Amelioration Effects Of CVB-D On Aldosterone-Induced Neonatal Rat Cardiac Myocytes Injury By Inhibiting Calcium Overload-Mitochondria Dysfunction Pathway

Objective:To investigate the regulatory mechanism of Ca²⁺-mitochondrial dysfuntion during aldosterone（ALD）induced neonatal rat cardiac myocytes（MRCMs）injury.To clarify the amelioration effect of CVB-D on ALD induced NRCMs injury,and to explore the mechanism is related to the inhibiting of Ca²⁺overload-mitochondria dysfunction.Methods:The heart tissue of 1-2 d SD rats digested with 0.08%trypsin,and the NRCMs was obtained for primary culture after1.5 h differential adherent,and the proliferation of non-cardiac cells was inhibited by5-Brdu.The growth state of NRCMs was observed by microscope and the changes of cell morphology were recorded by taking pictures.Immunocytochemistry was used to observe the cell number of the specific marker of cardiac troponin（cTnT）and calculate the cell purity.The optimal dose-effect and time-effect of ALD induced NRCMs injury were determined by MTT,and the effects of different dose of CVB-D,spirolactone（Spiro）,Intracellular calcium chelating agent（BAPTA-AM）and Bongkreic acid（BA）on ALD induced NRCMs injury were also determined by MTT.The experiment was divided into 8 groups:Control group（Control,no serum DMEM）,ALD group（ALD10μM）,Sprio group（Sprio 0.1μM+ALD 10μM）,CVB-D low dose group（CVB-D 0.1μM+ALD 10μM）,CVB-D high dose group（CVB-D H,CVB-D 0.5μM+ALD 10μM）,BA group（BA 0.5μM+ALD 10μM）,BAPTA-AM group（BAPTA-AM 0.05μM+ALD 10μM）,CVB H+BAPTA-AM group（CVB-D 0.5μM+BAPTA-AM 0.05μM+ALD 10μM）.NRCMs apoptosis were determined by Annexin V-FITC/PI.The pathological morphological changes of NRCMs were observed by Giemsa staining.The content of GSH and MDA,the activity of SOD and ATPase and the extracellular activity of LDH in NRCMs were determined by the commercial kit.The intracellular calcium,mitochondrial membrane potential（ΔΨm）and intracellular reactive oxygen species（ROS）were detected by fluorescent probes Flou-3 AM,JC-1 and DCFH-DA in NRCMs,respectively.The opening of mPTP was detected by Calcien AM/CoCl₂.Western blotting was used to detect the expressions of apoptosis pathway-associated proteins Bcl₂ and Bax,the mitochondrial protein Drp1 and Mfn1 expressions,and the autophagy pathway-associated proteins LC3-I/LC3-II and P62 expressions.Results:The isolated and purified NRCMs began to appear cluster and synchronous beated after primary cultured 72 h.The purity of NRCMs was 95%by immunocytochemistry.The results of MTT and Annexin V-FITC/PI showed that the cell survival rate was significantly reduced after ALD treated NRCMs for 24 h,while the cell survival rate was increased after pretreatment with CVB-D and Spiro.The results of LDH leakage showed that intracellular LDH leakage was significantly increased after ALD treated NRCMs for 24 h,while intracellular LDH leakage was decreased after pretreatment with CVB-D.Giemsa staining results showed that ALD could induce NRCMs to produce pathological morphology changes including pyknosis,perinuclear vacuoles,and CVB-D and Spiro could significantly attenuat the morphological changes induced ALD.Western blot results confirmed that the ratio of Bcl₂/Bax was significantly reduced after ALD treated NRCMs 24 h.After pretreatment with CVB-D and Spiro,the ratio of Bcl₂/Bax increased significantly.The results of fluorescence probe Flou-3AM,JC-1 and DCFH-DA showed that ALD significantly increased the concentration of Ca²⁺and ROS in NRCM cells,and significantly decreased the mitochondrial membrane potential,and CVB-D,Spiro and BAPTA-AM could significantly inhibit Ca²⁺overload,level of ROS increase and mitochondrial membrane potential decrease induced by ALD,and CVB-D and BAPTA-AM combined could significantly reverse the level of ROS and the change of mitochondrial membrane potential induced by ALD,the effects of no significantly difference with the BAPTA-AM.According to the results of commercial Kit,compared with the Control group,ALD group significantly increased MDA content and decreased GSH content,SOD and ATP activity;CVB-D and Spiro could ameliorate the content of GSH and MDA,the activity of SOD and ATPase induced by ALD in NRCMs,The effect of BAPTA-AM on the ATPase activity induced by ALD was similar to that of CVB-D and Spiro.The results of western blotting showed that after ALD treated NRCMs for 24 h,Drp1 was significantly up-regulated,Mfn1 was significantly down-regulated,P62 was significantly up-regulated,and the ratio of LC-3II/LC3-I was significantly decreased.Compared with ALD group,CVB-D,Spiro and BAPTA-AM significantly down-regulated Drp1,up-regulated Mfn1,down-regulated P62,and significantly increase the ratio of LC-3II/LC3-I.Moreover,there was no significant difference between the effects of CVB-D combined with BAPTA-AM on ALD induced Drp1,Mfn1,P62,LC-3II/LC3-I and the effects of BAPTA-AM.Conclusion:ALD can induce NRCMs injury,and its regulatory mechanism is related to Ca²⁺overload-mediated mitochondrial dysfunction.CVB-D ameliorate ALD induced NRCMs injury by inhibiting Ca²⁺overload-mitochondrial dysfunction pathway。...