Palmitate-induced apoptosis in cardiomyocytes is mediated through alterations in mitochondria: prevention by cyclosporin A

Palmitate, a C16 fatty acid found in high concentrations in the blood in acute myocardial infarction, induces apoptotic cell death. To more completely define the nature and mechanism underlying palmitate-induced cell death, cardiomyocytes were cultured from embryonic chick heart and were treated with palmitate. Concentration-dependent loss of cell viability was established by loss of the ability of palmitate-treated cells to exclude propidium iodide (PI), metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and retain fluorescein diacetate (FDA). Dual staining with PI and FDA and subsequent analysis by FACS established that palmitate-induced cell death was predominantly necrosis whereas apoptosis occurred in 13% of all dead cells. The low proportion of palmitate-induced apoptosis was confirmed by evaluation of the DNA content or PI fluorescent staining of the DNA of permeabilized cardiomyocytes. A critical role for mitochondria in the pathogenesis of palmitate-induced cell death was demonstrated, for the first time, based on palmitate-induced reduction of mitochondrial activity as assessed by the mitochondrial-selective dye chloromethyl-X-Rosamine and the presence of a greater amount of the mitochondrial marker cytochrome C in the cytosol of palmitate-treated cardiomyocytes than in control cells. Further, cyclosporin that inhibits the development of mitochondrial transition pores blocked palmitate-induced alteration in mitochondrial function and palmitate-induced cell death. We further demonstrated the selectivity of cyclosporin A for the prevention of apoptotic cell death in the heart as there was no alteration in necrotic cell death produced by palmitate with cyclosporin pretreatment. Our data demonstrate the nature of palmitate-induced cell death in cardiomyocytes (both apoptotic and necrotic), propose a mitochondrial basis for its pathogenesis and show that cyclosporin A prevents palmitate-induced apoptotic cardiomyocyte cell death.     

miR-24 Regulates Intrinsic Apoptosis Pathway in Mouse Cardiomyocytes

Numerous cardiac diseases, including myocardial infarction (MI) and chronic heart failure, have been associated with cardiomyocyte apoptosis. Promoting cell survival by inhibiting apoptosis is one of the effective strategies to attenuate cardiac dysfunction caused by cardiomyocyte loss. miR-24 has been shown as an anti-apoptotic microRNA in various animal models. In vivo delivery of miR-24 into a mouse MI model suppressed cardiac cell death, attenuated infarct size, and rescued cardiac dysfunction. However, the molecular pathway by which miR-24 inhibits cardiomyocyte apoptosis is not known. Here we found that miR-24 negatively regulates mouse primary cadiomyocyte cell death through functioning in the intrinsic apoptotic pathways. In ER-mediated intrinsic pathway, miR-24 genetically interacts with the CEBP homologous gene CHOP as knocking down of CHOP partially attenuated the induced apoptosis by miR-24 inhibition. In mitochondria–involved intrinsic pathway, miR-24 inhibits the initiation of apoptosis through suppression of Cytochrome C release and Bax translocation from cytosol to mitochondria. These results provide mechanistic insights into the miR-24 mediated anti-apoptotic effects in murine cardiomyocytes.     

Involvement of death receptor signaling in mechanical stretch-induced cardiomyocyte apoptosis

Recent evidences suggest that mechanical overload associated with abnormal blood pressure causes apoptosis in cardiovascular system. Still, the intracellular signaling leading to cardiomyocyte apoptosis has not been fully defined. Previous reports ascribed stretch-induced cardiomyocyte apoptosis to rennin-angiotensin-system (RAS) signaling and/or mitochondria-dependent apoptosis pathway. The present study shows the involvement of death receptor signaling in mechanical stretch-induced cardiomyocyte apoptosis. By employing a well-described in vitro stretch model, we studied stretch-induced apoptosis and found that the death receptor-mediated apoptotic signaling was activated in stretch-induced apoptosis in neonatal rat cardiomyocytes. The major finding are as following: (1) The mechanical stretch activated death receptor-mediated apoptotic signaling in cardiomyocytes, including activation of caspases 8, 9 and 3, up-regulation of Fas, FasL expression and cell surface trafficking of death ligands (FasL and TRAIL); (2) That exogenous death ligand (TRAIL) enhanced, while soluble death receptor (sDR5) neutralized, stretch-induced apoptosis; (3) Adenovirus-delivered dominant negative FADD (FADD-DN) significantly reduced apoptosis, caspases 8, 9, and 3 activation, and stretch-induced cyt c release from mitochondria. These data clearly suggested mechanical stretch activated death receptor-mediated apoptotic signaling in cardiomyocytes. In conclusion, our data suggest that the FADD-linked death receptor signaling may contribute to stretch-induced cardiomyocyte apoptosis, probably through activating mitochondria-dependent apoptotic signaling.     

Knockdown of endogenous RNF4 exacerbates ischaemia‐induced cardiomyocyte apoptosis in mice

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

Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction

Mouse mast cell protease-4 (mMCP4) is a chymase that has been implicated in cardiovascular diseases, including myocardial infarction (MI). This study tested a direct role of mMCP4 in mouse post-MI cardiac dysfunction and myocardial remodeling. Immunoblot and immunofluorescent double staining demonstrated mMCP4 expression in cardiomyocytes from the infarct zone from mouse heart at 28 day post-MI. At this time point, mMCP4-deficient Mcpt4^?/? mice showed no difference in survival from wild-type (WT) control mice, yet demonstrated smaller infarct size, improved cardiac functions, reduced macrophage content but increased T-cell accumulation in the infarct region compared with those of WT littermates. mMCP4-deficiency also reduced cardiomyocyte apoptosis and expression of TGF-β1, p-Smad2, and p-Smad3 in the infarct region, but did not affect collagen deposition or α-smooth muscle actin expression in the same area. Gelatin gel zymography and immunoblot analysis revealed reduced activities of matrix metalloproteinases and expression of cysteinyl cathepsins in the myocardium, macrophages, and T cells from Mcpt4^?/? mice. Immunoblot analysis also found reduced p-Smad2 and p-Smad3 in the myocardium from Mcpt4^?/? mice, yet fibroblasts from Mcpt4^?/? mice showed comparable levels of p-Smad2 and p-Smad3 to those of WT fibroblasts. Flow cytometry, immunoblot analysis, and immunofluorescent staining demonstrated that mMCP4-deficiency reduced the expression of proapoptotic cathepsins in cardiomyocytes and protected cardiomyocytes from H₂O₂-induced apoptosis. This study established a role of mMCP4 in mouse post-MI dysfunction by regulating myocardial protease expression and cardiomyocyte death without significant impact on myocardial fibrosis or survival post-MI in mice.     

Induced pluripotent stem cell‐conditional medium inhibits H9C2 cardiomyocytes apoptosis via autophagy flux and Wnt/β‐catenin pathway

Induced pluripotent stem cell‐derived conditioned medium (iPS‐CM) could improve cell viability in many types of cells and may be a better alternative for the treatment of myocardial infarction. This study aimed to examine the influence of iPS‐CM on anti‐apoptosis and the proliferation of H9C2 cardiomyocytes and investigate the underlying mechanisms. H9C2 cardiomyocytes were exposed to 200 μmol/L hydrogen peroxide (H₂O₂) for 24 hours with or without pre‐treatment with iPS‐CM. The ratio of apoptotic cells, the loss of mitochondrial membrane potential (△Ψm) and the levels of intracellular reactive oxygen species were analysed by flow cytometric analysis. The expression levels of BCL‐2 and BAX proteins were analysed by Western blot. Cell proliferation was assessed using cell cycle and EdU staining assays. To study cell senescence, senescence‐associated β‐galactosidase (SA‐β‐gal) staining was conducted. The levels of malondialdehyde, superoxide dismutase and glutathione were also quantified using commercially available enzymatic kits. The results showed that iPS‐CM containing basic fibroblast growth factor significantly reduced H₂O₂‐induced H9C2 cardiomyocyte apoptosis by activating the autophagy flux pathway, promoted cardiomyocyte proliferation by up‐regulating the Wnt/β‐catenin pathway and inhibited oxidative stress and cell senescence. In conclusion, iPS‐CM effectively enhanced the cell viability of H9C2 cardiomyocytes and could potentially be used to inhibit cardiomyocytes apoptosis to treat myocardial infarction in the future.     

Partial Correction of Abnormal Cardiac Development in Caspase-8-deficient Mice by Cardiomyocyte Expression of p35

Baculovirus p35 protein protects cells from apoptotic cell death by inhibiting caspase activation. We have established transgenic mouse lines specifically expressing p35 in cardiomyocytes, and primary cardiomyocytes isolated from these mice exhibit resistance to staurosporine-induced apoptosis. In a previous study, we observed defects in heart formation associated with abdominal hemorrhage and cardiomyocyte cell death in caspase-8-deficent animals. In order to better understand the etiology of the cardiac defects and embryonic lethality in caspase-8-deficient mice, we crossed these mice with the p35 transgenic animals. Although the newly generated mice still died in utero and exhibited some cardiac defects, cardiomyocyte apoptosis was suppressed and ventricular trabeculation was restored. Thus, cardiomyocyte expression of p35 prevented cell death induced by staurosporine or caspase-8 deficiency. Additionally, our data suggest that caspase-8 plays multiple roles in cardiac development.     

The 14-3-3tau phosphoserine-binding protein is required for cardiomyocyte survival

14-3-3 family members are intracellular dimeric phosphoserine-binding proteins that regulate signal transduction, cell cycle, apoptotic, and metabolic cascades. Previous work with global 14-3-3 protein inhibitors suggested that these proteins play a critical role in antagonizing apoptotic cell death in response to provocative stimuli. To determine the specific role of one family member in apoptosis, mice were generated with targeted disruption of the 14-3-3tau gene. 14-3-3tau(-/-) mice did not survive embryonic development, but haploinsufficient mice appeared normal at birth and were fertile. Cultured adult cardiomyocytes derived from 14-3-3tau(+/-) mice were sensitized to apoptosis in response to hydrogen peroxide or UV irradiation. 14-3-3tau(+/-) mice were intolerant of experimental myocardial infarction and developed pathological ventricular remodeling with increased cardiomyocyte apoptosis. ASK1, c-jun NH(2)-terminal kinase, and p38 mitogen-activated protein kinase (MAPK) activation was increased, but extracellular signal-regulated kinase MAPK activation was reduced, in 14-3-3tau(+/-) cardiac tissue. Inhibition of p38 MAPK increased survival in 14-3-3tau(+/-) mice subjected to myocardial infarction. These results demonstrate that 14-3-3tau plays a critical antiapoptotic function in cardiomyocytes and that therapeutic agents that increase 14-3-3tau activity may be beneficial to patients with myocardial infarction.     

Acute induction of autophagy as a novel strategy for cardioprotection

There is no question that necrosis and apoptosis contribute to cardiomyocyte death in the setting of myocardial ischemia-reperfusion. Indeed, considerable effort and resources have been invested in the development of novel therapies aimed at attenuating necrotic and apoptotic cell death, with the ultimate goal of applying these strategies to reduce infarct size and improve outcome in patients suffering acute myocardial infarction (MI) or ‘heart attack’. However, an issue that remains controversial is the role of autophagy in determining the fate of ischemic-reperfused cardiomyocytes: i.e., is induction of autophagy detrimental or protective? Recent data from our group obtained in the clinically relevant, in vivo swine model of acute MI provide novel evidence of a positive association between pharmacological upregulation of autophagy (achieved by administration of chloramphenicol succinate (CAPS)) and increased resistance to myocardial ischemia-reperfusion injury.     

Berberine Inhibits Doxorubicin-Triggered Cardiomyocyte Apoptosis via Attenuating Mitochondrial Dysfunction and Increasing Bcl-2 Expression

Cardiomyocyte apoptosis is an important event in doxorubicin (DOX)-induced cardiac injury. The aim of the present study was to investigate the protection of berberine (Ber) against DOX- triggered cardiomyocyte apoptosis in neonatal rat cardiomyocytes and rats. In neonatal rat cardiomyocytes, Ber attenuated DOX-induced cellular injury and apoptosis in a dose-dependent manner. However, Ber has no significant effect on viability of MCF-7 breast cancer cells treated with DOX. Ber reduced caspase-3 and caspase-9, but not caspase-8 activity in DOX-treated cardiomyocytes. Furthermore, Ber decreased adenosine monophosphate-activated protein kinase α (AMPKα) and p53 phosphorylation at 2 h, cytosolic cytochrome c and mitochondrial Bax levels and increased Bcl-2 level at 6 h in DOX-stimulated cardiomyocytes. Pretreatment with compound C, an AMPK inhibitor, also suppressed p53 phosphorylation and apoptosis in DOX-treated cardiomyocytes. DOX stimulation for 30 min led to a loss of mitochondrial membrane potential and a rise in the AMP/ATP ratio. Ber markedly reduced DOX-induced mitochondrial membrane potential loss and an increase in the AMP/ATP ratio at 1 h and 2 h post DOX exposure. In in vivo experiments, Ber significantly improved survival, increased stroke volume and attenuated myocardial injury in DOX-challenged rats. TUNEL and Western blot assays showed that Ber not only decreased myocardial apoptosis, caspase-3 activation, AMPKα and p53 phosphorylation, but also increased Bcl-2 expression in myocardium of rats exposed to DOX for 84 h. These findings indicate that Ber attenuates DOX-induced cardiomyocyte apoptosis via protecting mitochondria, inhibiting an increase in the AMP/ATP ratio and AMPKα phosphorylation as well as elevating Bcl-2 expression, which offer a novel mechanism responsible for protection of Ber against DOX-induced cardiomyopathy.     

Impedance-Based Monitoring of Ongoing Cardiomyocyte Death Induced by Tumor Necrosis Factor-α

Deregulated cardiomyocyte death is a critical risk factor in a variety of cardiovascular diseases. Although various assays have been developed to detect cell responses during cell death, the capability of monitoring cell detachment will enhance the understanding of death processes by providing instant information at its early phase. In this work, we developed an impedance-sensing assay for real-time monitoring of cardiomyocyte death induced by tumor necrosis factor-α based on recording the change in cardiomyocyte adhesion to extracellular matrix. Electrochemical impedance spectroscopy was employed in impedance data processing, followed by calibration with the electrical cell-substrate impedance-sensing technique. The adhesion profile of cardiomyocytes undergoing cell death processes was recorded as the time course of equivalent cell-substrate distance. The cell detachment was detected with our assay and proved related to cell death in the following experiments, indicating its advantage against the conventional assays, such as Trypan blue exclusion. An optimal concentration of tumor necrosis factor-α (20 ng/mL) was determined to induce cardiomyocyte apoptosis rather than the combinative cell death of necrosis and apoptosis by comparing the concentration-related adhesion profiles. The cardiomyocytes undergoing apoptosis experienced an increase of cell-substrate distance from 59.1 to 89.2 nm within 24 h. The early change of cell adhesion was proved related to cardiomyocyte apoptosis in the following TUNEL test at t = 24 h, which suggested the possibility of early and noninvasive detection of cardiomyocyte apoptosis.     

Cardiomyocyte specific ablation of p53 is not sufficient to block doxorubicin induced cardiac fibrosis and associated cytoskeletal changes

Doxorubicin (Dox) is an anthracycline used to effectively treat several forms of cancer. Unfortunately, the use of Dox is limited due to its association with cardiovascular complications which are manifested as acute and chronic cardiotoxicity. The pathophysiological mechanism of Dox induced cardiotoxicity appears to involve increased expression of the tumor suppressor protein p53 in cardiomyocytes, followed by cellular apoptosis. It is not known whether downregulation of p53 expression in cardiomyocytes would result in decreased rates of myocardial fibrosis which occurs in response to cardiomyocyte loss. Further, it is not known whether Dox can induce perivascular necrosis and associated fibrosis in the heart. In this study we measured the effects of acute Dox treatment on myocardial and perivascular apoptosis and fibrosis in a conditional knockout (CKO) mouse model system which harbours inactive p53 alleles specifically in cardiomyocytes. CKO mice treated with a single dose of Dox (20 mg/kg), did not display lower levels of myocardial apoptosis or reactive oxygen and nitrogen species (ROS/RNS) compared to control mice with intact p53 alleles. Interestingly, CKO mice also displayed higher levels of interstitial and perivascular fibrosis compared to controls 3 or 7 days after Dox treatment. Additionally, the decrease in levels of the microtubule protein α-tubulin, which occurs in response to Dox treatment, was not prevented in CKO mice. Overall, these results indicate that selective loss of p53 in cardiomyocytes is not sufficient to prevent Dox induced myocardial ROS/RNS generation, apoptosis, interstitial fibrosis and perivascular fibrosis. Further, these results support a role for p53 independent apoptotic pathways leading to Dox induced myocardial damage and highlight the importance of vascular lesions in Dox induced cardiotoxicity.     

Granulocyte colony-stimulating factor treatment enhances the efficacy of cellular cardiomyoplasty with transplantation of embryonic stem cell-derived cardiomyocytes in infarcted myocardium

We tested the hypothesis that granulocyte colony-stimulating factor (G-CSF) administration would enhance the efficacy of cellular cardiomyoplasty with embryonic stem (ES) cell-derived cardiomyocytes in infarcted myocardium. Three weeks after myocardial infarction by cryoinjury, Sprague-Dawley rats were randomized to receive either an injection of medium, ES cell-derived cardiomyocyte transplantation, G-CSF administration, or a combination of G-CSF administration and ES cell-derived cardiomyocyte transplantation. Eight weeks after treatment, the cardiac tissue formation, neovascularization, and apoptotic activity in the infarct regions were evaluated by histology and immunohistochemistry. The left ventricular (LV) dimensions and function of the treated heart were evaluated by echocardiography. Transplanted ES cell-derived cardiomyocytes survived and participated in the myocardial regeneration in the infarcted heart. A combination of G-CSF treatment and ES cell-derived cardiomyocyte transplantation significantly promoted angiogenesis and reduced the infarct area and cell apoptosis in the infarcted myocardium compared with ES cell-derived cardiomyocyte transplantation alone. The combination therapy also attenuated LV dilation, as compared with ES cell-derived cardiomyocyte transplantation alone. G-CSF treatment can enhance the efficacy of cellular cardiomyoplasty by ES cell-derived cardiomyocyte transplantation to treat myocardial infarction.     

LncRNA UCA1 protects cardiomyocytes against hypoxia/reoxygenation induced apoptosis through inhibiting miR-143/MDM2/p53 axis

BackgroundlncUCA1 is abundantly expressed in the heart, indicating it may be important in maintaining normal myocardial function. However, the underlying mechanism of lncUCA1 in heart disease, particularly myocardial infarction (MI), is still in its infancy.MethodsLncUCA1 and miR-143 expression were measured in hearts of MI models. Overexpression and knockdown of lncUCA1 in neonatal rat cardiomyocytes were performed to confirm the effects of lncUCA1 in hypoxia-induced apoptosis.ResultsThe expression of lncUCA1 decreased but miR-143 increased inversely in MI heart. Overexpressing lncUCA1 protected cardiomyocytes from H/R induced apoptosis via inhibiting miR-143, which regulates apoptosis by targeting MDM2/p53 pathway. While silencing lncUCA1 caused miR-143 upregulation and H/R-induced apoptosis increase. Moreover, miR-143 was proved to be a competitive target of lncUCA1.ConclusionslncUCA1 might protect cardiomyocyte against H/R induced apoptosis by suppressing miR-143 and modulated the following downstream MDM2/p53 signaling pathway, indicating the therapeutic potential of targeting lncUCA1 for MI.     

Nitric oxide and oxygen radicals induced apoptosis via bcl-2 and p53 pathway in hypoxia-reoxygenated cardiomyocytes

Twotypesofcellulardemisecanoccursimultaneouslyintissuesorculturedcellbynecrosisandapoptosis.Lossofmembraneintegrity,celledemaandbreak,andthecellcomponentsre-leasedoutarethecharacteristicsofnecrosis.Whilethecellapoptosisisaprogramcelldeathcodedbygeneandactivatedseriousendogenousenzymes[1].Recentstudieshavedemonstratedthatmyocardialischemia-reperfusioninjuryresultedinapoptoticcelldeathinadditiontotissuenecrosis[2—4].Oxygenstressisoneofthereasonsthatcausedcellapoptosisandtheoxygenradicalsinthest…     

Oxidative stress enhances phosphorylation of p53 in neonatal rat cardiomyocytes

p53 is an important regulator of cell growth and apoptosis and its activity is regulated by phosphorylation. Accordingly, in neonatal rat cardiomyocytes we examined the involvement of p53 in H₂O₂-induced apoptosis. Treatment with 50–100 μM H₂O₂ markedly induced apoptosis in cardiomyocytes, as assessed by gel electrophoresis of genomic DNA. To examine whether H₂O₂ increases p53 phosphorylation in cardiomyocytes, we utilized an antibody that specifically recognizes phosphorylated p53 at serine-15. The level of phosphorylated p53 was markedly increased by 100 μM H₂O₂ at 30 and 60 min. Using specific protein kinase inhibitors we examined the involvement of protein kinases in p53 phosphorylation in response to H₂O₂ treatment. However, staurosporine, a broad spectrum inhibitor of protein kinases, SB202190, a specific p38 kinase inhibitor, PD98059, a MAP kinase inhibitor, wortmannin, an inhibitor of DNA-PK and PI3 kinase, SP600125, a JNK inhibitor and caffeine,an inhibitor of ATM and ATR, failed to prevent the H₂O₂-induced phosphorylation of p53. cDNA microarray revealed that H₂O₂ markedly increased expression of several p53 upstream modifiers such as the p300 coactivator protein and several downstream effectors such as gadd45, but decreased the expression of MDM2, a negative regulator of p53. Our results suggest that phosphorylation of p53 at serine-15 may be an important signaling event in the H₂O₂-mediated apoptotic process.     

PUMA与心肌细胞凋亡的研究进展

p53上调的凋亡调节物(p53 upregulated modulator of apoptosis,PUMA)是新近发现的一种具有促凋亡作用的p53靶基因.与以往发现的其他p53靶基因比较,PUMA在促凋亡作用中有两个重要的特点:一是PUMA几乎介导p53依赖的所有凋亡信号;二是PUMA不仅介导p53依赖的凋亡信号,而且还可以介导p53非依赖的凋亡信号.也就是说,尽管PUMA是p53靶基因,但是其在p53非依赖细胞凋亡中也发挥重要作用.由此可见,PUMA是一个强大的促凋亡因子.在心肌细胞,PUMA参与缺血/再灌注、内质网应激、阿霉素等多种刺激诱导的细胞凋亡.因此,PUMA在心肌细胞凋亡中发挥重要作用.