion injury (MI/RI). An MI/RI model was established by left anterior descending coronary artery ligation in mice. The levels of miR-122-5p, suppressor of cytokine signaling-1 (SOCS1), phosphorylation of Janus kinase 2 (p-JAK2), and signal transducers and activators of transcription (p-STAT3) in the myocardial tissues of mice were measured. Downregulated miR-122-5p or upregulated SOCS1 recombinant adenovirus vectors were injected into mice before MI/RI modeling. The cardiac function, inflammatory response, myocardial infarction area, pathological damage, and cardiomyocyte apoptosis in the myocardial tissues of mice were evaluated. Cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) injury and cardiomyocyte biological function was tested upon transfection of miR-122-5p inhibitor. The target relation between miR-122-5p and SOCS1 was evaluated. miR-122-5p expression and p-JAK2 and p-STAT3 expression were high, and SOCS1 expression was low in the myocardial tissues of MI/RI mice. Decreasing miR-122-5p or increasing SOCS1 expression inactivated the JAK2/STAT3 pathway to alleviate MI/RI by improving cardiac function and reducing inflammatory reaction, myocardial infarction area, pathological damage, and cardiomyocyte apoptosis in mice. Silencing of SOCS1 reversed depleted miR-122-5p-induced cardioprotection for MI/RI mice. In vitro experiments revealed that the downregulation of miR-122-5p induced proliferative, migratory, and invasive capabilities of H/R cardiomyocytes while inhibiting apoptosis. Mechanically, SOCS1 was a target gene of miR-122-5p. Our study summarizes that inhibition of miR-122-5p induces SOCS1 expression, thereby relieving MI/RI in mice.

Abstract P1041: Redox Regulator MnTnBuOE-PyP5+ Protects Cardiomyocyte Against Oxidative Stress Mediated Myocardial Ischemia/reperfusion Injury

Background: Much evidence suggests that oxidative stress plays a major role in the pathogenesis of myocardial ischemia/reperfusion (I/R) injury and is a potential target for therapeutic interventions. We tested the hypothesis that the novel redox regulator, MnTnBUOE-2-PyP 5+ (BMX-001), mediates cardioprotection through reduction of oxidative stress in myocardial tissue following I/R injury. Methods and Results: BMX-001 (2mg/Kg, IP) or vehicle was administered in mice 4 hours before myocardial ischemia. Mice were then subjected to I/R injury by ligating the left anterior descending artery for 1 hour followed by 24 hours of reperfusion. Following 24 hours of reperfusion, infarct size, cardiomyocyte apoptosis, and SOD2 level were determined, and echocardiography was performed. Our study revealed that the redox regulator provided protection against I/R-induced injury in mice as evident by a significant reduction in infarct size, plasma LDH level, and oxidative stress. In addition, BMX-001 administration in mice markedly increased FoxO3a and SOD2 levels. BMX-001 treatment attenuated I/R induced cardiomyocyte apoptosis in-vivo and in-vitro as evident by a reduction in the number of TUNEL positive cells and improved cell viability. Echocardiography at 24 hours of reperfusion revealed improved ejection fraction and fractional shortening in mice treated with BMX-001. Furthermore, in-vitro treatment of BMX-001 (10μM) 24 hours before hypoxia/reoxygenation in H9c2 cells improved oxidative stress as evident by a reduction in cardiolipin peroxidation, mitochondrial superoxide level, and 4-HNE adducted proteins. Conclusion: Pre-treatment with redox regulator BMX-001 showed a beneficial effect in attenuating cardiomyocyte death, mitochondrial superoxide, and cardiolipin peroxidation following I/R injury in H9c2 cells. Pre-treatment with SOD2 mimetic improved heart function, decreased infarct size, and cardiomyocyte apoptosis in mice following I/R injury. Therefore, BMX-001 can be a novel cardioprotective agent against oxidative stress-induced myocardial I/R injury.

Omaveloxolone attenuates the sepsis-induced cardiomyopathy via activating the nuclear factor erythroid 2-related factor 2

Sepsis-induced cardiomyopathy (SIC) is a common complication of sepsis and is the main reason for the high mortality in sepsis patients. More recent studies have indicated that activating nuclear factor erythroid 2-related factor 2 (Nrf2) signaling plays a protective role in SIC. As a potent activator of Nrf2, Omaveloxolone plays a pivotal role in defending against oxidative stress and the inflammatory response. Thus, we examined the efficacy of omaveloxolone in SIC. In the present study, the mice were injected intraperitoneally with a single dose of LPS (10 mg/kg) for 12 h to induce SIC. The data in our study indicated that omaveloxolone administration significantly improved cardiac injury and dysfunction in LPS-induced SIC. In addition, omaveloxolone administration reduced SIC-related cardiac oxidative stress, the inflammatory response and cardiomyocyte apoptosis in mice. In addition, omaveloxolone administration also improved LPS-induced cardiomyocyte injury in an in vitro model using H9C2 cells. Moreover, knockdown of Nrf2 by si-Nrf2 abolished the omaveloxolone-mediated cardioprotective effects. In conclusion, omaveloxolone has potent cardioprotective potential in treating sepsis and SIC via activation of the Nrf2 signaling pathway.

Tetrahydrocurcumin improves lipopolysaccharide-induced myocardial dysfunction by inhibiting oxidative stress and inflammation via JNK/ERK signaling pathway regulation

BackgroundAcute myocardial dysfunction in patients with sepsis is attributed to oxidative stress, inflammation, and cardiomyocyte loss; however, specific drugs for its prevention are still lacking. Tetrahydrocurcumin (THC) has been proven to contribute to the prevention of various cardiovascular diseases by decreasing oxidative stress and inflammation. This study was performed to investigate the functions and mechanism of action of THC in septic cardiomyopathy. MethodsAfter the oral administration of THC (120 mg/kg) for 5 consecutive days, a mouse model of sepsis was established via intraperitoneal lipopolysaccharide (LPS, 10 mg/kg) injection. Following this, cardiac function was assessed, pathological section staining was performed, and inflammatory markers were detected. ResultsMyocardial systolic function was severely compromised in parallel with the accumulation of reactive oxygen species and enhanced cardiomyocyte apoptosis in mice with sepsis. These adverse changes were markedly reversed in response to THC treatment in septic mice as well as in LPS-treated H9c2 cells. Mechanistically, THC inhibited the release of pro-inflammatory cytokines, including tumor necrosis factor alpha, interleukin (IL)-1β, and IL-6, by upregulating mitogen-activated protein kinase phosphatase 1, to block the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK). Additionally, THC enhanced the levels of antioxidant proteins, including nuclear factor-erythroid 2-related factor 2, superoxide dismutase 2, and NAD(P)H quinone oxidoreductase 1, while decreasing gp91phox expression. Furthermore, upon THC treatment, Bcl-2 expression was significantly increased, along with a decline in Bax and cleaved caspase-3 expression, which reduced cardiomyocyte loss. ConclusionOur findings indicate that THC exhibited protective potential against septic cardiomyopathy by reducing oxidative stress and inflammation through the regulation of JNK/ERK signaling. The findings of this study provide a basis for the further evaluation of THC as a therapeutic agent against septic cardiomyopathy.

LncRNA LINC00461 exacerbates myocardial ischemia\u2013reperfusion injury via microRNA-185-3p/Myd88

ObjectiveLong non-coding RNAs (lncRNAs) play critically in the pathogenesis of myocardial ischemia–reperfusion (I/R) injury. Thus, it was proposed to investigate the mechanism of LINC00461 in the disease through mediating microRNA-185-3p (miR-185-3p)/myeloid differentiation primary response gene 88 (Myd88) axis.MethodsmiR-185-3p, LINC00461 and Myd88 expression in mice with I/R injury was measured. Mice with I/R injury were injected with the gene expression-modified vectors, after which cardiac function, hemodynamics, myocardial enzyme, oxidative stress, and cardiomyocyte apoptosis were analyzed.ResultsI/R mice showed LINC00461 and Myd88 up-regulation and miR-185-3p down-regulation. Down-regulating LINC00461 or up-regulating miR-185-3p recovered cardiac function, reduced myocardial enzyme levels, and attenuated oxidative stress and cardiomyocyte apoptosis in mice with I/R. miR-185-3p overexpression rescued the promoting effect of LINC00461 upregulation on myocardial injury in I/R mice.ConclusionLINC00461 knockdown attenuates myocardial I/R injury via elevating miR-185-3p expression to suppress Myd88 expression.

Palmatine attenuates the doxorubicin-induced inflammatory response, oxidative damage and cardiomyocyte apoptosis

BackgroundAs a natural isoquinoline alkaloid, palmatine (PLT) has been proven to play a protective role against a variety of cardiovascular diseases. However, little research on the effects of PLT on doxorubicin (DOX)-induced cardiotoxicity has been carried out. Thus, we investigated the potential functions of PLT in DOX-induced cardiotoxicity. In the present study, a single intraperitoneal injection of DOX (15 mg/kg) in mice was used to establish an acute cardiotoxicity model. Our study shows that PLT administration could reduce myocardial injury and improve cardiac dysfunction in DOX-treated mice. Further experiments showed that PLT administration suppressed the DOX-induced inflammatory response, oxidative damage and cardiomyocyte apoptosis in mice. Moreover, we found that the protective effect of PLT treatment was counteracted by sirtuin1 (Sirt1) knockdown. In summary, our study shows that PLT treatment can exert a protective effect against DOX-induced cardiotoxicity.

Percutaneous Intracoronary Delivery of Plasma Extracellular Vesicles Protects the Myocardium Against Ischemia-Reperfusion Injury in Canis.

[Figure: see text].

Circular RNA POSTN Promotes Myocardial Infarction-Induced Myocardial Injury and Cardiac Remodeling by Regulating miR-96-5p/BNIP3 Axis

Myocardial infarction (MI) is the most prevalent cardiac disease with high mortality, leading to severe heart injury. Circular RNAs (circRNAs) are a new type of regulatory RNAs and participate in multiple pathological cardiac progressions. However, the role of circRNAs Postn (circPostn) in MI modulation remains unclear. Here, we aimed to explore the effect of circPostn on MI-induced myocardial injury and cardiac remodeling. We identified that the expression of circPostn was elevated in the plasma of MI patients, MI mouse model, and hypoxia and reoxygenation (H/R)-treated human cardiomyocytes. The depletion of circPostn significantly attenuated MI-related myocardium injury and reduced the infarct size in MI mouse model. The circPostn knockdown obviously enhanced left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) and inhibited left ventricular anterior wall thickness at diastole (LVAWd) and left ventricular posterior wall thickness at diastole (LVPWd). The depletion of circPostn was able to decrease MI-induced expression of collagen 1α1 and collagen 3α1 in the ventricular tissues of mice. The protein expression of collagen and α-smooth muscle actin (SMA) was up-regulated in MI mice and was inhibited by circPostn knockdown. Meanwhile, the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was repressed by circPostn depletion in the ventricular tissues of MI mice. Besides, the circPostn depletion attenuated cardiomyocyte apoptosis in mice. Mechanically, circPostn served as a miR-96-5p sponge and miR-96-5p-targeted BNIP3 in human cardiomyocytes, in which circPostn up-regulated BNIP3 expression by targeting miR-96-5p. circPostn promoted H/R-induced cardiomyocyte injury by modulating miR-96-5p/BNIP3 axis. Thus, we conclude that circPostn contributes to MI-induced myocardial injury and cardiac remodeling by regulating miR-96-5p/BNIP3 axis. Our finding provides new insight into the mechanism by which circPostn regulates MI-related cardiac dysfunction. circPostn, miR-96-5p, and BNIP3 are potential targets for the treatment of MI-caused heart injury.

MicroRNA-425-3p inhibits myocardial inflammation and cardiomyocyte apoptosis in mice with viral myocarditis through targeting TGF-β1.

ObjectiveEmerging articles have profiled the relations between microRNAs and viral myocarditis. This research was unearthed to explore the capacity of miR‐425‐3p on cardiomyocyte apoptosis in mice with viral myocarditis and its mechanism.MethodsA total of 120 mice were classified into 4 groups in a random fashion (n = 30). The mice were intraperitoneally injected with coxsackievirus type B3 (CVB3) to induce myocarditis. On the 7th day after CVB3 infection, 10 mice in each group were euthanized to assess the heart function indices of mice, observe the pathological conditions, detect myocardial tissue apoptosis, and measure the inflammatory factor levels in myocardial tissues. Expression of miR‐425‐3p, transforming growth factor (TGF‐β1), and apoptosis‐associated proteins in myocardial tissues was determined. The remaining 20 mice in each group were used for survival observation. The luciferase activity assay was implemented to validate the relationship between miR‐425‐3p and TGF‐β1. miR‐425‐3p mimic was transfected into mouse cardiomyocytes HL‐1 and then infected with CVB3 to further verify the regulatory effect of miR‐425‐3p on the cardiomyocyte apoptosis in viral myocarditis.ResultsmiR‐425‐3p was lowly expressed in myocardial tissues of mice with viral myocarditis. Overexpressed miR‐425‐3p improved the cardiac function, alleviated pathological conditions, reduced cardiomyocyte apoptosis, decreased Bax and cleaved Caspase‐3 expression, elevated Bcl‐2 expression, decreased levels of inflammatory factors and improved survival rate of mice with viral myocarditis. Luciferase activity assay verified that miR‐425‐3p could bind to TGF‐β1, and overexpressed miR‐425‐3p suppressed TGF‐β1, p‐smad2/smad2 and p‐smad3/smad3 expression. In vitro experiments further verified that overexpression of miR‐425‐3p inhibited the apoptosis of CVB3‐HL‐1 cells, and the addition of TGF‐β1 would reverse this effect.ConclusionOur research indicates that miR‐425‐3p is poorly expressed in myocardial tissues of mice with viral myocarditis. Overexpressed miR‐425‐3p inhibits cardiomyocyte apoptosis and myocardial inflammation in mice with viral myocarditis as well as improves their survival rates through suppressing the TGF‐β1/smad axis.

Knockdown of endogenous RNF4 exacerbates ischaemia-induced cardiomyocyte apoptosis in mice.

RNF4, a poly‐SUMO‐specific E3 ubiquitin ligase, is associated with protein degradation, DNA damage repair and tumour progression. However, the effect of RNF4 in cardiomyocytes remains to be explored. Here, we identified the alteration of RNF4 from ischaemic hearts and oxidative stress‐induced apoptotic cardiomyocytes. Upon myocardial infarction (MI) or H2O2/ATO treatment, RNF4 increased rapidly and then decreased gradually. PML SUMOylation and PML nuclear body (PML‐NB) formation first enhanced and then degraded upon oxidative stress. Reactive oxygen species (ROS) inhibitor was able to attenuate the elevation of RNF4 expression and PML SUMOylation. PML overexpression and RNF4 knockdown by small interfering RNA (siRNA) enhanced PML SUMOylation, promoted p53 recruitment and activation and exacerbated H2O2/ATO‐induced cardiomyocyte apoptosis which could be partially reversed by knockdown of p53. In vivo, knockdown of endogenous RNF4 via in vivo adeno‐associated virus infection deteriorated post‐MI structure remodelling including more extensive interstitial fibrosis and severely fractured and disordered structure. Furthermore, knockdown of RNF4 worsened ischaemia‐induced cardiac dysfunction of MI models. Our results reveal a novel myocardial apoptosis regulation model that is composed of RNF4, PML and p53. The modulation of these proteins may provide a new approach to tackling cardiac ischaemia.

MiR-1/133 attenuates cardiomyocyte apoptosis and electrical remodeling in mice with viral myocarditis.

The role of miR-1 and miR-133 in regulating the expression of potassium and calcium ion channels, and mediating cardiomyocyte apoptosis in mice with viral myocarditis (VMC) is investigated herein. Male Balb/c mice were randomly divided into groups: control group, VMC group, VMC + miR-1/133 mimics group, or VMC + miR-1/133 negative control (NC) group. VMC was induced with coxsackievirus B3 (CVB3). MiR-1/133 mimics ameliorated cardiac dysfunction in VMC mice and was compared to the VMC+NC group. Hematoxylin and eosin staining showed a well-arranged myocardium without inflammatory cell infiltration in the myocardial matrix of the control group. However, in the VMC and VMC+NC groups, the myocardium was disorganized and swollen with necrosis, and the myocardial matrix was infiltrated with inflammatory cells. These changes were alleviated by miR-1/133 mimics. TUNEL staining revealed decreased cardiomyocyte apoptosis in the VMC + miR-1/133 mimics group compared with the VMC group. In addition, miR-1/133 mimics up-regulated the expression of miR-1 and miR-133, the potassium channel genes Kcnd2 and Kcnj2, as well as Bcl-2, and down-regulated the expression of the potassium channel suppressor gene Irx5, L-type calcium channel subunit gene a1c (Cacna1c), Bax, and caspase-9 in the myocardium of VMC mice. MiR-1/133 also up-regulated the protein levels of Kv4.2 and Kir2.1, and down-regulated the expression of CaV1.2 in the myocardium of VMC mice. MiR-1 and miR-133 decreased cardiomyocyte apoptosis by mediating the expression of apoptosis-related genes in the hearts of VMC mice.

E3-ubiquitin ligase TRIM6 aggravates myocardial ischemia/reperfusion injury via promoting STAT1-dependent cardiomyocyte apoptosis.

Cardiomyocyte apoptosis is a major cause of myocardial ischemia/reperfusion (MI/R) injury, in which the activation of the signal transducer and activator of transcription 1 (STAT1) plays an important role. The E3-ubiquitin ligase TRIM6 has been implicated in regulating STAT1 activity, however, whether it is associated with MI/R injury and the underlying mechanism are not determined. In this study, by investigating a mouse MI/R injury model, we show that TRIM6 expression is induced in mouse heart following MI/R injury. Additionally, TRIM6 depletion reduces and its overexpression increases myocardial infarct size, serum creatine phosphokinase (CPK) level and cardiomyocyte apoptosis in mice subjected to MI/R injury, indicating that TRIM6 functions to aggravate MI/R injury. Mechanistically, TRIM6 promotes IKKε-dependent STAT1 activation, and the inhibition of IKKε or STAT1 with the specific inhibitor, CAY10576 or fludarabine, abolishes TRIM6 effects on cardiomyocyte apoptosis and MI/R injury. Similarly, TRIM6 mutant lacking the ability to ubiquitinate IKKε and induce IKKε/STAT1 activation also fails to promote cardiomyocyte apoptosis and MI/R injury. Thus, these results suggest that TRIM6 aggravates MI/R injury through promoting IKKε/STAT1 activation-dependent cardiomyocyte apoptosis, and that TRIM6 might represent a novel therapeutic target for alleviating MI/R injury.

Leonurine alleviates LPS-induced myocarditis through suppressing the NF-кB signaling pathway

Myocarditis is a serious hazard to human life and is difficult to treat due to the proliferation of inflammatory lesions in the myocardium. Leonurine (LE) is a plant phenolic alkaloid extracted from Herba leonuri that has demonstrated cardioprotective effects in many preclinical experiments. However, whether LE can be used for myocarditis therapy has not been reported. We aimed to investigate the cardioprotective effects of LE on lipopolysaccharide (LPS)-induced myocarditis in vivo and vitro. The possible mechanism involved was also further elucidated. In vivo, C57BL/6 mice were exposed to LPS with or without LE. We found out that LE effectively improved cardiac function and attenuated cardiomyocyte apoptosis in mice with myocarditis. In addition, LPS-induced inflammatory and oxidative injuries in the myocardium were also reduced by LE administration. In vitro, LPS simultaneously induced apoptosis and reduced the H9c2 cells viability, followed by elevation of intracellular reactive oxygen species (ROS) generation. However, the abnormalities mentioned were preventable by LE pretreatment in a dose-dependent manner. Both in vivo and in vitro, LPS activated the nuclear factor kappa B (NF-кB) signaling pathway in myocarditis, and LE inhibited the increased expression of phosphorylated iκBα and p65 (p-iκBα, p-p65). Furthermore, the nuclear translocalization and nuclear protein expression of p65 in LPS-injured H9c2 cells were also suppressed by LE. Our results demonstrated that LE exerts potent cardioprotective effects against myocarditis via anti-inflammatory and antioxidative mechanisms, possibly through blocking the activation of NF-кB pathway.

Inhibition of TRPA1 Attenuates Doxorubicin-Induced Acute Cardiotoxicity by Suppressing Oxidative Stress, the Inflammatory Response, and Endoplasmic Reticulum Stress.

The transient receptor potential ankyrin 1 (TRPA1) channel is expressed in cardiomyocytes and involved in many cardiovascular diseases. However, the expression and function of TRPA1 in doxorubicin- (Dox-) induced acute cardiotoxicity have not been elucidated. This study aimed at investigating whether blocking the TRPA1 channel with the specific inhibitor HC-030031 (HC) attenuates Dox-induced cardiac injury. The animals were randomly divided into four groups: control, HC, Dox, and Dox + HC. Echocardiography was used to evaluate cardiac function, and the heart was removed for molecular experiments. The results showed that the expression of TRPA1 was increased in the heart after Dox treatment. Cardiac dysfunction and increased serum CK-MB and LDH levels were induced by Dox, but these effects were attenuated by HC treatment. In addition, HC mitigated Dox-induced oxidative stress, as evidenced by the decreased MDA level and increased GSH level and SOD activity in the Dox + HC group. Meanwhile, HC treatment lowered the levels of the proinflammatory cytokines IL-1β, IL-6, IL-17, and TNF-α induced by Dox. Furthermore, HC treatment mitigated endoplasmic reticulum (ER) stress and cardiomyocyte apoptosis induced by Dox. These results indicated that inhibition of TRPA1 could prevent Dox-induced cardiomyocyte apoptosis in mice by inhibiting oxidative stress, inflammation, and ER stress.

MAPK pathway regulated the cardiomyocyte apoptosis in mice with post-infarction heart failure.

To explore the role of the MAPK signaling pathway in the cardiomyocyte apoptosis of mice with post-infarction heart failure (HF). Mice were divided into sham and myocardial infarction (MI) groups. Before surgery, the MI group was divided into SB203580 and PBS subgroups. A post-infarction HF model was established by ligating the left anterior descending coronary artery. Ventricular dilatation and cardiac function were observed by small animal echocardiography. The growth of primary cardiomyocytes was observed under an inverted phase contrast microscope. The mRNA and protein expressions of endoplasmic reticulum stress (ERS) markers, GRP78 and CHOP, were detected by qRT-PCR and immunofluorescence assay, respectively. The MI group had enlarged left ventricle and decreased cardiac function. GRP78 and CHOP protein expressions in myocardial tissues, especially those of SB203580 subgroup, significantly increased (p < 0.05). The expressions of p-JNK and cleaved caspase 12 proteins, especially those of SB203580 subgroup, were significantly up-regulated. Cardiomyocytes of MI group were significantly more prone to apoptosis (p < 0.05), with SB203580 subgroup being more obvious. MI was accompanied by ERS, probably involving the MAPK signaling pathway. SB203580, a specific inhibitor of this pathway, can relieve cardiomyocyte apoptosis and protect the myocardium by suppressing such stress (Tab. 3, Fig. 7, Ref. 20).