The Effect Of Chloride Channel Blocker DIDS In Hyperglycemia Induced Apoptosis In Cardiomyocytes

With the increasing aging population and lifestyle modifications, the incidence ofdiabetes is rising gradually year by year in China. It is demonstrated that the number oftype2diabetes patients was up to92.4million in2010, which has become a major publichealth problem in our society. Epidemiological studies have shown that more than70%ofdiabetes patients died of cardiovascular diseases. The mortality rate of diabetic populationis2to4times more than a non-diabetic population. Thus, cardiovascular complications indiabetes have become the leading causes of death in diabetic patients, consistent with thenotion that diabetes is deemed as an independent risk factor for cardiovascular diseases,which is widely accepted by many experts all over the world. Hyperglycemia isconsidered to be the main reason for the heart and blood vessel injury, and the injury isclosely related to the duration and level of hyperglycemia. Special attention has been paidtowards diabetes research. Nonetheless, hyperglycemia remains as one of the mostimportant initiation factors for cardiovascular injuries in diabetic patients, long-termhyperglycemia can stimulate the activation of RAAS system, increase the generation ofROS, advanced glycation end products (AGEs) and its receptor (RAGE). It can also regulate the activation of inflammatory reaction and endoplasmic reticulum stressresponse, and accelerate the cardiomyocytes and vascular endothelial cells apoptosis andnecrosis. To explore the new mechanism of hyperglycemia injury to cardiomyocytes andblood vessels is of great importance for prevention and treatment for diabetes inducedcardiovascular complications.Chloride channels regulate cardiomyocyte volume to maintain balance state, cellresting membrane potential, and a variety of biological functions such as regulatingintracellular pH, cell proliferation and differentiation, and cell apoptosis. Chloridechannels are also involved in regulation of cell damage, such as the RAAS systemactivation, oxidative stress, endoplasmic reticulum stress, apoptosis, and inflammatoryreactions. Apoptosis-related study reported that: in the inevitable process of apoptosis cellshrinkage, called apoptotic volume decrease (apoptotic volume decrease, AVD),electrophysiological displayed along with the process of potassium, chloride ion currentoutflow, and chloride channel blockers can inhibit apoptosis of damaged cells. Ourprevious studies used STS and tunicamycin-induced myocardial apoptosis model, revealedthat the volume-sensitive chloride channels were involved in the process ofcardiomyocytes damage, and chloride channel blockers played an important role inprotecting cardiomyocytes.However, is the chloride channel also involved in myocardialinjury mechanism related to hyperglycemia? What is the chloride channel blockeraffectBased on this assumption, this study was to establish a hyperglycemia-inducedcardiomyocyte apoptosis model by detecting intracellular chloride ion concentration, cellviability, ROS and related apoptosis, and to explore how chloride channel affects highglucose-induced cardiomyocytes apoptosis, even ROS regulatory mechanism to chlorideion channel.Objectives:(1) To establish high glucose-induced cardiomyocyte apoptotic model in primarycultured neonatal rat and to determine the optimal treatment in both dose and timedependent manner. (2) To observe the effects of high glucose on cardiomyocyte chloride channels byintracellular chloride ion concentration changes and to observe the effect of chloridechannel blockers DIDS in cardiomyocyte apoptosis induced by high glucose in thecardiomyocytes apoptosis model.(3) To explore effect of reactive oxygen species (ROS) on chloride ion channels andeffect of chloride channel blockers DIDS on ROS levels in cardiomyocytes after the highglucose treatment.Methods:(1) Primary neonatal rat cardiomyocytes culture and model establishment:cardiomyocytes was acquired from neonatal SD rats (0-3days) through regular way, thecardiomyocytes purification used differential adherent method, primary cardiomyocyteswas cultured in vitro for48hours. Glucose concentration:5.5,11,22,33,44mmol/L inDMEM medium, different processing time is0,24,48,72and96h, to explore optimalexperimental conditions.(2) Group A: normal control group, mannitol group, high glucose group, high glucose+DIDS group and DIDS group. Cell viability was measured by MTT assay; Intracellularchanges of cell chloride ion concentration was detected by chloride ion fluorescent probe(MQAE).(3) Group B: normal control group, high glucose group, high glucose+DIDS groupand DIDS group. Flow cytometry and TUNEL apoptosis assay kit were used to detect theapoptosis rate in different groups; Western-blot was applied to test the expression ofpro-caspase-3protein; Caspase-3protein activity assay kit was used to detect theactivity of caspase-3protein; Superoxide anion fluorescent probes (DHE) were used todetect ROS.(4) Group C: normal control group, H2O2(adding exogenous ROS) group, highglucose group and high glucose+NAC (ROS scavenger) group. Intracellular changes ofcell chloride ion concentration was detected by chloride ion fluorescent probe (MQAE).Results: (1) Cardiomyocyte apoptosis model conditions: according to the concentrations ofglucose in5.5,11,22,33,44mmol/L in DMEM medium, different processing time is0,24,48,72and96hour,we found that the cardiomyocytes survival in high glucosewasconcentration-and time-dependent lowered, and the difference was statistically significant(P <0.05, n=6). Flow cytometry confirmed cardiomyocyte damage caused by highglucose was mainly through apoptosis process. Tip: glucose33mmol/L; time:72h;myocardial cell viability was73.2%±4.1%, which is the best experimental conditions.(2) The effects of high glucose on cardiomyocyte viability: MTT assay showednormal control group vs mannitol group, the cardiomyocyte survival is at the same level,there was no significant difference (P>0.05n=6); high glucose group compared with thenormal control group, the cardiomyocyte survival is increased significantly, the differencewas statistically significant (P <0.05, n=6); high glucose+DIDS group vs high glucosegroup, the cardiomyocyte survivalis increased significantly, the difference was statisticallysignificant (P <0.05, n=6);(3) Effects of hyperglycemia on intracellular cardiomyocytes chloride ion concentration:MQAE tests showed that normal control group vs mannitol group, the intracellularchloride ion concentration is at the same level, there was no significant difference(P>0.05,n=5); high glucose group vs normal control group, the intracellular chloride ionconcentration was decreased significantly (P <0.05, n=5); high glucose+DIDS group vshigh glucose group, the intracellular chloride concentration ion was increased significantly(P <0.05, n=5).(4) Effects of DIDS on cardiomyocytes apoptosis induced by hyperglycemia: Flowcytometry and TUNEL staining showed that the high glucose group compared withnormal control group, cell apoptosis rate was increased significantly,(P <0.05, n=5); highglucose+DIDS group compared with the high glucose group, cell apoptosis rate wasdecreased significantly, with statistical difference (P <0.05, n=5). Pro-caspase-3proteinexpression in the high glucose group was decreased significantly compared with normalcontrol group (P <0.05, n=5), the high glucose+DIDS group compared with the high glucose group, Pro-caspase-3protein expression was significantly increased (P <0.05,n=5). caspase-3protein activity in the high glucose group was increased significantlycompared with normal control group (P <0.05, n=5), the high glucose+DIDS groupcompared with the high glucose group, Pro-caspase-3protein activity was significantlydecreased(P <0.05, n=5).(5) Effects of ROS on cardiac chloride ion concentration: MQAE tests showed thatH2O2group and high glucose group compared with the control group, the chloride ionconcentration in cardiomyocytes were significantly decreased (P <0.05, n=5). Highglucose+NAC group compared with the high glucose group, the intracellular chlorideconcentration was significantly increased (P <0.05, n=5).(6) DIDS impact on the high glucose-induced ROS levels of cardiomyocyte:Dihydroethidium (DHE) probe test showed that high glucose group compared with thecontrol group, ROS levels was increased significantly in cardiomyocyte: the differencewas statistically significant (P <0.05, n=5); while high glucose+DIDS group comparedwith the high glucose group, a significant reduction of ROS generationin incardiomyocytes, the difference was statistically significant (P <0.05, n=5).Conclusion:(1) High glucose-induced cardiomyocyte apoptosis process is accompanied with theoutflow of intracellular chloride ions, while the chloride channel blockers DIDS inhibitoutflow of chloride ions of damaged cardiomyocyte to maintain the homeostasis ofintracellular environment.(2) Chloride channel blockers DIDS could inhibit the activation of caspase-3, throughthis process it can significantly inhibites high glucose-induced cardiomyocyte apoptosisand protect the myocardium.(3) High glucose stimulates a large number of intracellular ROS generation, whichmediate the activation of chloride channel of the damaged cardiomyocyte, following withthe increasing of outflow of chloride ions; the chloride channel blockers DIDS has an effect on cardiomyocyte by reducing ROS generation, suggesting that there may be a dualdirectional regulation mechanism between ROS and chloride channels.