HG-induced Cardiomyocyte Apoptosis Research Articles

Danlou tablet inhibits high-glucose-induced cardiomyocyte apoptosis via the miR-34a-SIRT1 axis

Diabetic cardiomyopathy (DCM) is highly prevalent and increases the risk of heart failure and sudden death. Therefore, proper and effective treatments for DCM are in urgent demand. Danlou tablet (Dan) is reported to confer protective effects on several heart diseases. However, to our knowledge, whether Dan provides protection against DCM is unclear. In this study, we explored the effect of Dan on DCM with the in vitro DCM model using AC16 cardiomyocytes. We found that Dan treatment significantly reduced cardiomyocyte apoptosis and oxidative stress in high-glucose (HG)-treated cardiomyocytes, as evidenced by decreased Annexin V-FITC+ cardiomyocytes, intracellular reactive oxygen species (ROS) levels, Bax/Bcl2 ratio, and cleaved-Caspase3/Caspase3 ratio. Interestingly, Dan treatment caused a decreased level of microRNA-34a (miR-34a), which could enhance cardiomyocyte apoptosis. Furthermore, miR-34a mimic blocked Dan's effect in apoptosis prevention. Finally, we observed that the miR-34a mimic effectively decreased the level of sirtuin 1 (SIRT1), while the miR-34a inhibitor increased the level of SIRT1. And downregulation of SIRT1 effectively reversed the effect of miR-34a inhibitor on cardiomyocyte apoptosis. Taken together, our study showed that Dan prevented HG-induced cardiomyocyte apoptosis through downregulating miR-34a and upregulating SIRT1. Our study has provided experimental support for the potential use of Dan in treating DCM. Further detailed study of Dan and the underlying mechanisms may shed light on the prevention and treatment of DCM.

MiR-29a regulates cardiomyocyte apoptosis by targeting Bak1 in diabetic cardiomyopathy.

This study sought to investigate the association between microRNA-29a (miR-29a) and cardiomyocyte apoptosis in diabetic cardiomyopathy (DCM). DCM rat model was established by treating rats with streptozotocin (STZ), followed by injection of NC or miR-29a-3p mimics into the myocardium of rats. High glucose (HG)-treated H9c2 cells were transfected with NC and miR-29a-3p mimics. DCM rats presented elevated levels of blood glucose, HbA1c, blood pressure, urine output, decreased body weight and cardiac contractile function after modeling. MiR-29a was lowly expressed in STZ-treated rats and HG-treated H9c2 cells. Upregulation of miR-29a improved cardiac structure and function and attenuated, alleviated myocardial histological abnormalities and fibrosis and lowered cardiomyocyte apoptosis in DCM rats. Meanwhile, HG promoted H9c2 cell apoptosis, while miR-29a overexpression attenuated the function of HG. Compared with control group, the protein expression of Bax, cleaved-caspase3 and Bak1 in DCM and HG groups were significantly upregulated, and the expression of Bcl-2 and Mcl-1 was downregulated, while miR-29a overexpression exerted opposite effect. Bioinformatics prediction method and western blot revealed that miR-29a directly targeted Bak1 and downregulated Bak1 expression. Overall, miR-29a regulated STZ- and HG-induced cardiomyocyte apoptosis by targeting Bak1, providing a novel understanding of the pathogenesis of DCM.

Elevated MST1 leads to apoptosis via depletion of YAP1 in cardiomyocytes exposed to high glucose

BackgroundGestational diabetes mellitus is a risk factor for congenital heart defects. The article aimed to investigate the expression and roles of MST1, YAP1, Last1/2 and Survivin in modulating HG-induced cardiomyocyte apoptosis and maternal diabetes-induced heart abnormality.MethodsDiabetes mellitus was induced in rats using streptozotocin. The protein expression and phosphorylation analysis in fetal heart tissue was assessed by western blot and immunohistochemical staining. Hoechst 33342 staining assay was performed to explore H9C2 apoptosis. The gene and protein expression in H9C2 cells was assessed by quantitative PCR and western blot. Knockdown of gene expression was assessed by RNA interference.ResultsOur results revealed that increased MST1 protein levels in the heart tissues of the offspring of diabetic rats in vivo and in H9C2 cardiomyocytes under HG treatment in vitro, respectively. Knockdown and overexpression experiments showed that MST1 played a key role in mediating HG-induced apoptosis in cardiomyocytes. Downregulation of YAP1 was associated with HG-induced, MST1-mediated cardiomyocyte apoptosis. Further study showed that MST1 downregulated the protein level of YAP1 through mediation of YAP1 phosphorylation on Ser127 and Ser397; this process also required LATS1/2 participation. MST1 overexpression increased the phosphorylation levels of LATS1/2, which were also shown to be increased in the heart tissues of diabetic offspring. We also found that YAP1 mediated the expression of Survivin during HG-induced apoptosis, and the Survivin-inhibitor YM155 partially inhibited the role of YAP1 in suppressing apoptosis induced by HG in cardiomyocytes.ConclusionThese findings reveal a regulatory mechanism of MST1/YAP1/Survivin signaling in modulating cardiomyocyte apoptosis in vitro and maternal diabetes-induced congenital heart defects in vivo.

Celastrol mitigates high glucose-induced inflammation and apoptosis in rat H9c2 cardiomyocytes via miR-345-5p/growth arrest-specific 6.

Celastrol (Cel) has been corroborated as an anti-inflammatory and anti-apoptotic agent in multiple cell damage models. However, the protective effect of Cel in high glucose (HG)-induced cardiomyocyte injury is still unclear. The present study aimed to determine whether Cel can mitigate HG-stimulated cardiomyocyte injury via regulating the miR-345-5p/growth arrest-specific 6 (Gas6) signaling pathway. Cardiomyocytes were exposed to normal glucose (NG; 5 mmol/l) or HG (30 mmol/l) and then administered with Cel. Cell counting kit-8 and flow cytometry assays were used to detect cell proliferative activity and apoptosis. mRNA and protein expression were analyzed using a quantitative reverse transcriptase-polymerase chain reaction and western blotting, respectively. A bioinformatics algorithm and a luciferase reporter gene assay were used to determine whether Gas6 is a direct target of miR-345-5p. The present study confirmed the inhibitory effects of Cel in HG-induced inflammation in cardiomyocytes. Moreover, Cel exhibited the ability to antagonize HG-induced cardiomyocyte apoptosis and suppress the elevated Bax/Bcl-2 ratio elicited by HG stimulation. Intriguingly, Cel treatment revoked the HG-triggered repression of Gas6 protein expression, and Gas6 loss-of-function accelerated HG-induced cardiomyocyte apoptosis. HG-triggered up-regulation of miR-345-5p expression was depressed following Cel treatment. Importantly, we validated that Gas6 is a direct target of miR-345-5p. Transfection with miR-345-5p inhibitors restrained HG-induced release of pro-inflammatory cytokines and cell apoptosis. The findings of the present study demonstrate that Cel administration antagonized HG-induced cardiomyocyte apoptosis and inflammation through up-regulating Gas6 expression by restraining miR-345-5p.

Inhibition of brain-type glycogen phosphorylase ameliorates high glucose-induced cardiomyocyte apoptosis via Akt-HIF-1α activation.

Brain-type glycogen phosphorylase (pygb) is one of the rate-limiting enzymes in glycogenolysis that plays a crucial role in the pathogenesis of type 2 diabetes mellitus. Here we investigated the role of pygb in high-glucose (HG)-induced cardiomyocyte apoptosis and explored the underlying mechanisms, by using the specific pygb inhibitors or pygb siRNA. Our results show that inhibition of pygb significantly attenuates cell apoptosis and oxidative stress induced by HG in H9c2 cardiomyocytes. Inhibition of pygb improved glucose metabolism in cardiacmyocytes, as evidenced by increased glycogen content, glucose consumption, and glucose transport. Mechanistically, pygb inhibition activates the Akt-GSK-3β signaling pathway and suppresses the activation of NF-κB in H9c2 cells exposed to HG. Additionally, pygb inhibition promotes the expression and the translocation of hypoxia-inducible factor-1α (HIF-1α) after HG stimulation. However, the changes in glucose metabolism and HIF-1α activation mediated by pygb inhibition are significantly reversed in the presence of the Akt inhibitor MK2206. In conclusion, this study found that inhibition of pygb prevents HG-induced cardiomyocyte apoptosis via activation of Akt-HIF-α.

GSK3β and MCL-1 mediate cardiomyocyte apoptosis in response to high glucose.

Gestational diabetes mellitus is a risk factor for congenital heart defects. Our previous results indicated that a decrease in myocardial cells and an increase in apoptotic cells leads to heart defects under hyperglycemia, but much work remains to elucidate this important mechanism of myocardial cell apoptosis induced by high glucose (HG). In this study, we found that a decrease in GSK3β phosphorylation on Ser9 occurred concomitantly with HG-induced cardiomyocyte apoptosis and in the heart tissues of the offspring of diabetic rats in vitro and in vivo. Decreases in GSK3β (Ser9) phosphorylation in response to HG were remarkably restored after treatment with SC79, an activator of the Akt signaling pathway. SB216763, an effective inhibitor of the GSK3β signaling pathway, suppressed HG-induced apoptosis in cardiomyocytes. Further studies showed a decrease in the expression of the anti-apoptotic protein MCL-1 was associated with GSK3β-mediated apoptosis. MCL-1 overexpression partly inhibits HG-induced apoptosis in cardiomyocytes. Herein, this study revealed the roles of GSK3β and MCL-1 in modulating HG-induced cardiomyocyte apoptosis and maternal diabetes-induced abnormalities.

Ulinastatin inhibits high glucose-induced cardiomyocyte apoptosis through activating Akt signaling.

Cardiomyocyte apoptosis is closely associated with the development of diabetic cardiomyopathy. Ulinastatin, a urinary trypsin inhibitor, exerts a protective effect on cardiac function. However, the molecular mechanism remains not fully clear. This study aims to explore the effect of ulinastatin on high glucose (HG)-induced cardiomyocyte apoptosis and the potential molecular mechanism. Neonatal rats cardiomyocytes were cultured and then treated with HG or/and ulinastatin. Cell viability was examined using a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Cell apoptosis was detected by flow cytometry. Mitochondrial membrane potential (MMP) was stained using a JC-1 probe and evaluated by fluorescence microscopy. Protein expressions of B-cell lymphoma 2 (Bcl-2) , BCL2-Associated X (Bax), cleaved caspase 3, p-Akt and Akt were determined by Western blot. Ulinastatin increased the HG-induced reduction in cell viability and MMP expression. Ulinastatin also inhibited HG-induced apoptosis. Ulinastatin decreased the Bax/Bcl-2 ratio and cleaved caspase 3 expression in cardiomyocyte treated with HG. Further, ulinastatin increased the phosphorylation level of Akt in cardiomyocyte treated with HG. These effects of ulinastatin were abrogated by LY294002, an Akt inhibitor. Ulinastatin inhibited HG-induced cardiomyocyte apoptosis through activating Akt signaling.

The RhoA/ROCK pathway mediates high glucose‐induced cardiomyocyte apoptosis via oxidative stress, JNK, and p38MAPK pathways

To understand the roles of the RhoA/ROCK and mitogen-activated protein kinase (MAPK) pathways in high glucose (HG)-induced apoptosis and oxidative stress in cardiomyocytes. Neonatal rat cardiomyocytes were cultured in Dulbecco's modified Eagle's medium, supplemented with 5.5 or 30mmol/L D-glucose, in the presence or absence of fasudil (50 or 100μM), SB203580, SP600125, or PD98059 (10μM, respectively). The percentage of early apoptotic cardiomyocytes was evaluated using flow cytometry. The superoxide dismutase activity and malondialdehyde contents in the cellular supernatants were measured. The Bax and Bcl-2 mRNA levels were determined by quantitative real-time PCR. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1), p38MAPK, JNK, and ERK as well as the protein levels of Bax, Bcl-2, and cleaved caspase-3 was analysed by Western blot. Fasudil, SB203580, and SP600125 effectively inhibited the HG-induced early apoptosis increase and decreased Bax mRNA expression, the Bax/Bcl-2 protein expression ratio, and cleaved caspase-3 protein levels in the cardiomyocytes; this was accompanied by upregulation of the Bcl-2 mRNA. Moreover, fasudil markedly increased the superoxide dismutase activity level and suppressed the elevation in HG-induced malondialdehyde content and the phosphorylation of MYPT1, p38MAPK and JNK. The RhoA/ROCK pathway mediates HG-induced cardiomyocyte apoptosis via oxidative stress and activation of p38MAPK and JNK in neonatal rats in vitro. Fasudil effectively ameliorates HG-induced cardiomyocyte apoptosis by suppressing oxidative stress and the p38MAPK and JNK pathways.

Attenuation of High Glucose-Induced Rat Cardiomyocyte Apoptosis by Exendin-4 via Intervention of HO-1/Nrf-2 and the PI3K/AKT Signaling Pathway

Exendin-4, a glucagon-like peptide-1 receptor agonist, demonstrated cytoprotective actions beyond glycemic control in recent studies. The aims of the present study were to investigate the effects of exendin-4 on high glucose (HG)-induced cardiomyocyte apoptosis and the possible mechanisms. Rat cardiomyocytes were divided into 3 groups: normal glucose group (NG group), HG group and HG +exendin-4 group (HG+Ex Group). Cardiomyocyte apoptosis was evaluated by double-staining with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) and flow cytometry. Intracellular reactive oxygen species (ROS) production was detected by 2’,7’-dichlorodihydrofluorescein diacetate (DCHF-DA) incubation and fluorescence microscopy. LY294002 (LY), a phosphoinositide 3-kinase (PI3K) pathway inhibitor, was added to the medium of the HG+Ex+LY Group for further western blot analysis. The proteins analyzed involved oxidative stress-associated proteins, heme oxygenase-1 (HO-1) and nuclear factor E2-related factor 2 (Nrf-2), and apoptosis-associated proteins, caspase-3, Bax/B-cell lymphoma 2 (Bcl-2) and p-AKT/AKT. HG treatment induced cardiomyocyte apoptosis (P = 0.00) and clearly upregulated ROS production (P = 0.00); exendin-4 co-incubation also ameliorated cardiomyocyte apoptosis (P = 0.004) and decreased ROS (P = 0.00) level significantly. HO-1 and Nrf-2 protein expression levels decreased significantly in the HG group (P < 0.05), but the levels were elevated by exendin-4 intervention (P < 0.05). Furthermore, exendin-4 attenuated HG-induced higher protein expression, including cleaved caspase-3 and Bax, increased the expression of Bcl-2 protein (P < 0.05). However, these impacts of exendin-4 were counteracted significantly by co-incubation with LY294002. In addition, exendin-4 ameliorated HG-induced p-AKT/AKT lower expression, and this impact was also suppressed by LY294002. Exendin-4 ameliorates HG-induced cardiomyocyte apoptosis, and the mechanisms may involve anti-oxidative stress via the HO-1/Nrf-2 system, as well as intervention of the PI3K/AKT signaling pathway.

Poly(ADP-ribose) polymerase 1 inhibition protects cardiomyocytes from inflammation and apoptosis in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is characterized by structural alterations such as cardiomyocyte hypertrophy, necrosis and focal fibrosis. Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme which can be activated by DNA damage and plays a critical role in various diseases. We hypothesized that PARP-1 may play an important role in DCM and that its inhibition may protect cardiomyocytes from inflammation and apoptosis in DCM. H9c2 cardiomyocytes were treated with normal glucose, mannitol or high glucose (HG). Male C57BL/6 mice or PARP-1−/− mice were treated with streptozotocin (STZ) by intraperitoneal injection for 5 consecutive days to induce diabetes. In vitro, HG stimulation induced oxidative stress and DNA damage and increased PARP-1 expression and activity. Compared with the control, pretreatment with PARP-1 siRNA significantly reduced HG-induced inflammatory response, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 secretion, and intercellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS) expression. PARP-1 inhibition reduced HG-induced cardiomyocyte apoptosis through downregulation of cleaved caspases and activation of IGF-1R/Akt pathway. In vivo, hyperglycemia increased the protein expression of nitrotyrosine and PARP-1 as well as PARP-1 activity. PARP-1 gene deletion significantly improved cardiac dysfunction and reduced inflammatory response and apoptosis. This work demonstrated the critical role of PARP-1 in diabetic heart injury, and suggested that PARP-1 inhibition may be a feasible strategy for the treatment of DCM.

HMGB1 mediates hyperglycaemia-induced cardiomyocyte apoptosis via ERK/Ets-1 signalling pathway.

Apoptosis is a key event involved in diabetic cardiomyopathy. The expression of high mobility group box 1 protein (HMGB1) is up-regulated in diabetic mice. However, the molecular mechanism of high glucose (HG)-induced cardiomyocyte apoptosis remains obscure. We aimed to determine the role of HMGB1 in HG-induced apoptosis of cardiomyocytes. Treating neonatal primary cardiomyocytes with HG increased cell apoptosis, which was accompanied by elevated levels of HMGB1. Inhibition of HMGB1 by short-hairpin RNA significantly decreased HG-induced cell apoptosis by reducing caspase-3 activation and ratio of Bcl2-associated X protein to B-cell lymphoma/leukemia-2 (bax/bcl-2). Furthermore, HG activated E26 transformation-specific sequence-1 (Ets-1), and HMGB1 inhibition attenuated HG-induced activation of Ets-1 via extracellular signal-regulated kinase 1/2 (ERK1/2) signalling. In addition, inhibition of Ets-1 significantly decreased HG-induced cardiomyocyte apoptosis. Similar results were observed in streptozotocin-treated diabetic mice. Inhibition of HMGB1 by short-hairpin RNA markedly decreased myocardial cell apoptosis and activation of ERK and Ets-1 in diabetic mice. In conclusion, inhibition of HMGB1 may protect against hyperglycaemia-induced cardiomyocyte apoptosis by down-regulating ERK-dependent activation of Ets-1.

Retinoic acid protects cardiomyocytes from high glucose‐induced apoptosis through inhibition of NF‐κB signaling Pathway

We have previously shown that retinoic acid (RA) has protective effects on high glucose (HG)-induced cardiomyocyte apoptosis. To further elucidate the molecular mechanisms of RA effects, we determined the interaction between nuclear factor (NF)-κB and RA signaling. HG induced a sustained phosphorylation of IKK/IκBα and transcriptional activation of NF-κB in cardiomyocytes. Activated NF-κB signaling has an important role in HG-induced cardiomyocyte apoptosis and gene expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein-1 (MCP-1). All-trans RA (ATRA) and LGD1069, through activation of RAR/RXR-mediated signaling, inhibited the HG-mediated effects in cardiomyocytes. The inhibitory effect of RA on NF-κB activation was mediated through inhibition of IKK/IκBα phosphorylation. ATRA and LGD1069 treatment promoted protein phosphatase 2A (PP2A) activity, which was significantly suppressed by HG stimulation. The RA effects on IKK and IκBα were blocked by okadaic acid or silencing the expression of PP2Ac-subunit, indicating that the inhibitory effect of RA on NF-κB is regulated through activation of PP2A and subsequent dephosphorylation of IKK/IκBα. Moreover, ATRA and LGD1069 reversed the decreased PP2A activity and inhibited the activation of IKK/IκBα and gene expression of MCP-1, IL-6, and TNF-α in the hearts of Zucker diabetic fatty rats. In summary, our findings suggest that the suppressed activation of PP2A contributed to sustained activation of NF-κB in HG-stimulated cardiomyocytes; and that the protective effect of RA on hyperglycemia-induced cardiomyocyte apoptosis and inflammatory responses is partially regulated through activation of PP2A and suppression of NF-κB-mediated signaling and downstream targets.

High Glucose‐induced repression of RAR/RXR in cardiomyocytes is mediated through oxidative stress/JNK signaling

The biological actions of retinoids are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We have recently reported that decreased expression of RARα and RXRα has an important role in high glucose (HG)-induced cardiomyocyte apoptosis. However, the regulatory mechanisms of HG effects on RARα and RXRα remain unclear. Using neonatal cardiomyocytes, we found that ligand-induced promoter activity of RAR and RXR was significantly suppressed by HG. HG promoted protein destabilization and serine-phosphorylation of RARα and RXRα. Proteasome inhibitor MG132 blocked the inhibitory effect of HG on RARα and RXRα. Inhibition of intracellular reactive oxidative species (ROS) abolished the HG effect. In contrast, H(2)O(2) stimulation suppressed the expression and ligand-induced promoter activity of RARα and RXRα. HG promoted phosphorylation of ERK1/2, JNK and p38 MAP kinases, which was abrogated by an ROS inhibitor. Inhibition of JNK, but not ERK and p38 activity, reversed HG effects on RARα and RXRα. Activation of JNK by over expressing MKK7 and MEKK1, resulted in significant downregulation of RARα and RXRα. Ligand-induced promoter activity of RARα and RXRα was also suppressed by overexpression of MEKK1. HG-induced cardiomyocyte apoptosis was potentiated by activation of JNK, and prevented by all-trans retinoic acid and inhibition of JNK. Silencing the expression of RARα and RXRα activated the JNK pathway. In conclusion, HG-induced oxidative stress and activation of the JNK pathway negatively regulated expression/activation of RAR and RXR. The impaired RAR/RXR signaling and oxidative stress/JNK pathway forms a vicious circle, which significantly contributes to hyperglycemia induced cardiomyocyte apoptosis.