Apoptosis In Neonatal Rat Cardiomyocytes Research Articles

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.

Ginsenoside Rg5 attenuates hypoxia-induced cardiomyocyte apoptosis via regulating the Akt pathway.

Ginsenoside Rg5 has been implicated in a variety of diseases. However, it is unknown whether Ginsenoside Rg5 can protect against hypoxia-induced neonatal rat cardiomyocytes (NRMs). The purpose of this study was to look into the effect of Ginsenoside Rg5 on hypoxia-induced NRMs apoptosis as well as the underlying molecular mechanism. In this study, following isolation and culture of ventricular myocardial cells from neonatal rats, the appropriate concentration of Rg5 was determined using the MTT assay, the effect of Rg5 on apoptosis was assessed employing TUNEL staining and flow cytometry assays. Levels of apoptosis-related proteins and phosphorylated level of Akt (ser 473 and ser 308) were analyzed using the western blot analysis. Finally, the experimental results shown that Ginsenoside Rg5 significantly inhibited hypoxia-induced NRMs apoptosis, decreased the expression pro-apoptotic protein Bax, increased the expression of anti-apoptotic protein Bcl-2 ratio and the level of cleaved caspase 3. Akt signaling activation was found to be the mechanism of Ginsenoside Rg5s protective effect on hypoxia-induced NRMs apoptosis, as an Akt inhibitor eliminated the anti-apoptotic effects of Ginsenoside Rg5. Various analyses were performed and verified, ginsenoside Rg5 suppressed hypoxia-induced apoptosis in NRMs via activation of the Akt signaling.

PPARγ Mediates the Cardioprotective Roles of Danlou Tablet After Acute Myocardial Ischemia-Reperfusion Injury.

Ischemic heart disease is one of the biggest threats to human life in the world. Reperfusion therapy is an effective strategy to reduce infarct size and ischemic injury. However, reperfusion process may cause secondary myocardial injury which is defined as ischemia-reperfusion injury (IRI). Exploring potential therapeutic strategy to attenuate IRI is extremely important. Danlou tablet (Dan), a Chinese herbal compound consisting of ten herbs, has been identified to be protective for the heart. However, the mechanism of Dan-induced cardioprotection after acute reperfusion was unelucidated. In this study, to investigate the role and mechanism of Dan in myocardial IRI, we performed acute IRI modeling in mice and oxygen-glucose deprivation–reperfusion (OGD/R)-induced apoptosis in primary neonatal rat cardiomyocytes (NRCMs). We found that Dan had protective effect against acute IRI in mice, as evidenced by reduced infarct size, TUNEL-positive cardiomyocytes (CMs), and Bax/Bcl2 ratio and cleaved-caspase 3/caspase 3 ratio in vivo. Meanwhile, Dan inhibited OGD/R-induced apoptosis of NRCMs in vitro. Mechanistically, Dan could activate proliferator-activated receptor gamma (PPARγ) in both IRI hearts and OGD/R-stressed NRCMs, while inhibition of PPARγ attenuated the protective effect of Dan against IRI in vivo and OGD/R-induced CM apoptosis in vitro. These data reveal that Dan attenuates acute myocardial IRI and CM apoptosis through activating PPARγ. Our findings may extend the knowledge of Chinese medicine and provide potential strategy for the precise treatment of ischemic heart diseases.

SH3-Binding Glutamic Acid Rich-Deficiency Augments Apoptosis in Neonatal Rat Cardiomyocytes.

Congenital heart disease (CHD) is one of the most common birth defects in humans, present in around 40% of newborns with Down’s syndrome (DS). The SH3 domain-binding glutamic acid-rich (SH3BGR) gene, which maps to the DS region, belongs to a gene family encoding a cluster of small thioredoxin-like proteins sharing SH3 domains. Although its expression is confined to the cardiac and skeletal muscle, the physiological role of SH3BGR in the heart is poorly understood. Interestingly, we observed a significant upregulation of SH3BGR in failing hearts of mice and human patients with hypertrophic cardiomyopathy. Along these lines, the overexpression of SH3BGR exhibited a significant increase in the expression of hypertrophic markers (Nppa and Nppb) and increased cell surface area in neonatal rat ventricular cardiomyocytes (NRVCMs), whereas its knockdown attenuated cellular hypertrophy. Mechanistically, using serum response factor (SRF) response element-driven luciferase assays in the presence or the absence of RhoA or its inhibitor, we found that the pro-hypertrophic effects of SH3BGR are mediated via the RhoA–SRF axis. Furthermore, SH3BGR knockdown resulted in the induction of apoptosis and reduced cell viability in NRVCMs via apoptotic Hippo–YAP signaling. Taking these results together, we here show that SH3BGR is vital for maintaining cytoskeletal integrity and cellular viability in NRVCMs through its modulation of the SRF/YAP signaling pathways.

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

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Effects of Shenfu Qiangxin Drink on H2O2-induced oxidative stress, inflammation and apoptosis in neonatal rat cardiomyocytes and possible underlying mechanisms.

The aim of the present study was to investigate the effects of Shenfu Qiangxin Drink (SFQXD) on acute myocardial infarction (AMI) and identify the possible underlying mechanisms. Levels of reactive oxygen species (ROS) and inflammatory factors, including interleukin (IL)-6, IL-1β and tumor necrosis factor-α (TNF-α) in the blood samples of patients with AMI were measured using commercially available kits by visible spectrophotometry after SFQXD administration. The contents of phosphorylated (p-) forkhead box O3a (FOXO3a) was examined using an ELISA kit. In addition, a hydrogen peroxide (H2O2)-induced myocardial injury model was established in vitro using neonatal rat cardiomyocytes. Following treatment with SFQXD, the levels of intracellular ROS, cell apoptosis, oxidative stress- and inflammation-related markers were measured using commercially available kits by visible spectrophotometry. Additionally, western blot analysis was used to measure the expression of sirtuin-4 (SIRT4), p-FOXO3a, acetylated FOXO3a (ace-FOXO3a) and apoptosis-related genes (Bcl-2, Bax, BIM and cleaved caspase-3). Subsequently, to investigate the possible underlying regulatory mechanisms, SIRT4 expression was silenced by transfection with small hairpin RNA against SIRT4, following which changes in the extent of oxidative stress, inflammation and apoptosis were assessed. The levels of ROS and interleukin (IL)-1β were found to be significantly reduced, whilst FOXO3a phosphorylation was markedly increased following administration with SFQXD. In vitro, SFQXD dose-dependently inhibited H2O2-induced oxidative stress, inflammation and apoptosis in neonatal rat cardiomyocytes. In addition, FOXO3a phosphorylation was markedly upregulated whilst FOXO3a acetylation was downregulated following treatment of H2O2-induced primary neonatal cardiomyocytes with SFQXD. SIRT4 knockdown also markedly reversed the effects of SFQXD on oxidative stress, inflammation and apoptosis in neonatal rat cardiomyocytes. In conclusion, these findings demonstrated that SFQXD may alleviate oxidative stress-induced myocardial injury by potentially regulating SIRT4/FOXO3a signaling, suggesting that SFQXD may be of clinical value for the treatment of AMI.

Protective Effects of Shenfuyixin Granule on H2O2-Induced Apoptosis in Neonatal Rat Cardiomyocytes.

Shenfuyixin granule (SFYXG, i.e., Xinshuaikang granule) is a prescription, commonly used in the clinical experience, which plays a significant role in the treatment of heart failure. The purpose of this present research was to investigate the protective effect of SFYXG, and the mechanism about anti-H2O2-induced oxidative stress and apoptosis in the neonatal rat cardiomyocytes. Myocardial cells, as is well known, were divided into 4 groups: normal, model, SFYXG, and coenzyme Q10 group, respectively. Cells viability was determined by MTT assay. Flow cytometry and AO/EB staining were implemented to test the apoptosis rate and intracellular reactive oxygen species (ROS) level. Mitochondrion membrane potential (MMP) was evaluated by JC-1 fluorescence probe method. The myocardial ultrastructure of mitochondrion was measured by electron microscope. The related mRNA expression levels of Bax, Bcl-2, Caspase-3, caspase-8, and caspase-9 were detected by real-time polymerase chain reaction (PCR). Also, the expression levels of Bax and Bcl-2 protein were detected by Western blot, and the expression levels of caspase-3, caspase-8, and caspase-9 protein were tested by caspase-Glo®3 Assay, caspase-Glo®8 Assay, and caspase-Glo®9 Assay, respectively. GAPDH was used as the internal reference gene/protein. The results revealed that SFYXG (0.5 mg/ml) raised the viability of myocardial cell, weakened the apoptosis rate and ROS level, corrected the mitochondrion membrane potential stability, and improved cell morphology and ultrastructure of myocardial mitochondrion. Furthermore, SFYXG upregulated the antiapoptosis gene of Bcl-2, but downregulated the proapoptosis genes of Bax, caspase-3, and caspase-9. In conclusion, SFYXG could appear to attenuate myocardial injury by its antioxidative and antiapoptosis effect.

Differential expression of miR-142-3p protects cardiomyocytes from myocardial ischemia-reperfusion via TLR4/NFkB axis.

Our research aims to explore the impact of miR-142 on myocardial apoptosis in the mouse ischemia and reperfusion (IR) model and investigate the underlying mechanisms at the molecular level. A considerable downregulation of miR-142 was observed in the cardiac area of mice post IR modeling. To understand the regulatory function of IR-induced miR-142 downregulation, the animals were categorized into four groups: IR model group; IR + agomir-142 group (IR mice treated with agomir-142); IR + antagomir-142 group (IR mice treated with antagomir-142); IR + agomir-142 + negative control (NC) group (IR mice processed with agomir-NC). The results indicated that agomir-142 upregulation was capable of shrinking IR damage-triggered infarction of the ventriculus sinister, strengthening myocardial function, and guarding against cardiomyocyte apoptosis, whereas further decreased miR-142 with antagomir-142 infection displayed negative influence of miR-142 against mice IR damage. In the cellular assay, miR-142 overexpression significantly improved proliferation and inhibited the apoptosis of neonatal rat cardiomyocytes (NRCs). Moreover, we found that miR-142 reduced the Bcl-2/Bax ratio and upregulated hydrogen peroxide (H2 O2 )-induced caspase-3 expression. Furthermore, transfection with an miR-142 mimic prevented the upregulation of TLR4/NFkB expression and activation in H2 O2 -treated NRCs. Our findings also revealed that miR-142 is linked to the 3'-untranslated area of the TLR4 gene. In addition, TLR4 overexpression considerably ablated the protective effects of miR-142 in terms of the cell viability of H2 O2 -treated NRCs. Taken together, miR-142 agomir injection in mice and miR-142 mimic transfection in NRCs plays a role in protecting the heart from IR damage and malfunction via the TLR4/NFkB axis both in vivo and in vitro.

P5399microRNA-486 mediates exercise-induced cardiac growth and prevents cardiac ischemia-reperfusion injury

Abstract Background Molecules mediating exercise-induced physiological cardiac growth may be beneficial to prevent cardiac injury and remodeling. MicroRNA-486 (miR-486) is a muscle-enriched microRNA which was found to be increased in exercised heart. However, the role of miR-486 in exercise-induced cardiac growth is largely unknown. Purpose We aimed to study the function of miR-486 in exercise-induced cardiac growth, and to investigate its potential role in protection against cardiac ischemia/reperfusion (I/R) injury. Methods Male C57BL/6 adult mice were subjected to 3-week swimming exercise to induce physiological cardiac growth. Knockdown of miR-486 was performed by intramyocardial injection of miR-486 sponge to evaluate the role of miR-486 in exercise-induced cardiac growth. Moreover, intramyocardial injection of miR-486 overexpression lentivirus was performed to investigate whether miR-486 could reduce infarct size in mice suffered from acute I/R injury (30 min ligation and 24 hr reperfusion of left anterior descending coronary artery). Meanwhile, the functional role of miR-486 was evaluated in neonatal rat cardiomyocytes (NRCMs) treated with oxygen glucose deprivation/reperfusion (OGDR) using Tunel staining. Finally, function-rescue assays were performed to identify target genes of miR-486. Results Exercise significantly upregulated miR-486 in the heart, which was reduced by intramyocardial injection of miR-486 sponge. Exercise induced-cardiac growth was abolished in mice with intramyocardial injection of miR-486 sponge, as evidenced by reduced heart weight/body weight ratio and heart weight/tibia length ratio compared to control exercised-mice, suggesting a potential role of miR-486 in mediating exercise-induced cardiac growth. Meanwhile, we found that intramyocardial injection of miR-486 overexpression lentivirus was able to reduce the infarct size as determined by TTC staining. Moreover, in OGDR-induced apoptosis of NRCMs, miR-486 mimics was able to reduce cardiomyocyte apoptosis, while miR-486 inhibitor performed the opposite. Finally, PTEN and FoxO1 were identified as target genes of miR-486 in the control of cardiomyocyte apoptosis. Conclusions miR-486 mediates exercise-induced cardiac growth and protects against cardiac I/R injury. Increasing miR-486 might serve as a potential target to reduce myocardial apoptosis upon I/R injury. Acknowledgement/Funding This work was supported by the grants from National Natural Science Foundation of China 81722008, 91639101 and 81570362 to JJ Xiao, 81770401 to Y Bei

MicroRNA-155 inhibition attenuates endoplasmic reticulum stress-induced cardiomyocyte apoptosis following myocardial infarction via reducing macrophage inflammation

Endoplasmic reticulum stress (ERS)-induced cardiomyocyte apoptosis plays an important role in the pathological process following myocardial infarction (MI). Macrophages that express microRNA-155 (miR-155) mediate cardiac inflammation, fibrosis, and hypertrophy. Therefore, we investigated if miR-155 regulates ERS-induced cardiomyocyte apoptosis after MI using a mouse model, lipopolysaccharide (LPS)-induced rat bone marrow derived macrophages (BMDMs)and hypoxia-induced neonatal rat cardiomyocytes (NRCMs). In vivo, miR-155 levelswere significantly higher in the MI group compared to the sham group. MI increasedmacrophage infiltration, nuclear factor-κB (NF-κB) activation, ERS induced-apoptosis, and SOCS1 expression, all of which were attenuated by the miR-155 antagomir, with the exception of SOCS1 expression. Additionally, post-MI cardiac dysfunction was significantly improved by miR-155 inhibition. In vitro, LPS upregulated miR-155 expression in BMDMs, and the miR-155 antagomir decreased LPS-induced macrophage inflammation and NF-κB pathway activation, but increased expression of SOCS1. Hypoxia increased NF-κB pathway activation, ERS marker expression, and apoptosis in NRCMs. Interestingly, conditioned medium from LPS-induced macrophages in combination with the miR-155 antagomir decreased, while the miR-155 agomir increased, the hypoxia-induced effects in NRCM's. The miR-155 agomir effects were reversed by inhibiting the NF-κB pathway in cardiomyocytes. Moreover, SOCS1 knockdown in LPS-induced macrophages promoted NF-κB pathway activation and ERS–induced cardiomyocyte apoptosis in the hypoxia-induced NRCMs, but the SOCS1-siRNA-induced effects were markedly decreased by miR-155 antagomir treatment. These data suggest that miR-155 inhibition attenuates ERS-induced cardiomyocyte apoptosis after MI via reducing macrophage inflammation through the SOCS1/NF-κB pathway.

Liraglutide, a glucagon-like peptide-1 analog, inhibits high glucose-induced oxidative stress and apoptosis in neonatal rat cardiomyocytes.

Cardiomyocyte apoptosis serves an important role in diabetic cardiomyopathy. Liraglutide, a glucagon-like peptide-1 analog, has been indicated to exert a cardioprotective effect. However, the role of liraglutide on cardiomyocyte apoptosis in hyperglycemia is not fully understood. The aim of the current study was to assess whether liraglutide protects against high glucose (HG)-induced cardiomyocyte apoptosis in vitro. Sprague-Dawley neonatal rat cardiomyocytes were cultured in Dulbecco's modified Eagle's medium, supplemented with 5.5 or 25 mmol/l D-glucose or 5.5 mmol/l D-glucose + 19.5 mmol/l mannitol, in the presence or absence of liraglutide (10 or 100 nmol/l). Cell viability was assessed via an MTT assay and early apoptosis rates were assessed via flow cytometry. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in cell supernatants were measured. Bcl-2 associated X (Bax), B-cell lymphoma-2 (Bcl-2) and cleaved/full caspase-3 protein levels were determined via western blotting. The results revealed that liraglutide effectively inhibited the HG-induced increase in early apoptosis and MDA content and markedly increased SOD activity. Furthermore, liraglutide markedly inhibited the HG-induced increase in Bax and cleaved caspase-3 protein expression, and upregulated the expression of Bcl-2. The present study demonstrated that liraglutide suppressed HG-induced oxidative stress and cardiomyocyte apoptosis. Thus, the anti-apoptotic actions of liraglutide may be attributable, in part, to the inhibition of Bax, the inhibition of caspase-3 activation and the upregualtion of Bcl-2.

Resveratrol alleviates hypoxia/reoxygenation injury‑induced mitochondrial oxidative stress in cardiomyocytes.

Resveratrol (RES) is a naturally occurring antioxidant compound found in red wine. Although it has been demonstrated to have a cardioprotective effect, the mechanism underlying this effect remains to be fully elucidated. The aim of the present study was to determine whether RES exerts a protective effect against mitochondrial oxidative stress and apoptosis in neonatal rat cardiomyocytes (NRCMs) induced by hypoxia/reoxygenation (H/R) injury. Primary cultured NRCMs were used as a model system and were divided into four experimental groups: Control, H/R, H/R + DMSO (H/R with 0.2% DMSO) and H/R + RES (H/R with 100 µM RES) groups. Mitochondrial oxidative stress was determined by measuring the alteration in the mitochondrial membrane potential (ΔΨm) of NRCMs, the release of lactate dehydrogenase (LDH) and the ratio of B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) from NRCMs. Cell apoptosis was assessed by measuring cell apoptotic rates and the activity of caspase 3. In the H/R+RES group, RES significantly alleviated structural impairment, including disordered α-actin and F-actin, in the NRCMs induced by H/R injury. RES attenuated H/R injury-induced mitochondria oxidative stress. RES also attenuated H/R injury-induced cell apoptosis; it decreased the NRCM apoptotic rate from 84.25±7.41% (H/R) to 46.39±5.43% (H/R+RES) (P<0.05, n=4), rescued the decrease in the Bcl2/Bax ratio induced by H/R from 0.53±0.08-fold (H/R) to 0.86±0.06-fold (H/R+RES) (P<0.05, n=5) and alleviated the increased activity of caspase 3 induced by H/R from 1.32±0.06-fold to 1.02±0.04-fold (P<0.05, n=5). Furthermore, RES significantly attenuated the increment of LDH release induced by H/R injury in NRCMs from 1.41±0.03-fold (H/R) to 1.02±0.06-fold (H/R+RES) (P<0.01, n=4) and alleviated the depolarization of ΔΨm induced by H/R, shifting the ratio of JC-1 monomer from 62.39±1.82% (H/R) to 35.31±8.63% (H/R+RES) (P<0.05, n=4). RES alleviated the decrease in sirtuin 1 induced by H/R injury from 0.61±0.06-fold (H/R) to 1.01±0.05-fold (H/R+RES) (P<0.05, n=5). In conclusion, the present study is the first, to the best of our knowledge, to demonstrate that RES provides cardioprotection against H/R injury through decreasing mitochondria-mediated oxidative stress injury and structural impairment in NRCMs. These results provide scientific evidence for the clinical application of RES in the treatment of cardiac conditions.

Shengmai injection alleviates H2O2‑induced oxidative stress through activation of AKT and inhibition of ERK pathways in neonatal rat cardiomyocytes

Ethnopharmacological relevanceShengmai injection (SMI) is a classical traditional Chinese medicine (TCM) officially recorded in Pharmacopoeia of the People's Republic of China (version 2015) and has long been used to treat heart failure in China. However scientific evidence for the anti-oxidative stress potential of SMI used in traditional medicine is lacking. Aim of studyThe present study aimed to evaluate the efficacy of SMI in alleviating H2O2‑induced Oxidative Stress the underlying mechanisms Materials and methodsH2O2-induced oxidative stress model of cardiomyocytes was established with primary cultured neonatal rat cardiomyocytes. CCK8 cell viability assay and lacatate dehydrogenase cytotoxicity assay were performed to ensure the safety dose and lowest effective dose for the mode employing CCK-8 cell viability assay kit and lactate dehydrogenase cytotoxicity assay kit. ROS levels were determined using CM-H2DCFDA fluorescent probe in cardiomyocytes with H2O2-induced oxidative stress. The change of NAD(P)H level in cardiomyocytes was evaluated during the process of oxidative stress. The content of myocardial cytosolic Ca2+ and Ca2+ was determined using Fura-2/AM and Rhod 2-AM fluorescent probe in mitochondrial in the process of oxidative stress. Annexin V-FITC/PI double staining was applied to examine the apoptotic cells in cardiomyocytes with oxidative stress. To identify the apoptosis after oxidative stress myocardial cells with the application of Annexin V-FITC/PI double staining apoptosis detection kit. Quantitative polymerase chain reaction (RT-PCR) was applied to measure the expression of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSR). Western blot was performed to observe the phosphorylation of AKT and ERK1/2. ResultsSMI was shown to significantly attenuate oxidative stress-induced cell proliferation arrest and apoptosis in neonatal rat cardiomyocytes. In addition, SMI treatment could decrease the production of reactive oxygen species (ROS), nicotinamide adenine dinucleotide (NADH) and malondialdehyde (MDA), and reduce the overloads of cytoplasmic Ca2+ and mitochondrial Ca2+ induced by H2O2. SMI could also restore the mRNA expression and activities of SOD, GSR, and CAT suppressed by H2O2. Mechanistically, SMI upregulated intracellular AKT phosphorylation and downregulate ERK1/2 phosphorylation in H2O2-treated cardiomyocytes. Pretreatment with LY294002, an AKT phosphorylation inhibitor, suppressed the protective role of SMI in cardiomyocytes, while pretreatment with PD98059, an ERK1/2 phosphorylation inhibitor, enhanced the effect of SMI. ConclusionsIn conclusion, SMI may attenuate oxidative stress-induced damage in cardiomyocytes potentially through the AKT and ERK1/2 pathway and can function as a promising injectable traditional Chinese medicine to treat oxidative stress-induced injury.

Palmitic acid, but not high-glucose, induced myocardial apoptosis is alleviated by N\u2011acetylcysteine due to attenuated mitochondrial-derived ROS accumulation-induced endoplasmic reticulum stress

Pharmacological inhibition of reactive oxygen species (ROS) is a potential strategy to prevent diabetes-induced cardiac dysfunction. This study was designed to investigate precise effects of antioxidant N‑acetylcysteine (NAC) in alleviating diabetic cardiomyopathy (DCM). Echocardiography and histologic studies were performed 12 weeks after streptozocin injection. Protein levels involved in endoplasmic reticulum stress (ERS) and apoptosis were analyzed by western blotting in diabetic hearts or high-glucose (HG, 30 mM)- and palmitic acid (PA, 300 μM)-cultured neonatal rat cardiomyocytes (NRCMs). ROS generation and structural alterations of mitochondria were also assessed. We report that NAC alleviated diabetes-induced cardiac abnormality, including restored ejection fraction (EF %), fraction shortening (FS %), peak E to peak A ratio (E/A) and reduced cardiac hypertrophy and fibrosis. These effects were concomitant with blocked ERS and apoptosis, as evidenced by inactivation of phosphorylated inositol-requiring enzyme-1α (IRE1α)/spliced X-box binding protein 1 (XBP1), phosphorylated protein kinase-like kinase (PERK)/phosphorylated eukaryotic initiation factor 2α (eIF2α) and glucose-regulated protein 78 (GRP78)/activating transcription factor 6 (ATF6α)/C/EBP homologous protein (CHOP) pathways, as well as suppressed Bcl-2-associated X protein (BAX)/B-cell lymphoma-2 (Bcl-2) and cleaved caspase 3 expressions. Mechanistically, PA mediated excessive mitochondrial ROS generation and oxidative stress, which were antagonized by NAC and Mito-TEMPO, a mitochondrial ROS inhibitor. No effects were noted by addition of apocynin, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, and NADPH oxidase 4 (NOX 4) and NOX 2 expressions were not altered, indicating that PA-induced ROS generation is independent of NADPH oxidases. Most intriguingly, HG failed to promote ROS production despite its ability to promote ERS and apoptosis in NRCMs. Collectively, these findings indicate that NAC primarily abrogates PA-mediated mitochondrial ROS through ERS and therefore alleviates myocardial apoptosis but has little effect on HG-induced cardiac injury. This uncovers a potential role for NAC in formulating novel cardioprotective strategies in DCM patients.

Dexmedetomidine attenuates H2O2-induced neonatal rat cardiomyocytes apoptosis through mitochondria- and ER-medicated oxidative stress pathways

Dexmedetomidine (DEX), an α2 adrenoceptor agonist, has sedative and analgesic properties and myocardial protective effects. However, the mechanism underlying the protective effects of DEX on the myocardium remain unclear. The present study aimed to determine whether DEX serves an important role on cardioprotection through the endoplasmic reticulum (ER)- and mitochondria-mediated apoptosis signaling pathways. Neonatal rat cardiomyocytes (NRCMs) were cultured and divided four groups: i) Normal culture medium with 10% fetal bovine serum (control group); ii) H2O2 at 500 µM (H2O2 group); iii) DEX at 5 µM (DEX group); and iv) H2O2 plus DEX (H2O2 + DEX group). The levels of apoptosis and oxidative stress of NRCMs were investigated by ELISA, western blotting, flow cytometry and cell immunofluorescence. DEX significantly suppressed H2O2-induced apoptosis, and increased activity of caspases 3, 8 and 9 of NRCMs. DEX inhibited mitochondria-mediated oxidative stress and apoptosis, as evidenced by decreased levels of reactive oxygen species and lactic dehydrogenase, alleviated mitochondrial membrane potential depolarization, and increased Bcl-2-associated X protein/B-cell lymphoma 2 ratio. In addition, DEX decreased the activity of caspase 12, and the expression levels of glucose-regulated protein 78 kDa and serine/threonine-protein kinase/endoribonuclease IRE1, three major signaling molecules involved in the ER stress-mediated apoptosis pathway. Preventive treatment with DEX alleviates cardiomyocyte against H2O2-induced oxidative stress injury through attenuating the mitochondria- and ER-mediated apoptosis pathways.

Orientin Reduces Myocardial Infarction Size via eNOS/NO Signaling and Thus Mitigates Adverse Cardiac Remodeling.

Orientin is a flavonoid extracted from Chinese traditional herb, Polygonum orientale L. Previous study has reported that orientin protected myocardial from ischemia reperfusion injury. However, whether orientin could protect against cardiac remodeling after myocardial injury remains unclear. The aim of our study is to investigate the effects of orientin in the progression of cardiac remodeling after myocardial infarction (MI). Mice cardiac remodeling model was established by left coronary artery ligation surgery. Experimental groups were as follows: vehicle-sham, orientin-sham, vehicle-MI, and orientin-MI. Animals were treated with vehicle or orientin (40 mg/kg) for 25 days starting 3 days after surgery. After 4 weeks of MI, mice with orientin treatment had decreased mortality and improved cardiac function. Significantly, at 4 weeks post-MI, orientin treatment decreased fibrosis, inflammatory response, and cardiomyocyte apoptosis. Furthermore, orientin treatment attenuated the hypoxia-induced neonatal rat cardiomyocyte apoptosis and increased cell viability. Additionally, orientin supplementation mitigated oxidative stress in remodeling heart tissue and cardiomyocytes exposed to hypoxia as measured by 2′,7′-dichlorodihydrofluorescein diacetate fluorescent probe. Mechanistically, orientin promotes cardioprotection by activating the eNOS/NO signaling cascades, which was confirmed by eNOS inhibitor (L-NAME) in vitro and in vivo. Inhibition of oxidative stress by orientin via eNOS/NO signaling cascades in the heart may represent a potential therapy for cardiac remodeling.

Ibutilide protects against cardiomyocytes injury via inhibiting endoplasmic reticulum and mitochondrial stress pathways

Atrial fibrillation (AF) is a complex disease with multiple inter-relating causes culminating in rapid atrial activation and atrial structural remodeling. The contribution of endoplasmic reticulum and mitochondria stress to AF has been highlighted. As the class III antiarrhythmic agent, ibutilide are widely used to AF. This study was designed to explore whether ibutilide could treat AF by inhibiting endoplasmic reticulum stress pathways and mitochondria stress. The neonatal rat cardiomyocytes were isolated and exposed to H2O2, ibutilide was add to the culture medium 12 h. Then the cell viability, oxidative stress levels and apoptotic rate were analyzed. In addition, endoplasmic reticulum stress related protein (GRP78, GRP94, CHOP), mitochondria-dependent protein (Bax, Bcl-2) and caspase-3/9/12 were identified by real-time PCR and western blot analysis. In our results, remarkable decreased cell viability and oxidative stress levels were detected in cardiomyocytes after treating with H2O2. The apoptotic rate and the expression of proteins involved in mitochondrial stress and endoplasmic reticulum stress pathways increased. While ibutilide significantly inhibited these changes. These data suggested that ibutilide serves a protective role against H2O2-induced apoptosis of neonatal rat cardiomyocytes, and the mechanism is related to suppression of mitochondrial stress and endoplasmic reticulum stress.

MiR-22 regulates starvation-induced autophagy and apoptosis in cardiomyocytes by targeting p38α

microRNAs (miRNAs) are short noncoding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression. They play critical regulatory roles in many cardiovascular diseases, including ischemia-induced cardiac injury. Here, we report microRNA-22, highly expressed in the heart, can protect cardiomyocytes from starvation-induced injury through promoting autophagy and inhibiting apoptosis. Quantitative real-time PCR (qPCR) demonstrated that the expression of miR-22 in starvation-treated neonatal rat cardiomyocytes (NRCMs) was markedly down-regulated. Over-expression of miR-22 significantly promoted starvation-induced autophagy and inhibited starvation-induced apoptosis in NRCMs. In contrast, reduction of miR-22 suppressed autophagy and accelerated apoptosis in starving NRCMs. Immunohistochemistry and TUNEL staining results also provided further evidence that miR-22 promoted autophagy and inhibited apoptosis in myocardial cells. Furthermore, both luciferase reporter assay and western blot analysis were performed to identify p38α as a direct target of miR-22. Taken together, miR-22 plays an important role in regulating autophagy and apoptosis in ischemic myocardium through targeting p38α. miR-22 may represent a potential therapeutic target for the treatment of ischemic heart diseases.

Abstract 12613: Apurinic/apyrimidinic Endonuclease/redox Factor-1 Gene Enhances Anti-apoptotic Function of Cardiac Progenitor Cells via TAK1-Activation and Promotes Cardiac Regeneration in Myocardial Infarction

Introduction: Overcoming the poor survival of cell grafts is an indispensable mission in cell therapy. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1) is known as a multifunctional enzyme to encourage cell survival, whereas the role of APE1 in cardiac cell therapy is still unknown. Hypothesis: APE1 overexpression in cardiac progenitor cells (CPC) ameliorates the effect of cardiac cell therapy. Methods: CPCs from 8-10 week-old C57BL/6 mice hearts were transfected with APE1-DsRed gene (APE1-CPC) or DsRed gene (Control [Ct]-CPC). The apoptosis induced by oxidative stress was assessed in APE1-/Ct-CPCs, and in neonatal rat cardiomyocytes (NRCM) within the co-culture system of APE1-/Ct-CPCs. Western blot analysis indicated the cellular signal to protect CPC via APE1 enzyme. To evaluate the effect of APE1 overexpression in cell therapy, we transplanted APE1-CPCs and Ct-CPCs into the mice myocardial infarction (MI) model and assessed the pathophysiological role of APE1 with functional and histological analysis. Results: Under the oxidative stress condition, APE1 overexpression inhibited the apoptosis of CPCs and accelerated TAK1 activation (Ct-CPC : APE1-CPC = 1.5±0.4 : 3.3±1.6 fold, p&lt;0.05), and consequently NfKB phosphorylation in CPCs. In the co-culture system, the apoptosis of NRCMs was inhibited with APE1-CPCs compared to that with Ct-CPCs. In vivo, in the mice MI model, the number of total CPC grafts and cardiac α-actinin-positive graft CPCs were significantly larger in APE1-CPC injected mice (APE1 mice) compared to Ct-CPC injected mice (Ct mice) at 7 days after implantation. Eventually, the left ventricular ejection fraction of APE1 mice was significantly improved compared to Ct mice (Ct mice : APE1 mice = +3.1±6.7 : +11.3±4.0%, p&lt;0.05) and was accompanied with the attenuation of fibrosis at 28 days after implantation. Conclusions: APE1 gene inhibited the apoptosis of CPCs and host cells against oxidative stress via the activation of TAK1-NFkB pathway, which is a novel insight into the stress response of APE1 enzyme. Furthermore, APE1-CPC grafts that effectively survived in the ischemic heart restored cardiac dysfunction and attenuated myocardial infarct size, and may be an innovative strategy to reinforce cardiac cell therapy.

Differential behaviour of epicardial adipose tissue-secretomes with high and low orosomucoid levels from patients with cardiovascular disease in H9C2 cells

Epicardial adipose tissue releases orosomucoid (ORM), an acute phase protein with multiple modulatory and protective properties. We aimed to identify the effect of EAT-supernatants according to their ORM levels on H9C2 cells. H9C2 were cultured with EAT-secretomes or ORM protein itself on a Real-Time Cell Analyser. Secretome proteins identification was performed by LC-mass spectrometry according to their ORM levels. Two of them were validated by ELISA in EAT-supernatants from 42 patients. ORM effect on H9C2 and neonatal rat cardiomyocytes apoptosis under hypoxia with or without fatty acid treatment was determined by Annexin-V flow cytometry measurement. Caspase-3 expression levels were determined by western blot in H9C2. Our results showed a differential effect of EAT-secretomes according their ORM levels. Although additional secreted proteins can contribute to their beneficial effects, ORM reduced hypoxia-induced apoptosis through caspase-3 inhibition. Our data showed the cardioprotective role of ORM and suggest that its quantification on EAT secretomes might help us to find new secreted factors with a cardioprotective role.