The Susceptibility To Angiotensin Ⅱ-induced Apoptosis And Its Underlying Mechanisms In Hypertrophic Cardiomyocytes

Because of the high mortality, heart failure has been one of the most serious diseases threatening human health. An important character of heart failure is the progressive worsening of ventricular function over months or years. The mechanisms responsible for this hemodynamic deterioration are not known, but may be related to progressive intrinsic contractile dysfunction and/or to ongoing cardiomyocytes loss. The intrinsic contractile dysfunction in heart failure may be involved in the suppression of myocardial excitation-contraction coupling processes and has been given much emphasis* Supported by Foundation for University Key Teacher by the Ministry of Education, People's Republic of ChinaVIon in the past. However, the ongoing cardiomyocyte loss caused by cardiomyocyte apoptosis is paid more and more attention to recently. Though considerable researches have suggested that cardiomyocyte apoptosis play a key role in the transition from compensative myocardial hypertrophy to heart failure, a lot of questions are still open. For example, is there any increase in apoptotic rate in hypertrophic cardiomyocyte? What is the relationship between the hypertrophic extent and the susceptibility to apoptosis in cardiomyocytes? if the susceptibility to apoptosis has been enhanced in hypertrophic cardiomyocytes, what is the underlying mechanism for it?To answer those questions mentioned above, we set up an experimental model of hypertrophic cardiomyocytes by exposing primary cultured neonatal cardiac myocytes to endothelin-1 (ET-1) for 24 h. The hypertrophic extent of cardiomyocytes was qualitatively evaluated by myofibril organization and quantitatively evaluated by the surface area of cardiomyocyte. The myofibril organization in cardiomyocytes was observed on fluorescent microscope by staining the cells with phalloidin conjugated FITC. The surface area of cardiomyocyte stained by cosine was measured by Adobe Photoshop version 6.0. Repeated experiments proved this experimental hypertrophic model is reliable. Based on this model, we further studied on apoptosis induced by Angiotensin II (Ang II) in control cardiomyocytes and hypertrophic cardiomyocytes. Different concentration of Ang II were added to both normal and hypertrophic cardiomyocytes for 24 h, then cardiomyocyte nuclei were stained with either fluorescent DNA-binding dye Hoechst 33258 or terminal deoxynucleotidyl transferase mediated 2'-deoxyuridine 5'triphosphate (dUTP) nick end labeling (TUNEL) technique to detect apoptosis. In addition, WesternVIIblot were taken to investigate caspase-9 and PKC8 expression in cultured cardiomyocytes. Finally, the activity of caspase-3 was measured by substrate analyzing. The main results are as follows:1) ET-1 has a pro-hypertrophic, but no pro-apoptotic effect on cardiomyocytes. Myocytes treated with 10 or 100 nmol/L ET-1 for 24 h showed a 168 or 184 % significant increase in surface area compared with the control group (P<0.01). However, the two ET-1 treatment groups showed no difference in apoptotic rate (10.94+0.61 % and 11.71 ?.73 %, respectively) from the control group (10.66 ?.15 %) (P>0.05).2) Ang II has both pro-hypertrophic and pro-apoptosis effects on cardiomyocytes. Myocytes treated with 0.1, 1.0, or 10.0 nmol/L Ang II for 24 h showed significant increases in surface area to 146, 162 or 224 % respectively, compared with the control group (P<0.01). The apoptotic rate increased to 12.64?.53 %, 17.21 ?.43 %, or 16.15?.34 % in the three Ang II treatment groups respectively. There were significant differences in each Ang II treatment group compared with the control group( 10.66 ?.43 %) (P<0.05orP<0.01).3) The pro-apoptotic effect of Ang II is mediated by its ATj receptor. Cultured cardiomyocytes treated with 10.0 nmol/L Losartan or 10.0 nmol/L PD 123319 showed no noticeable difference in apoptotic rate( 10.94?.37% or 11.02 ?.85% respectively ) from the control group (P>0.05). 1.0 nmol/L Ang II increased cardiomyocytes apoptotic rate to 17.22 ?.43 %, but this incr...