Mechanisms Of The Transition From Hypertrophy To Apoptosis In Cardiac Myocytes

Heart failure is a kind of important clinical diseases with progressive myocardial dysfunction. Recently accumulating evidences suggest that apoptosis contributes to heart failure as the loss of viable cardiac myocytes. In clinical, heart failure is mainly caused by chronic pressure or volume overload. Therefore, the importance of apoptosis in the transition form compensatory hypertrophy to decompensation of the heart has been recognized. To understand the molecular and cellular mechanisms involved in the regulation of cardiac myocyte hypertrophy and apoptosis may provide a new idea in the therapy to prevent and cure this kind of heart failure. However* Supported by Foundation for University Key Teacher by the Ministry of Education People's Republic of Chinavmthis research is only in the beginning and there are three questions to be considered firstly: 1) Whether the action of various pro-hypertrophic factors may increase the apoptotic rate in combination or succession? 2) Whether hypertrophic cardiac myocytes are more susceptible to apoptosis than normal myocytes? 3) What are the critical signal transduction pathways during the transition from hypertrophy to apoptosis in cardiac myocytes?Based on these questions, Primary cardiac myocytes were cultured in serum-free media. First we established methods to detect hypertrophy and apoptosis in cardiac myocytes. To detect myocyte hypertrophy, we measured profile area of cardiac myocytes stained with HE and observed cardiac myocyte myofibril stained with FITC conjugated phalloidin under fluorescent microscope. To detect myocyte apoptosis, cardiac myocyte nuclei were stained with fluorescent DNA-binding dyes Hoechst 33258 and propidium iodide and viewed under fluorescent microscope, fragmentation of genomic DNA was isolated from the neonatal rat cardiac myocytes by electrophoresis on 2% agarose gel to observe DNA ladder and cardiac myocytes were stained with terminal deoxy-nucleotidyl transferase mediated UTP end labeling (TUNEL). We investigated and compared the effects of NE, ET-1 and PGF2a on hypertrophy and apoptosis in neonatal rat cardiac myocytes. The main results of present work are as follows:I) A stable method for the culture of primary neonatal rat cardiac myocytes: The cultured neonatal rat cardiac myocytes in 10% PCS media survived over 10 days and the cultured myocytes in serum-free media survived over 10 days too. The morphological features and beat rate of myocytes were in good condition during their growth. During 4-6 days, the beat rates areDC80-100 beats/min. The cardiac myocyte were identified by immunocytochemical staining with actin and cTnl monoclonal antibodies. The purity of cultured neonatal rat cardiac myocyte was 90%.II) Normal ranges of profile area of myocytes and the percentage of apoptotic rate in cultured cardiac myocytes: normal range of profile area of myocytes cultured for 6 days was 900~3000 urn2 and the mean profile area was 1924 + 74 um2. With Hoechst 33258 and PI, we know the percentage of apoptosis in myocytes cultured in serum-containing for 6 days was 9.67 + 0.47 % and the percentage of apoptosis in myocytes cultured in serum-free for 6 days was 11.05 ?.70%. There was no significant difference between serum-containing and serum-free conditions (.P>0.05) .III) Enhancement of NE-induced apoptosis in cardiac myocytes by ET-1: 1 umol/L of NE treatment caused an increase in the percentage of apoptotic cells (18.39 ?.80% versus 11.05 ?.70% in control cultures, PO.01); 10 umol/L of NE induced 30.27?.32 % apoptotic cells. ET-1 treatment did not increase the percentage of apoptotic cells (P>0.05 versus CON). The combination of 10 nmol/L of ET-1 and 1 umol/L of NE caused a marked increase in the percentage of apoptotic cells (35.20?.37% versus CON, PO.01). BQ-123 (the antagonist of ET-1 receptor A) inhibited this effect of ET-1 on enhancing NE-induced apoptosis in cardiac myocytes (20.5 ?1.9%, PO.01 versus 10 nmol/L of ET-1+1 umol/L of NE).IV) Increased susceptibility of hypertrophic...