The Effects Of Chloride Channel Inhibitor On STS-induced Cardiomyocyte Apoptosis And Signaling Transduction Involved

Cardiomyocyte apoptosis contributes to a variety of heart diseases such as heart failure, myocardial infarction, cardiomyopathy, arrhythmias as well as cardiac remodeling, and is also a major contributor to ischemia-reperfusion injury[1, 2]. As cardiomyocyte is terminal differentiation without differentiation potential, so inhibition of cardiomyocyte apoptosis is emerging as a potential therapeutic strategy for various forms of cardiovascular diseases.Apoptosis is also named as programmed cell death, is a gene-controlled and active process with a variety of morphological and biochemical changes. Ionic homeostasis and cell volume regulation are essential for the survival of cells. The early cell shrinkage is an important morphological characteristic, which was largely ignored in the past. In recent years, apoptotic volume decrease (AVD) was demonstrated playing important roles in cell apoptosis as a perquisite of cell apoptosis. The activated Cl- channels might be a pivotal candidate for AVD and cell apoptosis with Cl- is the most abundant anion in organisms. Both mitochondrion-mediated intrinsic and death receptor-mediated extrinsic apoptosis stimuli have been reported to rapidly activate Cl- conductance in various cell types. Furthermore, apoptotic cell death in a substantive variety of cell types can be rescued by blocking the Cl- currents. However, the underlying mechanisms responsible for the anti-apoptotic effect of Cl- channel inhibition remain obscure. So far the relationship between the PI3-kinase/Akt/eNOS pathway,Bcl-2/Bax and chloride channel inhibition in cell apoptosis has not been previously investigated. Therefore, the aims of the present study were to explore detailed molecular mechanisms involved in chloride channel inhibition in STS-induced cardiomyocyte apoptosis.Aims:1. Primary cultures of neonatal rat cardiomyocytes and apoptosis induction with a potent mitochondrial apoptotic inducer staurosporine (STS), and investigating the effects of chloride channel blockers on cardiomyocyte apoptosis.2. The effects of chloride channel blocker DIDS on cell signaling pathway PI3-kinase/Akt and its downstream molecules eNOS and Nitric oxide (NO) in STS-treated cardiomyocyte apoptosis.3. The effects of chloride channel blocker DIDS on Bcl-2/Bax levels in STS-treated cardiomyocyte apoptosis.Methods:1. STS is a potent mitochondrial apoptotic inducer and is used to induce apoptosis, a parallel group were simultaneously treated with chloride blockers. To further examine the role of the PI3K/Akt pathway, another two groups were pretreated with the specific PI3K inhibitor LY294002 (20 μmol/L) or a non-specific eNOS inhibitor L-NAME (100μmol/L) for 60 min before other additions and kept the same concentration during following studies. Cardiomyocytes cultured in normal medium served as a control group.2. Cell shape of cardiomyocytes was observed under a phase-contrast microscope. To detect nuclear morphological changes accompanying apoptosis, the cultured cardiomyocytes were exposed to Hoechst-33258 and examined under a fluorescence microscope.3. Cell viability was assessed by the colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and caspase-3 activity was assayed using the caspase-3 Assay Kit/Fluorimetric. In addition, cell apoptosis was also confirmd by DNA fragmentation (DNA ladder).4. The effect of chloride channel blockers such as DIDS, NPPB and phloretin on cell viability and apoptosis exposed to STS.5. The related prosurvival signaling pathway PI3-kinase/Akt and eNOS/ NO were determined by Western blotting. Total NO production (NOX)in culture medium was determined by measuring the concentration of nitrite with a modified Griess reaction method.6. Bax translocation was assessed by double immunofluorescence labeling and Western blotting.Results:1. Exposure of cardiomyocytes to STS resulted in a significant decrease in cell viability and a significant increase in caspase-3 activity in a concentration- and time-dependent manner. After treatment with STS (4μmol/L) for 8 h, the cell viability was significantly decreased with a significant increase of caspase-3 activity and obvious DNA ladder formation as compared with control group.The primary neonatal rat cardiomyocyte has a regular morphology and automatism. In contrast, STS treatment induced morphological alterations, such as rounding up of the cells and a loss of cell-cell contacts. Moreover, automatism usually observed in normal cardiomyocytes was markedly weakened or abolished, accompanied by typical apoptotic changes, such as segmented and irregularly shaped nuclei.2. The impact of chloride channel blockers DIDS (250μmol/L), NPPB (100μmol/L), phloretin (30μmol/L) on STS-induced alterations in cardiomyocytes was investigated. STS-induced cell death was significantly reduced (77.4%, 70.1%, 74.4%; all P<0.01 vs. STS) and cell shape changes were largely prevented in the presence of chloride channel blockers. DIDS, NPPB and phloretin prevented the alterations in cellular and nuclear morphology, reduced the activation of caspase-3 by 60% and DNA ladder formation.3. The levels of Akt in cardiomyocytes were consistent in the presence of STS or STS plus DIDS. Akt phosphorylation (p-Akt) has been demonstrated to reflect Akt activation. The levels of p-Akt were not clearly changed after challenging with STS. However, in the presence of DIDS, Akt was significantly activated, resulting in a 2.1-fold increase of p-Akt in STS-treated cardiomyocytes (P<0.01 vs. STS). Time-matched application of DIDS alone (direct application of DIDS to control cells) did not significantly influence p-Akt levels. In addition, the effect of DIDS on Akt activity was completely abolished by treatment with LY294002 (P<0.01 vs. STS+DIDS). Treatment with LY294002 also completely abolished the protective effects of DIDS on STS-induced cardiomyocyte apoptosis. Specifically, cell viability was significantly diminished (32.0±5.3% compared to the control, P<0.01 vs. STS+DIDS). Furthermore, the inhibition of DNA ladder formation and caspase-3 activation were also reversed by LY294002.4. eNOS is a novel substrate for Akt. Phosphorylation of eNOS by Akt and the increased NOx are important for its anti-apoptotic effect. We therefore attempted to determine whether eNOS phosphorylation (p-eNOS) and NOx contribute to the protective effects of DIDS. Cardiomyocytes treated with STS for 8 h, presented a significant decrease in eNOS and p-eNOS levels (51.4%, 43.9% of those in control cells respectively, P<0.01 vs. control). Most importantly, DIDS partly, but significantly reversed the STS-induced reduction in eNOS and p-eNOS (both P<0.01 vs. STS). To explore whether the increases in eNOS and p-eNOS were related to PI3K/Akt pathway, cardiomyocytes were treated with LY294002. The results showed that LY294002 completely abrogated the increase of p-eNOS, and also abolished DIDS-induced increase in eNOS levels (P<0.01 vs. STS+DIDS). In contrast, pretreatment with L-NAME for 1 h before and during the co-application of STS and DIDS, had no effect on eNOS and p-eNOS levels.In line with the enhanced p-eNOS, DIDS also resulted in a marked increase in NOx content, which was effectively blocked by LY294002. Similar to LY294002, L-NAME also completely inhibited the increased NOx. Most interestingly, L-NAME only partly abolished the cytoprotective effects of DIDS, indicating that the protective effect of DIDS is not exclusively related to the enhancement of NOx formation via the PI3K/Akt pathway, but also related to other targets modulated by PI3K/Akt.5. The balance of Bcl-2/Bax levels determines, in part, cell fate to apoptotic stimuli and previous studies have demonstrated that Bax translocation is involved in the process of STS-induced apoptosis. These prompted us first to investigate whether DIDS exerts its anti-apoptotic effect through regulating Bcl-2/Bax levels and our results demonstrated that neither STS nor DIDS altered Bcl-2/Bax levels.6. By double immunofluorescence labeling technique, we found that in normal cardiomyocytes, a diffuse localization of Bax is primarily found in the cytoplasm. In contrast, STS resulted in Bax translocation from cytoplasm to mitochondria as revealed by the localization of Bax at mitochondria visualized by a specific mitochondrial probe. Interestingly, treatment with DIDS significantly inhibited STS-induced Bax translocation. Pretreatment with LY294002 effectively blocked the effects of DIDS and restored the STS-induced Bax translocation.The immunofluorescence findings were confirmed by Western blotting. After exposing cardiomyocytes to STS, the mitochondrial Bax content was increased whereas cytosolic Bax content decreased (P<0.01 vs. control), suggesting the increased Bax translocation. When cells were simultaneously treated with DIDS, the content of Bax in the mitochondria did not significantly increase, and Bax only slightly decreased in the cytosolic fraction compared to the control cells, but significant differences were found as compared with STS-treated cardiomyocytes (P<0.01 vs. STS). The effect of DIDS on Bax translocation was similarly reversed by LY294002.Conclusions:1. Chloride channel blockers such as DIDS, NPPB and phloretin prevented cardiomyocyte apoptosis induced by STS (4μmol/L for 8 h) and improved cell viability. 2. DIDS prevents cell apoptosis of primary neonatal rat cardiomyocytes exposed to STS in a PI3K-Akt-pathway-dependent manner. Phosphorylation of eNOS by Akt with subsequent increase in NOx, partly but significantly contributes to the anti-apoptotic effect of DIDS.3. In addition, we have provided the first evidence that DIDS had no effect on Bcl-2/Bax levels, but significantly inhibited STS-induced Bax translocation from cytosol to mitochondria, which may also be related to PI3K/Akt signaling pathway and the anti-apoptotic effect of DIDS.