Study On Apoptosis Susceptibility Of Various Hypertrophic Cardiomyocytes

Heart failure is a terminal stage of all kinds of heart diseases. Chronic pressure and/or volume overload is one of the basic causes of heart failure. Compensatory cardiac myocyte hypertrophy is an adaptive response to chronic pressure and/or volume overload to maintain cardiac function, and finally will convert into decompensatory phase. There are various hypotheses about the conversion mechanism while it is still obscure. More attention has been paid to contribution of the loss of viable cardiomyocytes due to apoptosis to heart failure in recent years, and it is also suggested that apoptosis susceptibility of hypertrophic cardiomyocyte is increased. It is helpful to prevent and treat heart failure if the relation between cardiomyocyte hypertrophy and apoptosis is elucidated.The aim of our research was to explore the changes of apoptosis susceptibility of various hypertrophic cardiomyocytes. Above all, we established an effective method for measuring surface area and viability of neonatal rat cardiomyocytes in primary culture. Then we observed the effects of cell seedingdensity, culture time and serum on cardiac myocyte surface area and viability and analyzed the relation between surface area and viability. Based on these results, we observed morphological features of hypertrophic cardiomyocytes induced by ET-1, IGF-I or T3. The dose-effect of ET-1, IGF-I and T3 on myocyte surface area was also studied. Finally, using AngII as an apoptotic stimulus, we observed the changes of apoptosis susceptibility of hypertrophic cardiomyocytes induced by ET-1,IGF-I or T3. The main results are as follows:1) An effective method for measurement of myocyte surface area and viability The profile of cultured myocytes by rapid eosine staining was more clear than that by regulating eosine staining, so myocyte surface area could be measured exactly. After comparing several kinds of methods for measuring cell viability, we defined cardiomyocyte viability as metabolic ability of a single cardiomyocyte. According to this definition, cardiomyocyte viability was represented by A490/total cell numbers in our research.2) Effects of cell seeding density, culture time and serum on myocyte surface area and viability The cardiac myocyte surface area was increased in 2~7 d culture when cell seeding density was 2.5 X 104 cells/cm2(P<0.01). Surface area of cells cultured in serum-containing medium was bigger than that cultured in serum-free medium(P<0.01). When cell seeding density increased from 1.5 X 104 cells /cm2 to 6.5 X 104 cells /cm2, myocyte surface area decreased gradually.The A490/total cell numbers increased when cell seeding density ranged from 1.5X104 /cm2 to 4.5X104 /cm2, then the ratiodecreased when cell seeding density ranged from 4.5X10 /cm to 6.5X104 /cm2, ,A490/total cell numbers did not change significantly in 3-8 d culture at the seeding density of 2.5X104 /cm2(P>0.05). A490/total cell numbers of cells cultured in serum-free medium were lower than that in serum-containing medium(P<0.01).From the above results, we infered that there is no relation between cardiac myocyte surface area and viability under different culture conditions.3) Dose-effects of ET-1, IGF-I or T3 on myocyte surface area Myocytes treated with 1 nmol/L, 10 nmol/L, or 100 nmol/L ET-1 for 48 h showed 124 + 4% ,151 +6% or 160 + 7% increase, respectively, in surface area compared with control cells(P<0.01). Myocytes treated with 1 nmol/L, 10 nmol/L, or 100 nmol/L IGF-I for 48 h showed 121 + 5%, 135+7% or 161+9% increase in surface area compared with control cells(P<0.01). Myocytes treated with 1 nmol/L, 10 nmol/L or 100 nmol/L T3 for 48 h showed 123 + 3%, 149 + 5%, or 151 + 6% increase in surface area compared with control cells(P<0.01).4) Changes of apoptosis susceptibility of hypertrophic cardiomyocytes induced by ET-1, IGF-I or T3 Compared with control cells, h...