| Object:Because of less research on the cardiac sarcoplasmic reticulum calcium cycle produced by CGP37157, as specific mitochondrial Sodium-Calcium exchanger (mNCX), it is employed to investigate its influence on ventricular myocyte calcium cycling.Methods:(1)The measurement of cardiac ATP content: 32 SD rats were randomly divided into four groups. After perfusing heart with 60μmol/L calcium Tyrode's solution(control group) and CGP37157-Tyrode's solution(experiment group), a few cardiac tissue is chosen to measure the ATP content in different groups with ATP Assay Kit.(2)Morphological observation: the former steps like rats groups and perfusion follows (1), then a ventricular tissue is taken away and used as transmission electron microscope(TEM) experiments.(3)The measurement of cAMP content: the former steps like rats groups and perfusion follows (1), a few ventricular tissue is used to measure cAMP content in different groups with enzyme linked immunoassay(ELISA) cAMP Kit.(4)Recording diastolic calcium cycling in ventricular myocytes: 20 SD rats were equally and randomly divided into two groups, and isolated ventricular myocytes with collagenase. After incubating myocytes with Fluo-3/AM, Laser scanning confocal microscope(LSCM) is employed to record the calcium transient. (5)Recording calcium transient in systolic ventricular myocytes: the former steps like rats groups and isolation follows (4), after incubation with Fluo-3/AM, whole cell patch-LSCM system chronically records L-type calcium current(ICa,L) and cytosolic calcium transient. (6)statistic analysis: SPSS11.5 is employed and all the data are presented as mean±SEM, and t-test and x~2 test was used for the statistical analysis. P-values of <0.05 were considered significant.Results:(1)Morphological observation with TEM: CGP37157 caused structural changes in ventricular myocytes, leading to the lysis of mitochondrial cristae and the injury was more serious after longer perfusion. (2)ATP content in ventricular tissue: after perfusion with CGP37157, ATP content significantly decreased(0.75±0.01μM/L,0.44±0.02μM/L,0.17±0.01μM/L VS 1.23±0.02μM/L, n=8, P<0.05); 60min group greatly decreased than 30min (0.17±0.01μM/L VS 0.44±0.02μM/L, n=8, P<0.01); (3)cAMP content in ventricular tissue:CGP37157 leads cAMP content increased, the content as follows: 791±35pMol/g,1101±55pMol/g,1525±40pMol/g,2017±60pMol/g; 60 min group cAMP increased significantly(n=8, P<0.05); (4)Diastolic calcium cycling in ventricular myocytes:calcium efflux was significantly increased in experiment group and control group(5.02±0.015 VS 1.01±0.006, n=10, P<0.01); calcium influx was not changed(1.08±0.15VS 0.99±0.09,n=10,P>0.05); calcium storage was decreased(20.2±0.8 VS 0.3±0.3, n=10, P<0.01 ); (5)Systolic calcium cycling in ventricular myocytes: ICa,L was significantly decreased in experiment group than in control group(2.02±0.16pA/pF VS 0.69±0.07pA/pF, n=10, P<0.01); the amplitude of calcium transient was significantly decreased (45.5±5.2 VS 23.2±4.8,n=10,P<0.01 );Conclusions:CGP37157 caused abnormal ventricular calcium cycling, which may due to mitochondrial injury and is associated with abnormal PKA signal pathway.(1) CGP37157 caused mitochondrial injury, with damaged structure and decreased ATP production;(2) CGP37157-mediated mitochondrial injury caused abnormal diastolic calcium cycling, along with increased calcium efflux;(3) CGP37157 leads increased cAMP in ventricular myocytes, and then activated PKA activity, it might lead to systolic calcium leak;(4) CGP37157 caused decreased current density of ICa,L, and thus leaded to lower calcium transient amplitude.
|
PDF Full Text Request