| BackgroundCardiac hypertrophy is generally recognized as an adaptive response of the myocardium to various intrinsic and extrinsic, mechanical and hormonal stimuli, which leads to the heart working harder under conditions of increased workload. Initially, cardiomyocytes in cardiac hypertrophy expand in size and increase their protein synthesis. However, prolonged hypertrophy causes myocyte disarray, apoptosis and ventricular fibrosis, which results in heart remodeling and ceases its compensatory function. Eventually, the cardiac remodeling progresses to congestive heart failure (CHF), which is one of the leading causes of mortality in the world. Despite the widespread use of β-blockers and inhibitors of the renin-angiotensin-aldosterone system clinically in the past few decades, the incidence and the mortality of CHF still remain high, highlighting the need for additional understanding of remodeling events and early drugs intervention.Hypercholesterolemia is regarded as one of the major risk factors for cardiovascular disease and increases all-cause mortality in different epidemiological studies. The relationship between hypercholesterolemia and cardiovascular mortality has been known for decades. Previous studies shown that hypercholesterolemia in normotensive, non-diabetic adults is independently associated with mild, concentric LV geometry. These clinical studies suggest that hypercholesterolemia plays a role in the origin of myocardial remodeling. The apolipoprotein E-deficient (ApoE-/-) mouse has been the most widely used animal model for hyperlipidemia and atherosclerosis because it has substantially elevated total plasma cholesterol, i.e. hypercholesterolemia, and rapidly develops atherosclerotic lesions similar to those observed in humans. Apart from the obvious vascular phenotype in the ApoE-/-mouse, the cardiac phenotypes, such as cardiac hypertrophy featured by an increase in cardiac weights and LV wall thickness, myocardial hypertrophy, cardiac fibrosis and cardiac dysfunction, were also noticed. Despite certain controversies in these animal researches, studies have provided evidence that hypercholesterolemia is closely related to aberrant cardiac hypertrophy. Nevertheless, the mechanisms involved in the cardiac hypertrophy and the impact of hypercholesterolemia on cardiomyocytes regeneration, aging, apoptosis and P53 pathway in the ApoE-/- mouse are poorly understood.Astragaloside IV (AST IV), is a monomeric and natural saponin isolated from Astragalus membranaceus (Fisch.) Bge, which is a traditional Chinese herb widely used in clinical practice and has been demonstrated to be effective in treating cardiovascular diseases. Accumulating evidences have demonstrated that AST IV possessed diverse cardiovascular protective properties against cardiac hypertrophy, cardiomyocyte apoptosis, myocardial fibrosis, oxidative stress and so on. However, the effects of AST IV on blood lipid levels and cardiac alterations in hypercholesterolemic ApoE-/-mice have not yet been established.ObjectiveTo investigate the hypothesis that hypercholesterolemia might induce the decrease of myocyte regeneration, increase of senescence, apoptosis, cardiac fibrosis and the unwanted activation of P53 signaling in heart, which lead to the development of adverse cardiac remodeling characterized by cardiac weights increase, ventricular chamber dilation, ventricular function decrease. The intervention of AST IV would be likely to play a positive role in interfering with the acquisition of adverse cardiac remodeling and rescue the impaired ventricular function in ApoE-/- mice.Materials and Methods1. AST-IV preparationThe purity of AST-IV was shown to be> 98%. It was completely dissolved in hydroxypropyl-beta-cyclodextrin (HPBCD) to obtain solutions at concentrations of 0.1 mg/ml and 1 mg/ml, respectively.2. AnimalsMale ApoE-/-mice (KO) and its genetic background C57BL/6J mice (WT) (body weight 18 ± 2 g) at 6-7 weeks of age were used. WT mice served as control group (n= 7). Simultaneously, KO mice were divided randomly into 3 groups:model group (KO, n= 7), AST IV low dose (AST-LD, n= 7) and high dose (AST-HD, n= 8) treatment group. The latter two groups received intraperitoneal injection of 1 mg/kg and 10 mg/kg of AST IV respectively every other day over a period of 8 weeks. Meanwhile, control and model mice were injected with dissolvent. Animals were sacrificed at 16 weeks after intervention of AST IV to assess the therapeutic effects and another WTs (n= 8) and KOs (n= 7) at the age of 8 weeks were killed to obtain baseline parameters.3. Echocardiography, Plasma Lipid Levels and Cardiac AnatomyMice were anesthetized with isoflurane. M-mode echocardiographic parameters of left ventricular (LV) in the short axis view at the level of the papillary muscles were measured. The results of the LV diameter and posterior wall thickness were collected in diastole and systole, and then fractional shortening (FS) and ejection fraction (EF) were calculated. After keeping from feeding the chow diet but water for 12 hours, mice were weighed and sacrificed. Plasma was isolated from blood sampled. After sacrificing, the heart and LV were dissected and weighed. Then, the LV was sectioned equally into 2 parts perpendicular to the longitudinal axis of the heart, one was embedded in OCT to obtain frozen sections for histology study, and the other was frozen rapidly in liquid nitrogen and preserved until use.4. Histology, Immunofluorescent Staining, Myocyte Size and NumberFrozen sections were used for immunolabeling, as well as Masson trichrome staining. Myocytes were labeled by cardiac troponin I mouse monoclonal antibody with a M.O.M. immunodetection kit. Oxidative stress related proteins NADPH oxidase 4 (NOX4), superoxide dismutase 1 and superoxide dismutase 2 (SOD 1/2) were detected. Cycling cells were marked by Ki67. To test cellular senescence, sections were exposed to P16INK4a antibodies. Secondary antibody was used to combine with the above-mentioned primary antibodies. DAPI was used to identify the nuclei. Detection of myocyte size and number were based on the methods published before.5. In Situ Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling (TUNEL)In situ cell death detection kit with fluorescein was employed and performed principally according to the manufacturer’s instructions to detect and quantify apoptosis at single cell level based on labeling of DNA stand breaks.6. Quantitative Real Time-PCRTotal RNA was extracted from tissue with RNAiso Plus and was reverse-transcribed into cDNA using the PrimeScriptTM RT reagent kit, and then the cDNA was amplified. The mRNA expressions of a series of genes related to the cell cycle, proliferation, senescence, P53-dependent apoptosis pathway and antioxidants were determined by QRT-PCR.Results1. Plasma lipid levels:ApoE-/- mouse displayed severe hypercholesterolemia rather than hypertriglyceridemia at 8 weeks of age. The concentrations of TC and LDL-C in KOs were 3.7-fold and 8.7-fold higher, respectively, than those of WT mouse. Conversely, HDL-C level decreased 23% compared with that of WTs, While, no significant difference in TG between the two groups was detected. A similar trend of these four plasma lipid levels in KOs was also observed at 16 weeks. After administrated with AST IV for 8 weeks, the mice in AST-LD and the AST-HD groups showed significantly higher serum TC and LDL-C in comparison with the KOs. Overall, ApoE-/- mice developed hypercholesterolemia, the treatment of AST IV for 8 weeks couldn’t decrease the TC and LDL-C levels of ApoE-/- mouse.2. Cardiac mass and size:At 8 weeks, heart weight (HW), left ventricular weight (LVW), HW to body weight (BW) ratio (HW/BW), and LVW-to-BW ratio (LVW/BW), LVW-to-HW ratio (LVW/HW) increased in KOs. In KO animals, LV longitudinal axis (LAX) also increased consistently, resulting in a significant expansion in LV. Moreover, differences of these variables still maintained at 16 weeks. All these parameters were reduced in AST-HD (HW 24%, LVW 28%, HW/BW 8%, LVW/BW 10%, LVW/HW 5%, LAX 10%) compared with KOs. But there were no significant alterations in AST-LD group except a 20% decrease in LVW compared with KOs.3. Echocardiographic measurements:At 8 weeks, compared with WTs, the increase of LV end-diastolic dimension (LVDd,14%) and LV end-systolic dimension (LVDs,11%), in combination with the growth of LAX, resulted in an increase in LV end-diastolic volume (LVEDV,39%) and LV end-systolic volume (LVESV,30%), respectively, in KOs with hypercholesterolemia. Furthermore, at 16 weeks, besides the increase of LVDs, KOs was also characterized by a 35%rise in LVESV, and importantly, a decrease in ejection fraction (EF,11%), fractional shortening (FS, 13%), LV posterior wall at end systole (LVPWs,6%) and LV posterior wall at end diastole (LVPWs,13%) compared with WTs. Taken together, further expansion and systolic function impairment suggested adverse cardiac remodeling in KOs with hypercholesterolemia. In contrast, the changes of EF and FS in AST-HD were significantly improved (EF:60% vs.54%, FS:31% vs.28%) in comparison with those of KOs. No significant improvement in echocardiographic measurements was detected in AST-LD at 16 weeks compared with KOs.4. Cardiac fibrosis:No difference was shown in LV collagen content between KOs and WTs at 2 months of age. The collagen volume fraction increased over twice in hypercholesterolemic KOs compared with that of WTs at 16 weeks, and declined nearly in half in AST-HD animals.5. Myocyte size and number:A 19% increase in myocyte size was detected in hypercholesterolemic KOs at 8 weeks, whereas myocyte number was almost the same as WTs. At 16 weeks, a greater increase (38%) in myocyte volume and a 14% drop in myocyte number were found in hypercholesterolemic KOs compared with WTs. Whereas, myocyte size was markedly suppressed in AST-LD (19%) and AST-HD (27%). Meanwhile, the number of ventricular myocytes was greatly recovered after high dose of AST IV administration compared with KOs.6. Cell growth and senescence:The fraction of ki67-positive myocytes of total myocytes in KOs was close to WTs at 8 weeks. But it was less than nearly 80% compared with WTs at 16 weeks, and was increased markedly more than three times after high dose treatment of AST IV. Meanwhile, the fraction of ki67-positive cells to total heart cells presented similar changes in this experiment. The percentage of P16INK4a-positive myocytes in hypercholesterolemic KOs did not differ from WTs at 8 weeks. However, this rate in KOs exceeded 2.9-fold to WTs at 16 weeks. Decreases in the proportion of P16INK4a-positive myocytes were noted in AST-LD (39%) and AST-HD (60%) groups, compared to KOs at 16 weeks. Correspondingly, the proportion of P16INK4a-positive cells in total heart cells decreased 55% in AST-HD mice compared with KOs..7. Cell apoptosis:In KO group at 8 weeks, few myocyte apoptosis was occurred (< 2%), and it was not different from WTs. Apoptotic myocytes were detected extensively in KOs, and the fraction of apoptotic myocytes in total myocytes was almost 23 folds more than WTs at 16 weeks. After intervention of AST IV for 8 weeks, the extent of apoptotic cell death was suppressed in AST-LD (87%) and AST-HD (90%) groups. With respect to the proportion of apoptotic cells in total cardiac cells, as demonstrated above, it traveled the identical variety track.8. Oxidative stress and antioxidant defense:NADPH oxidase 4 (NOX4) related to superoxide metabolism, SOD1 and SOD2 involved in reactive oxygen species (ROS) metabolism, were determined by immunofluorescent staining in this study. There were no differences in the fraction of NOX4-positive cells in total heart cells and the positive expression areas of SOD 1/2 between KOs and WTs at 8 weeks. Nevertheless, at 16 weeks, the fraction of NOX4-positive cells in total heart cells in KOs was higher than WTs, and the positive expression areas of SOD2 in KOs were significantly smaller than that in WTs. No difference was existed in the relative expression of SOD1 between KOs and WTs. Under the exposure of AST IV, the fraction of NOX4-positive cells was significantly decreased in the hearts of AST-LD (16%) and AST-HD (17%) groups. Whereas, the expressions of SOD1 and SOD2 were not altered in the hearts of AST-LD and AST-HD groups.9. Changes of transcriptional profile:The expressions of a sets of genes linked to the cell cycle, proliferation, senescence, p53-dependent apoptosis pathway and antioxidants were determined by QRT-PCR.1) At-8 weeks, most of the cell cycle related genes did not altered in KOs except the expressions of Cyclin D 1(2.2 fold) and MCM5 (2.7 fold) up-regulated, and the CDKN1A and CDKN1B decreased significantly 70% and 28%, respectively, compared with those of WTs. At 16 weeks, the increase in CDKN1A (7 fold), CDKN1B (2.7 fold), especially CDKN2A (9.6 fold), which may reflect the cell cycle arrest in untreated KOs with hypercholesterolemia, were consistent with the observed increase protein expression of P16INK4a above. The gene expressions of CDK4 (2.2 fold), Cyclin B1 (11.3 fold), Ki67 (4.7 fold) and MCM5 (3.6 fold) were higher in KOs at 16 weeks. The transcription of Ki67 did not consistent with decrease of its translation protein. Transcripts for CDKN1A, CDKN1B, CDKN2A, MCM5, Cyclin B1, D1, E1, A2 and CDK4 were decreased in KOs exposed to the low and/or high doses of AST.2) A series of genes involved in p53-dependent apoptosis pathway, including P53, TP53, Bax, Bcl2, Mcll, caspase-3, Mdm2, ATM and GADD45A, were studied. The transcription of most of above-mentioned genes in KOs was not affected at 8 weeks. Nevertheless, the 1.6 fold up-regulation of P53 and 27% down-regulation of Mcll were the exceptions. Most of genes in P53-dependent apoptosis pathway, excluding Bax and GADD45A, were increased more than 2.5 times at transcription level in KOs at 16 weeks. The transcriptional profiles of these genes were similar in AST-LD and AST-HD groups, which presented marked decreasing mRNA expressions of genes related to P53-dependent apoptosis pathway. These data were highly consistent with the massive cell apoptosis observed in KOs and small amount cell apoptosis under the exposure of AST Ⅳ. Summarily, these findings at the transcriptional level indicated that hypercholesterolemia triggered P53-dependent apoptosis pathway, and AST Ⅳ administration prevented this signaling pathway activating.3) No alteration in genes associated with oxidative stress and antioxidant defense including Duox1, Srxn1, NOX1, NOX4, GPX2, SOD1, SOD2 was detected at 8 weeks. At 16 weeks, most of genes except Duox1 and SOD1 were up-regulated at transcription level in KOs compared with WTs. However, the mRNA expressions of these genes were lowered in AST treated groupsConclusionsHypercholesterolemia induces the decrease of myocyte regeneration, increase of senescence, apoptosis, cardiac fibrosis and the unwanted activation of P53 signaling in heart, which lead to the development of adverse cardiac remodeling characterized by cardiac weights increase, ventricular chamber dilation, ventricular function decrease. Importantly, AST Ⅳ treatments prevent cardiac remodeling and impaired ventricular function induced by hypercholesterolemia even thought it has no effects on decreasing plasma lipid levels. The results suggested that AST Ⅳ may substantially exert cardioprotection effects in hypercholesterolemia by modulating P53 pathway, decreasing cardiac apoptosis and senescence, and increasing the cardiac regeneration ability. This study provided further supporting evidences for the applications of AST IV to treat cardiac remodeling associated with hypercholesterolemia. |
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