Effects Of Hypoxia-inducible Factor-1 On The Intracellular Calcium Transport In Fetal Rat Cardomyocytes Under Hypoxia

Research Background Congenital heart disease?CHD?is the most common birth defects in China,which seriously affects the population quality and increases the burden of family and society.With the development of the diagnosis and treatment of CHD,fetal intervention could be carried out for some complex CHD.However,the technical barriers of fetal cardiopulmonary bypass and fetal myocardial protection were not solved,which hindered the development of fetal cardiac surgery.Fetus lives in the intrauterine hypoxia environment.The mechanism of fetal myocardium protection under hypoxic conditions is an important part of the research of fetal cardiac surgery.Objectives This study aimed to investigate the effect of hypoxia-inducible factor-1?HIF-1?on intracellular calcium(Ca²⁺)transport in fetal rat cardiomyocytes under hypoxia and to explore new methods of myocardial protection for the fetus.Methods The cardiomyocytes of Sprague-Dawley?SD?rat fetuses with a gestational age of 14?18 d were isolated and placed in an incubator containing 5%carbon dioxide,3%oxygen,and 92%nitrogen.Cell culture was maintained at 37?for 24 hours.All the cardiomyocytes were then divided into six groups for study,which were the control group without drugs?1st group?,the group treated with 10?M phosphocreatine?2nd group?,the group treated with 100 ?M HIF-1 agonist?DMOG??3id group?,the group treated with 10 ?M phosphocreatine and 100?M DMOG?4th group?,the group treated with 10 ?M HIF-1 inhibitor?Acriflavine??5th group?,and the group treated with 10 ?M phosphocreatine and 10 p,M Acriflavine?6th group?.Real-time fluorescent quantitative PCR?RT-PCR?and Western blot analysis were employed 24 h after the treatment to respectively detect the mRNA transcription and protein expression levels of HIF-1?,L-type Ca²⁺ channels?LCa?,T-type Ca²⁺ channels?TCa?,sodium-calcium exchanger?NCX?,ryanodine receptors,and Ca²⁺-ATPase of sarcoplasmic reticulum?SERCA2??inside the cardiomyocytes of rat fetuses.The change in the intracellular Ca²⁺ concentration of the cardiomyocytes was monitored under a laser scanning confocal microscope.Results The RT-PCR showed that there was significant interaction between drugs administration and phosphocreatine in the mRNA level of HIF-1?,TCa,NCX and Ryanodine receptor.Phosphocreatine inhibited the drugs effects on the HIF-1 and Ca+2 channels.Without phosphocreatine,the mRNA levels of HIF-1?,TCa,NCX,and Ryanodine receptor in the 3rd group were higher than those in the 5th group?P<0.05,n=5?.With phosphocreatine,the mRNA levels of HIF-1? and Ryanodine receptor in the 4th group were higher than those in the 6th group?P<0.05,n=5?.There was no interaction between drugs administration and phosphocreatine in the mRNA level of LCa and SERCA2a.The mRNA levels of SERCA2a in the 5th group and 6th group were significantly higher than those in the 1st group,2nd group,3rd group and 4th group?P<0.05,n=5?.On the other hand,Western blot analysis showed that there was no interaction between drugs administration and phosphocreatine in the protein expression levels of HIF-1?,LCa,NCX and SERCA2a.The protein expression of LCa both in the 1st group and 2nd group were higher than those in the 3rd group,4th group,5th group and 6th group?P<0.05,n=5?.The protein expression of SERCA2? in the 2nd group was significantly higher than those in the 3rd group?P<0.05,n=5?.The results of calcium fluorescence measurement showed that there was interaction between drugs administration and phosphocreatine.Phosphocreatine inhibited the drugs effects on the calcium fluorescence?P<0.05,n=10?.Conclusions Under hypoxic conditions,HIF-1 took part in the regulation of Ca²⁺ transport in fetal rat cardiomyocytes.Activation of HIF-1 enhanced the mRNA level of TCa,NCX and Rynaodine receptor,decreased the mRNA level of SERCA2a,and decreased the protein expression of LCa,and thus increased intracellular Ca²⁺overload.Phosphocreatine inhibited the mRNA levels of HIF-1 and Ca²⁺ channels that regulated by HIF-1,and thus reduced the intracellular Ca²⁺ overload.So the inhibition of HIF-1 and energy supply might be effective in fetal myocardial protection under hypoxia.