| Objective:Sophoridine is one of the alkaloids extracted from Sophora alopecuroides L. It has been reported that sophoridine has pharmacologic actions and application perspective on anti-arrhythmia and enhancing cardiac contractility. In this study, the models of chronic heart failure (CHF) were induced by permanent ligation on the left anterior descending coronary artery and sophoridine as intervention agents. We studied abnormal calcium cycle and ultrastructure in cardiomyocytes of CHF rats, and expected to provide background experimental data of sophoridine clinical application.Methods:(1) SD rats were randomly divided into five groups:sham operated rats; chronic heart failure rats; low dose treated group (rats with5mg/kg sophoridine); middle dose treated group (rats with10mg/kg sophoridine), and high dose treated group (rats with20mg/kg sophoridine).(2) CHF models were induced by permanent ligation on the left anterior descending coronary artery, sham operated rats went the same procedure without ligation of the LAD. Four weeks later, hemodynamic and heart-body weight indices were assessed.(3) Cardiomyocytes stained by Fluo-3/AM and Fluo-5N/AM were isolated by an enzymatic dissociation method, Ica·L,Ca2+transients and SR content were recorded by patch-clamp and laser scanning confocal microscope.(4) Ca2+handling proteins SERCA2a,PLB,NCX were determined by western-blot and semiquantitative analysis.(5) Ultrastructures of myocardium were observed by transmission electron microscope.(6) SPSS11.0software was used for the statistical analyses. Datum were expressed as mean±D for all experimental results. Statistical analysis were made with ANOVA and followed by LSD test for individual comparisons of means. P-values of <0.05were considered significant.Results:(1) Compared with sham operated group,±dp/dtmax in CHF group was obviously decreased, and LVEDP and HW/BW were remarkably increased. Compared with CHF group,±dp/dtmax were increased, and LVEDP and HW/BW were decreased markedly in middle and high dose treated group;+dp/dtmax were increased markedly in low dose treated group, but there was no significance in-dp/dtmax between low dose treated and CHF group. (2) Compared with sham, the amplitude of Ica·L was reduced in CHF cardiomyocytes. Compared with CHF group, the amplitude of Ica·L was enhanced in each sophoridine treated group.(3) The incidence of calcium spark and amplitude of Ca2+transients were dramatically reduced in CHF group, and spatial averages of Ca2+transients (ΔF/F0) of CHF group (17.05±1.25, n=12cardiomyocytes) were reduced markedly compared with sham group (49.64±2.68, n=12cardiomyocytes, P<0.01); the incidence of calcium spark and amplitude of Ca2-transients were increased in each sophoridine treated gpoup, and ΔF/F0of low dose treated group (18.96±1.01, n=12cardiomyocytes) were increased compared with CHF group (17.05±1.25, n=12cardiomyocytes, P<0.05), in addition, AF/Fo of middle (45.79±2.52, n=12cardiomyocytes) and high dose treated group (47.21±2.07, n=12cardiomyocytes) were markedly increased compared with CHF group (17.05±1.25, n=12cardiomyocytes, P<0.01).(4) The flourescence of cardiomyocytes stained with Fluo-5N/AM were dramatically reduced in CHF group, and spatial averages of Ca2+transients (ΔF/F0) of CHF group (21.4±2.54, n=12cardiomyocytes) were reduced markedly compared with sham group (56.4±3.01, n=12cardiomyocytes, P<0.01); the flourescence of cardiomyocytes stained with Fluo-5N/AM were increased in each sophoridine treated gpoup, and AF/Fo of low dose treated group (31.1±2.93, n=12cardiomyocytes), middle (53.8±3.38, n=12cardiomyocytes) and high dose treated group (56.2±3.28, n=12cardiomyocytes) were increased remarkably compared with CHF group (21.4±2.54, n=12cardiomyocytes, P<0.01).(5)①The expression of SERCA2a and SERCA2a/GAPDH (0.041±0.003, n=6) was dramatically reduced in CHF group compared with sham group (0.25±0.02, n=6, P<0.01); SERCA2a/GAPDH in middle (0.082±0.006, n=6) and high dose treated group (0.074±0.005, n=6) were dramatically increased compared with CHF group (0.041±0.003, n=6, P<0.01); there was no significance between low dose group (0.042±0.002, n=6) and CHF group.②There was no significance of PLB/GAPDH between CHF group (0.63±0.03, n=6) and sham group (0.66±0.03, n=6), and there was also no significance among CHF and drug treated groups.③There was no significance of NCX/GAPDH between CHF group (0.099±0.004, n=6) and sham group (0.099±0.005, n=6); NCX/GAPDH in low dose treated group (0.083±0.003, n=6) were dramatically decreased compared with CHF group (0.099±0.004, n=6, P<0.01); NCX/GAPDH in high dose treated group (0.116±0.009, n=6) were increased markedly compared with CHF group (0.099±0.004, n=6, P<0.01); there was no significance between middle dose treated group (0.093±0.004, n=6) and CHF group.④Compared with sham group (2.66±0.20, n=6), PLB/SERCA2a in CHF group (15.13±0.94, n=6) was increased markedly (P<0.01); compared with CHF group, PLB/SERCA2a in low dose group (13.85±1.88, n=6) was decreased (P<0.05), in addition, middle (7.69±0.48, n=6) and high dose group (8.57±0.87, n=6) were dramatically decreased (P<0.01).⑤Compared with sham group (0.40±0.03, n=6), NCX/SERCA2a in CHF group (2.40±0.23, n=6) was increased markedly (P<0.01); compared with CHF group, NCX/SERCA2a in low (1.98±0.12, n=6), middle (1.14±0.04, n=6) and high dose group (1.58±0.19, n=6) were dramatically decreased (P<0.01).(6) Effects of sophoridine on ultrastructure of myocardium:myofilaments in myocardium of the sham operated group were lined up in line; structures of mitochondria were intact and cristae were clear. The ultrastructure of myocardium in CHF rats displayed that myofilaments were dissolved, and most of myofilaments were disordered, in addition, some cristae were dissolved and their outlines were vague. In low dose treated group, myofilaments were in good order and the dissolution was recovered slightly. Furthermore, myofilaments in middle dose group ranked regularly, and moreover, structures of mitochondria were complete and most of cristae were clear. In high dose treated group, the dissolution of myofilaments disappeared and ranked tightly, which light band and dark band were distinct, besides, structures of mitochondria were approximately complete.Conclusion:Sophoridine can improve cardiac function, abnormal calcium cycle and myocardium ultrastructures of CHF rats, which can provide theoretical support for clinical application of sophoridine. |
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