Apoptosis In Neonatal Cardiomyocytes Research Articles

Buyang Huanwu decoction inhibits cardiomyocyte apoptosis after myocardial infarction by enhancing aldehyde dehydrogenase-2 activity and protein expression

ObjectiveTo investigate the potential mechanisms of the protective effects of Buyang Huanwu decoction (BYHWD) on cardiomyocyte apoptosis after myocardial infarction (MI) and to provide the foundation for the clinical application of BYHWD in preventing and treating cardiomyocyte apoptosis-related disorders after MI. MethodsThe MI rat model was established by ligation of the left anterior descending (LAD) coronary artery. After the 20-week treatment with BYHWD, the following parameters were assessed to investigate the protective effects of BYHWD on cardiomyocyte apoptosis after MI, both in vivo and in vitro: mortality rate; heart weight/body weight (HW/BW); transthoracic echocardiography measurements; histopathological analysis; immunohistochemistry (IHC) analysis; TUNEL apoptosis assay; caspase-3 activity assay; aldehyde dehydrogenase-2 (ALDH2) enzymatic activity assay; malondialdehyde (MDA) content analysis; western blot analysis; MTT assay; and Hoechst 33342/PI apoptosis assay. ResultsThe mortality of the MI rats was significantly reduced by BYHWD treatment. Furthermore, BYHWD treatment significantly improved the heart function and significantly reduced the HW/BW ratio. Histopathological analysis showed that BYHWD treatment significantly relieved MI-induced cardiac injury and significantly decreased collagen formation. The TUNEL apoptosis assay showed that treatment with BYHWD reduced the cardiomyocyte apoptosis of MI rats in a dose-dependent manner. Moreover, BYHWD treatment could significantly inhibit oxidative stress-induced neonatal cardiomyocyte apoptosis, which is consistent with the results of animal experiments in vivo. Interestingly, the ALDH2 activity and protein expression was significantly reduced in the MI model group, while the content of toxic aldehydes, including 4-HNE and MDA, was increased. Importantly, BYHWD enhanced the activity of ALDH2 and the expression of ALDH2 protein and attenuated the content of toxic aldehydes in a dose-dependent manner. Furthermore, we found that co-treatment with an ALDH2 inhibitor, disulfiram, partially inhibited the cardiomyocyte protective effects of BYHWD. ConclusionsCollectively, our results suggest that BYHWD exerted anti-apoptotic effects in cardiomyocytes after MI by enhancing the ALDH2 activity and protein expression and decreasing the toxic aldehyde content.

Calycosin protects against oxidative stress-induced cardiomyocyte apoptosis by activating aldehyde dehydrogenase 2.

Myocardial infarction (MI) is the leading cause of death worldwide, and oxidative stress is part of the process that causes MI. Calycosin, a naturally occurring substance with cardioprotective properties, is one of the major active constituents in Radix Astragali. In this study, effect of Calycosin was investigated in vivo and in vitro to determine whether it could alleviate oxidative stress and oxidative stress-induced cardiac apoptosis in neonatal cardiomyocytes (NCMs) via activation of aldehyde dehydrogenase 2 (ALDH2). Calycosin protected against oxidative stress and oxidative stress-induced apoptosis in NCMs. Molecular docking revealed that the ALDH2-Calycosin complex had a binding energy of -9.885 kcal/mol. In addition, molecular docking simulations demonstrated that the ALDH2-Calycosin complex was stable. Using BLI assays, we confirmed that Calycosin could interact with ALDH2 (KD =1.9 × 10-4 M). Furthermore, an ALDH2 kinase activity test revealed that Calycosin increased ALDH2 activity, exhibiting an EC50 of 91.79 μM. Pre-incubation with ALDH2 inhibitor (CVT-10216 or disulfiram) reduced the cardio-protective properties Calycosin. In mice with MI, Calycosin therapy substantially reduced myocardial apoptosis, oxidative stress, and activated ALDH2. Collectively, our findings clearly suggest that Calycosin reduces oxidative stress and oxidative stress-induced apoptosis via the regulation of ALDH2 signaling, which supports potential therapeutic use in MI.

RNA-binding protein CELF1 promotes cardiac hypertrophy via interaction with PEBP1 in cardiomyocytes.

Cardiac hypertrophy is considered as a common pathophysiological process in various cardiovascular diseases. CUG triplet repeat-binding protein 1 (CELF1) is an RNA-binding protein that has been shown to be an important post-transcription regulator and involved in several types of cancer, whereas its role in cardiac remodeling remains unclear. Herein, we found that the expression of CELF1 was significantly increased in pressure overload-induced hypertrophic hearts and angiotensin II (Ang II)-induced neonatal cardiomyocytes. Based on transverse aortic constriction-induced cardiac hypertrophy model, CELF1 deficiency markedly ameliorated cardiac hypertrophy, cardiac fibrosis, oxidative stress, and apoptosis. Accordingly, CELF1 deficiency alleviated the production of reactive oxygen species (ROS) and apoptosis of neonatal cardiomyocytes via inhibition of Raf1, TAK1, ERK1/2, and p38 phosphorylation. Mechanistically, depletion or overexpression of CELF1 negatively regulated the protein expression of phosphatidylethanolamine-binding protein 1 (PEBP1), while the mRNA expression of PEBP1 remained unchanged. RNA immunoprecipitation revealed that CELF1 directly interacted with PEBP1 mRNA. Biotin pull-down analysis and dual-luciferase assay showed that CELF1 directly bound to the fragment 1 within 3'UTR of PEBP1. Moreover, knockdown of PEBP1 partially enhanced the production of ROS and apoptosis of neonatal cardiomyocytes inhibited by CELF1 deficiency. In conclusion, CELF1 binds to the 3'UTR of PEBP1 and acts as an endogenous activator of MAPK signaling pathway. Inhibition of CELF1 attenuates pathological cardiac hypertrophy, oxidative stress, and apoptosis, thus could be a potential therapeutic strategy of pathological cardiac hypertrophy.

Tom70 protects against diabetic cardiomyopathy through its antioxidant and antiapoptotic properties.

Mitochondrial dysfunction plays a critical role in the pathogenesis of diabetic cardiomyopathy. Translocase of mitochondrial outer membrane 70 (Tom70) primarily facilitates the import of mitochondrial preproteins that may be involved in the regulation of oxidative stress and mitochondrial function. This study aimed to investigate the role of Tom70 in the development of myocardial injury in leptin receptor-deficient (db/db) diabetic mice. Tom70 siRNA or an overexpressing lentivirus was intramuscularly injected into mouse hearts or used to treat cultured neonatal cardiomyocytes. We found that Tom70 was downregulated in the diabetic hearts compared with the level in the wild-type hearts and that knocking down Tom70 exacerbated cardiac hypertrophy, fibrosis, and ventricular dysfunction in the db/db mice. Similarly, the in vitro data demonstrated that silencing Tom70 enhanced high-glucose and high-fat (HGHF) medium treatment-induced mitochondrial superoxide production, decreased ATP production and the mitochondrial membrane potential, and enhanced cell apoptosis in neonatal cardiomyocytes. Importantly, overexpression of Tom70 alleviated HGHF medium-induced oxidative stress, mitochondrial dysfunction, and cell apoptosis. Furthermore, in vivo data confirmed that reconstitution of Tom70 ameliorated cardiac hypertrophy, interstitial fibrosis, and ventricular dysfunction in the db/db mice. In addition, Tom70 overexpression mitigated mitochondrial fragmentation and dysfunction in the hearts of the db/db mice. Taken together, these findings suggest that downregulation of Tom70 contributes to the development of diabetic cardiomyopathy and that reconstitution of Tom70 may be a new therapeutic strategy for the prevention and treatment of diabetic cardiomyopathy.

Specific ablation of CD4+ T-cells promotes heart regeneration in juvenile mice.

Unlike adult cardiomyocytes, neonatal cardiomyocytes can readily proliferate that contributes to a transient regenerative potential after myocardial injury in mice. We have recently reported that CD4+ regulatory T-cells promote this process; however, the role of other CD4+ T-cell subsets as well as CD8+ T-cells in postnatal heart regeneration has been less studied. Methods: by comparing the regenerating postnatal day (P) 3 and the non-regenerating P8 heart after injury, we revealed the heterogeneity of CD4+ and CD8+ T-cells in the myocardium through single cell analysis. We also specifically ablated CD4+ and CD8+ T-cells using the lytic anti-CD4 and -CD8 monoclonal antibodies, respectively, in juvenile mice at P8 after myocardial injury. Results: we observe significantly more CD4+FOXP3- conventional T-cells in the P8 heart when compared to that of the P3 heart within a week after injury. Surprisingly, such a difference is not seen in CD8+ T-cells that appear to have no function as their depletion does not reactivate heart regeneration. On the other hand, specific ablation of CD4+ T-cells contributes to mitigated cardiac fibrosis and increased cardiomyocyte proliferation after injury in juvenile mice. Single-cell transcriptomic profiling reveals a pro-fibrotic CD4+ T-cell subset in the P8 but not P3 heart. Moreover, there are likely more Th1 and Th17 cells in the P8 than P3 heart. We further demonstrate that cytokines of Th1 and Th17 cells can directly reduce the proliferation and increase the apoptosis of neonatal cardiomyocytes. Moreover, ablation of CD4+ T-cells can directly or indirectly facilitate the polarization of macrophages away from the pro-fibrotic M2-like signature in the juvenile heart. Nevertheless, ablation of CD4+ T-cells alone does not offer the same protection in the adult heart after myocardial infarction, suggesting a developmental change of immune cells including CD4+ T-cells in the regulation of age-related mammalian heart repair. Conclusions: our results demonstrate that ablation of CD4+ but not CD8+ T-cells promotes heart regeneration in juvenile mice; and CD4+ T-cells play a distinct role in the regulation of heart regeneration and repair during development.

Increased right atrial appendage apoptosis is associated with differential regulation of candidate MicroRNAs 1 and 133A in patients who developed atrial fibrillation after cardiac surgery

Atrial fibrillation (AF) following on-pump coronary artery bypass grafting (CABG) is a common condition associated with increased morbidity and mortality. We investigated the possibility that miRs may play a contributory role in postoperative AF and associated apoptosis. A total of 42 patients (31 males and 11 females, mean age 65.0 ± 1.3 years) with sinus rhythm and without a history of AF were prospectively enrolled. We examined the levels of the muscle-specific miRs 1 and 133A and markers of apoptosis including TUNEL staining, caspase-3 activation, Bcl2 and Bax mRNAs in right atrial appendage (RAA) biopsies and blood plasma taken before aortic cross-clamping and after reperfusion. After reperfusion, indices of apoptosis increased the RAA. There was no change in tissue or plasma miR −1 and -133A levels compared to pre CABG. However, in patients who postoperatively developed AF (n = 14, 7 males and 7 females), compared to patients that remained in SR (n = 28, 24 males and 4 females) post CABG, tissue miR-1 increased whereas miR-133A decreased and negatively correlated with RAA apoptosis. Mechanistically, overexpression of miR-133A inhibited hypoxia-induced rat neonatal cardiomyocyte apoptosis and phosphorylated pro-survival Akt, responses abolished by a miR-133A antisense inhibitor oligonucleotide or by pre-treatment with an Akt inhibitor. In postoperative AF, differential regulation of pro- and anti-apoptotic miRs-1 and -133A respectively in the RAA, may contribute to postoperative apoptosis. These results provide new insights into molecular mechanisms of postoperative AF with potential therapeutic implications.

The long non-coding RNA H19 promotes cardiomyocyte apoptosis in dilated cardiomyopathy.

In the previous study, we generated a rat model of dilated cardiomyopathy (DCM) induced by adriamycin and found that the expression of lncRNA H19 was significantly upregulated in myocardial tissue. The present study was aimed to investigate the potential role of H19 in the pathogenesis of adriamycin-induced DCM. H19 knockdown in the myocardium of DCM rats attenuated cardiomyocyte apoptosis and improved left ventricular structure and function. Adriamycin treatment was associated with elevated H19 and miR-675 expression and increased apoptosis in neonatal cardiomyocytes. Enforced expression of miR-675 was found to induce apoptosis in cardiomyocytes with adriamycin treatment and H19-siRNA transfection. The 3′-untranslated region of PA2G4 was cloned downstream of a luciferase reporter construct and cotransfected into HEK293 cells with miR-675 mimic. The results of luciferase assay showed that PA2G4 was a direct target of miR-675. The expression of PA2G4 was reduced in cardiomyocytes transfected with miR-675 mimic. Moreover, H19 knockdown was found to increase PA2G4 expression and suppress apoptosis in cardiomyocytes exposed to adriamycin. In conclusion, our study suggests that H19/miR-675 axis is involved in the promotion of cardiomyocyte apoptosis by targeting PA2G4, which may provide a new therapeutic strategy for the treatment of adriamycin-induced DCM.

Antiphospholipid antibodies enhance rat neonatal cardiomyocyte apoptosis in an in vitro hypoxia/reoxygenation injury model via p38 MAPK

A significant amount of myocardial damage during a myocardial infarction (MI) occurs during the reperfusion stage, termed ischaemia/reperfusion (I/R) injury, and accounts for up to 50% of total infarcted tissue post-MI. During the reperfusion phase, a complex interplay of multiple pathways and mechanisms is activated, which ultimately leads to cell death, primarily through apoptosis. There is some evidence from a lupus mouse model that lupus IgG, specifically the antiphospholipid (aPL) antibody subset, is pathogenic in mesenteric I/R injury. Furthermore, it has previously been shown that the immunodominant epitope for the majority of circulating pathogenic aPLs resides in the N-terminal domain I (DI) of beta-2 glycoprotein I (β2GPI). This study describes the enhanced pathogenic effect of purified IgG derived from patients with lupus and/or the antiphospholipid syndrome in a cardiomyocyte H/R in vitro model. Furthermore, we have demonstrated a pathogenic role for aPL containing samples, mediated via aPL–β2GPI interactions, resulting in activation of the pro-apoptotic p38 MAPK pathway. This was shown to be inhibited using a recombinant human peptide of domain I of β2GPI in the fluid phase, suggesting that the pathogenic anti-β2GPI antibodies in this in vitro model target this domain.

Rap1-mediated nuclear factor-kappaB (NF-κB) activity regulates the paracrine capacity of mesenchymal stem cells in heart repair following infarction.

Paracrine effect is the major mechanism that underlies mesenchymal stem cells (MSC)-based therapy. This study aimed to examine how Rap1, telomeric repeat-binding factor 2-interacting protein 1 (Terf2IP), which is a novel modulator involved in the nuclear factor-kappaB (NF-κB) pathway, regulates the paracrine effects of MSC-mediated heart repair following infarction. NF-κB activity of stromal cells was increased by Rap1 as measured by pNF-κB-luciferase reporter activity, and this was abolished by IkB-dominant-negative protein. Knockdown of Rap1 with shRap1 resulted in diminished translocation of p65-NF-κB from the cytoplasm to nuclei in response to tumor necrosis factor-α (TNF-α) stimulation. Compared with BM-MSCs, Rap1−/−-BM-MSCs displayed a significantly reduced ratio of phosphorylated NF-κB to NF-κB-p65 and of Bax to Bcl-2, and increased resistance to hypoxia-induced apoptosis by the terminal deoxynucleotidal transferase-mediated dUTP nick end labeling (TUNEL) assay. In contrast, re-expression of Rap1 in Rap1−/−-BM-MSCs resulted in loss of resistance to apoptosis in the presence of hypoxia. Moreover, absence of Rap1 in BM-MSCs led to downregulation of NF-κB activity accompanied by reduced pro-inflammatory paracrine cytokines TNF-α, IL (interleukin)-6 and monocyte chemotactic protein-1 in Rap1−/−-BM-MSCs compared with BM-MSCs. The apoptosis of neonatal cardiomyocytes (NCMCs) induced by hypoxia was significantly reduced when cocultured with Rap1−/−-BM-MSC hypoxic-conditioned medium (CdM). The increased cardioprotective effects of Rap1−/−-BM-MSCs were reduced when Rap1−/−-BM-MSCs were reconstituted with Rap1 re-expression. Furthermore, in vivo study showed that transplantation of Rap1−/−-BM-MSCs significantly improved heart function, decreased infarct size, prevented cardiomyocyte apoptosis and inhibited inflammation compared with controls and BM-MSCs (P<0.01). This study reveals that Rap1 has a critical role in the regulation of MSC paracrine actions. Compared with BM-MSCs, Rap1−/−-BM-MSCs decreased NF-κB sensitivity to stress-induced pro-inflammatory cytokine production and reduced apoptosis. Selective inhibition of Rap1 in BM-MSCs may be a novel strategy to enhance MSC-based therapeutic efficacy in myocardial infarction.

Abstract 175: Pro-survival Function of Mef2 in Cardiomyocytes is Enhanced by β-blockers

β1-adrenergic receptor (β1-AR) stimulation increases apoptosis in cardiomyocytes via activation of cAMP/ protein kinase A (PKA) signaling. β-adrenergic receptor antagonists, or β-blockers, oppose the action of PKA signaling by blocking the β1-receptor and effectively inhibit apoptosis and heart failure. The Myocyte Enhancer Factor 2 (MEF2) proteins have been implicated as nuclear targets for signaling cascades involved in muscle-gene expression and have important roles in proliferation, apoptosis and survival in multiple cell types. We previously reported that PKA signaling represses MEF2 activity. Here, we assessed whether β-blockers can inhibit neonatal cardiac myocyte apoptosis by interfering with PKA dependent MEF2 repression. We show that siRNA mediated MEF2 loss of function induced cardiomyocyte apoptosis. β1AR activation by isoproterenol treatment represses MEF2 transcriptional activity and promotes apoptosis in neonatal cardiomyocytes and, importantly, this effect was reversed in cells expressing a PKA resistant form of MEF2D (S121/190A), as indicated by FACS analysis. We also report that a β-blocker, Atenolol, antagonizes isoproterenol induced apoptosis and also acutely enhanced MEF2 transcriptional activity. We observed that β-adrenergic stimulation modulated MEF2 cellular localization in neonatal cardiomyocytes and this was reversed by atenolol treatment. In addition, we also document that Kru[[Unable to Display Character: &amp;#776;]]ppel-like factor 6 (KLF6) is an important MEF2 target gene and loss of function analysis using siRNA-mediated knockdown of KLF6 expression resulted in cardiomyocyte apoptosis. Collectively, these observations, establish that MEF2 plays an important pro-survival role in cardiomyocytes which can be modulated by β-adrenergic signaling. These observations have important clinical implications and may contribute to novel strategies for preventing cardiomyocyte apoptosis associated with heart pathology.

Вплив флокаліну на розвиток апоптозу та некрозу при аноксії–реоксигенації ізольованих неонатальних кардіоміоцитів

The influence of the new cardioprotector flocalin was investigated on the culture of rat's neonatal cardiomyocytes during anoxia-reoxygenation modelling. The mechanisms of apoptosis and necrosis were investigated under influence of ATP-sensitive potassium (K(ATP)) channels activation and in conditions of blocking of the L-type calcium (VGCCs) channels. Flocalin was added in the culture medium in the dose 5 and 20 microM at 2 minutes before anoxia (30 minutes) and following reoxygenation (60 minutes). These doses are near to: the first dose means the opening of K(ATP) channels and the second one means the IC50 block of VGCCs. It is discovered that in dose 5 microM of flocalin drew the change of correlation of living, necrotizing and apoptizing cells drew side-shifting living. The number of live cells was almost the same like in control (experiments without anoxia-reoxygenation modelling). The opening of K(ATP) channels decreases necrosis in two times and fully prevented development of apoptosis which was induced anoxia-reoxygenation modelling. Flocalin depressed the apoptosis of neonatal cardiomyocytes so that he was on to 36% less than in control group (without anoxia-reoxygenation). But in the high dose (20 microM) that provokes not only K(ATP) channels opening but also IC50 block of VGCCs cardioprotection was not detected after modelling of anoxia-reoxygenation. The last can be investigation both enough strong activating of the potassium channels and by investigation of both factors are opening of potassium and inhibition of VGCCs channels and, accordingly, substantial diminishing of level of introcellular Ca2+ and violation of metabolic processes yet to anoxia-reoxygenation. Thus, small doses of flocalin, that induce moderate opening of K(ATP) channels significantly decrease the number of necrotic and apoptotic cells in culture of rat neonatal cardiomyocytes induced by anoxia-reoxygenation.

Role of δ2 Opioid Receptor in Cardioprotection Against Hypoxia–Reoxygenation Injury

Several lines of in vivo evidence demonstrated that activation of δ-opioid receptors (ORs) with agonists mimics the cardioprotective effect of ischemic preconditioning. However, the subtypes of ORs involved and the molecular and cellular mechanisms are not entirely clear. To investigate the significance of the contribution by δ ORs to cardiomyocyte survival, we used an in vitro model of hypoxia/reoxygenation (H/R) in primary cultures of neonatal rat cardiomyocytes to study the role of different δ ORs in cardiomyocyte apoptosis and the relevant downstream signaling pathway. The results showed that apoptosis in neonatal cardiomyocytes induced by H/R was reversed by δ2 OR agonist, deltorphin E but not by δ1 OR agonist DPDPE; the deltorphin E-induced cytoprotection was totally abrogated by the MEK inhibitor PD98059 and overexpression of dominant interfering form of MEK1; in contrast, overexpression of constitutive active form of MEK1 exerted a similar protective effect as deltorphin E. These results suggest that δ2 OR, but not δ1 OR, plays a key role in preventing cardiomyocytes from apoptosis during H/R injury, which is mainly mediated by the MEK/ERK1/2 pathway.

Effects of bpV(pic) and bpV(phen) on H9c2 cardiomyoblasts during both hypoxia/reoxygenation and H2O2-induced injuries

Reactive oxygen species (ROS) are involved in myocardial injury. ROS are known to inactivate lipid phosphatase and tension homolog on chromosome 10 (PTEN), an enzyme that increases apoptosis in neonatal cardiomyocytes. BpV(pic) and bpV(phen), two bisperoxovanadium molecules and PTEN inhibitors, may be involved in limiting myocardial infarction. To compare the protective effects of bpV(pic) and bpV(phen) on ROS-induced cardiomyocyte injury and their possible mechanisms, we selected two popular models of hypoxia/reoxygenation (H/R) and H2O2-induced injury in H9c2 cardiomyoblasts to investigate their effects against injury. We found that pre-treatment with bpV(pic) and bpV(phen) increased the viability and protected the morphology of H9c2 cells under the conditions of H/R and H2O2 by inhibiting LDH release, apoptosis and caspases 3/8/9 activities. However, their respective inhibitory abilities in the two models were different, suggesting that the quantity of ROS from the two models might be different. However, the conflict between ROS and PTEN may affect the action of bpV(pic) and bpV(phen). Taken together, the results demonstrate that bpV(pic) and bpV(phen) have inhibitory effects on oxidative stress-induced cardiomyocyte injury that may be partially modulated by the action of ROS on PTEN.

Impact of N-Acetylcysteine on Neonatal Cardiomyocyte Ischemia-Reperfusion Injury

Reactive oxygen species (ROS) are hypothesized to play a key role in myocardial ischemia-reperfusion (IR) injury after cardiopulmonary bypass in children. Clinical studies in adults and several animal models suggest that myocardial IR injury involves cardiomyocyte apoptosis and necrosis. This study investigated a potential relationship between IR-induced ROS production and neonatal cardiomyocyte apoptosis using both in vitro and ex vivo techniques. For in vitro experiments, embryonic rat cardiomyocytes (H9c2 cells) exposed to hypoxia-reoxygenation (HR) showed a time-dependent increase in gp91 phox (a marker for ROS production by NADPH oxidases), caspase-3 (a key mediator of apoptosis) expression, and a decrease in the glutathione redox ratio. N-acetylcysteine (NAC; 0.25-2 mM), a potent antioxidant, decreased gp91 phox and caspase-3 expression, inhibited apoptosis and restored the glutathione redox ratio. For ex vivo study, IR injury significantly reduced left ventricular (LV) function and increased the expression of gp91 phox and caspase-3 in Langendorff-perfused neonatal (7-14 d) rabbit hearts. NAC (0.4 mM) treatment completely attenuated LV dysfunction after IR. In summary, neonatal myocardial IR injury is associated with an increase in cardiomyocyte oxidative stress and apoptosis. NAC attenuates apoptosis in an in vitro embryonic rat cardiomyocyte model of HR, and myocardial dysfunction in an ex vivo neonatal rabbit model of myocardial IR injury.

Rad GTPase induces cardiomyocyte apoptosis through the activation of p38 mitogen-activated protein kinase

Rad is a member of a subclass of small GTP-binding proteins, the RGK family. In the present study we investigated the role of Rad protein in regulating cardiomyocyte viability. DNA fragmentation and TUNEL assays demonstrated that Rad promoted rat neonatal cardiomyocyte apoptosis. Rad silencing fully blocked serum deprivation induced apoptosis, indicating Rad is necessary for trigger cardiomyocyte apoptosis. Rad overexpression caused a dramatic decrease of the anti-apoptotic molecule Bcl-xL, whereas Bcl-xL overexpression protected cardiomyocytes against Rad-induced apoptosis. Rad-triggered apoptosis was mediated by the activation of p38 MAPK. The p38 blocker SB203580 effectively protected cardiomyocytes against Rad-evoked apoptosis.

RNA Binding Protein QKI Inhibits the Ischemia/reperfusion-induced Apoptosis in Neonatal Cardiomyocytes

Backgrounds: RNA-binding protein QKI is abundantly expressed in the brain and heart. The role of QKI in the nervous system has been well characterized, but its function in cardiac muscle is still poorly understood. The present study was to investigate the role of QKI in ischemia/reperfusion-induced apoptosis in cardiomyocytes. Methods: A simulated ischemia/reperfusion model was established in neonatal cardiomyocytes and adult rat heart. After QKI5 or QKI6 was expressed by adenovirus and QKI was knocked down QKI by RNAi in the cardiomyocytes, RT-PCR, western blot and immunofluorescence staining were applied to detect gene expression alterations. Apoptosis was evaluated by PARP degradation, DNA fragmentation (DNA laddering) and flow cytometry. Results: Our study demonstrated that both QKI5 and QKI6 were present in cardiomyocytes, while QKI5 expression was greatly inhibited by simulated ischemia/reperfusion. Knocking down endogenous QKI by RNAi enhanced cell susceptibility to apoptosis, whereas overexpression of either QKI5 or QKI6 suppressed IR-induced apoptosis substantially. The pro-apoptotic transcription factor FoxO1, a potential QKI target, was induced by ischemia/reperfusion at both total amount and nuclear distribution. Accordingly, FOXO1 downstream target genes were negatively affected by the presence of QKI with IR treatment. Conclusion: In summary, our study supports that both QKI-5 and 6 are anti-apoptotic proteins in cardiomyocytes, favoring cardiac survival via antagonizing the elevation of some pro-apoptotic factors in cardiac injury.

Atrogin-1/MAFbx Enhances Simulated Ischemia/Reperfusion-induced Apoptosis in Cardiomyocytes through Degradation of MAPK Phosphatase-1 and Sustained JNK Activation

Atrogin-1/MAFbx is a major atrophy-related E3 ubiquitin ligase that is expressed specifically in striated muscle. Although the contribution of atrogin-1 to cardiac and muscle hypertrophy/atrophy has been examined extensively, it remains unclear whether atrogin-1 plays an essential role in the simulated ischemia/reperfusion-induced apoptosis of primary cardiomyocytes. Here we showed that atrogin-1 markedly enhanced ischemia/reperfusion-induced apoptosis in cardiomyocytes via activation of JNK signaling. Overexpression of atrogin-1 increased phosphorylation of JNK and c-Jun and decreased phosphorylation of Foxo3a. In addition, atrogin-1 decreased Bcl-2, increased Bax, and enhanced the activation of caspases. Furthermore, JNK inhibitor SP600125 markedly blocked the effect of atrogin-1 on cell apoptosis and the expression of apoptotic-related proteins and caspases. Importantly, atrogin-1 induced sustained activation of JNK through a mechanism that involved degradation of MAPK phosphatase-1 (MKP-1) protein. Atrogin-1 interacted with and triggered MKP-1 for ubiquitin-mediated degradation. In contrast, proteasome inhibitors markedly blocked the degradation of MKP-1. Taken together, these results demonstrate that atrogin-1 promotes degradation of MKP-1 through the ubiquitin-proteasome pathway, thereby leading to persistent activation of JNK signaling and further cardiomyocyte apoptosis following ischemia/reperfusion injury.

Abstract 3785: An Involvement of a Novel G-protein Activator on Hypoxia-Induced Apoptosis of Cardiomyocytes and its Interaction with Connexin 43

Recently, we identified a novel receptor-independent G-protein activator, Activator of G-protein signaling 8 (AGS8), from a cDNA library of rat hearts subjected to repetitive transient ischemia. AGS8 mRNA was induced in the ischemic myocardium and it regulated Gβγ signaling. In the face of myocardial ischemia and/or ischemia/reperfusion, AGS8 may be involved in signaling events associated with adaptation of the cardiomyocyte to ischemic events. As an initial step to address this issue, we examined the influence of AGS8 on survival of cultured cardiomyocyte following hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia for 6h, 24 h or hypoxia (6h)/reoxygenation (18h) following transfection of AGS8 siRNA. Apoptosis was determined by DNA end-labeling or detection of changes of mitochondrial membrane potential. Interestingly, silencing of AGS8 inhibited apoptosis of cardiomyocytes induced by hypoxia alone (57.8±3.4%, p&lt;0.05 vs. control-siRNA, at 24 h, mean±SEM) as well as hypoxia/reoxygenation (36.4±4.0%, p&lt;0.05 vs. control-siRNA, mean±SEM). Transfection of pcDNA::AGS8 by virus envelope increased apoptosis of NCM (151%±9.6%, p&lt;0.05 vs. vector group, mean±SEM) following hypoxia/reoxygenation, but not NCM cultured in normoxia. Affinity purified AGS8 antibody recognized immunoreactive signals enriched at the cell-cell interface of ventricular cardiomyocytes and NCM, regions also enriched in the gap junction protein connexin 43 (CX43). Indeed, AGS8 and CX43 co-immunoprecipitated from left ventricle lysates. The level of CX43 protein was decreased by overexpression of AGS8 in NCM, whereas suppression of AGS8 expression increased the level of CX43. Subsequent studies indicated that AGS8 stimulated phosphorylation of CX43 in a Gβγ dependent manner following transient expression in COS7 cells. As the level and/or phosphorylation of CX43 may influence apoptosis of cardiomyocytes, these studies suggest a potential mechanism by which AGS8 induction in the face of ischemia may provide a component of the signaling events involved in adaptation and suggest that targeted disruption of the AGS8 cascade may protect the myocardium from ischemic injury.

Protective effect of eicosapentaenoic acid on palmitate-induced apoptosis in neonatal cardiomyocytes

Long chain polyunsaturated fatty acids (PUFAs) play an important role in cardioprotection. These effects have been largely attributed to membrane docosahexaenoic acid. Conversely, saturated fatty acids trigger apoptosis in cardiomyocytes, with modifications of mitochondrial properties including cardiolipin loss, cytochrome c release and caspase-3 activation. The purpose of this study was to investigate the chronic effect of eicosapentaenoic acid (EPA) on mitochondrial apoptosis induced by palmitate treatment and the associated signalling pathways. Confluent cultures of rat neonatal cardiomyocytes were treated for 2 days in media enriched with either EPA or arachidonic acid (AA) and then exposed to palmitate (0.5 mM) to induce apoptosis, in the absence of PUFA supplements. The EPA treatment resulted in significant membrane enrichment in n − 3 PUFAs, especially in docosapentaenoic acid (DPA), and a large decrease in AA. Both AA and EPA treatments prevented caspase-3 activation, translocation of Bax to the mitochondria and release of cytochrome c induced by palmitate treatment. Furthermore, EPA, but not AA prevented the loss of mitochondrial cardiolipin due to apoptosis. These results suggest that EPA supplementation is able to protect cardiomyocytes against palmitate-induced apoptosis via an implication of different mitochondrial elements, possibly through its elongation to DPA, which is very efficient in cardiomyocytes.

The Pro-angiogenic Cytokine Pleiotrophin Potentiates Cardiomyocyte Apoptosis through Inhibition of Endogenous AKT/PKB Activity

Pleiotrophin is a development-regulated cytokine and growth factor that can promote angiogenesis, cell proliferation, or differentiation, and it has been reported to have neovasculogenic effects in damaged heart. Developmentally, it is prominently expressed in fetal and neonatal hearts, but it is minimally expressed in normal adult heart. Conversely, we show in a rat model of myocardial infarction and in human dilated cardiomyopathy that pleiotrophin is markedly up-regulated. To elucidate the effects of pleiotrophin on cardiac contractile cells, we employed primary cultures of rat neonatal and adult cardiomyocytes. We show that pleiotrophin is released from cardiomyocytes in vitro in response to hypoxia and that the addition of recombinant pleiotrophin promotes caspase-mediated genomic DNA fragmentation in a dose- and time-dependent manner. Functionally, it potentiates the apoptotic response of neonatal cardiomyocytes to hypoxic stress and to ultraviolet irradiation and of adult cardiomyocytes to hypoxia-reoxygenation. Moreover, UV-induced apoptosis in neonatal cardiomyocytes can be partially inhibited by small interfering RNA-mediated knockdown of endogenous pleiotrophin. Mechanistically, pleiotrophin antagonizes IGF-1 associated Ser-473 phosphorylation of AKT/PKB, and it concomitantly decreases both BAD and GSK3beta phosphorylation. Adenoviral expression of constitutively active AKT and lithium chloride-mediated inhibition of GSK3beta reduce the potentiated programmed cell death elicited by pleiotrophin. These latter data indicate that pleiotrophin potentiates cardiomyocyte cell death, at least partially, through inhibition of AKT signaling. In conclusion, we have uncovered a novel function for pleiotrophin on heart cells following injury. It fosters cardiomyocyte programmed cell death in response to pro-apoptotic stress, which may be critical to myocardial injury repair.