Apoptosis In Human Cardiomyocytes Research Articles

Abstract 13757: Suppression of Ischemia-Hypoxia Reperfusion Injury in Human iPS Cell-Derived Cardiomyocytes Through Downregulation of EGR1 Mediated by miR-124-3p

Background: Ischemic heart diseases are one of the leading causes of death globally. Although timely revascularization is performed, some cases may still result in heart failure due to ischemia-hypoxia reperfusion (IH/R) injury. Early growth response 1 (EGR1) was reported to involved in reperfusion injury, but its specific role in IH/R-induced apoptosis in human cardiomyocytes (CMs) remains unexplored. Hypothesis: EGR1 is involved in the exacerbation of the IH/R-mediated apoptosis in human CMs, and miR-124-3p acts as an antagonist of EGR1. Methods: Human induced pluripotent stem cell (hiPSC)-derived CMs were cultured in serum-free low-glucose medium with a hypoxic chamber. EGR1 expression and the extent of apoptosis were evaluated by qRT-PCR, Western blotting (WB), and immunofluorescent assay (IFA). RNA-seq was conducted to identify specific pathways. SiRNA targeting EGR1 and miR-124-3p mimic were used to downregulate EGR1 under IH/R. Results: qRT-PCR (Control vs IH/R: 1.000 ± 0.000 vs 5.749 ± 2.947; P<0.01) and WB (0.037 ±0.038 vs 0.953 ± 0.306; P<0.0001) analyses revealed higher expression of EGR1 in IH/R group. IFA demonstrated a higher ratio of cleaved Caspase-3-positive apoptotic cells in IH/R group (0.202 ±0.206 vs 0.377 ± 0.252; P<0.001). RNA-seq analysis indicated significant downregulation of pathways related to cell survival in IH/R. The siRNA downregulated EGR1 successfully, as confirmed by qRT-PCR, WB, and IFA (IH/R Water vs IH/R siRNA: 0.612 ±0.307 vs 0.257 ±0.122; P<0.01), which led to the suppression of apoptosis (0.288 ±0.143 vs 0.142 ±0.049; P<0.05). miR-124-3p mimic downregulated EGR1 under IH/R conditions, as shown by PCR (IH/R Water vs IH/R miRNA mimic: 3.731 ±1.126 vs 2.568 ±0.959; P<0.0001) and WB (0.947 ±0.438 vs 0.478 ±0.277; P<0.05). IFA proved the suppression of EGR1 by miR-124-3p mimic (0.414 ±0.101 vs 0.062 ±0.064; P<0.0001), which subsequently reduced CM apoptosis (0.234 ±0.127 vs 0.092 ±0.047; P<0.001). Conclusion: EGR1 participate in exacerbating apoptosis mediated by IH/R in human CMs. The downregulation of EGR1 suppresses IH/R-induced apoptosis. Treatment with miR-124-3p targeting EGR1 may potentially serve as a novel therapeutic approach for cardioprotection in the future.

Silica nanoparticles induce cardiac injury and dysfunction via ROS/Ca2+/CaMKII signaling.

Interest is growing to better comprehend the interaction of silica nanoparticles (SiNPs) with the cardiovascular system. In particular, the extremely small size, relatively large surface area and associated unique properties may greatly enhance its toxic potentials compared to larger-sized counterparts. Nevertheless, the underlying mechanisms still need to be evaluated. In this context, the cardiotoxicity of nano-scale (Si-60; particle diameter about 60 nm) and submicro-scale silica particles (Si-300; 300 nm) were examined in ApoE-/- mice via intratracheal instillation, 6.0 mg/kg·bw, once per week for 12 times. The echocardiography showed that the sub-chronic exposure of Si-60 declined cardiac output (CO) and stroke volume (SV), shorten LVIDd and LVIDs, and thickened LVAWs of ApoE-/- mice in compared to the control and Si-300 groups. Histological investigations manifested Si-60 enhanced inflammatory infiltration, myocardial fiber arrangement disorder, hypertrophy and fibrosis in the cardiac tissue, as well as mitochondrial ultrastructural injury. Accordingly, the serum cTnT, cTnI and ANP were significantly elevated by Si-60, as well as cardiac ANP content. In particular, Si-60 greatly increased cardiac ROS, Ca2+ levels and CaMKII activation in comparison with Si-300. Further, in vitro investigations revealed silica particles induced a dose- and size-dependent activation of oxidative stress, mitochondrial membrane permeabilization, intracellular Ca2+ overload, CaMKII signaling activation and ensuing myocardial apoptosis in human cardiomyocytes (AC16). Mechanistic analyses revealed SiNPs induced myocardial apoptosis via ROS/Ca2+/CaMKII signaling, which may contribute to the abnormalities in cardiac structure and function in vivo. In summary, our research revealed SiNPs caused myocardial impairments, dysfunction and even structural remodeling via ROS/Ca2+/CaMKII signaling. Of note, a size-dependent myocardial toxicity was noticed, that is, Si-60 greater than Si-300.

S100 calcium-binding protein A12 knockdown ameliorates hypoxia-reoxygenation-induced inflammation and apoptosis in human cardiomyocytes by regulating caspase-4-mediated non-classical pyroptosis.

Ischemic heart disease, especially myocardial infarction, poses a serious risk to human health. S100 calcium-binding protein A12 (S100A12) expression was previously reported to be upregulated in ST-segment elevation myocardial infarction. Therefore, the present study investigated the role of S100A12 in hypoxia/reoxygenation (H/R)-induced cardiomyocytes injury and the associated mechanism. An in vitro H/R-induced cardiomyocyte injury model was first established using AC16 cells. The expression level was found to be hugely upregulated in H/R-induced AC16 cells. Subsequently, cell transfection was conducted to knock down the expression level of S100A12, and the following cellular biological assays revealed that S100A12 knockdown could not only inhibit H/R-induced AC16 cell injury by improving cell viability and decreasing the release of lactate dehydrogenase, as well as reducing apoptotic cells, but also reduce the production of inflammatory cytokines (TNF-α, IL-1β and IL-6), restore the balance of oxidation-antioxidant factors (malondialdehyde, superoxide dismutase and glutathione), and inhibit the activated pyroptosis upon H/R induction. Then, co-immunoprecipitation was used to verify the interaction between S100A12 and caspase-4 (CASP4). CASP4 overexpression reversed the inhibitory effects of S100A12 downregulation on H/R-induced cardiomyocyte injury. In conclusion, these results suggest that S100A12 knockdown can ameliorate H/R-induced cardiomyocyte injury by regulating CASP4 expression. Therefore, S100A12 serves as a potential therapeutic target for the treatment of myocardial ischemia/reperfusion injury.

Circular RNA circACSL1 aggravated myocardial inflammation and myocardial injury by sponging miR-8055 and regulating MAPK14 expression

Myocarditis (MC) is a common, potentially life-threatening inflammatory disease of the myocardium. A growing body of evidence has shown that mitogen-activated protein kinase 14 (MAPK14) participates in the pathogenesis of MC. However, the upstream regulators of MAPK14 remain enigmatic. Circular RNAs (circRNAs) have been identified to play vital roles in the pathophysiology of cardiovascular diseases. Nevertheless, the clinical significance, biological function, and regulatory mechanisms of circRNAs in MC remain poorly understood. In this study, we determined a novel circRNA, circACSL1 (ID: hsa_circ_0071542), which was significantly upregulated in the acute phase of MC, and its dynamic change in expression was related to the progression of MC. We used lipopolysaccharide (LPS) to induce the inflammatory responses in the human cardiomyocytes (HCM) line for in vitro and in cellulo experiments. The pro-inflammatory factors (IL-1β, IL-6, and TNF-α), myocardial injury markers (cTnT, CKMB, and BNP), cell viability, and cell apoptosis were measured to evaluate the extent of myocardial inflammation and myocardial injury level. Functional experiments, including gain-of-function and loss-of-function, were then performed to investigate the pro-inflammatory roles of circACSL1. The results revealed that circACSL1 could aggravate inflammation, myocardial injury, and apoptosis in HCM. Mechanistically, circACSL1 acted as a sponge for miR-8055-binding sites to regulate the downstream target MAPK14 expression. Furthermore, overexpression of miR-8055 rescued the pro-inflammatory effects of circACSL1 on HCM, and the upregulation of MAPK14 induced by circACSL1 was attenuated by miR-8055 overexpression. Knockdown of circACSL1 or overexpression of miR-8055 reduced myocardial inflammation and myocardial injury level and these effects were rescued by overexpression of MAPK14. In summary, our study demonstrated that circACSL1 could aggravate myocardial inflammation and myocardial injury through competitive absorption of miR-8055, thereby upregulating MAPK14 expression. Moreover, circACSL1 may represent a potential novel biomarker for the precise diagnosis of MC and offer a promising therapeutic target for MC treatment.

Ginsenoside Rg2 protects cardiomyocytes against trastuzumab-induced toxicity by inducing autophagy.

Trastuzumab (TZM) significantly improves the outcomes of patients with breast cancer; however, it is associated with severe cardiotoxicity. Ginsenoside Rg2 was reported to exert protective effects against myocardial injury and apoptosis in human cardiomyocytes (HCMs). However, whether ginsenoside Rg2 protects HCMs against TZM-induced toxicity remains unclear. The present study investigated the proliferation of HCMs using a Cell Counting Kit-8 assay and Ki67 immunofluorescence staining. Apoptotic cells were detected by Annexin V/propidium iodide staining and flow cytometry. Furthermore, monodansylcadaverine staining was performed to detect cell autophagy. In addition, western blotting was used to detect the expression levels of phosphorylated (p)-Akt, p-mTOR, beclin 1, microtubule associated protein 1 light chain 3α (LC3) and autophagy protein 5 (ATG5) in HCMs. Pretreatment with ginsenoside Rg2 significantly protected HCMs against TZM-induced cytotoxicity by inhibiting apoptosis. Furthermore, pretreatment with ginsenoside Rg2 induced autophagy in HCMs by upregulating the expression levels of p-Akt, p-mTOR, beclin 1, LC3 and ATG5. The results obtained in the present study suggested that ginsenoside Rg2 could protect HCMs against TZM-induced cardiotoxicity by activating autophagy. Therefore, ginsenoside Rg2 may serve as a potential therapeutic agent to prevent TZM-related cardiotoxicity in patients with breast cancer.

Activation of forkhead box O3a by mono(2-ethylhexyl)phthalate and its role in protection against mono(2-ethylhexyl)phthalate-induced oxidative stress and apoptosis in human cardiomyocytes.

Mono(2-ethylhexyl)phthalate (MEHP), the active metabolite of di(2-ethylhexyl)phthalate (DEHP), is known to exert cardiotoxicity. The aim of the present study was to investigate the role of forkhead box O3a (FOXO3a) in MEHP-induced human AC16 cardiomyocyte injuries. MEHP reduced cell viability and mitochondrial membrane potential (ΔΨm), whereas it increased lactate dehydrogenase (LDH) leakage, production of reactive oxygen species (ROS), and apoptosis in cardiomyocytes. The expression of FOXO3a and its target genes, mitochondrial superoxide dismutase (Mn-SOD) and apoptosis repressor with caspase recruitment domain (ARC), increased after MEHP exposure, but the expression of p-FOXO3a protein was decreased. Overexpression of FOXO3a decreased the production of ROS and the apoptosis rate induced by MEHP, and the expression of Mn-SOD and ARC was further increased after MEHP exposure. In contrast, knockdown of FOXO3a resulted in increased ROS production and apoptosis and suppressed the expression of Mn-SOD and ARC in the presence of MEHP. However, overexpression or knockdown of FOXO3a did not affect MEHP-induced loss of ΔΨm. In conclusion, the loss of ΔΨm and apoptosis are involved in MEHP-induced cardiomyocyte toxicity. Activation of FOXO3a defends against MEHP-induced oxidative stress and apoptosis by upregulating the expression of Mn-SOD and ARC in AC16 cardiomyocytes.

Inhibition of EIF-5A prevents apoptosis in human cardiomyocytes after malaria infection.

In this study, a determination of Troponin I and creatine kinase activity in whole-blood samples in a cohort of 100 small infants in the age of 2-5years from Uganda with complicated Plasmodium falciparum malaria suggests the prevalence of cardiac symptoms in comparison to non-infected, healthy patients. Troponin I and creatine kinase activity increased during infection. Different reports showed that complicated malaria coincides with hypoxia in children. The obtained clinical data prompted us to further elucidate the underlying regulatory mechanisms of cardiac involvement in human cardiac ventricular myocytes. Complicated malaria is the most common clinical presentation and might induce cardiac impairment by hypoxia. Eukaryotic initiation factor 5A (eIF-5A) is involved in hypoxia induced factor (HIF-1α) expression. EIF-5A is a protein posttranslationally modified by hypusination involving catalysis of the two enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase. Treatment of human cardiomyocytes with GC7, an inhibitor of DHS, catalyzing the first step in hypusine biosynthesis led to a decrease in proinflammatory and proapoptotic myocardial caspase-1 activity in comparison to untreated cardiomyocytes. This effect was even more pronounced after co-administration of GC7 and GPI from P. falciparum simulating the pathology of severe malaria. Moreover, in comparison to untreated and GC7-treated cardiomyocytes, co-administration of GC7 and GPI significantly decreased the release of cytochrome C and lactate from damaged mitochondria. In sum, coadministration of GC7 prevented cardiac damage driven by hypoxia in vitro. Our approach demonstrates the potential of the pharmacological inhibitor GC7 to ameliorate apoptosis in cardiomyocytes in an in vitro model simulating severe malaria. This regulatory mechanism is based on blocking EIF-5A hypusination.

MiR-885 mediated cardioprotection against hypoxia/reoxygenation-induced apoptosis in human cardiomyocytes via inhibition of PTEN and BCL2L11 and modulation of AKT/mTOR signaling.

Ischemia/reperfusion (I/R) injury could cause the enhanced cell apoptosis of cardiomyocytes, which is one of key contributors for the development of ischemic heart disease. Recent studies emphasized the role of microRNAs (miRNAs) in regulating cardiomyocyte apoptosis. The study planned to elucidate the molecular actions of miR-885 on mediating human cardiomyocytes (HCMs) apoptosis induced by hypoxia/reoxygenation (H/R) and to explore the potential molecular mechanisms. The present data revealed that H/R stimulation inhibited HCM viability and potentiated HCM apoptosis, and more importantly, the expression of miR-885 in HCMs was markedly repressed after H/R stimulation. Further experimental examinations demonstrated that overexpression of miR-885 attenuated H/R-induced increased in HCM apoptotic rates, while miR-885 knockdown impaired HCM viability and increased HCM apoptotic rates. Moreover, the mechanistic studies showed that miR-885 inversely regulated the expression of phosphatase and tensin homolog (PTEN) and BCL2 like 11 (BCL2L11) in HCMs, and enforced expression of PTEN and BCL2L11 partially antagonized the protective actions of miR-885 overexpression on H/R-induced HCM injury. Moreover, H/R suppressed AKT/mTOR signaling, which was attenuated by miR-885 overexpression in HCMs. In conclusion, the present study for the first time showed the downregulation of miR-885 induced by H/R in HCMs, and provided the evidence that miR-885 attenuated H/R-induced cell apoptosis via inhibiting PTEN and BLC2L11 and modulation of AKT/mTOR signaling in HCMs.

Overexpressing microRNA-150 attenuates hypoxia-induced human cardiomyocyte cell apoptosis by targeting glucose-regulated protein-94.

MicroRNA (miR)-150 has been demonstrated to protect the heart from ischemic injury. However, the protective effect of miR-150 in hypoxia-injured cardiomyocytes remains unclear. The present study aimed to investigate the target gene of miR-150 and the underlying molecular mechanisms of miR-150 in hypoxia-induced cardiomyocyte apoptosis. Using the hypoxia model of human cardiomyocytes (HCMs) in vitro, it was demonstrated that miR-150 was markedly inhibited in HCMs after hypoxia treatment. Overexpressing miR-150 significantly decreased hypoxia-induced HCM death and apoptosis. In addition, GRP94 was revealed to be a direct target of miR-150. Additionally, GRP94 was demonstrated to be involved in hypoxia-induced HCM apoptosis, and the protein expression levels of GRP94 were increased in HCMs in the presence of hypoxia. These findings demonstrated that miR-150 is involved in hypoxia-mediated gene regulation and apoptosis in HCMs. Furthermore, GRP94 knockout increased the cell viability of hypoxia-impaired HCMs with miR-150 mimic or miR-150 inhibitor transfection. In conclusion, miR-150 may serve a protective role in cardiomyocyte hypoxia injury, and the underlying mechanism was mediated, at least partially, by inhibiting GRP94 expression. These findings may provide a novel insight for the therapy of hypoxia-induced myocardial I/R injury.

The Long Non-Coding RNA SNHG1 Attenuates Cell Apoptosis by Regulating miR-195 and BCL2-Like Protein 2 in Human Cardiomyocytes

Background/Aims: Long non-coding RNAs (lncRNAs) are theorized to play key roles in the development of heart diseases. However, the role of lncRNAs in cardiomyocyte apoptosis is largely unknown. The present study examined the role of lncRNA SNHG1 in the human cardiomyocytes (HCMs) apoptosis and explored the underlying molecular mechanisms. Methods: SNHG1, miR-195 and mRNA expression was detected by qRT-PCR; protein level was determined by western blot; cell viability was detected by MTT assay; cell apoptosis was evaluated by flow cytometry and caspase-3 activity assay; the interaction between SNHG1 and miR195 was examined by using luciferase reporter assay. Results: Hydrogen peroxide (H₂O₂) treatment significantly suppressed cell viability and increased cell apoptotic rate and caspase-3 activity in HCMs. Overexpression of SNHG1 attenuated the effects of H₂O₂ on HCMs viability and apoptosis; while SNHG1 exerted the opposite effects. SNHG1 was found to sponge miR-195 and suppress the expression of miR-195 in HCMs. Overexpression of miR-195 suppressed cell viability and induced apoptosis in HCMs, and miR-195 was found to negatively regulate the expression of BCL-2 like protein 2 (BCL2L2) via targeting its 3’ untranslated region. Overexpression of BCL2L2 partially reversed the effects of miR-195 overexpression on cell viability and cell apoptosis of HCMs. MiR-195 overexpression or BCL2L2 knockdown attenuated the effects of SNHG1 overexpression on cell viability, cell apoptosis and protein levels of cleaved caspase-3, cleaved caspase-9 and Bax in H₂O₂-treated HCMs. Conclusion: Our results suggest a novel SNHG1/miR-195/BCL2L2 axis in the regulation of cardiomyocyte apoptosis. Modulation of SNHG1 may represent a novel strategy to treat cardiomyocyte apoptosis-related heart diseases.

Aleglitazar, a Balanced Dual PPARα and -γ Agonist, Protects the Heart Against Ischemia-Reperfusion Injury.

To evaluate whether aleglitazar (Ale), a dual PPARα/γ agonist, has additive effects on myocardial protection against ischemia-reperfusion injury. Human cardiomyocytes (HCMs), cardiomyocytes from cardiac-specific PPARγ knockout (MCM-PPARγ (CKO) ) or wild type (MCM-WT) mice were incubated with different concentrations of Ale, and subjected to simulated ischemia-reperfusion (SIR) or normoxic conditions (NSIR). Cell viability, apoptosis and caspase-3 activity were determined. HCMs were transfected with siRNA against PPARα (siPPARα) or PPARγ (siPPARγ) followed by incubation with Ale. PPARα/γ DNA binding capacity was measured. Cell viability, apoptosis and levels of P-AKT and P-eNOS were assessed. Infarct size following 30min coronary artery occlusion and 24h reperfusion were assessed in WT and db/db diabetic mice following 3-day pretreatment with vehicle, Ale or glimeperide. Ale (at concentrations of 150-600nM) increased cell viability and reduced apoptosis in HCMs, MCM-WT and MCM-PPAR (CKO) exposed to SIR. In HCM, the protective effect was partially blocked by siPPARα alone or siPPARγ alone, and completely blocked by siPPARα+siPPARγ. Ale increased P-Akt/P-eNOS in HCMs. P-Akt or P-eNOS levels were decreased when PPARα alone, PPARγ alone and especially when both were knocked down. Peritoneal GTTs revealed that db/db mice had developed impaired glucose tolerance and insulin sensitivity, which were normalized by Ale or glimepiride treatment. Ale, but not glimepiride, limited infarct size in both WT and diabetic mice after ischemia-reperfusion. Ale protects against myocardial apoptosis caused by hypoxia-reoxygenation in vitro and reduces infarct size in vivo.

Silencing of hepcidin enforces the apoptosis in iron-induced human cardiomyocytes

BackgroundIron is essential not only for erythropoisis but also for several bioenergetics’ processes in myocardium. Hepcidin is a well-known regulator of iron homeostasis. Recently, researchers identified low hepcidin was independently associated with increased 3-year mortality among systolic heart failure patients. In addition, our previous in vivo study revealed that the left ventricular mass index increased in chronic kidney disease patients with lower serum hepcidin. We hypothesize that hepcidin interacts with the apoptotic pathway of cardiomyocytes during oxidative stress conditions.MethodsTo test this hypothesis, human cardiomyocytes were cultured and treated with ferrous iron. The possible underlying signaling pathways of cardiotoxicity were examined following knockdown studies using siRNAs of hepcidin (siRNA1 was used as a negative control and siRNA2 was used to silence hepcidin).ResultsWe found that ferrous iron induces apoptosis in human cardiomyocytes in a dose-dependent manner. This iron-induced apoptosis was linked to enhanced caspase 8, reduced Bcl-2, Bcl-xL, phosphorylated Akt and GATA-4. Hepcidin levels increased in human cardiomyocytes pretreated with ferrous iron and returned to non-iron treated levels following siRNA2 transfection. In iron pretreated cardiomyocytes, the siRNA2 transfection further increased caspase 8 expression and decreased the expression of GATA-4, Bcl-2, Bcl-xL and phosphorylated Akt than iron pretreatment alone, but caspase 9 levels remained unchanged.ConclusionsOur findings suggest that hepcidin can rescue human cardiomyocytes from iron-induced apoptosis through the regulation of GATA-4/Bcl-2 and the extrinsic apoptotic pathway.

Cyclosporine-Induced Apoptosis in Human Cardiomyocytes Through P53-Dependent Pathway

Cyclosporine A (CsA) is the most effective and widely used immunosuppressant drug in heart, lung and kidney transplantation. However, the effect of CsA is limited by the significant toxicity. The mechanism of CsA-induced toxicity is remaining controversial. Cellular apoptosis is being suggested as a possible mediator of CsA toxicity. To date, regarding the effects of CsA on apoptosis and apoptosis-related gene regulation in cardiomyocytes remain unclear. Therefore, the current study was designed to investigate the effect of CsA on apoptosis and apoptosis-related gene p53 expression in human cardiomyocytes. We hypothesized that CsA induces apoptosis in human cardiomyocytes through p53-dependent pathway.Human cardiac atrial tissue was obtained from open-heart surgery (n=5). The cardiac tissue was minced and incubated in the special tissue culture system for 24 hours in the absence or presence of CsA (10-7 M). To detect the DNA fragmentation, in situ terminal deoxymucleotidyl transferase dUTP nick end labeling (TUNEL) was performed.