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Aims/introduction: Angiotensin-(1-7) (Ang-[1-7]), recognized as a new bioactive peptide in the renin-angiotensin system, shows biological and pharmacological properties in diabetic cardiovascular diseases. The leptin-induced p38 mitogen-activated protein kinase (MAPK) pathway has been reported to contribute to high glucose (HG)-induced injury. In the present study, we showed the mechanism of how Ang-(1-7) can protect against HG-stimulated injuries in H9c2 cells., Materials and Methods: H9c2 cells were treated with 35 mmol/L glucose (HG) for 24 h to establish a model of HG-induced damage. Apoptotic cells were observed by Hoechst 33258 staining. Cell viability was analyzed by cell counter kit-8. The expression of protein was detected by western blot. Reactive oxygen species was tested by 2',7'-dichlorodihydrofluorescein diacetate staining. Mitochondrial membrane potential was measured by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide staining., Results: The present results showed that treating H9c2 cells with HG obviously enhanced the expressions of both the leptin and phosphorylated (p)-MAPK pathway. However, the overexpression levels of leptin and p-p38 MAPK/p-extracellular signal-regulated protein kinase 1/2 (ERK1/2), but not p-c-Jun N-terminal kinase, were significantly suppressed by treatment of the cells with Ang-(1-7). Additionally, leptin antagonist also markedly suppressed the overexpressions of p38 and ERK1/2 induced by HG, whereas leptin antagonist had no influence on the overexpression of c-Jun N-terminal kinase. More remarkable, Ang-(1-7), leptin antagonist, SB203580 or SP600125, respectively, significantly inhibited the injuries induced by HG, such as the increased cell viability, decreased apoptotic rate, reduction of ROS production and increased mitochondrial membrane potential. Furthermore, the overexpressions of p38 MAPK, ERK1/2 and leptin were suppressed by N-actyl-L-cystine., Conclusions: The present findings show that Ang-(1-7) protects from HG-stimulated damage as an inhibitor of the reactive oxygen species-leptin-p38 MAPK/ERK1/2 pathways, but not the leptin-c-Jun N-terminal kinase pathway in vitro., (© 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Recently, a novel mechanism known as 'programmed necrosis' or necroptosis has been shown to be another important mechanism of cell death in the heart. In this study, we investigated the role of necroptosis in high glucose (HG)-induced injury and inflammation, as well as the underlying mechanisms. In particular, we focused on the interaction between necroptosis and reactive oxygen species (ROS) in H9c2 cardiac cells. Our results demonstrated that the exposure of H9c2 cardiac cells to 35 mM glucose (HG) markedly enhanced the expression level of receptor-interacting protein 3 (RIP3), a kinase which promotes necroptosis. Importantly, co-treatment of the cells with 100 µM necrostatin-1 (a specific inhibitor of necroptosis) and HG for 24 h attenuated not only the increased expression level of RIP3, but also the HG-induced injury and inflammation, as evidenced by an increase in cell viability, a decrease in ROS generation, the attenuation of the dissipation of mitochondrial membrane potential and a decrese in the secretion levels of inflammatory cytokines, i.e., interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Furthermore, treatment of the cells with 1 mM N-acetyl‑L‑cysteine (a scavenger of ROS) for 60 min prior to exposure to HG significantly reduced the HG-induced increase in the RIP3 expression level, as well as the injury and inflammatory response described above. Taken together, the findings of this study clearly demonstrate a novel damage mechanism involving the positive interaction between necroptosis and ROS attributing to HG-induced injury and inflammation in H9c2 cardiac cells.

It has been reported that exogenous hydrogen sulfide (H2S) protects against high glucose (HG)-induced cardiac injury and has a modulatory effect on heat shock protein (HSP) and Akt, which play a cardioprotective role. In this study, we examined whether the HSP90/Akt pathway contributes to the protective effects of exogenous H2S against HG-induced injury to H9c2 cardiac cells. Our results revealed that the exposure of H9c2 cardiac cells to 35 mM glucose (HG) for 1 to 24 h decreased the expression of HSP90 and markedly reduced the expression level of phosphorylated (p)-Akt in a time-dependent manner. Co-exposure of the cells to HG and geldanamycin (GA; an inhibitor of HSP90) aggravated the inhibition of the p-Akt expression level by HG. Of note, treatment of the cells with 400 µM NaHS (a donor of H2S) for 30 min prior to exposure to HG significantly attenuated the HG-induced decrease in the expression levels of both HSP90 and p-Akt, along with inhibition of HG-induced cell injury, as indicated by the increase in cell viability and superoxide dismutase (SOD) activity, and by a decrease in the number of apoptotic cells, reactive oxygen species (ROS) generation, as well as by the decreased dissipation of mitochondrial membrance potential (MMP). Importantly, treatment of the cells with GA or LY294002 (an inhibitor of Akt) prior to exposure to NaHS and HG considerably blocked the cardioprotective effects of NaHS against the HG-induced injury mentioned above. On the whole, the findings of this study demonstrate that the inhibition of the HSP90/Akt pathway may be an important mechanism responsible for HG-induced cardiomyocyte injury. We also provide novel evidence that exogenous H2S protects H9c2 cells against HG-induced injury by activating the HSP90/Akt pathway.

Background/aims: Hyperglycemia activates multiple signaling molecules, including reactive oxygen species (ROS), toll-like receptor 4 (TLR4), receptor-interacting protein 3 (RIP3, a kinase promoting necroptosis), which mediate hyperglycemia-induced cardiac injury. This study explored whether inhibition of ROS-TLR4-necroptosis pathway contributed to the protection of ATP-sensitive K+ (KATP) channel opening against high glucose-induced cardiac injury and inflammation., Methods: H9c2 cardiac cells were treated with 35 mM glucose (HG) to establish a model of HG-induced insults. The expression of RIP3 and TLR4 were tested by western blot. Generation of ROS, cell viability, mitochondrial membrane potential (MMP) and secretion of inflammatory cytokines were measured as injury indexes., Results: HG increased the expression of TLR4 and RIP3. Necrostatin-1 (Nec-1, an inhibitor of necroptosis) or TAK-242 (an inhibitor of TLR4) co-treatment attenuated HG-induced up-regulation of RIP3. Diazoxide (DZ, a mitochondrial KATP channel opener) or pinacidil (Pin, a non-selective KATP channel opener) or N-acetyl-L-cysteine (NAC, a ROS scavenger) pre-treatment blocked the up-regulation of TLR4 and RIP3. Furthermore, pre-treatment with DZ or Pin or NAC, or co-treatment with TAK-242 or Nec-1 attenuated HG-induced a decrease in cell viability, and increases in ROS generation, MMP loss and inflammatory cytokines secretion. However, 5-hydroxy decanoic acid (5-HD, a mitochondrial KATP channel blocker) or glibenclamide (Gli, a non-selective KATP channel blocker) pre-treatment did not aggravate HG-induced injury and inflammation., Conclusion: KATP channel opening protects H9c2 cells against HG-induced injury and inflammation by inhibiting ROS-TLR4-necroptosis pathway., (© 2017 The Author(s)Published by S. Karger AG, Basel.)

Hyperglycemia, as well as diabetes mellitus, has been shown to impair ATP-sensitive K+ (KATP) channels in human vascular smooth muscle cells. Hydrogen sulfide (H2S) is also known to be an opener of KATP channels. We previously demonstrated the cardioprotective effects exerted by H2S against high-glucose (HG, 35 mM glucose)-induced injury in H9c2 cardiac cells. As such, we hypothesized that KATP channels play a role in the cardioprotective effects of H2S against HG-induced injury. In this study, to examine this hypothesis, H9c2 cardiac cells were treated with HG for 24 h to establish a model of HG-induced insults. Our findings revealed that treatment of the cells with HG markedly decreased the expression level of KATP channels. However, the decreased expression of KATP channels was reversed by the treatment of the cells with 400 µM sodium hydrogen sulfide (NaHS, a donor of H2S) for 30 min prior to exposure to HG. Additionally, the HG-induced cardiomyocyte injuries, including cytotoxicity, apoptosis, oxidative stress and mitochondrial damage, were ameliorated by treatment with NaHS or 100 µM diazoxide (a mitochondrial KATP channel opener) or 50 µM pinacidil (a non-selective KATP channel opener) for 30 min prior to exposure to HG, as indicated by an increase in cell viability, as well as a decrease in the number of apoptotic cells, the expression of cleaved caspase-3, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial membrane potential (MMP). Notably, treatment of the H9c2 cardiac cells with 100 µM 5-hydroxydecanoic acid (5-HD, a mitochondrial KATP channel blocker) or 1 mM glibenclamide (Gli, a non-selective KATP channel blocker) for 30 min prior to treatment with NaHS and exposure to HG significantly attenuated the above-mentioned cardioprotective effects exerted by NaHS. Notably, treatment of the cells with 500 µM N-acetyl‑L‑cysteine (NAC, a scavenger of ROS) for 60 min prior to exposure to HG markedly reduced the HG-induced inhibitory effect on the expression of KATP channels. Taken together, our results suggest that KATP channels play an important role in the cardioprotective effects of exogenous H2S against HG-induced injury. This study also provides novel data demonstraring that there is an antagonistic interaction between ROS and KATP channels in HG-exposed H9c2 cardiac cells.

Hyperglycemia has been reported to activate the nuclear factor-κB (NF-κB) pathway. We have previously demonstrated that exogenous hydrogen sulfide (H2S) protects cardiomyocytes against high glucose (HG)-induced injury by inhibiting the activity of p38 mitogen-activated protein kinase (MAPK), which can activate the NF-κB pathway and induce interleukin (IL)-1β production. In the present study, we aimed to investigate the hypothesis that exogenous H2S protects cardiomyocytes against HG-induced injury and inflammation through the inhibition of the NF-κB/IL-1β pathway. H9c2 cardiac cells were treated with 35 mM glucose (HG) for 24 h to establish a model of HG-induced damage. Our results demonstrated that treatment of the cells with 400 µM sodium hydrogen sulfide (NaHS, a donor of H2S) or 100 µM pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB) for 30 min prior to exposure to HG markedly attenuated the HG-induced increase in the expression levels of the phosphorylated (p)-NF-κB p65 subunit. Notably, pre-treatment of the H9c2 cardiac cells with NaHS or PDTC significantly suppressed the HG-induced injury, including cytotoxicity, apoptosis, oxidative stress and mitochondrial insults, as evidenced by an increase in cell viability, as well as a decrease in the number of apoptotic cells, the expression of cleaved caspase-3, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial membrane potential (MMP). In addition, pre-treatment of the cells with NaHS or PDTC ameliorated the HG-induced inflammatory response, leading to a decrease in the levels of IL-1β, IL-6 and tumor necrosis factor-α (TNF-α). Importantly, co-treatment of the H9c2 cells with 20 ng/ml IL-1 receptor antagonist (IL-1Ra) and HG markedly reduced the HG-induced increase in p-NF-κB p65 expression, cytotoxicity, the number of apoptotic cells, as well as the production of TNF-α. In conclusion, the present study presents novel mechanistic evidence that exogenous H2S protects H9c2 cardiac cells against HG-induced inflammation and injury, including cytotoxicity, apoptosis, overproduction of ROS and the dissipation of MMP, by inhibiting the NF-κB/IL-1β pathway. We also provide new data indicating that the positive interaction between the NF-κB pathway and IL-1β is critical in HG-induced injury and inflammation in H9c2 cardiac cells.

Hyperglycemia is a risk factor for the development of diabetic cardiovascular complications, which are associated with the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we demonstrate the inhibitory effects of exogenous hydrogen sulfide (H₂S) on the activation of the MAPK pathway. The aim of the present study was to determine whether exogenous H₂S prevents high glucose (HG)-induced injury by inhibiting the activation of the p38 MAPK and extracellular signal-regulated kinase (ERK)1/2 (members of MAPK) pathways in cardiomyoblasts (H9c2 cells). The findings of the present study demonstrated that the treatment of H9c2 cells with HG (35 mM glucose) for 24 h not only significantly induced injury, including cytotoxicity, apoptosis, overproduction of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (MMP), but also upregulated the expression levels of phosphorylated (p)-p38 MAPK and p-ERK1/2. The increased expression levels of p-p38 MAPK and p-ERK1/2 were markedly reduced by pre-treatment of the H9c2 cells with 400 µM sodium hydrogen sulfide (NaHS; a donor of H2S) prior to exposure to 35 mM glucose. Importantly, pre-treatment of the cells with 400 µM NaHS or 3 µM SB203580 (a selective inhibitor of p38 MAPK) or 15 µM U0126 (a selective inhibitor of ERK1/2) attenuated the HG-induced cardiomyocyte injury, leading to an increase in cell viability and a decrease in the number of apoptotic cells, preventing ROS generation, as well as the loss of MMP. In addition, pre-treatment of the cells with 1,000 µM N‑acetyl‑L‑cysteine (a ROS scavenger) prior to exposure to HG ameliorated the HG-induced cytotoxicity. Taken together, the data from the present study demonstrate for the first time, to our knowledge, that exogenous H2S exerts a protective effect against HG‑induced injury by inhibiting the activation of the p38 MAPK and ERK1/2 pathways and preventing oxidative stress in H9c2 cells.

We previously demonstrated the protective effect of hydrogen sulfide (H2S) against doxorubicin (DOX)-induced cardiotoxicity through inhibition of endoplasmic reticulum stress. The aim of the present study was to explore the role of p38 mitogen-activated protein kinase (MAPK) in DOX-induced cardiotoxicity and ascertain whether exogenous H2S protects DOX-induced injury by inhibiting p38 MAPK in cardiomyoblasts (H9c2). We observed that exposure of H9c2 cells to 5 µM DOX not only markedly induced injuries, including cytotoxicity, apoptosis, overproduction of reactive oxygen species (ROS) and dissipation of mitochondrial membrane potential (MMP), but also enhanced the expression level of phosphorylated (p)-p38 MAPK. The DOX-induced increase in expression of p-p38 MAPK was significantly attenuated by pretreatment of H9c2 cells with either 400 µM sodium hydrogen sulfide (NaHS) (a donor of H2S) or 1,000 µM N-acetyl-L-cysteine (NAC, an ROS scavenger) prior to exposure to DOX. Pretreatment with either 400 µM NaHS or 3 µM SB203580, a selective inhibitor of p38 MAPK, ameliorated DOX-induced cardiomyocyte injuries, as evidenced by an increase in cell viability, and decreases in the number of apoptotic cells, ROS generation as well as dissipation of MMP. In conclusion, the findings of the present study demonstrated that the activation of p38 MAPK contributes to DOX-induced injuries, including cytotoxicity, apoptosis, mitochondrial damage and oxidative stress in H9c2 cells. We also provide novel evidence that exogenous H2S protects H9c2 cells against DOX-induced cardiotoxicity by inhibition of the p38 MAPK pathway.

Heat shock proteins (HSPs) are critical for adaptation to hypoxia and/or ischemia. Previously, we demonstrated that cobalt chloride (CoCl2), a well-known hypoxia mimetic agent, is an inducer of HSP90. In the present study, we tested the hypothesis that CoCl₂-induced upregulation of HSP90 is able to provide cardioprotection in serum and glucose-deprived H9c2 cardiomyocytes (H9c2 cells). Cell viability was detected using a CCK-8 assay, while HSP90 expression was detected via western blotting. The findings of this study showed that serum and glucose deprivation (SGD) induced significant cytotoxicity, overproduction of reactive oxygen species (ROS) and a loss of mitochondrial membrane potential (MMP) in H9c2 cells. In addition, SGD downregulated the expression of HSP90 in a time-dependent manner. The selective inhibitor of HSP90 17-allylamino-17-demethoxygeldanamycin (17-AAG) aggravated SGD-induced cytotoxicity. CoCl₂ at 100 µM time-dependently enhanced the expression of HSP90. Treatment with CoCl₂ from 50 to 200 µM significantly attenuated cytotoxicity and the downregulation of HSP90 expression induced by SGD for 24 h, respectively. Notably, pretreatment of H9c2 cells with 17-AAG at 2 µM for 60 min before exposure to both CoCl2 (100 µM) and SGD significantly blocked the CoCl2-induced cardioprotective effect, demonstrated by decreased cell viability and MMP loss, as well as increased ROS generation. Taken together, these results suggest that HSP90 may be one of the endogenous defensive mechanisms for resisting ischemia-like injury in H9c2 cells, and that HSP90 plays an important role in chemical hypoxia-induced cardioprotection against SGD-induced injury by its antioxidation and preservation of mitochondrial function.

The present study evaluated potential mechanisms of hydrogen sulfide (H2S)-mediated cardioprotection using an in vitro chemical hypoxia-induced injury model. We have demonstrated that H2S protects H9c2 cardiomyoblasts (H9c2) against chemical hypoxia-induced injuries by suppressing oxidative stress and preserving mitochondrial function. The aim of this study was to investigate the role of heat shock protein 90 (HSP90) in cardioprotection of H2S in H9c2 cells. The findings of the present study showed that cobalt chloride (CoCl2), a chemical hypoxia agent, significantly enhanced the expression of HSP90 and that 17-allylamino-17-demethoxy geldanamycin (17-AAG), a selective inhibitor of HSP90, aggravated concentration-dependent cytotoxicity induced by CoCl2. Exogenous administration of NaHS (a donor of H2S) augmented not only HSP90 expression under normal conditions, but also CoCl2-induced overexpression of HSP90. Pre-treatment with 17-AAG significantly blocked the cardioprotection of H2S against CoCl2-induced injuries, leading to increases in cytotoxicity and apoptotic cells. Furthermore, pre-treatment with 17-AAG also antagonized the inhibitory effects of NaHS on overproduction of reactive oxygen species (ROS), a loss of mitochondrial membrane potential (MMP) and ATP depletion induced by CoCl2. In conclusion, these results demonstrate that the increased expression of HSP90 may be one of the endogenous defensive mechanisms for resisting chemical hypoxia-induced injury in H9c2 cells. We also provide novel evidence that HSP90 mediates the cardioprotection of H2S against CoCl2-induced injuries by its antioxidant effect and preservation of mitochondrial function in H9c2 cells.