| Objective:To observe the protective effect of cyclovirobuxine D (CVB-D) on the heart failure rats, to clear the influence of the drug on the calcium transient in the myocytes derived from adult rats and neonatal rats, to discover its effect on the expression of the four calcium cycling pathway proteins and their genes:LTCC, RYR2, SERCA2a and NCX. The results of the studies would show the experimental base for the clinic application of CVB-D.Methods:The studies included four parts.1. Heart failure rats were induced by left coronary artery occlusion and verified usingechocardiography30days later (EF≤45%). The heart failure rats were divided into five groups and were orally treated with Cyclovirobuxine D (0.5,1.0and2.0mg/kg), verapamil, or vehicle respectively for30days. Thirty days later, the following indices of the rats were detected:the appearance, mortality, M-mode echocardiography parameters, spectral doppler echocardiography parameters, hemodynamics, microcirculation, left ventricular microcirculation, ANP, AngII and ET content in plasma, left ventricular mass (LVM), heart mass (HM), left ventricular mass index (LVMI), heart mass (HM), histology examination and scoring, collagen examination and interstitial collagen volume fraction (ICVF), ultrastructure assessments, Na+-K+-ATPase and Ca2+-Mg2+-ATPase in heart tissue. The indices above might define the protective effect of the drug and supply the relationship between the dose and effect.2. The establish of rats heart failure model, grouping and administration method were equal with above. Treated with drugs for30days, the myocytes were isolated and the the content of calcium was detected by laser confocal technology. Calcium transient, Tpeak<and Texhaustion were obtained.3. The neonatal rat cardiomyocytes were primarily cultured and the calcium content in the cells were detected by laser confocal technology. The extent of calcium transient, velocity reaching the peak, Tpeak, Texhaustion and the velocity of calcium exhaustion were seemed as target indices. The cells were randomly divided into4groups, after determination the calcium transient of spontaneous activity, diltiazem (10umol/L), ruthenium red (10umol/L), NiCl2(5mol/L) and caffeine (10mol/L) were added into the cell culture dishes respectively and the calcium transient was detected. After CVB-D (10mol/L) was added into the dishes, the calcium transient was detected once again. The indices above might define the effect of CVB-D on the function of calcium cycling protein.4. Heart failure rats were induced by left coronary artery occlusion. The heart failure rats were divided into four groups and were orally treated with Cyclovirobuxine D (1.0and2.0mg/kg), verapamil, or vehicle respectively for30days. At the same time, the sham group was treated by vehicle. Each group comprised8rats. Thirty days later, the expression of LTCC, RYR2, SERCA2a and NCX in the myocyte were detected by immunohistofluorescence, western blot and the related genes were detected by RT-PCR. The changes of the expression of these proteins might explain the protective effect of CVB-D on HF derived from cardiac ischemia.Result:1. Comparing with sham group, the survival rate, LVPWd, IVS, EF, LVFS, CO, SV, CI, A peak, LVSP,±p/dtmax, LV microcirculation, Na+-K+-ATPase and Ca2+-Mg2+-ATPase in heart tissue decreased in vehicle group (P<0.05,0.01), while LVIDs, LVESV, HR, E peak, E/A, HR, LVEDP, t-dp/dtmax, ejection time, the content of ANP, Ang II and ET in plasma, LVM, HM, LVMI, HMI increased obviously in vehicle group (P<0.05,0.01). The HE stain of the vehicle group displayed the myocardial hypertrophy, derangement of muscle fibers, disorder of the myocardial cells and increase of histology scoring (P<0.05,0.01). In masson stain, there was no collagen tissue in sham group, while the death of myocardial cell, appearance of collagen fibers and increase of ICVF were shown in vehicle group (P<0.05,0.01). In ultrastructure assessments, the vehicle group showed significant disorganization and cardiac muscle disarray, rupture of the mitochondria and stretching of the intercalated disks of myocardial cells. These changes in vehicle group showed the formation of the heart failure model. Three doses of CVB-D (0.5.1.0and2.0mg/kg) decreased LVIDs, LVESV, HR, E/A, LVM, HM, LVMI, HMI, ANP in plasma and increased CO. CI. SV. EF. LVFS. Ca2+-Mg2+-ATPase in heart tissue (vs model group, P<0.05,0.01). CVB-D of1.0mg/kg and2.0mg/kg might decrease E peak, the content of Ang II and ET in plasma and increase the activity of Na+-K+-ATPase (vs model group, P<0.05,0.01). CVB-D of2.0mg/kg would increase A peak, SAP, LVSP,±dp/dtmax and LV microcirculation, decrease HR, LVEDP, t-dp/dtmax and ejection time (vs model group, P<0.05,0.01). HE stain indicated that CVB-D treating groups showed muscle fibers arranged in an orderly, mild myocardial hypertrophy, interstitial infiltration of inflammatory cells, mild interstitial fibrous tissue hyperplasia, normal stain and decrease in pathological score (vs model group, P<0.05,0.01). Masson staining showed CVB-D could significantly increase the myocardial cells, arrange the cells in orderly rules, reduce collagen fibers significantly and reduce ICVF significant ly. In ultrastructure examination, CVB-D groups showed the normalization of the myocardial fibers, arrangement in an order, the improvement of the shape of the nucleus, reduce of mitochondria vacuoles and the structure of cell organelles close to normal.2. In the determination of [Ca2+]i in acutely isolated cardiomyocytes from HF adult rats, the CVB-D (0.5,1.0and2.0mg/kg) could increase the extend of calcium transient, shorten the Tpeak and Texhaustion(v5model group, P<O.05,0.01).3. In the determination of [Ca2+]i in the cardiomyocytes from neonatal rats, the extent of calcium transient and the velocity reaching the peak decreased obviously, Texhaustion was shortened (vs spontaneous activity, P<0.05,0.01) after ruthenium red blocked the RYR2channel and the calcium transient parameters showed no change with the treatment of CVB-D (vs ruthenium red treatment, P>0.05). After the diltiazem blocked LTCC, the extent of calcium transient decreased and Texhaustion was shortened (vs spontaneous activity, P<0.01), and the extent of calcium transient and velocity reaching the peak increased significantly with the treatment of CVB-D (vs ruthenium red treatment, P<0.01). After the NiCl2blocked NCX, Texhaustion was prolonged and the velocity of calcium exhaustion slowed down (vs spontaneous activity, P<0.05,0.01) and extent of calcium transient, velocity reaching the peak and the velocity of calcium exhaustion increased markedly with the treatment of CVB-D (vs ruthenium red treatment, P<0.01). In addition, there was a trend to be prolonged on Texhaustion-After caffeine blocked SERCA2a, the extent of calcium transient, velocity reaching the peak and the velocity of calcium exhaustion increased markedly, Tpeak and Texhaustion were prolonged (vs spontaneous activity, P<0.05). With the treatment of CVB-D, Tpeak was further prolonged (vs ruthenium red treatment, P<0.05).4. In vehicle group, the expression of RYR2and SERCA2a in the myocyte decreased significantly vs sham group in western blot and RT-PCR detection. In addition, the expression of NCX increased significantly vs sham group in western blot and RT-PCR detection (P<0.05,0.01). CVB-D1.0,2.0mg/kg could inhibit the decrease of the expression of RYR2and SERCA2a and the increase of NCX (P<0.05,0.01). Yet, there was no significant change in the expression of LTCC in each group.Conclusion:For heart failure derived from cardiac ischemia, CVB-D might adjust the effect of the last three proteins of LTCC^RYR2> SERCA2a> NCX, which ensured that there was enough calcium to be released in myocardial contraction period and that the calcium was transported into the SR to decrease the content of calcium in cytoplasm in myocardial diastolic period. It might help the cycle and utilization of the intracellar calcium, prevent the loss of calcium and, ultimately, improve the systolic and diastolic function of the falling heart. The improvement of CVB-D was beneficial not only to the heart ejection function, but also to the myocyte itself. |
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