The Role Of Mitochondrial Fission Protein Drp1 In Mice Embryonic Heart Development

The heart is the first organ to form during embryonic development.The abnormal development of the heart can cause congenital heart diseases.Congenital heart diseases are the most common inborn defects in humans,and its incidence can reach 1%to 5%.Congenital heart diseases not only endanger human health in the cardiovascular system,they also contribute to diseases in other systems,including arrhythmia,sudden death,pulmonary dysfunction and other diseases.Understanding and exploring the molecular and genetic mechanisms in cardiac embryonic development can provide important clues for better prevention and treatment of congenital heart disease,and thus is highly significant for clinical and basic scientific research.Mitochondria are very important organelles in eukaryotic cells originated 2billion years ago.Mitochondria have two double-layered lipid membrane structures,the outer membrane and the inner membrane.The inner membrane is folded into a ridge called cristae,with the purpose of increasing the inner membrane surface area.The outer membrane is the boundary between the mitochondria and the cell matrix,and contains multiple receptors on it.These receptors mediate communication between mitochondria and other organelles.Oxidative phosphorylation?OXPHOS?complexes I to V are located on the inner membrane.The main function of these complexes is to generate ATP.Mitochondrial permeability transition pores?mPTP?are located on the mitochondrial membrane,and its exact location is currently unknown.Between the inner and outer membranes is the inter membrane space,which contains cytochrome C,which plays the role of electron transfer in the oxidized phosphate complex.In addition,it can also promote apoptosis.Inside the inner membrane is the mitochondrial matrix,which contains mitochondrial DNA,ribosomes,enzymes,and various ions.In accordance with current studies,mitochondria are believed playing an essential role in mice heart development.Including mitochondrial biogenesis,ROS,apoptosis,mitochondrial fusion and fission,which are all important for mice heart development.Mitochondria are highly dynamic organelles that undergo a coordinated cycle of fission and fusion called"mitochondrial dynamics"to maintain their shape,distribution and size in different cells.Dynamin-related protein 1?Drp1?is a member of the protein activin superfamily.It consists of GTPase and GTPase effector domains,which are separated from each other by helical amino acid segments.Drp1 acts on the outer mitochondrial membrane to induce mitochondrial division.Previous studies have shown that dynamin-2,which belongs to the same GTPase,can also affect mitochondrial division,but recent studies have shown that only Drp1 is essential for mitochondrial division while Dynamin1-3 are optional for mitochondrial division.The role of Drp1 in mouse heart development has been studied using the Cre-Loxp system to conditionally knock down Drp1 in mouse heart cardiomyocytes.These studies include the use of Mhy-6-Cre and Mck-Cre to knock out Drp1 in cardiomyocytes.Studies have found that knockout of Drp1 can cause death in neonatal mice.Drp1-deficient mice showed dilated cardiomyopathy with abnormal morphology.In the published studies,Drp1 is inactivated at the perinatal stage.The role of Drp1 in the cardiac cardiomyocytes during mid-gestation remains unclear.In our current study,we inactivated Drp1 during mid-gestation and examined its role of Drp1 during heart development.Methods:1.To investigate the effect of Drp1 conditional knockout on cardiac morphology in cardiomyocytes at the different embryonic stages.Mef2C-Cre;Drp1^loxp/+male mice were crossed with Drp1^loxp/loxpoxp/loxp female mice to obtain mutants(Mef2C-Cre;Drp1^loxp/loxp)and control wild-type(Drp1^loxp/+)mice.The mutant embryos at E9.5 and E10.5 and their littermate control embryos were sagittal sectioned,and then immunofluorescent staining was performed to detect the expression of Drp1 in embryo hearts.Heart sections of E11.5 to E16.5 wild type and mutants were HE stained to examine heart morphology.2.To investigate the effect of Drp1 conditioned knockout on cardiomyocyte proliferation,apoptosis and cell orientation in the ventricular wall during embryonic development;The control and mutant heart of E12.5 were sectioned,and immunofluorescence staining was performed to detect cell proliferation,apoptosis,and orientation of cardiac muscle cells in the ventricular wall.3.To investigate the causes of different orientation defect in control and mutant ventricles;Frozen sections of control and mutant hearts of E12.5 were used to detect the expression of N-cadherin on the surface of cardiomyocytes by immunofluorescence staining.Total proteins were extracted from control and mutant hearts and expression of N-cadherin was detected by Western blot.4.To investigate the distribution and morphological changes of mitochondria in cardiomyocytes after Drp1 conditional knockout;E10.5 embryos were paraffin sectioned,and immunofluorescence staining was performed to observe the distribution of mitochondria in cardiomyocytes in cardiomyocytes with Drp1-deleted.E14.5 cardiac cardiomyocytes were cultured in vitro and immunostained to detect the distribution of mitochondria in control and mutant cardiomyocytes.Transmission electron microscopy?TEM?was performed on sections of E11.5 and E14.5 hearts to compare mitochondrial ultrastructure between control and mutant samples.5.To investigate the changes of oxidative phosphorylation complexes function on mitochondrial inner membrane of cardiomyocytes after Drp1 conditional knockout;Frozen sections of fresh E14.5 control and mutant hearts were used to detect the function of oxidative phosphorylation complex I and complex IV in mitochondria using DAB staining and NADH staining techniques.We dissected embryonic hearts from control and mutant embryos in E14.5 and examined the function of mitochondrial respiratory by respiratory system.6.To investigate the difference in RNA expression profiles between mutant and control cardiomyocytes after conditional knockout of Drp1.Cardiomyocytes were extracted the right ventricle of E13.0 mutant and wild-type hearts.After sorting,single-cell RNA sequencing technology was used to detect the expression of genes in different cell populations.Through analysis of RNA sequencing,differential expressed genes between and control and mutant samples were identified.Result:1.Immunofluorescence analysis showed that Drp1 was partially inactivated in the right ventricle and outflow tract of the E9.5 mutant embryonic heart,and was completely inactivated in the right ventricle and outflow tract of the E10.5 mutant embryonic heart.Starting from E12.5,heart morphology began to be observed.Development of the right ventricle of mutant hearts is retarded,and the volume of the right ventricle was smaller than that of the control heart.At E17.5,all mutant embryos died.2.At E12.5,immunofluorescence analysis showed that cardiomyocyte proliferation in right ventricles of mutant hearts was significantly lower than that of the control,while the apoptosis of the mutant right ventricle was significantly increased than the control.Cardiomyocytes orientation in the right ventricle wall of mutant is different from that of myocardial cells in of control.No defect was observed in the left ventricle where Drp1 is not deleted.3.Immunofluorescence analysis showed that the expression of N-cadherin in cardiomyocytes of mutant right ventricles decreased significantly compared to control samples.Western blot confirmed that the reduction in expression of N-cadherin in mutant right ventricles.4.Immunofluorescence analysis showed that the distribution of mitochondria in mutant right ventricular cardiomyocytes was different from that of control heart at E10.5.Staining of in vitro cultured cardiomyocytes derived from E14.5 hearts showed that the distribution of mitochondria around nuclei in mutant is different from control.In mutant samples,mitochondria appear to be hyperfusion,as expected from reduced mitochondrial fission.Electron microscopic results show that at E11.5 and E14.5,the mitochondrial ultrastructure in mutant cardiomyocytes is altered.5.The function of oxidative phosphorylation complex I and complex IV in mutant heart is reduced compared to control hearts.The ability of coupling between mitochondrial respiratory chain electron transfer and ATP production in mutant hearts is lower than that in control hearts.6.At E13.0,single cell sequencing revealed expression of 240 genes is significantly changed by Drp1 deletion.Bioinformatic analysis suggested that 35functional pathways were significantly changed.The most significantly changed group of genes are the ones encoding ribosome structures,suggesting the cross talk between mitochondrial fission and translation.This is the first study the reveals the direct connection between the two processes.Conclusion:Drp1 plays a crucial role during mouse heart development.Conditional knockout of Drp1 in the right ventricle cause embryonic lethality with full penetrance,and severe abnormalities in heart morphology,structure,and function.Cardiomyocyte proliferation is reduced in mutant hearts,while apoptosis is increased.The cardiomyocytes in mutant are abnormally oriented during development,and this abnormality is likely controlled by the N-Cadherin protein expression.The morphology of mitochondria in cardiomyocytes was changed in mutant at E10.5,and the ultrastructure of mitochondria was altered in mutant at E11.5 and E14.5.OXPHOS activity is impaired in mutant cardiomyocytes.By single-cell RNA sequencing,many genes were identified to be expressed differently mutant and control cardiomyocytes at E13.0.Bioinformatic analysis revealed that multiple pathways were impaired in mutants.Among them,expression of a group of genes encoding ribosomal structures were most significantly altered.Our result suggests that abnormal mitochondrial fission may have a strong effect on the transcription of genes encoding ribosomal proteins,indicating that there is a cross talk between mitochondrial dynamics and protein translation.