Inhibitory Effect Of Zacopride On AngiotensinⅡ-induced Cardiac Remodeling

Objective:To investigate the effect of inward rectifier potassium channels(I_K1)agonist zacopride（Zac）on angiotensinⅡ（AngⅡ）-induced cardiac remodeling and explore the underlying mechanism.MethodsThe ventricular cardiomyocytes（CMs）and cardiacfibroblasts（CFb）from neonatal SD rats were isolated and cultured by tissue digestion and differential adherence methods.The activation model of rat cardiomyocytes and cardiac fibroblast induced by angiotensinⅡwere established.The cells were randomly divided into control group,AngⅡ（1μmol/L,24h）model group,AngⅡ+Zac（1μmol/L,24h）group,AngⅡ+Zac+BaCl₂（1μmol/L,24h）group,AngⅡ+Zac+chloroquine（Chlo,0.3μmol/L,24h）group and AngⅡ+captopril（Cap,10μmol/L,24h）group.BaCl₂ and chloroquine are applied as I_K11 atagonists,respectively.Captopril is introduced as anti-remodeling positive control.The surface area and intracellular Ca²⁺concentration of cardiomyocytes were detected by laser confocal microscopy.The protein expression of Kir2.1（inward rectifier potassium channel protein）and cleaved caspase-3 was determined by Western blot.The changes in the number of cells in each group were observed under the microscope.CCK8method was used to detect the effect of zacopride on the cell activity of CFb.The amount of collagen secreted by CFb was determined by ELISA.The apoptosis of the CFb was analyzed by flow cytometry.Results:Part 1 Zacopride inhibited AngⅡ-induced cardiomyocyte hypertrophy and apoptosis1.Compared with the AngⅡgroup,zacopride treatment obviously restored the cardiomyocyte morphology and intracellular Ca²⁺concentration（P<0.05）.Low-dose BaCl₂ and chloroquine as an I_K11 blocker reversed the effect of zacopride（P<0.05）.2.Western blot results showed that the expression of Kir2.1 protein in ventricular myocytes up-regulated after zacopride intervention（P<0.05）,and the expression of activated caspase-3 down-regulated.Selective blockade of I_K11 by BaCl₂ or chloroquine eliminated the effects of zacopride（P<0.05）.Part 2 Zacopride inhibited AngⅡ-induced proliferation and activation of cardiac fibroblasts1.Compared with control group,the fibroblasts in AngⅡgroup were significantly proliferated,flame-shaped growing.The cell activity of the CFb and collagen synthesis were significantly increased（P<0.05）.Compared with AngⅡmodel group,zacopride treatment reduced the number of cells,inhibited the cell activity and collagen synthesis of the CFb.I_K11 blockers BaCl₂ and chlo reversed the effect of zacopride.2.Compared with the control group and AngⅡgroup,zacopride treatment induced apoptosis of cardiac fibroblasts,and the effects of zacopride could be blocked by low-dose BaCl₂ and Chloroquine.3.Western blot results showed that compared with the control group,AngⅡsignificantly inhibited Kir2.1 expression,and zacopride treatment up-regulated Kir2.1expression.Application of low-dose BaCl₂ and Chlo blocked the above effects of zacopride.Captopril didn’t enhance Kir2.1 expression in AngⅡ-incubated cardiac fibroblasts.Conclusion:Via enhancing I_K1（Kir2.1）expression,zacopride attenuated AngⅡ-induced intracellularcalcium-overloadandrepressedapoptosisandhypertrophyin cardiomyocytes.Via enhancing I_K1（Kir2.1）expression,zacopride inhibited AngⅡ-induced the proliferation and activation of fibroblasts,and facilitates apoptosis.These may be the main mechanisms of zacopride against ventricular remodeling.