| Objective: To investigate the change of extracellular and/or intracellular chloride concentration on primary cultured rat ventricular myocytes subjected to A/R injury. Methods: We performed 180 minutes simulated ischemia in cultured neonatal SD rat ventricular myocytes, followed by 120 minutes simulated reperfusion. Cultured myocardial cells of neonatal SD rats have been randomly divided into fourteen groups: Control group, Cl--free control group, DIDS control group, SITS control group, Ca2+-free control group, A/R group, Cl--free group, DIDS group, SITS group, Ver group, Cl--free+SITS group, Ver+ Cl--free group, Ca2+-free group, Ca2+-free+Cl--free group. Myocardial cells beating rate were counted under inverted microscope, cell viability and the activity of LDH in culture were measured at the end of experiment. The morphology or ultrastructure assessment of ventricular myocytes was observed by inverted microscope or transmission electron microscope, respectively. Results: ①The beating rate (98±16 beat·min-1)of ventricular myocytes in Control group was consistent and regular, cell viability(92.2±5.6%)and LDH activity(2.17±1.47 IU·L-1)was considered normal, control myocytes had well-preserved ultrastructure. Mitochondria were elongated or oval in shape, mitochondria membrane was intact and clear.②There was no significant about cell beating rate, cell viability and LDH activity in Cl--free Control, DIDS Control, SITS Control or Ca2+-free Control group (p>0.05), compared with that of Control group. ③the beating rate(13±3 beat·min-1) became significantly slow, myocytes beating rhythm became obviously inconsistent, few of myocytes no longer beat spontaneously. the cell viability (38±3.6%) decreased markedly and LDH activity (107.0±24.8 IU·L-1) increased sharply in A/R group compared with that of Control group(p<0.01), A/R myocytes had showed that most of the mitochondria were markedly abnormal in shape and greater in size, with abnormal cristae or areas of loss of matrix. In some mitochondria, the cristae and matrix was cleared out.③The cell beating rate (21±5 beat·min-1), the cell viability (44.2±3.2%)and LDH release (89.0±23.3 IU·L-1) in DIDS group were similar to those of A/R group (p>0.05).The change of mito- chondria in DIDS group was similar to that of A/R group. ④the beating rates of myocytes in Cl--free (77±10 beat·min-1), Cl--free+Ver (88±10 beat·min-1), Ver (74±9 beat·min-1), SITS (95±10 beat·min-1), Ca2+-free(94±10 beat·min-1),Ca2+-free+Cl--free(94±10 beat·min-1)and Cl--free+SITS(95±10 beat·min-1) was similar to each other(p>0.05),The beating rate in above seven groups was significantly increased ,Compared with that of A/R(p<0.05 or p<0.01).The cell viability of Cl--free(60.8±5.2%),Cl--free+Ver(74.8±6.3%),SITS(68.2±6.6%),Ver(65.2±5.3%), Ca2+-free(74.2±5.3%),Ca2+-free+Cl--free(85.3±13.5%)or SITS+ Cl--free (78.3±12.5%)increased significantly ,compared with that of A/R (p<0.05 or p<0.01),respectively, LDH activity of Cl--free(41.8±13.2 IU·L-1),Cl--free+Ver(20.7±6.7IU·L-1),SITS(14.0±2.6 IU·L-1) ,Ver(31.8±8.5 IU·L-1) ,Ca2+-free(22.6±5.4 IU·L-1),Ca2+-free+Cl--free(12.0±5.5 IU·L-1)or SITS+Cl--free(15.2±2.6 IU·L-1 )significantly decreased, compared with A/R respectively(p<0.05 or p<0.01),The cellular structures were extremely well preserved in SITS, Cl--free or SITS+Cl--free cells subjected to the A/R. ⑤The cell viability of Cl--free +Ver ,Ca2+-free+ Cl--free or SITS+ Cl--free increased significantly, compared with Cl--free(p<0.05),LDH activity of Cl--free +Ver,Ca2+-free+ Cl--free or SITS+ Cl--free decreased significantly compared with Cl--free(p<0.05).⑥the cell viability in Ca2+-free+ Cl--free group increased, compared with that of Ca2+-free(p<0.05). the cell viability in Cl--free+Ver group increased, compared with that of Ver(p<0.05).the cell viability in Cl--free+SITS group was increased, compared with that of SITS(p<0.05).LDH activity in Ca2+-free+ Cl--free group declined, compared with that of Ca2+-free(p<0.05)... |
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