Effects of Astragaloside IV on heart failure in rats

MI in Wistar rats was induced by coronary ligation. Through a thoracotomy on the left side, the heart was exteriorized and extensive MI was induced by ligation of the left coronary artery according to Tonnessen et al. [17]. Animals in the sham group were subjected to the same surgical procedure, except that the coronary artery was not ligated. Echocardiography was performed three weeks later in the surviving rats anaesthetized by ether; those with heart failure according to echocardiographic criteria were randomly divided into five groups [18, 19] where each group had ten animals. Group one (model) received NS; group two to four received ASI at 0.1, 0.5, 1.0 mg/kg respectively; group five received quinapril at 1.0 mg/kg. The animals in the sham group received NS. The treatment, which started three weeks after coronary ligation, was carried out via intravenous injection once a day for 14 days. The experiment lasted for five weeks.

We used a commercially available echocardiography (VIVID 3 Pro, GE, USA) equipped with a 10 MHz linear transducer. The animals, with shaved chests, were placed in supine position. The transducer was placed gently on the left. Two-dimensional (2D) images of the left ventricle (LV) were obtained in both parasternal long-axis and short-axis views at a frame rate of 130 Hz. M-mode tracings were recorded at the level of the papillary muscles with 2D guidance. LV wall thickness and cavity diameters were measured outside the infarcted area by M-mode, through the largest diameters of the ventricle. Each image consisted of 24 consecutive heart cycles and the focus area was 0–40 mm from the transducer. Fractional shortening (FS), in percentage, was calculated according to the following formula:

where LVIDd is the LV internal diameter in diastole, and LVIDs is the LV internal diameter in systole [20]. LV internal diameters were measured according to the guidelines of the American Society of Echocardiography [21]. All measurements were obtained on at least three beats. LV systolic wall stress = 1.36 × (aortic systolic pressure × LVIDs)/(IVSs + LVPWs), where IVSs is the interventricular septum thickness at end-systole and LVPWs is the posterior wall thickness at end-systole. The tracings were analyzed by one author (WTW) who had no prior knowledge of the group assignment.

An F-2 (0.7 mm in internal diameter) catheter with pressure-transducer was inserted through the right carotid artery into the aorta to determine the mean arterial blood pressure. The catheter was subsequently advanced further into the LV, and end-diastolic pressure (LVEDP) and systolic pressure (LVSP) were recorded. The transducer was connected to a polygraph (model 6300, Nihon Kohden, Japan) and a data acquisition workstation (model MP150, BIOPAC Systems, USA) to a computer running Acknowledge (version 3.7.1). LVEDP was read directly from the pressure curve as being the diastolic pressure immediately preceding the pressure rise associated with LV contraction. Peak positive and peak negative first derivatives of the LV pressure (+LVdp/dt and -LVdp/dt) were calculated with the Acknowledge software.

After haemodynamic measurements, the rats were subsequently euthanized by excision of the heart. The atria and ventricles were sectioned, separated and weighted. The infarcted area (scar tissue) was excised with care from the non-infarcted LV to avoid contamination of viable myocardial tissue by necrotic tissue. The infarction size was estimated by weighing the excised scar tissue. The non-infarcted LV from the CHF rats was further sectioned by separating the tissue contiguous to the infarcted area (viable LV free wall) and the tissue distal to the infarcted area (interventricular septum). A piece of the apical part of the nonischemic interventricular septum was processed separately for histochemical analysis of apoptosis. The right tibia of each rat was isolated and its length measured [22]. Myocardial tissue from interventricular septum was microtomed into 5 μm sections. Apoptotic cell death was analyzed by terminal-deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) method. The Bcl-2 associated X protein (Bax) related to apoptosis was determined with biotin SP-HRP.

Three weeks after coronary artery ligation, the FS of the animals in the model group markedly decreased and further decreased after the animals were given NS for two more weeks. The values of +dp/dt and -dp/dt decreased 36% and 48% respectively two weeks after NS administration. One week after the administration of ASI (0.1, 0.3 and 1.0 mg/kg/day), the decline of the FS was attenuated (P < 0.05, P < 0.001 and P < -0.001). Two weeks after the administration of ASI (at a dose of 1.0 mg/kg/day), the decline of the FS was attenuated (P < 0.05). The dp/dt in the ASI-treated groups (0.3 and 1.0 mg/kg/day) significantly increased compared with the model group (P < 0.05 and P < 0.01) (Tables 1 and 2).

LV geometry in various groups was analyzed with echocardiography (Table 3). The findings demonstrated the progression of LV remodelling during a 5-week period after coronary artery ligation in the absence of treatment. Both LVIDd and LVIDs increased significantly and progressively three weeks after surgery. Both LVIDd and LVIDs decreased significantly two weeks after ASI treatment (0.3 and 1.0 mg/kg/day).

LVSP in the model group was 21% lower than that in the sham group, while LVEDP in the model group increased markedly, suggesting severe cardiac dysfunction in the CHF rats after coronary artery ligation (Table 4). ASI (at doses of 0.1 and 0.3 mg/kg/day) attenuated the decrease of LVSP, while LVEDP decreased 55.4% (P < 0.01) compared with the model group. The rats in the model group displayed higher wall stress of LV. ASI (1.0 mg/kg/day) significantly lowered the wall stress by 22% (P < 0.05) compared with the model group.

LA, LV, RA and RV weight-to-tibial-length ratios decreased significantly after ASI (1.0 mg/kg/day) treatment compared with the model group (Table 5).

Assessment of apoptotic cell death was performed with in situ TUNEL staining of mycardial sections. Apoptosis was observed in the CHF rats with an increased level of Bax protein. Compared with the model group, both apoptotic cells and Bax proteins were reduced markedly after two weeks of ASI treatment (1.0 mg/kg/day) (Table 6).