Protective Role In Cardiomyocytes Research Articles

Pharmacokinetic Characteristics of Bisdemethoxycurcumin and Its Mechanism of Reversing Cardiomyocyte Apoptosis

To investigate the pharmacokinetic characteristics of bisdemethoxycurcumin in vivo and the reversal effect of phosphatidylinositol 3-kinase/protein kinase B/caspase-9 signaling pathway on thapsigargininduced cardiomyocyte apoptosis in neonatal rat. Demethoxycurcumin and bisdemethoxycurcumin were intragastrically administered to adult Sprague–Dawley rats. Plasma was collected and the drug concentration was measured by high performance liquid chromatography. The pharmacokinetic parameters of the drug were calculated by Drug and Statistics 2.1.1 pharmacokinetic software. After that, primary cardiomyocytes from Sprague–Dawley neonatal rats were isolated and cultured, and they were divided into three groups; control (conventional culture without any drug treatment), model (endoplasmic reticulum stress model obtained by treatment with 1 mg/ml thapsigargin for 24 h), demethoxy curcumin group (treated with 1 μmol/l thapsigargin and 10 μmol/l demethoxy curcumin for 24 h at the same time) and bisdemethoxycurcumin group (treated with 1 μmol/l thapsigargin and 10 μmol/l bisdemethoxycurcumin for 24 h at the same time). Cell survival activity was measured by cell counting kit-8 assay. Western blot was used to detect the expression of intracellular apoptotic factors Bcl-2-associated X, B-cell lymphoma protein 2 and B-cell lymphoma protein 2/Bcl-2-associated X protein and the expression levels of phosphatidylinositol 3-kinase/protein kinase B/caspase-9 signaling pathway molecules phosphatidylinositol 3-kinase, protein kinase B, caspase-3 and caspase-9 protein. The results showed that the main pharmacokinetic parameters area under the timeconcentration curve 0-12 h, mean residency time 0-12 h, maximum plasma concentration, time to maximum plasma concentration of demethoxycurcumin and bisdemethoxycurcumin were (1.35±0.36) μg/ml•h and (1.01±0.14) μg/ml•h, (4.05±0.53) h and (3.43±0.21) h, (0.46±0.09) μg/ml and (0.45±0.11) μg/ml, (0.14±0.00) h and (0.20±0.00) h, respectively. In contrast with the model group, the survival activity of demethoxycurcumin and bisdemethoxycurcumin groups was significantly increased, the apoptosis rate was significantly decreased; the lactate dehydrogenase, malondialdehyde content and reactive oxygen species level were decreased, the superoxide dismutase activity was increased, the Bcl-2-associated X, caspase-3 and caspase-9 protein expression was decreased and the B-cell lymphoma protein 2/Bcl-2-associated X, phosphatidylinositol 3-kinase and protein kinase B protein expression was increased (p<0.05). However, bisdemethoxycurcumin was slightly superior to demethoxycurcumin in improving the above indicators, but the difference was not statistically meaningful (p>0.05). In summary, demethoxy curcumin and bisdemethoxycurcumin can mediate phosphatidylinositol 3-kinase/protein kinase B/caspase-9 signaling pathway to reduce thapsigargin-induced endoplasmic reticulum stress injury in cardiomyocytes, which in turn plays a protective role in cardiomyocytes.

HSP70 protects H9C2 cells from hypoxia and reoxygenation injury through STIM1/IP3R

Hypoxia/reoxygenation (H/R) is used as an in vivo model of ischemia/reperfusion injury, and myocardial ischemia can lead to heart disease. Calcium overload is an important factor in myocardial ischemia–reperfusion injury and can lead to apoptosis of myocardial cells. Therefore, it is of great clinical importance to find ways to regulate calcium overload and reduce apoptosis of myocardial cells, and thus alleviate myocardial ischemia–reperfusion injury. There is evidence that heat shock protein 70 (HSP70) has a protective effect on the myocardium, but the exact mechanism of this effect is not completely understood. Stromal interaction molecule 1 and inositol 1,4,5-triphosphate receptor (STIM/1IP3R) play an important role in myocardial ischemia–reperfusion injury. Therefore, this study aimed to investigate whether HSP70 plays an anti-apoptotic role in H9C2 cardiomyocytes by regulating the calcium overload pathway through STIM1/IP3R. Rat H9C2 cells were subjected to transient oxygen and glucose deprivation (incubated in glucose-free medium and hypoxia for 6 h) followed by re-exposure to glucose and reoxygenation (incubated in high glucose medium and reoxygenation for 4 h) to simulate myocardial ischemia reperfusion-induced cell injury. H9C2 cell viability was significantly decreased, and lactate dehydrogenase (LDH) release and apoptosis were significantly increased after oxygen and glucose deprivation. Transfection of HSP70 into H9C2 cells could reduce the corresponding effect, increase cell viability and anti-apoptotic signal pathway, and reduce the apoptotic rate and pro-apoptotic signal pathway. After hypoxia and reoxygenation, the expression of STIM1/IP3R and intracellular calcium concentration of HSP70-overexpressed H9C2 cells were significantly lower than those of hypoxia cells. Similarly, direct silencing of STIM1 by siRNA significantly increased cell viability and expression of anti-apoptotic protein Bcl-2 and decreased apoptosis rate and expression of pro-apoptotic protein BAX. These data are consistent with HSP70 overexpression. These results suggest that HSP70 abrogates intracellular calcium overload by inhibiting upregulation of STIM1/IP3R expression, thus reducing apoptosis in H9C2 cells and playing a protective role in cardiomyocytes.

Abstract 13219: ER-Selective Autophagy Alleviates ER Stress-Mediated Myocardial Injury

Introduction: Increasing lines of evidence suggest endoplasmic reticulum-selective autophagy (ER-phagy) as a cell-protective pathway to mediate degradation of damaged ER. However, the role of ER-phagy in cardiomyocytes and its molecular mechanisms remain unsolved. Hypothesis: Considering that various cardiac loads cause ER stress which contributes to the development of heart failure, we hypothesized that ER-phagy would be a compensatory system for protecting cardiomyocytes against ER stress. Methods and Results: To evaluate ER-phagy in cardiomyocytes, we generated lentivirus (LV) and cardiac-specific transgenic mice (Tg-mice) harboring ss-RFP-GFP-KDEL sequence, an ER-phagy reporter possessing an N-terminal ER signal sequence followed by tandem fluorescence sequences and ER retention signal. Fluorescent microscopic analyses and immunoblotting demonstrated that amino acid deprivation of LV-ss-RFP-GFP-KDEL-tranduced H9c2 (tfH9c2) cells markedly elevated the amount of RFP fragments, suggesting that ER-phagy is increased in tfH9c2 cells. 48 hours of food starvation markedly induced ER-phagy in Tg-mice hearts, as well. Treatment with Doxorubicin (Dox), a cardiotoxic compound inducing ER stress, activated ER-phagy in tfH9c2 cells as evidenced by the increase in RFP fragments detected by immunoblotting (Control vs. Dox: 1.0±0.2a.u. vs. 31.6±6.3a.u.; p&lt;0.05). Consistently, treatment with Dox (20 mg/kg) significantly induced ER-phagy in myocardium of Tg-mice compared with those of saline-treated ones (Saline vs. Dox: 1.0±0.05a.u. vs. 2.5±0.2a.u.; p&lt;0.05). qRT-PCR analyses revealed that treatment with Dox enhanced the expression of CCPG1, an ER-phagy receptor, in tfH9c2 cells (Control vs. Dox: 1.0±0.05a.u. vs. 2.1±0.2a.u.; p&lt;0.05). Furthermore, LV-CCPG1 transduction-mediated silencing CCPG1 of tfH9c2 cells resulted in the reduction of ER-phagy activity, accumulation of pro-apoptotic proteins including cleaved caspase 3 and deterioration of cell survival (Cell viability: Control vs. Knockdown: 79.0±3.3% vs. 62.4±2.7%; p&lt;0.05) in response to Dox treatment. Conclusions: These results suggest that ER-phagy plays a protective role in cardiomyocytes against Dox toxicity, possibly through CCPG1-mediated signaling.

LncRNA SNHG1 protects the cardiac muscle cells from hypoxia/ re-oxygenation injury in vitro by targeting microRNA-21-5p and miR-30a-5p

Purpose: Cardiovascular diseases are responsible for numerous deaths globally. Long noncoding RNA SNHG1 has presented its protective role in cardiomyocytes previously. Herein, we examined the underlying molecular mechanisms of SNHG1 in cardiac muscle cells from hypoxia and re-oxygenation (H/R) in vitro.Methods: RT-qPCR measured expression of SNHG1, miR-21-5p and miR-30a-5p in rat cardiac muscle cell line HL-1 before and after H/R treatment and cell transfection, which was applied to regulate expression of SNHG1, miR-21-5p and miR-30a-5p for further use. The flow cytometry method was used to compare changes in cellular apoptosis, and cell viability was measured by CCK-8 method.Bioinformatics predicted the bindings ofSNHG1 and miR-21-5p / miR-30a-5p while the luciferase reporter assays further verified this.Results: The outcomes revealedthat SNHG1 was downregulated and meanwhile miR-21-5p / miR-30a5p was elevated that enhanced apoptosis and reduced cell viability in HL-1 cells. However, overexpressed SNHG1 inhibited cell apoptosis and increased cell viability brought by H/R. In addition, SNHG1 targeted at miR-21-5p/ miR-30a-5p, which contributed to the inter-regulation in between. Furthermore, interactive experiments revealed that upregulation of miR-21-5p/ miR-30a-5p added to the cell apoptosis which was induced by H/R and partially counteracted by the upregulation of SNHG1.Conclusion: In this study we have demonstrated the protective role of SNHG1 in the moderation of H/R-induced HL-1 apoptosis and viability through suppression of miR-21-5p/miR-30a-5p. This offers new perspective into the molecular interpretation of cardiovascular diseases such as ischemic reperfusion injury.&#x0D; Keywords: SNHG1; apoptosis; Hypoxia/Re-oxygenation injury;miR-21-5p; miR-30a-5p; Cardiovascular diseases

Bisoprolol, a β1 antagonist, protects myocardial cells from ischemia–reperfusion injury via PI3K/AKT/GSK3β pathway

The aim of this work was to explore whether bisoprolol plays a protective role in cardiomyocytes against ischemia-reperfusion injury via PI3K/AKT/ GSK3β pathway. We pretreated male Sprague Dawley (SD) rats with bisoprolol by oral administration prior to 0.5h ischemia/4h reperfusion. Myocardial infarct size and serum levels of cTnI and CK-MB were measured. In vitro, H9c2 cells were treated with hypoxia and reoxygenation, followed by measurement of cell viability, apoptosis, ROS production, cytometry, activities of AKT, GSK3β, and p-38 in the presence and absence of GSK3β siRNA. We found that bisoprolol reduced infarct size from 44% in I/R group to 31% in treated group (P<0.05). The levels of cTnI and CK-MB were decreased from 286±7pg/mL and 32.2±2ng/mL in I/R group to 196±2pg/mL and 19.6±0.9ng/mL in the treated group, respectively (P<0.05). Bisoprolol also increased cell viability while decreased apoptosis and ROS production in the treatment of hypoxia/ reoxygenation. Furthermore, bisoprolol increased AKT and GSK3β phosphorylation, an effect that was immediately eliminated by LY294002. GSK3β-specific siRNA experiment further confirmed that bisoprolol protected the myocardium against hypoxia/reoxygenation-induced injury via suppressing GSK3β activity. In conclusion, bisoprolol protected myocardium against ischemia-reperfusion injury via the PI3K/AKT/ GSK3β pathway.

Low concentration of rutin treatment might alleviate the cardiotoxicity effect of pirarubicin on cardiomyocytes via activation of PI3K/AKT/mTOR signaling pathway.

Cancer is the leading cause of deaths around the world, especially in low- and middle- income countries. Pirarubicin (THP) is an effective drug for treatment of cancer, however, there still exists cardiotoxic effects of THP. Rutin is a kind of antioxidative compound extracted from plants, and might be a protective compound for cardiomyocytes. Phosphatidylinositol 3-hydroxy kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is critical for cellular survival, proliferation and metabolism, and thus we speculated rutin might perform a protective role in cardiomyocytes via PI3K/AKT/mTOR signaling pathway. And in this experiment, we first established a cardiotoxicity model of THP in mice model and cell models, and then found that rutin treatment could increase the proliferation of cells at low concentration. Then we explored the possible mechanism of the protective effect of rutin using Western blotting, quantitative polymerase chain reaction (qPCR) and ELISA methods, and found that the activation of PI3K/AKT/mTOR/nuclear factor-κB (NF-κB) signaling pathway was increased, and expression of downstream molecules involved in antioxidative stress were also increased. We further noticed that concentration of angiogenesis promoting factors were also increased in medium of cultured cells. Thus, we speculated that rutin could increase the activation of PI3K/AKT/mTOR signaling pathway, further decrease the oxidative stress level via increasing the expression of antioxidative stress enzymes with the increasing concentration of angiogenesis promoting factors, resulting in the protective role in cardiomyocytes and cardiac function.

Atorvastatin protects cardiac progenitor cells from hypoxia-induced cell growth inhibition via MEG3/miR-22/HMGB1 pathway.

Heart failure (HF) induced by ischemia myocardial infarction (MI) is one of the major causes of morbidity and mortality all around the world. Atorvastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, has been demonstrated to benefit patients with ischemic or non-ischemic-induced HF, but the mechanism is still poorly understood. Increasing evidence indicates that lncRNAs play important role in variety of human disease. However, the role and underlying molecular mechanisms remain largely unclear. In our work, we applied 0.5% O2 to generate a hypoxia cardiac progenitor cell (CPC) model. Then, CCK8 and EdU assays were employed to investigate the role of atorvastatin in hypoxia CPC cell model. We found that hypoxia inhibits CPC viability and proliferation through modulating MEG3 expression, while atorvastatin application can protect CPCs from hypoxia-induced injury through inhibiting MEG3 expression. Then, we demonstrated that repression of MEG3 inhibited the hypoxia-induced injury of CPCs and overexpression of MEG3 inhibited the protective effect of atorvastatin in the hypoxia-induced injury of CPCs. Furthermore, our study illustrated that atorvastatin played its role in CPC viability and proliferation by modulating the expression of HMGB1 through the MEG3/miR-22 pathway. Our study, for the first time, uncovered the molecular mechanism of atorvastatin's protective role in cardiomyocytes under hypoxia condition, which may provide an exploitable target in developing effective therapy drugs for MI patients.

Downregulation of miR-130a, antagonized doxorubicin-induced cardiotoxicity via increasing the PPAR\u03b3 expression in mESCs-derived cardiac cells

Doxorubicin (Dox) is a widely used powerful chemotherapeutic component for cancer treatment. However, its clinical application has been hampered due to doxorubicin-induced cardiomyopathy upon the cessation of chemotherapy. Previous studies revealed that PPARγ plays a crucial protective role in cardiomyocytes. Modulation of miRNA expression is an applicable approach for prohibition of toxicity induction. Therefore, the aim of present study is uprising of PPARγ transcript levels via manipulation of miRNAs to limit Dox-induced cardiotoxicity in mESCs-derived cardiac cells, as in vitro model cell to provide a simple direct approach for further clinical therapies. Based on bioinformatics data mining, eventually miR-130a was selected to target PPARγ. This miRNA is highly expressed in heart. The expression of miR-130a increases sharply upon Dox treatment while specific antagomiR-130a reverses Dox-induced reduced expression of PPARγ, cellular apoptosis, and inflammation. Our data strongly suggest that antagomiR-130a limits Dox-induced cellular toxicity via PPARγ upregulation and may have clinical relevance to limit in vivo Dox toxicity.

Integrin β3 inhibits hypoxia-induced apoptosis in cardiomyocytes.

Hypoxia-induced apoptosis plays an important role in cardiovascular diseases. Integrin β3 is one of the main integrin heterodimer receptors on the surface of cardiac myocytes. However, despite the important role that integrin β3 plays in the cardiovascular disease, its exact role in the hypoxia response remains unclear. Hence, in the present investigation we aimed to study the role of integrin β3 in hypoxia-induced apoptosis in H9C2 cells and primary rat myocardial cells. MTT assay, flow cytometry and TUNEL assay results showed that hypoxia inhibited cardiomyocyte proliferation and induced cardiomyocyte apoptosis. The expression levels of integrin β3 and HIF1α were upregulated in hypoxia-induced cardiomyocytes as revealed by real-time PCR and western blot analysis. Furthermore, knockdown of integrin β3 expression by siRNA increased hypoxia-induced cardiomyocyte apoptosis. In addition, integrin β3 overexpression weakened hypoxia-induced cardiomyocyte apoptosis. The protein expressions of integrin β3 and HIF1α were upregulated in acute myocardial infarction rat cardiac tissues compared with the control rat cardiac tissues. Our data suggest that integrin β3 plays a protective role in cardiomyocytes during hypoxia-induced apoptosis.

The Protective Role of Paraoxonase 2 in Cardiomyocytes Against Myocardial Ischemia‐Reperfusion Injury

IntroductionAcute myocardial infarction (AMI) induced ischemic injury results in ion accumulation, particularly calcium overload, ATP depletion and cell death. Following AMI, restoration of blood flow, or reperfusion, to the myocardium exacerbates cell death, primarily through the generation of reactive oxygen species (ROS) and the opening of the mitochondrial permeability transition pore (mPTP). Paraoxonase 2 (PON2), an intracellular protein with antioxidant, anti‐inflammatory properties, is localized in the mitochondria and is known to protect against mitochondrial oxidative stress and dysfunction.HypothesisPON2 protects cardiomyocytes against myocardial ischemia‐reperfusion (IR) injury by mitigating mitochondrial oxidative stress and dysfunction.MethodsMale PON2 deficient (PON2‐def) and C57BL6/J (WT) mice were subjected to in vivo IR injury via left anterior descending coronary artery occlusion (30‐minute) and reperfusion (24‐hours), and PON2 gene expression and infarct sizes were evaluated. Excised PON2‐def and WT hearts were subjected to ex vivo Langendorff IR injury (20‐minute ischemic episode, 5‐minute reperfusion). Following ex vivo IR injury, cardiomyocytes were isolated and mitochondrial calcium retention capacity (CRC), generation of ROS and cell apoptosis (TUNEL assay) were measured (n=3–4/group). Stable cell lines were generated in H9c2 cells, a rat ventricular cardiomyocyte cell line, which overexpressed human PON2 (H9c2‐hPON2) and empty vector (H9c2‐EV). H9c2 cell lines were subjected to hypoxia‐reoxygenation (HR) injury [3‐hours in 2% O2 (hypoxia), followed by 2‐hours in normoxic conditions (reperfusion)]. The cells were also subjected to HR injury with and without LY294002, a potent PI3K inhibitor. Following HR, CRC and ROS generation were measured (n=3–4/group). Data is expressed as mean±SEM. T‐test and two‐way ANOVA are used for statistical analysis, with probability values less than 0.05 considered statistically significant.ResultsFollowing in vivo IR injury, PON2‐def mice exhibited a ~2‐fold larger infarct than WT mice, and PON2 gene expression was ~2‐fold upregulated in the WT mice. Cardiomyocytes from PON2‐def mice subjected to ex vivo IR injury had reduced CRC (160±32 in WT, 80±0 in PON2‐def, nmoles calcium/6.0×104 cells, p&lt;0.05), 2‐fold increased ROS production (p&lt;0.05) and increased apoptosis compared to WT controls (25% of TUNEL positive nuclei in PON2‐def vs 15% in WT, p&lt;0.05). H9c2‐hPON2 cells subjected to HR injury exhibited increased CRC compared to H9c2‐EV cells (141.7±16.67 in H9c2‐EV, 200±14.43 in H9c2‐hPON2, nmoles calcium/1.5×106 cells, p&lt;0.05), indicating that PON2 overexpression increases the threshold for the opening of the mPTP in response to calcium overload. H9c2‐hPON2 cells demonstrated a 40% reduction in ROS generation versus the H9c2‐EV control cells post HR injury (p&lt;0.05). Treatment with LY294002 abolished the protective effects of overexpressing PON2 with significantly reduced CRC in the H9c2‐hPON2 group (105±5 in H9c2‐EV+PI3Ki, 60±10 in H9c2‐hPON2+PI3Ki, nmoles calcium/1.5×106 cells, p&lt;0.05) and a 60% increase in ROS (p&lt;0.05) production versus H9c2‐EV.ConclusionPON2 plays a protective role in cardiomyocytes against myocardial IR injury by inhibiting mPTP opening and reducing ROS generation and functions by way of the PI3K/Akt/GSK‐3β pathway. Our results suggest that PON2 is a potential therapeutic candidate to ameliorate myocardial IR injury.Support or Funding InformationThis work is supported by NHLBI grant R01 HL071776‐01.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-210 protects cardiomyocytes from OGD/R injury by inhibiting E2F3.

To detect the change in miRNA-210 expression of cardiomyocytes under hypoxia/reoxygenation status. Also, the effect of miR-210 on the apoptosis of cardiomyocytes induced by oxygen-glucose deprivation/reperfusion (OGD/R) and its mechanism through establishing the OGD/R injury model of primary cardiomyocytes in this experiment were investigated. The cell model of OGD/R injury was established. The cell apoptosis in each group was detected by methyl thiazolyl tetrazolium (MTT) assay and detection of Caspase-3 activity. The change in miR-210 expression in each group was detected by Real-time fluorescence quantitative polymerase chain reaction (PCR). The high-expression and low-expression miR-210 models were established through the transient transfection of miR-210 mimic and inhibitor to detect the relevant indexes of cell apoptosis. At the same time, changes in mRNA and protein expressions of E2F3 were detected by RT-PCR and Western blotting, respectively. The E2F3 overexpression vector was constructed, and the overexpression vector plasmid and miR-210 mimic were jointly transfected into the cells to detect the relevant indexes of cell apoptosis. After OGD/R treatment, the activity of Caspase-3 was increased, the survival of cardiomyocytes was significantly inhibited and the expression level of miR-210 was up-regulated in OGD/R injury. Transfection of miR-210 mimic for miR-210 overexpression could alleviate the OGD/R-induced cardiomyocyte injury, while the decrease of miR-210 expression could aggravate the apoptosis of cardiomyocytes. In addition, the high expression of miR-210 could inhibit the protein expression of E2F3, and co-transfection of E2F3 plasmid and miR-210 mimic could reverse the inhibiting effect of miR-210 on the apoptosis of cardiomyocytes. We confirmed that miR-210 can inhibit the OGD/R-induced apoptosis of cardiomyocytes, and miR-210, as an upstream factor, plays a protective role in cardiomyocytes through directly inhibiting the protein expression of its target gene E2F3.

Calcitonin Gene-Related Peptide Improves Hypoxia-Induced Inflammation and Apoptosis via Nitric Oxide in H9c2 Cardiomyoblast Cells

Objectives: The aim of this work was to investigate whether calcitonin gene-related peptide (CGRP) plays a protective role in cardiomyocytes against hypoxia-induced inflammation and apoptosis via an NO-mediated pathway. Methods: H9c2 cardiac cells were exposed to hypoxia for 2 h to establish a model of myocardial hypoxic-ischemic injury. The cells were pretreated with either CGRP or nitric oxide synthase (NOS) inhibitor (<smlcap>L</smlcap>-NAME) before being exposed to hypoxia for 30 min. Cell viability was analyzed using a cell counter kit 8 (CCK-8). The levels of IL-6 and TNF-α were determined by the corresponding enzyme-linked immunosorbent assay. The expression levels of several apoptosis proteins (p53, caspase-3, cytochrome C) and NOS were detected by Western blot assays. An NO kit was used to evaluate the production of NO. Results: Pretreatment of H9c2 cardiac cells with CGRP for 30 min prior to exposure to hypoxia markedly improved cell viability (83.57 ± 3.21 vs. 62.83 ± 8.30%, p < 0.001); the same effect was observed following pretreatment with the NOS inhibitor <smlcap>L</smlcap>-NAME (89.34 ± 5.95 vs. 75.01 ± 5.61%, p < 0.01). Pretreatment with CGRP also significantly attenuated the inflammatory responses induced by hypoxia, as evidenced by decreases of the levels of both IL-6 (193.21 ± 13.54 vs. 293.38 ± 56.49%, p < 0.001) and TNF-α (207.71 ± 44.27 vs. 281.46 ± 64.88%, p < 0.001). Additionally, CGRP significantly decreased the hypoxia-induced overexpression of the apoptotic proteins (p53: 0.27 ± 0.10 vs. 0.87 ± 0.30, p < 0.001; caspase-3: 0.65 ± 0.15 vs. 0.98 ± 0.26, p < 0.001; cytochrome C: 1.51 ± 0.39 vs. 2.80 ± 0.69, p < 0.001) and enhanced the expression of both endothelial NOS (eNOS; 0.59 ± 0.24 vs. 0.37 ± 0.14, p < 0.05) and phosphorylated eNOS (0.60 ± 0.13 vs. 0.40 ± 0.07, p < 0.05). Furthermore, the application of both <smlcap>L</smlcap>-NAME and CGRP attenuated the hypoxia-induced expression of inducible NOS (iNOS; p < 0.05) and enhanced a hypoxia-mediated decrease in NO (p < 0.01). Interestingly, the expression levels of cell apoptosis (p < 0.05), iNOS and eNOS (p < 0.05) were decreased with <smlcap>L</smlcap>-NAME and CGRP cotreatment following 2 h of acute hypoxia, but the apoptotic factors (p < 0.05) were increased compared with only CGRP pretreatment. Conclusion: CGRP protects cardiomyocytes from hypoxia-induced inflammation and apoptosis by modulating NO production.

Silencing of uncoupling protein 2 by small interfering RNA aggravates mitochondrial dysfunction in cardiomyocytes under septic conditions.

Uncoupling protein 2 (UCP2) regulates the production of mitochondrial reactive oxygen species (ROS) and cellular energy transduction under physiological or pathological conditions. In this study, we aimed to determine whether mitochondrial UCP2 plays a protective role in cardiomyocytes under septic conditions. In order to mimic the septic condition, rat embryonic cardiomyoblast-derived H9C2 cells were cultured in the presence of lipopolysaccharide (LPS) plus peptidoglycan G (PepG) and small interfering RNA (siRNA) against UCP2 (siUCP2) was used to suppress UCP2 expression. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR), western blot analysis, transmission electron microscopy (TEM), confocal microscopy and flow cytometry (FCM) were used to detect the mRNA levels, protein levels, mitochondrial morphology and mitochondrial membrane potential (MMP or ΔΨm) in qualitative and quantitative analyses, respectively. Indicators of cell damage [lactate dehydrogenase (LDH), creatine kinase (CK), interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the culture supernatant] and mitochondrial function [ROS, adenosine triphosphate (ATP) and mitochondrial DNA (mtDNA)] were detected. Sepsis enhanced the mRNA and protein expression of UCP2 in the H9C2 cells, damaged the mitochondrial ultrastructure, increased the forward scatter (FSC)/side scatter (SSC) ratio, increased the CK, LDH, TNF-α and IL-6 levels, and lead to the dissipation of MMP, as well as the overproduction of ROS; in addition, the induction of sepsis led to a decrease in ATP levels and the deletion of mtDNA. The silencing of UCP2 aggravated H9C2 cell damage and mitochondrial dysfunction. In conclusion, our data demonstrate that mitochondrial morphology and funtion are damaged in cardiomyocytes under septic conditions, while the silencing of UCP2 using siRNA aggravated this process, indicating that UCP2 may play a protective role in cardiomyocytes under septic conditions.

The IκB Kinase β/Nuclear Factor κB Signaling Pathway Protects the Heart From Hemodynamic Stress Mediated by the Regulation of Manganese Superoxide Dismutase Expression

Cardiomyocyte death plays an important role in the pathogenesis of heart failure. The nuclear factor (NF)-kappaB signaling pathway regulates cell death, however, the effect of NF-kappaB pathway on cell death can vary in different cells or stimuli. The purpose of the present study was to clarify the in vivo role of the NF-kappaB pathway in response to pressure overload. First, we subjected C57Bl6/J mice to pressure overload by means of transverse aortic constriction (TAC) and examined the activity of the NF-kappaB pathway in response to pressure overload. IkappaB kinase (IKK) and NF-kappaB were activated after TAC. Then, we investigated the role of the activation using cardiac-specific IKKbeta-deficient mice (CKO). CKO displayed normal global cardiac structure and function compared with control littermates. We subjected CKO and control mice to pressure overload. One week after TAC, CKO showed cardiac dilation, dysfunction, and lung congestion, which are characteristics of heart failure. The number of apoptotic cells in the hearts of CKO mice increased significantly after TAC. The levels of manganese superoxide dismutase mRNA and protein expression in CKO after TAC were significantly attenuated compared with control mice. The levels of oxidative stress and c-Jun N-terminal kinase (JNK) activation in CKO after TAC were significantly greater than those in control mice. Isoproterenol-induced cell death of isolated adult CKO cardiomyocytes was inhibited by treatment with either a manganese superoxide dismutase mimetic or a JNK inhibitor. Thus, the IKKbeta/NF-kappaB signaling pathway plays a protective role in cardiomyocytes because of the attenuation of oxidative stress and JNK activation in a setting of acute pressure overload.

P38 MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) IS ACTIVATED BY NORADRENALINE AND SERVES A CARDIOPROTECTIVE ROLE, WHEREAS ADRENALINE INDUCES p38 MAPK DEPHOSPHORYLATION

1. The aim of the present study was to investigate the role of p38 mitogen-activated protein kinases (MAPK) in mediating the effect of noradrenaline (NA) on cardiomyocyte cell viability. 2. Cardiomyocytes from embryonic chick heart were treated with various concentrations of NA, phenylephrine or isoproterenol and p38 MAPK activation was determined by western blotting. Total cell death was assessed by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was determined by specific DNA fragmentation. 3. At 100 micromol/L, NA produced a significant increase in cell death that was associated with microscopic changes and DNA fragmentation indicative of apoptosis. The p38 MAPK inhibitor SB202190 (at 1 micromol/L beginning 1 h before NA), reduced NA-induced p38 MAPK activation and significantly accentuated NA-induced cell death. In contrast, the mitogen-activated protein kinase kinase ERK1/2 inhibitor PD98059 (at 1 micromol/L beginning 1 h before NA) did not significantly alter NA-induced cell death. These effects of NA were mediated, in part, through alpha-adrenoceptor because phenylephrine (100 micromol/L), like NA, also induced p38 MAPK activation. However, 100 micromol/L isoproterenol produced a sustained dephosphorylation of p38 MAPK. 4. These data show that NA-induced p38 MAPK activation, through alpha-adrenoceptor, has a protective role in cardiomyocytes to antagonize NA-induced cell death. In contrast, beta-adrenoceptor stimulation produces dephosphorylation of p38 MAPK.

Forced expression of the cell cycle inhibitor p57Kip2 in cardiomyocytes attenuates ischemia-reperfusion injury in the mouse heart

BackgroundMyocardial hypoxic-ischemic injury is the cause of significant morbidity and mortality worldwide. The cardiomyocyte response to hypoxic-ischemic injury is known to include changes in cell cycle regulators. The cyclin-dependent kinase inhibitor p57Kip2 is involved in cell cycle control, differentiation, stress signaling and apoptosis. In contrast to other cyclin-dependent kinase inhibitors, p57Kip2 expression diminishes during postnatal life and is reactivated in the adult heart under conditions of cardiac stress. Overexpression of p57Kip2 has been previously shown to prevent apoptotic cell death in vitro by inhibiting stress-activated kinases. Therefore, we hypothesized that p57Kip2 has a protective role in cardiomyocytes under hypoxic conditions. To investigate this hypothesis, we created a transgenic mouse (R26loxpTA-p57k/+) that expresses p57Kip2 specifically in cardiac tissue under the ventricular cardiomyocyte promoter Mlc2v.ResultsTransgenic mice with cardiac specific overexpression of p57Kip2 are viable, fertile and normally active and their hearts are morphologically indistinguishable from the control hearts and have similar heart weight/body weight ratio. The baseline functional parameters, including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LVdp/dtmax, heart rate (HR) and rate pressure product (RPR) were not significantly different between the different groups as assessed by the Langendorff perfused heart preparation. However, after subjecting the heart ex vivo to 30 minutes of ischemia-reperfusion injury, the p57Kip2 overexpressing hearts demonstrated preserved cardiac function compared to control mice with higher left ventricular developed pressure (63 ± 15 vs 30 ± 6 mmHg, p = 0.05), rate pressure product (22.8 ± 4.86 vs 10.4 ± 2.1 × 103bpm × mmHg, p < 0.05) and coronary flow (3.5 ± 0.5 vs 2.38 ± 0.24 ml/min, p <0.05).ConclusionThese data suggest that forced cardiac expression of p57Kip2 does not affect myocardial growth, differentiation and baseline function but attenuates injury from ischemia-reperfusion in the adult mouse heart.