| ObjectiveHypoxia is the key pathology factor in ischemia heart disease.It is believe that apoptosis induced by hypoxia play important role in post-infarction ventricular remodeling and end-stage heart failure.Then,the mechanisms about cardiac myocytes apoptosia is unclear.Patiente of coronary heart disease and with diabetes mellitus are usually in more serious condition.The reason also is one hot spot.Hyperglycemia can induce apoptosis by oxidative stress,we postulate that hyperglycemia affect patient's condition and prognosis by some effect on apoptosis in patient of coronary heart disease.Hypoxia inducible factor 1(HIF-1) is a key regulatory factor in hypoxia response,it can modulate glucose and iron and energy metabolism,modulate vascular active and acid-base equilibrium,it also regulate cell survival and apoptosis.So HIF-1 might play role in cardiac myocytes apoptosia induced by hypoxia and affect the effect of hyperglycemia on ischemia heart disease.The study about those is little and showing different results.At present,the role of is unclarity,and the study about the role of HIF-1 on ischemia injury of cardiac myocytes by hyperglycemia is little.Here we will discuss the effect of HIF-1 on cardiac myocytes apoptosia induced by hypoxia and the effects of hyperglycemia on HIF-1 signal and apoptosia induced by hypoxia in primary neonatal rat cardiac myocytes.Methods1.To culture the primary neonatal rat cardiac myocytes,Neonatal cardiomyocytes were prepared from 1～2-day-old Wistar rats.At the 4th day in culture,cells were incubated in condition of normoxia,hypoxia and hypoxia with 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole(YC-1,one chemical inhibitor of hypoxia inducible factor 1alpha) for 24h.The hypoxia environment was achieved by treating cells with cobalt chloride.Cell viability was measured by Trypan blue staining.Cardiomyocyte apoptosis was detected with terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling,Hoechst33258 DNA binding assay and Annexin V/PI binding assay.Semi-quantitative reverse transcription-polymerase chain reaction was used to study the expression of hypoxia inducible factor lalpha and Bcl2/adenovirus EIB 19kD-interacting protein 3.Their protein levels were detected by Western blot.2.At the 4th day in control culture,cells were exposured to condition of normoxia and hypoxia in medium containing 33 mmol/L glucose(high glucose group) and 5.5 mmol/L glucose(normal glucose group).The hypoxia environment was achieved by treating cells with cobalt chloride.Cell viability was measured by Trypan blue staining.Cardiomyocyte apoptosis was detected with terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling,Hoechst33258 DNA binding assay and Annexin V/PI binding assay.Semi-quantitative reverse transcription-polymerase chain reaction was used to study the expression of hypoxia inducible factor 1alpha and Bcl2/adenovirus EIB 19kD-interacting protein 3.Their protein levels were detected by Western blot.immuncytochemical staining was used to assay HIF-1αprotein expression and its translocation to nucleus.Results1.The roles of hypoxia inducible factor 1alpha on apoptosis induced by hypoxia:Compared with normoxia condition,the mRNA and protein level of HIF-1αand BNIP3 significantly increased after exposure of rat cardiac cells to hypoxia(P＜0.01, respectively),and cells apoptosia rate increased(P＜0.01).However,when HIF-1αwas inhibited by treating cells with 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole,all mRNA expression and protein level of BNIP3 were decreased(P＜0.01,respectively), and cells apoptosia rate significantly decreased(P＜0.01).2.The effects of hyperglycemia on apoptosis induced by hypoxia and HIF-1αsignal pathway:In normoxia condition,Cell survival rate in high glucose group was lower (P＜0.01) and apoptosic rate was higher(P＜0.01) than those in normal glucose group. However,in hypoxia condition,cell survival rate in high glucose group was higher (P＜0.01) and apoptosic rate was lower(P＜0.05) than those in normal glucose group. In normoxia condition,the expression of HIF-1αmRNA is similar in normal glucose group and high glucose group,but the protein level of HIF-1 in high glucose group was higher than in normal glucose group(P＜0.05).Then,in hypoxia condition,there were no obviously difference in the mRNA and protein level of HIF-1αbetween normal glucose group and high glucose group(P＞0.05).Anti-HIF-1αimmuncytochemical staining showing that HIF-1αaccumulate and translocation to nucleus in normoxia with normal glucose and in hypoxia with normal glucose,but just accumulate in cytoplasm in hypoxia with high glucose,and BNIP3 between high glucose group and high glucose group.In normoxia condition,there were no obviously difference in the mRNA and protein level of BNIP3 between normal glucose group and high glucose group (P＞0.05),but in hypoxia condition,all the mRNA and protein level of BNIP3 in high glucose group were lower than those in normal glucose group(P＜0.01).Conclusion1.Hypoxia inducible factor 1alpha media cardiac myocytes apoptotis in cultured primary neonatal rat role in chemical hypoxia condition.Pro-apoptotic protein BNIP3 may be one of the basis molecule.2.In cultured primary neonatal rat cardiac myocytes,hyperglycemia plays an anti-apoptotic role in hypoxia condition contrast with pro-apoptotic role in normxia condition.Hyperglycemia coule affected apoptosis by the effect on HIF-1 signal pathway.
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