Cardiac Cell Apoptosis Research Articles

Effects of cardiac biological activities on low-intensity physical training in doxorubicin-induced cardiotoxicity rat models

Objective: In the present study, we investigated the protective effects of low-intensity treadmill training in doxorubicin-induced cardiotoxicity rat models. Design: Randomized controlled trial. Methods: In this study, we randomly divided them into four groups. The normal group included non-cardiotoxicity normal control (n=10), the control group included non-treadmill training after doxorubicin-induced cardiotoxicity (n=10), the experimental group I included low-intensity treadmill training (3 m/min) after doxorubicin-induced cardiotoxicity (n=10), and the experimental group II included low-intensity treadmill training (8 m/min) after doxorubicin-induced cardiotoxicity (n=10). Rats in the treadmill training group underwent treadmill training, which began at 2 weeks after first intraperitoneal injection. We determined the body weight change for each rat on days 1 and 21. Biochemical markers (lactate dehydrogenase [LDH], creatine kinase [CK], glutathion, aspartate transaminase [AST], and alanine transaminase [ALT]) concentration in the serum change of rats from all four groups was examined at the end of the experiment. Results: The results showed that the experimental group I and II showed a significant increase in body weight as compared with that of the control group (p<0.05). We observed that the biochemical markers (LDH, CK, glutathion, AST, and ALT) were improved in the experimental group I than the experimental group II (p<0.05). There was no difference between the experimental groups. Conclusions: In conclusion, our data suggest that low-intensity treadmill training applied after doxorubicin treatment protects against cardiotoxicity following treatment, possibly by enhancing antioxidant defenses and inhibiting cardiac muscle cell apoptosis.

Abstract 17481: MicroRNAs Mediate the Protective Effect of Angiotensin Converting Enzyme Inhibition (ACEi) on the Heart in Rat Model of Acute Kidney Injury

Introduction: Cardiovascular complications are common in patients with kidney disease. MicroRNAs play an important role in regulating cell death pathways and cardiac hypertrophy but it is not known whether cardiac microRNAs contribute to cardio-renal cross talk. microRNA-1 and microRNA-133 are associated with cardiac fibrosis and apoptosis after myocardial infarction, whilst microRNA-212/132 is associated with cardiac hypertrophy. Aim: Using a rat model we investigated whether acute kidney injury leads to changes in cardiac microRNAs (1,133,212 and 132) and their gene targets. We also tested the effect of treatment with the angiotensin converting enzyme inhibitor (ACEi) ramipril on these cardiac microRNAs. Methods: Heart tissues were collected from rats 10 days after subtotal nephrectomy (STNx) surgery, where one kidney was removed and the other partially ligated (n=9), from sham animals (n=9), and from STNx rats treated with ramipril (n=9). RNA was extracted from the left ventricle and quantitative real time PCR was used to measure microRNA and their target gene expression levels. Results: In rats with renal injury, there was a significant increase in left ventricular hypertrophy (P&lt;0.05 vs sham). There was also a significant increase in expression of cardiac microRNA-212 (2 fold, P&lt;0.05 vs Sham) and microRNA-132 (3 fold, P&lt;0.05 vs Sham). Ramipril treatment in STNx rats attenuated the increase in microRNA-212 and caused a significant increase in expression of microRNA-133 (P&lt;0.001 vs Sham, P&lt;0.001 vs STNx+Veh) and microRNA-1 (P&lt;0.01 vs Sham, P&lt;0.01 vs STNx+Veh). Renal injury induced left ventricular hypertrophy was attenuated in ramipril treated STNx rats (P&lt;0.001 vs STNx+Veh). We also found altered expression of caspase 9, fibronectin 1, collagen 1A1 and forkhead box O3, all known for their involvement in the regulation of apoptosis, fibrosis and hypertrophy in cardiac cells, whilst being targets for the above microRNAs. Conclusions: The involvement of microRNAs in cardiac pathologies associated with kidney injury is a novel finding of our study. Our finding suggests an involvement of microRNA-1, microRNA-133 and microRNA-212/132 in the cardioprotective action of ACE inhibition in acute kidney injury.

GW25-e3243 Sanguinarine Inhibited Ang II Induced Apoptosis in H9c2 Cardiac Cells via Restoring ROS-decreased Mitochondrial Membrane Potential

GW25-e3243 Sanguinarine Inhibited Ang II Induced Apoptosis in H9c2 Cardiac Cells via Restoring ROS-decreased Mitochondrial Membrane Potential

GW25-e5361 Traditional Chinese medicine Tongxinluo inhibits ischemia/reperfusion induced apoptosis of human cardiac microvascular endothelial cells via activating the RISK pathways

GW25-e5361 Traditional Chinese medicine Tongxinluo inhibits ischemia/reperfusion induced apoptosis of human cardiac microvascular endothelial cells via activating the RISK pathways

α-Lipoic acid protected cardiomyoblasts from the injury induced by sodium nitroprusside through ROS-mediated Akt/Gsk-3β activation

It has been long noted that cardiac cell apoptosis provoked by excessive production of nitric oxide (NO) plays important roles in the pathogenesis of variant cardiac diseases. Attenuation of NO-induced injury would be an alternative therapeutic approach for the development of cardiac disorders. This study investigated the effects of α-lipoic acid (LA) on the injury induced by sodium nitroprusside (SNP), a widely used NO donor, in rat cardiomyoblast H9c2 cells. SNP challenge significantly decreased cell viability and increased apoptosis, as evidenced by morphological abnormalities, nuclear condensation and decline of mitochondrial potential (ΔΨm). These changes induced by SNP were significantly attenuated by LA pretreatment. Furthermore, LA pretreatment prevented the SNP-triggered suppression of Akt and Gsk-3β activation. Blockade of Akt activation with triciribin (API) completely abolished the cytoprotection of LA against SNP challenge. In addition, LA moderately increased intracellular ROS production. Interestingly, inhibition of ROS with N-acetylcysteine abrogated Akt/Gsk-3β activation and the LA-induced cytoprotection following SNP stimulation. Taken together, the results indicate that LA protected the SNP-induced injury in cardiac H9c2 cells through, at least in part, the activation of Akt/Gsk-3β signaling in a ROS-dependent mechanism.

Maslinic acid activates mitochondria-dependent apoptotic pathway in cardiac carcinoma.

Cardiac carcinoma is the most common subtype of gastric cancer and its incidence has increased in recent years. The current chemotherapeutic drugs exhibit limited effectiveness and significant side effects in patients. Maslinic acid (MA) exerts an anti-tumor activity on a wide range of cancers and has no significant side effect; however, the anti-tumor effect of MA on cardiac carcinoma has not yet been explored. MTT assays, tumor xenograft animal model, immunoblotting, MMP assessment and flow cytometry were performed in this study. MA was able to suppress the viability of cardiac carcinoma cells in both a time- and dose-dependent manner. This natural compound exhibited no cytotoxicity in normal cells. Its inhibitory effect on tumor growth was further confirmed in a mouse model. Mechanistically, MA induced the activation of p38 MAPK in cardiac carcinoma cells and, in turn, changed their mitochondrial membrane potential (MMP). Finally, caspase cascades were activated by a series of cleavages, leading to apoptosis in cardiac cancer cells. Inhibition of p38 MAPK signaling was able to rescue the effect of MA on cardiac carcinoma cells. Our data demonstrated that natural compound, MA, suppressed the growth of cardiac carcinoma by inducing apoptosis via the p38 MAPK/mitochondria/caspase pathway. MA and its derivatives may be promising anti-tumor agents for cardiac carcinoma treatment in the future. (Supplemental Figures available here.).

Increased concentration of circulating angiogenesis and nitric oxide inhibitors induces endothelial to mesenchymal transition and myocardial fibrosis in patients with chronic kidney disease

BackgroundSudden cardiovascular death is increased in chronic kidney disease (CKD). Experimental CKD models suggest that angiogenesis and nitric oxide (NO) inhibitors induce myocardial fibrosis and microvascular dropout thereby facilitating arrhythmogenesis. We undertook this study to characterize associations of CKD with human myocardial pathology, NO-related circulating angiogenesis inhibitors, and endothelial cell behavior. MethodsWe compared heart (n=54) and serum (n=162) samples from individuals with and without CKD, and assessed effects of serum on human coronary artery endothelial cells (HCAECs) in vitro. Left ventricular fibrosis and capillary density were quantified in post-mortem samples. Endothelial to mesenchymal transition (EndMT) was assessed by immunostaining of post-mortem samples and RNA expression in heart tissue obtained during cardiac surgery. Circulating asymmetric dimethylarginine (ADMA), endostatin (END), angiopoietin-2 (ANG), and thrombospondin-2 (TSP) were measured, and the effect of these factors and of subject serum on proliferation, apoptosis, and EndMT of HCAEC was analyzed. ResultsCardiac fibrosis increased 12% and 77% in stage 3–4 CKD and ESRD and microvascular density decreased 12% and 16% vs. preserved renal function. EndMT-derived fibroblast proportion was 17% higher in stage 3–4 CKD and ESRD (Ptrend=0.02). ADMA, ANG, TSP, and END concentrations increased in CKD. Both individual factors and CKD serum increased HCAEC apoptosis (P=0.02), decreased proliferation (P=0.03), and induced EndMT. ConclusionsCKD is associated with an increase in circulating angiogenesis and NO inhibitors, which impact proliferation and apoptosis of cardiac endothelial cells and promote EndMT, leading to cardiac fibrosis and capillary rarefaction. These processes may play key roles in CKD-associated CV disease.

Transforming growth factor-β-activated kinase 1 enhances H2O2-induced apoptosis independently of reactive oxygen species in cardiomyocytes

Heat shock protein 70 (HSP70) protects against cardiac diseases such as ischemia/reperfusion injury and myocardial infarction. However, the underlying mechanisms have not yet been fully characterized. In this study, we investigated the effects of reactive oxygen species (ROS) and transforming growth factor-β-activated kinase 1 (TAK1) on HSP70-regulated cardiomyocyte protection. Cultured cardiomyocytes of neonatal rats were transfected with HSP70, TAK1 or both of them before exposure to H2O2, and the ROS generation, p38 mitogen-activated protein kinase (p38) activity and apoptosis were examined. H2O2 significantly enhanced intracellular ROS generation and apoptosis as expected, and all these cellular events were greatly abolished by overexpression of HSP70. However, H2O2-induced increments in p38 phosphorylation and cardiac cell apoptosis were largely enhanced by TAK1 overexpression, whereas the similar transfection did not affect the ROS generation in the cardiomyocytes. Moreover, inhibition of H2O2-increased ROS generation, p38 phosphorylation, and cardiomyocytes apoptosis by overexpression of HSP70 tended to disappear when the cells were cotransfected with TAK1. Our data suggest that HSP70 protects cardiomyocytes from apoptosis under oxidative stress through downregulation of intracellular ROS generation and inhibition of p38 phosphorylation. Although TAK1 itself has no effect on intracellular ROS accumulation, it may affect the inhibitory effects of HSP70 on ROS generation, p38 activity and cardiomyocyte injury.

Adrenergic Signaling Regulates Mitochondrial Ca2+ Uptake Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter

Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells.

Effect of glutahione S-transferases on cardiac microvascular endothelial cells

Objective To detect the effect of glutahione S-transferases (GSTP1) expression on the migration,proliferation and apoptosis of cardiac microvascular endothelial cells (CMECs).Methods After obtaining CMECs from SD rats by enzymatic digestion method,the special plasmid of GSTP was transfected.The ability of migration,proliferation and apoptosis of CMECs were examined by using Transwell assay,methyl thiazol tetrazolium (MTT) assay and flow cytometry,respectively.Results The ability of migration of CMECs was significantly reduced in GSTP1 over-expression group as compared with control group (9.0 ±2.7 vs.12.8 ±2.5,P ＜0.05),and the ability of proliferation of CMECs was also obviously reduced in comparison with control group (0.26 ± 0.02 vs.0.05 ± 0.01,P ＜ 0.05).But the apoptotic index was increased significantly (P ＜ 0.05).Conclusion GSTP1 expression can effectively inhibit migration and proliferation,and promote apoptosis of CMECs. Key words: Glutahione S-transferases; Cardiac microvascular endothelial cells; Migration; Proliferation

Haemodynamic unloading increases the survival and affects the differentiation of cardiac stem cells after implantation into an infarcted heart

It has been anticipated that stem cell therapy is capable of repairing an injured heart but is currently limited by its marginal efficacy. We believe that mechanical stress due to haemodynamic loading may negate the therapeutic potency of stem cells and therefore investigated how haemodynamic unloading affects the survival and differentiation of stem cells after implantation into an infarcted heart. A left ventricular (LV) haemodynamic unloading model was implemented by heterotopic transplantation of an infarcted donor heart into another healthy mouse. An in situ infarcted heart with general haemodynamic loading was used as control. A total of 5 million cardiac stem cells expanded from green fluorescence protein (GFP)-transgenic mouse were intramyocardially implanted into the infarcted LVs of haemodynamically unloaded donor heart or general haemodynamic loaded heart. The survival and differentiation of the implanted cardiac stem cells were evaluated by histological analyses at 3 and 21 days after cell implantation (n = 5-6 in each time points per group). Compared with the general haemodynamic loading condition, haemodynamic unloading of the infarcted hearts significantly improved the survival, increased the proliferation and inhibited the apoptosis of cardiac stem cells at 21 days after cell implantation (P < 0.05). In addition, the number of GFP(+)/Sca-1(+) cells was much higher in the unloaded hearts than in the loaded hearts at 21 days after cell implantation, although the difference was not statistically significant (5.67 ± 5.10 vs 0.75 ± 0.50, P = 0.051). Among the surviving GFP(+) donor cells 21 days after implantation, the expressions of platelet endothelial cell adhesion molecule-1, smooth muscle actin and sarcomeric alpha actin were ~7, 38 and 27% in the loaded heart and ~19, 14 and 55% in the unloaded heart, respectively. Haemodynamic unloading favours the survival/engraftment of donor stem cells and affects their differentiation after implantation into an infarcted heart. Although further studies in a large animal model are required to investigate the functional benefits of haemodynamic unloading on stem cell therapy, we may temporarily unload the damaged heart to enhance cell engraftment and then load the heart again to induce the differentiation of stem cells.

Animal Models in Studies of Cardiotoxicity Side Effects from Antiblastic Drugs in Patients and Occupational Exposed Workers

Cardiotoxicity is an important side effect of cytotoxic drugs and may be a risk factor of long-term morbidity for both patients during therapy and also for staff exposed during the phases of manipulation of antiblastic drugs. The mechanism of cardiotoxicity studied in vitro and in vivo essentially concerns the formation of free radicals leading to oxidative stress, with apoptosis of cardiac cells or immunologic reactions, but other mechanisms may play a role in antiblastic-induced cardiotoxicity. Actually, some new cytotoxic drugs like trastuzumab and cyclopentenyl cytosine show cardiotoxic effects. In this report we discuss the different mechanisms of cardiotoxicity induced by antiblastic drugs assessed using animal models.

Correction: FoxO3a Modulates Hypoxia Stress Induced Oxidative Stress and Apoptosis in Cardiac Microvascular Endothelial Cells

[This corrects the article DOI: 10.1371/journal.pone.0080342.].

Abstract 18531: Adrenergic Stimulation Enhances Mitochondrial Ca 2 + Uptake and Cell Death Signaling Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca 2 + Uniporter

Introduction: Mitochondrial Ca 2+ homeostasis is crucial for balancing cell survival and death. The recent breakthrough discovery of the molecular identity of the mitochondrial Ca 2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study the role of mitochondrial Ca 2+ regulation in cardiac cells. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca 2+ entry through post-translational modifications of MCU are completely unknown. Hypothesis: Persistent adrenergic signaling induces mitochondrial Ca 2+ overload and activates cell death signaling through MCU tyrosine phosphorylation. Methods: Tyrosine kinase activation and tyrosine phosphorylation of MCU were observed in rat neonatal cardiomyocytes and H9C2 cardiac myoblasts. Mitochondrial Ca 2+ and reactive oxygen species (ROS) were measured using mitochondrial matrix-targeted Ca 2+ -sensitive inverse pericam and MitoSOX, respectively. Mitochondrial permeability transition pore (mPTP) activity was observed by monitoring the release of GFP-tagged mitochondrial protein, Smac-GFP, using confocal microscopy. Results: α 1 -adrenoceptor (α 1 -AR) signaling activates mitochondria-localized Ca 2+ -dependent tyrosine kinase named proline-rich tyrosine kinase 2 (Pyk2) and accelerates mitochondrial Ca 2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, persistent α 1 -AR stimulation increases ROS production, mPTP activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca 2+ overload. Moreover, pretreatment of a potent Pyk2 inhibitor PF-431396 or overexpression of dominant-negative mutant of MCU effectively inhibited α 1 -AR-mediated ROS generation and mPTP activation. Conclusion: The α 1 -AR-Pyk2 dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca 2+ entry and apoptosis in cardiac cells. Pyk2-MCU signaling may become novel potent therapeutic targets for preventing mitochondrial Ca 2+ overload, oxidative stress and cardiac injury under pathophysiological states such as heart failure, where catecholamine levels highly increase.

FoxO3a Modulates Hypoxia Stress Induced Oxidative Stress and Apoptosis in Cardiac Microvascular Endothelial Cells

Cardiac microvascular endothelial cells (CMECs) dysfunction induced by hypoxia is an important pathophysiological event in myocardium ischemic injury, whereas, the underlying mechanism is not fully clarified. FoxO transcription factors regulate target genes involved in apoptosis and cellular reactive oxygen species (ROS) production. Therefore, the present study was designed to elucidate the potential role of FoxOs on the hypoxia-induced ROS formation and apoptosis in CMECs. Exposure to low oxygen tension stimulated ROS accumulation and increased apoptosis in CMECs within 6–24 h. Hypoxia also significantly increased the expressions of HIF-1α and FoxO3a. However, hypoxia decreased the phosphorylation of Akt and FoxO3a, correlated with increased nuclear accumulation. Conversely, the expression of FoxO1 was not significantly altered by hypoxia. After inhibition of HIF-1α by siRNA, we observed that hypoxia-induced ROS accumulation and apoptosis of CMECs were decreased. Meanwhile, knockdown of HIF-1α also inhibited hypoxia induced FoxO3a expression in CMECs, but did not affect FoxO1 expression. Furthermore, hypoxia-induced ROS formation and apoptosis in CMECs were correlated with the disturbance of Bcl-2 family proteins, which were abolished by FoxO3a silencing with siRNA. In conclusion, our data provide evidence that FoxO3a leads to ROS accumulation in CMECs, and in parallel, induces the disturbance of Bcl-2 family proteins which results in apoptosis.

High Glucose Induced Oxidative Stress and Apoptosis in Cardiac Microvascular Endothelial Cells Are Regulated by FoxO3a

AimCardiac microvascular endothelial cells (CMECs) dysfunction contributes to cardiovascular complications in diabetes, whereas, the underlying mechanism is not fully clarified. FoxO transcription factors are involved in apoptosis and reactive oxygen species (ROS) production. Therefore, the present study was designed to elucidate the potential role of FoxO3a on the CMECs injury induced by high glucose.Materials and MethodsCMECs were isolated from hearts of adult rats and cultured in normal or high glucose medium for 6 h, 12 h and 24 h respectively. To down-regulate FoxO3a expression, CMECs were transfected with FoxO3a siRNA. ROS accumulation and apoptosis in CMECs were assessed by dihydroethidine (DHE) staining and TUNEL assay respectively. Moreover, the expressions of Akt, FoxO3a, Bim and BclxL in CMECs were assessed by Western blotting assay.ResultsROS accumulation in CMECs was significantly increased after high glucose incubation for 6 to 24 h. Meanwhile, high glucose also increased apoptosis in CMECs, correlated with decreased the phosphorylation expressions of Akt and FoxO3a. Moreover, high glucose incubation increased the expression of Bim, whereas increased anti-apoptotic protein BclxL. Furthermore, siRNA target FoxO3a silencing enhanced the ROS accumulation, whereas suppressed apoptosis in CMECs. FoxO3a silencing also abolished the disturbance of Bcl-2 proteins induced by high glucose in CMECs.ConclusionOur data provide evidence that high glucose induced FoxO3a activation which suppressed ROS accumulation, and in parallel, resulted in apoptosis of CMECs.

MicroRNA‐214 Protects Cardiac Myocytes Against H2O2‐Induced Injury

Reactive oxygen species (ROS)-induced cardiac myocyte injury resulting from changes in the expression levels of multiple genes plays a critical role in the pathogenesis of numerous heart diseases. The purpose of this study was to determine the potential roles of microRNA-214 (miR-214) in hydrogen peroxide (H2O2)-mediated gene regulation in cardiac myocytes. In this study, we used quantitative real-time RT-PCR (qRT-PCR) to demonstrate that miR-214 was upregulated in cardiac myocytes after treatment with H2O2. We transfected cells with pre-miR-214 to upregulate miR-214 expression and transfected cells with a miR-214 inhibitor (anti-miR-214) to downregulate miR-214 expression. H2O2-induced cardiac cell apoptosis was detected by flow cytometry. The level of apoptosis was increased by the miR-214 inhibitor and decreased by pre-miR-214. Therefore, we believe that miR-214 plays a positive role in H2O2-induced cardiac cell apoptosis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is constitutively active and is considered to be the primary downregulator of the pro-oncogenic PI3K/Akt pathway. Western blot analysis revealed that the expression of the PTEN protein in cardiac myocytes decreased after H2O2 induction. Anti-miR-214 increased PTEN protein expression level, in contrast, pre-miR-214 decreased the PTEN protein expression level in cultured cardiac myocytes. These results indicate that PTEN is regulated by miR-214 and serves as an important target of miR-214 in cardiac myocytes. In conclusion, miR-214 is sensitive to H2O2 stimulation, and miR-214 protects cardiac myocytes against H2O2-induced injury via one of its targets, PTEN.

MicroRNA-150 aggravates H&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;O&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;-induced cardiac myocyte injury by down-regulating &amp;lt;italic&amp;gt;c-myb&amp;lt;/italic&amp;gt; gene

MicroRNAs (miRNAs) are one class of non-coding RNAs that play an important role in post-transcriptional regulation via the degradation or translational inhibition of their target genes. MicroRNA-150 (miR-150) plays a vital role in regulating the development of B and T lymphocytes. Although the dysregulation of miR-150 was confirmed in human myocardial infarction, little is known regarding the biological functions of miR-150 in response to reactive oxygen species (ROS)-mediated gene regulation in cardiac myocytes. Using quantitative real-time reverse transcription-polymerase chain reaction, we demonstrated that the level of miR-150 was up-regulated in cardiac myocytes after treatment with hydrogen peroxide (H2O2). To identify the potential roles of miR-150 in H2O2-mediated gene regulation, we modulated expression of miR-150 using miR-150 inhibitor and miR-150 mimics. Results showed that silencing expression of miR-150 decreased H2O2-induced cardiac cell death and apoptosis. In lymphocytes, c-myb was a direct target of miR-150. In cardiac myocytes, we found that c-myb was also involved in miR-150-mediated H2O2-induced cardiac cell death. These results suggested that miR-150 participates in H2O2-mediated gene regulation and functional modulation in cardiac myocytes. MiR-150 may play an essential role in heart diseases related to ROS, such as cardiac hypertrophy, heart failure, myocardial infarction, and myocardial ischemia/reperfusion injury.

Abstract 060: CCN1/a 6 β 1 Mediates Myocardial Injuries Induced by Work Overload or by Doxorubicin in Mice

Introduction: Matricellular protein CCN1 is expressed in myocardial infarction, pressure overload, and ischemia in mice, and in patients with a failing heart. Despite its well-documented angiogenic activities, CCN1 promotes fibroblast apoptosis in some contexts. The role of CCN1 in an injured heart was not clear. We assessed the hypothesis that CCN1 plays a detrimental role and mediates cardiac injury through its proapoptotic activities. Methods and Results: To test the role of CCN1 in cardiac injury, we employed two different myocardial injury models in mice, including a work-overload-induced injury created by isoproterenol treatment (ISO; 100 mg/kg/day; s.c. for 5 days; n= 6 for each group) and an injury induced by the cardiotoxicity of doxorubicin (DOX, single dose of 15 mg/kg; i.p. sacrificed after 14 days). Ccn1 expression was induced in the damaged myocardium in both injury models. A line of knock-in mice carrying an apoptosis-defective Ccn1 mutant allele, Ccn1-dm , which has disrupted integrin α 6 β 1 binding sites, were tested in the ISO- or DOX -induced cardiac injury. Myocardial damage was seen in tissues from wile-type (WT) hearts after receiving ISO. Ccn1 dm/dm (DM) mice possessed remarkable resistance against ISO or DOX treatments and exhibited no tissue damage or fibrosis compared to WT mice after H&amp;E or Masson’s trichrome stainings. DM mice were resistant to both ISO- and DOX-induced cardiac cell apoptosis, indicating that CCN1 is critically mediating cardiomyocyte apoptotic death in cardiac injury. Moreover, we found that death factor Fas ligand (FasL) and its receptor Fas were upregulated in WT mice receiving ISO or DOX treatments by immunohistochemical staining, compared with the PBS-control. 8-OHdG-positive, a marker for oxidative stress, cardiomyocytes were increased by ISO or DOX treatments as well. In contrast, the expression of Fas/FasL, and the 8-OHdG-positive cardiomyocytes in the myocardium of DM mice were not changed by ISO or DOX. Conclusions: We identify CCN1 as a novel pathophysiological regulator of cardiomyocyte apoptosis in cardiac injury. Blocking apoptotic function of CCN1 effectively prevents myocardial injury in mice. CCN1 and its receptor α 6 β 1 represent promising future therapeutic targets in cardiac injury.

Synthetic liver X receptor agonist T0901317 attenuates high glucose-induced oxidative stress, mitochondrial damage and apoptosis in cardiomyocytes

The aim of the present study was to investigate the protective effects of T0901317 (T0), a potent agonist of liver X receptors (LXRs), on high glucose-induced oxidative stress and apoptosis in H9c2 cardiac cells. Exposure of H9c2 cells to high glucose alone, not only caused a significant increase in apoptosis and reactive oxygen species (ROS) generation, but also led to a decrease in mitochondrial membrane potential (ΔΨm), release of cytochrome c, decrease in Bcl-2, increase in Bax expression and the activation of caspase-3, caspase-9, poly (ADP-ribose) polymerase (PARP) and nuclear factor (NF)-κB. However, pretreatment with T0 effectively decreased apoptosis, reduced the levels of ROS, abrogated ΔΨm, inhibited cytochrome c release and NF-κB activation, increased Bcl-2 and decreased Bax expression. In conclusion, our data suggest that T0 exerts protective effects against high glucose-induced apoptosis in H9C2 cardiac muscle cells via inhibition of ROS production, mitochondrial death and NF-κB activation.