Evaluation Of Cardiac Function In Fetuses With Ventricular Hypoplasia And Mechanism Of Myocardial Injury

Part Ⅰ: Evaluation of Cardiac Function in Fetuses with Ventricular Hypoplasia by Two-Dimensional Speckle Tracking ImagingObjective: This study was aimed to evaluate myocardial function changes of the nonhypoplastic sided ventricle in fetuses with ventricular hypoplasia(VH)by two-dimensional speckle tracking imaging(2D-STI).Methods: A total of 132 singleton fetuses diagnosed with VH were retrospectively included from January 2016 to June 2022 at our center,and 132 gestational age(GA)-matched(1:1)normal fetuses were selected as control group.According to prenatal echocardiographic results,the cohort were divided into left ventricular hypoplasia group(LVH,n=81)and right ventricular hypoplasia group(RVH,n=51).Conventional echocardiography parameters including left(LV)and right ventricular(RV)end-diastolic dimension(EDD),end-diastolic length(EDL),sphericity index(SI),and diameters of aorta valve(AV),ascending aorta(AAo),pulmonary valve(PV)and main pulmonary artery(MPA).All conventional measurements were normalized by GA and converted to z-score values.Fetal nonhypoplastic ventricular function parameters including global longitudinal strain(GLS)and segmental longitudinal strain were acquired by 2D-STI.The variables affecting RVGLS in LVH group were screened by XGBOOST model and Shapley Additive ex Planations(SHAP)values.Multiple linear regression was used to analyze the influencing factors of LVGLS in fetuses with RVH.Results:(1)Conventional echocardiographic parameters in VH fetuses and controls:Compared with controls,LVH fetuses presented lower left heart measurements including z-scores of LVEDD,LVEDL,AV and AAo diameters(p<0.001).Increased RVEDD z-score and decreased RVEDL z-score caused lower RVSI,indicating a more spherical-shaped RV(p<0.001).Z-score values of left and right pulmonary arteries(LPA,RPA)diameters were reduced,while ductus arteriosus z-score(DA z-score)increased(p<0.001).There were no significant differences between LVH and control group in peak systolic velocities(PSV)of AV,PV and DA.RVH fetuses showed lower z-scores of RVEDD,RVEDL,RVSI,PV,MPA,LPA,RPA and DA,when compared with control group(p<0.01).Meanwhile,z-scores of LVEDD and AAo diameter were increased,while LVEDL z-score and LVSI decreased,indicating a more spherical-shaped LV(p <0.05).PV and DA displayed higher PSVs in RVH group(p<0.01).(2)Myocardial strain parameters in VH fetuses and controls:LVH fetuses had significantly lower RVGLS than controls(p<0.001),and lower longitudinal strains in the basal and middle segments of RV free wall and interventricular septum(p<0.05),but no significant differences were detected in the apical segmental strains.Differ from controls,whose segmental RV strains decreased progressively from basal to apical segments(p<0.05),LVH fetuses presented similar segmental RV strains without significant differences.RVH fetuses showed significantly lower left ventricular global(LVGLS)and segmental longitudinal strains than controls(p<0.001).Similar to controls,RVH group presented higher strain values in LV apical segments than those in middle or basal segments(p<0.001).(3)Variables affecting myocardial strain in VH fetuses:In LVH group,RVGLS was associated with RV/LV ratio,LVSI,LVEDD z-score and accompanied with double outlet right ventricle(DORV).In RVH group,RVEDD z-score and accompanied with ventricular septal defect(VSD)were independent determinants of LVGLS.Conclusion: Non-hypoplastic sided ventricles in VH fetuses presented morphological remodeling and impaired myocardial function.RV segmental strain gradient was changed in LVH fetuses,while LV segmental gradient preserved in RVH fetuses.Myocardial function of non-hypoplastic sided ventricle is associated with the size of hypoplastic sided ventricle and the combined intra-cardiac malformations.Part Ⅱ Evaluation of Myocardial Microstructure in Fetuses with Ventricular Hypoplasia by Diffusion Tensor ImagingObjective: This study was aimed to assess myocardial microstructural changes of nonhypoplastic sided ventricle in fetuses with ventricular hypoplasia(VH)by diffusion tensor imaging(DTI),and to explore the correlation between those changes and ventricular systolic function.Methods: A total of 8 fetal specimens diagnosed with VH were included in our study from January 2016 to June 2022 at our center.In the same period,3 fetal specimens with normal hearts due to inevitable abortion or stillbirth were enrolled as the control group.According to fetal echocardiographic results,study objects were divided into left ventricular hypoplasia group(LVH,n=4)and right ventricular hypoplasia group(RVH,n=4).All specimens were donated with the informed consent of the pregnancies.The specimens were dissected to observe and record the malformations of cardiovascular system.Cardiac chambers and great vessels were isolated,and placed in 4% formalin solution fixed for 48 hours.Fetal heart specimens were scanned using diffusion tensor cardiovascular magnetic resonance(DTCMR),and the images were postprocessed by DSI-studio software.Myocardial microstructure was assessed by qualitative observation of the arrangements and orientations of myocardial fibers,and quantification analysis of fraction anisotropy(FA),mean diffusion coefficient(MD)and helix angle(HA).Correlation analysis was performed for relationship between cardiac function and myocardium microstructure.Results:(1)Diffusion tensor imaging parameters in VH fetuses and controls:(ⅰ)Fraction anisotropy(FA):(a)In control group,the global FA of left(LV)and right ventricle(RV)was 0.19±0.02 and 0.18±0.01,respectively.Segmental analysis showed FA values of LV and RV apical segments were higher than those of basal and middle segments(p<0.001).(b)Compared with controls,RV global and segmental FA values were higher in LVH group(p<0.01),and segmental analysis showed the incremental gradient from basal to apical segment(p<0.001).(c)RVH group presented similar LV global FA with controls.However,segmental analysis showed lower FAs in basal segments(p<0.001),but higher FAs in middle and apical segments(p<0.001).It also displayed an increasing gradient from basal to apical segment(p<0.001).(ⅱ)Mean diffusion coefficient(MD):(a)In control group,the global MD of left(LV)and right ventricle(RV)was 1.34±0.04 and 1.28 ± 0.03,respectively.Segmental analysis showed segmental changes in LV and RV,and MD in the middle,apical and basal segments presented different values from high to low(p<0.001).(b)RV global and segmental MDs in LVH group were lower than those in controls(p<0.001),and segmental analysis found apical MD was lowest(p<0.001).(c)Compared with controls,LV global and segmental MDs were reduced in RVH group(p<0.001),and showed decreasing gradient from basal to apical segment(p<0.001).(ⅲ)Myocardium helix angle(HA)configuration:(a)In control group,LV myocardial fibers were distributed in three layers from epicardium to endocardium,that was,lefthanded myocardium(LHM)at the epicardium,circumferential myocardium(CM)at the mid-wall,and right-handed myocardium(RHM)at the endocardium;however,there was no typical three-layer distribution in RV.Segmental analysis of myocardial fibers in LV and RV all showed highest proportion in CM,followed by RHM and LHM.In addition,every segment had different proportions of LHM,RH and CM.The content of right-handed spiral myocardium(RHM)was the highest in the basal segment of left and right ventricles,the content of circular myocardium(CM)was the highest in the middle segment,and the content of left-handed spiral myocardium(LHM)was the highest in the apical segment.(b)LVH fetuses had no three-layer distribution of myocardial fibers: mainly CM and LHM at epicardium,and myofibers with three directions interleaved at endocardium.Segmental analysis showed similar distribution patterns in all RV segments as controls,which is CM,RHM and LHM from high to low.But the proportion of CM in basal,middle and apical RV segments all decreased,while RHM and LHM increased in those segments(p<0.001).(c)LV in RVH fetuses still can be detected with three-layer myocardial fibers arranged distinguishably: RHM at the endocardium,CM at the mid-wall and LHM at the epicardium.Quantitative analysis for LV in RVH fetuses showed myocardial proportions in each segment followed the distribution pattern: CM,LHM and RHM from high to low.Compared with control group,RHM proportions decreased in basal,middle and apical segment(p<0.001),while LHM increased in basal and middle segment(p<0.001),CM increased in basal and apical segment(p<0.001),and decreased in middle segment(p<0.001).(2)Correlation analysis between cardiac function and myocardial microstructure in nonhypoplastic sided ventricle:RV global longitudinal strain(RVGLS)in LVH fetuses was positively correlated with LHM proportions in RV basal and middle segment(R2=0.903,p=0.04;R2=0.95,p=0.01);LV global longitudinal strain(LVGLS)in RVH fetuses was positively correlated with RHM proportion in LV middle segment(R2=0.99,p= 0.002).Conclusion: Myocardium microstructure of non-hypoplastic sided ventricle altered in VH fetuses,which was also correlated with ventricular systolic function.Part Ⅲ Comparative Proteomics Study of Myocardium in Fetuses with Ventricular HypoplasiaObjective: This study was aimed to compare non-hypoplastic side ventricular myocardium proteomics profile in ventricular hypoplasia(VH)fetuses with normal fetuses,and bioinformatics analysis was performed for differentially expressed proteins to investigate mechanisms of myocardial function impairment.Method: A total of 6 fetuses diagnosed with VH were included from January 2016 to June 2022 in our center.According to fetal echocardiography,the cohort were divided into left ventricular hypoplasia group(LVH,n=3)and right ventricular hypoplasia group(RVH,n=3).All above specimens were from the second part of this study.After routine dissection,the corresponding ventricular myocardial tissue was taken for protein quantitative analysis based on liquid chromatography-mass spectrometry.DDA and DIA mapping were established,and characteristic Peak area of ion pairs were extracted by Spectronaut software.The protein expression of left/right ventricular myocardium in normal fetuses was used as the control group.Expression of myocardial proteins in control fetuses and non-hypoplastic side ventricles in LVH and RVH group were compared,respectively.Differential proteins were selected by fold change≥ 1.5 and p<0.05.Gene Ontology(GO)database and Kyoto encyclopedia of genes and genomes(KEGG)database were used for functional analysis and pathway enrichment analysis of differential proteins,respectively.The protein-protein interaction(PPI)network was analyzed by STRING database.In addition,the correlation between non-hypoplastic side ventricular strains in VH fetuses and expression quantity of differential proteins were analyzed.Results:(1)Differentially expressed proteins in LVH group:A total of 144 differential proteins in right ventricle(RV)of LVH fetuses were screened out,in which 68 proteins were up-regulated,76 were down-regulated.GO analysis showed that these differential proteins mainly constituted the structures of cell outer capsule,collagen extracellular matrix,endoplasmic reticulum lumen,chromosomal telomeres,contractile fiber,cytoskeleton,DNA replication licensing factor complex protein(MCM),etc.;participated in actin fiber depolymerization,endoderm formation,cell division,regulation of cell cycle G2-M transition and other biological processes;related to RNA binding,cell adhesion molecule binding,cytoskeletal protein and other molecular functions.KEGG analysis showed that differential proteins were involved in extracellular matrix receptor interaction,cell cycle and other signaling pathways.PPI analysis showed that MCM and NUMB protein homologues had the highest degree of inter-protein connectivity.(2)Differentially expressed proteins in RVH group:A total of 95 differential proteins were screened out in left ventricle(LV)for RVH fetuses,in which 66 proteins were up-regulated,29 were down-regulated.GO analysis showed that differential proteins mainly constituted the structures of secretion vesicles,intercellular junctions,plasma membrane complexes,collagen-containing extracellular matrix,contractile fibers;participated in biological processes such as muscle contraction,cytoskeletal aggregation,and regulation of cardiac contraction,binding to signaling receptors,and mediated molecular function including lipid binding;related to signal receptor binding,lipid binding,chaperone binding,actin binding and other molecular functions.KEGG analysis showed that differential proteins were involved in c-CMG-PKG signaling pathway,lipid signaling pathway,epinephrine signaling pathway in cardiomyocytes and other signaling pathways.The results of PPI showed that nucleolin(NCL)and muscular dystrophin(DMD)had the highest protein connectivity.(3)Correlation analysis between differential proteins and global longitudinal strains:The expressions of microtubule-binding protein(NEXN)(r=0.96)and myosin(MYH1)(r=0.91)were positively correlated with RVGLS in right ventricle of LVH fetuses.The expressions of DMD(r=0.93),voltage-dependent type I calcium channel subunit α-1s(CNCA1S)(r=0.74)and sodium-potassium transport ATP enzyme subunit α-2(ATP 1A2)(r=0.81)were positively correlated with LVGLS in left ventricle of RVH fetuses.Conclusion: The differential proteins in the non-hypoplastic sided of VH fetus are involved in many biological processes,such as cell cycle and myocardial contraction.The expression of microtubule binding protein(NEXN)and dystrophin(DMD)are related to the ventricular function of the non-hypoplastic sided of VH fetuses.