Clinical And Therapeutic Translational Research Of Cardiomyopathy In Children With Diastolic Dysfunction

PART Ⅰ CLINICAL AND GENE CHARACTERISTICS AND PROGNOSIS OF PEDIATRIC CARDIOMYOPATHY WITH DIASTOLIC DYSFUNCTIONObjective: Cardiomyopathy is a heterogeneous group of myocardial diseases and one of common causes of diastolic dysfunction in children.Due to the lack of effective treatments,the prognosis is poor.This study was aimed to analyze the incidence,clinical and gene characteristics,prognosis and risk factors of cardiomyopathy children with diastolic dysfunction,compared to cardiomyopathy children with systolic dysfunction cardiomyopathy,by collecting the data of Children’s Hospital of Chongqing Medical University during recent 10 years.Methods:(1)Data on 317 patients with cardiomyopathy who were at admitted in Children’s Hospital of Chongqing Medical University during November 2007 to December 2017 were retrospectively analyzed.(2)Survival function was estimated by Kaplan–Meier curve analysis.(3)Clinical characteristics and prognostic factors associated with mortality were evaluated by Cox proportional hazards regression analysis.(4)A total of 21 HCM and RCM children with diastolic dysfunction who were followed up in the past 2 years were tested by Whole Exon Sequencing.Results:(1)Of 317 studied patients,40.1%(127),25.2%(80),24.6%(78),and 10.1%(32)were diagnosed with hypertrophic cardiomyopathy(HCM),restrictive cardiomyopathy(RCM),dilated cardiomyopathy(DCM)and left ventricular noncompaction cardiomyopathy(LVNC),respectively.(2)168 were boys(53%)and 51 were girls(47%),with a mean age of 4.5 ± 4.4 years.Age at diagnosis was significantly different between subgroups(P ? 0.01).11(3.5%)patients with cardiomyopathy had family history.(3)For all patients,the most common symptom was dyspnea(84.2%),followed by hepatomegaly(59.3%),cyanosis(58.4%),cardiomegaly(50.8%),cardiac murmur(43.2%),peripheral edema(37.2%)and so on.Except for HCM,the majority of patients(62.2% DCM,59% LVNC,75% RCM)were in NYHA/Ross class III or IV.(4)35% cardiomyopathy experienced abnormal ECG.HCM and RCM were characterized with diastolic dysfunction,along with prolonged isovolumetric relaxation time(IVRT),abnormal E/A ratio and reduced left ventricular end diastolic dimension(LVEDD).DCM and LVNC were characterized with systolic dysfunction,along with reduced ejection fraction(EF)and increased LVEDD.(5)The cumulative survival rates among four groups were statistically different(log-rank P ? 0.05)according to Kaplan–Meier survival analysis.The five-and ten-year survival rates were 67.3% and 56.1%,51.1% and 41.3%,75.5% and 60.1%,74.1% and 57.2% in HCM,RCM,DCM and LVNC,respectively.At follow-up,92(29%)patients with CM died: 23(28.8%)in HCM,16(50%)in RCM 30(23.6%)in DCM,and 23(29.5%)in LVNC.(6)In HCM patients,the Cox repression analysis identified eight predictors of survival: jugular venous distension,hepatomegaly,NYHA/Ross class III or IV,P-R interval,EF,left ventricular posterior wall(LVPW)thickness,pericardial effusion,and WPW syndrome.The prognosis of RCM was related to male,family history,orthopnea,NYHA/Ross grade III or more,P-R interval,and LVEDD.For DCM patients,the risk of death was significantly associated with NYHA/Ross class III or IV,QTc interval,EF,faction shortening,and moderate-severe tricuspid regurgitation.QRS duration,right ventricular end diastolic dimension(RVEDD),and noncompaction to compaction ratio(N/C)were risk factors for LVNC.Survival was adversely related to NYHA/Ross class III or IV in patients with DCM,HCM or RCM.(7)In this group,17(80.9%)of 21 children with HCM or RCM were found to have genetic abnormalities.Among the 9 cases of RCM,5 cases were detected with gene mutation,and 3 cases were carried with TNNI3 gene mutation at amino acid site 192.Mutations were detected in all(12)children with HCM,and 5 children with HCM carried MYH7 mutations.Conclusion:(1)In this study,the proportion of HCM and RCM was relatively low,RCM was the lowest,and DCM was the highest.(2)HCM and RCM were characterized with diastolic dysfunction,HCM had relatively mild clinical symptoms.DCM and LVNC were characterized with systolic dysfunction.(3)The prognosis of DCM was relatively good,whereas that of RCM was the worst.Several factors were related to the prognosis of cardiomyopathy,and NYHA/Ross class III or IV is an increased risk of death in patients with DCM,LVNC,and RCM.(4)Most of the cardiomyopathy children with diastolic dysfunction carried gene mutations,mainly located in the gene encoding the myosin protein.MYH7 was the most common in HCM and TNNI3 was the most common in RCM.(5)there is overlap between genotypes and phenotypes,and some HCM cases may be related to innate metabolic factors,which needs early gene and related detection.PART Ⅱ PATHOLOGY AND CTNI PROTEIN ANALYSIS OF RESTRICTEIVE CARDIOMYOPATHYObjective: PART I study revealed,children with RCM have the highest mortality and the worst prognosis and cardiac troponin I(c Tn I)mutation is a common cause of RCM.Myocardial histopathological analysis is important for the diagnosis,differential diagnosis,analysis of disease progression and histological features of RCM.In this study,tissues of RCM patients with diastolic heart failure were collected for pathological comparative analysis,and the associated pathogenic protein c Tn I was detected.Methods:(1)Clinical data and heart samples were collected.(2)RCM pathological changes,including heart,lung,liver and kidney,were analyzed by HE,Masson,PAS and Congo red staining.(3)Ultrastructure of hearts was observed by transmission electron microscope.(4)The heart tissues of 1y,40 y,52y and RCM were extracted into protein,and the expression level of c Tn I is detected by WB.Results:(1)In the RCM patient with c Tn I R192 C mutation,echocardiographic manifestations were bi-atrial enlargement and restrictive filling disorder.(2)Compared to control group,RCM heart showed enlarged bi-atriums,stenotic ventricles and ventricular endometrial thickening.HE and Masson staining showed cardiac fibrils were irregular and subintimal collagen fibers were significantly proliferated,respectively.PAS and Congo red staining did not reveal any abnormality in the RCM heart.Transmission electron microscopy showed that left ventricular myocardial muscle filaments were disordered and dissolved.Z-lines were distorted and mitochondrial arrangement was disordered and damaged in the RCM heart.(3)In RCM,the lung demonstrated its volume was increased,the capsule was tense,the cut surface was dark red,the texture became hard,and the whole lung was scattered with rust spots.HE staining showed hemosiderin particles and heart failure cells and Masson staining showed that subintimal collagen fibers were significantly proliferated in the lung.(4)In RCM,the liver demonstrated its volume was increased,the capsule was tense,the cut surface was dark red and the texture became hard.HE staining showed disordered hepatic lobular structure,and hepatocellular atrophy,degeneration and necrosis.Masson staining showed that subintimal collagen fibers were significantly proliferated in the liver.(5)In RCM,the kidney showed its volume was increased,the capsule was tense,the cut surface was dark red and the texture became hard.HE staining revealed blurred nephron structure and dilatated glomerular capillary,and hypertrophic renal tubular cells and Masson staining showed that subintimal collagen fibers were significantly proliferated in the kidney.(6)c Tn I protein expression levels of RCM and elderly heart left ventricular and ventricular septal myocardial tissue were lower than that of young adults.The c Tn I protein level of left ventricular and ventricular septal myocardial tissues in RCM was lower than that in right ventricle.Conclusion:(1)RCM mainly manifested as enlarged bi-atriums,increased stiffness of the ventricular wall,and restrictive filling disorders,which could eventually develop into diastolic heart failure,which may be related to the destruction of myocardial myofilaments and interstitial fibrosis.(2)Severe chronic heart failure can cause congestion and lesions in lung,liver,kidney and other tissues.(3)c Tn I protein expression level was decreased in the left ventricular and ventricular septal with c Tn I mutation,which was similar to the elderly heart.PART Ⅲ EXPERIMENTAL TREATMENT OF DIASTOLIC DYSFUNCTION IN RESTRICTEIVE CARDIOMYOPATHY MICEObjective: PART I and II demonstrated RCM mainly manifested as diastolic dysfunction,and finally developed into diastolic HF.The treatment of diastolic dysfunction is mainly symptomatic treatment with limited means.In this study,two kinds of small molecule compounds with different mechanisms were used(The first one is the compound acting on myosin,namely M-compound;The second is catechin EGCG(T-compound),which acts on cardiac troponin,to conduct experimental treatment on RCM transgenic(TG)mice,and compare the therapeutic effects of these two small molecule compounds on heart function of RCM TG mice.Methods:(1)Wild-type(WT)and RCM TG mice were randomly divided into the solvent 0.3mg/kg,and 1mg/kg groups of M-compound,and solvent,50mg/kg,200mg/kg groups of T-compound.All mice were tested with high-frequency echocardiography before intervention.(2)M-compound was injected subcutaneously at the back neck,and high-frequency echocardiography was performed after 2h.(3)T-compound EGCG 50mg/kg/day was intraperitoneally injected for 3 months(from the age of 2 months),high-frequency echocardiography was performed after the intervention.EGCG 200mg/kg was performed to observe the effects.Results:(1)Compared with WT mice,IVRT of TG mice was prolonged,tissue Doppler E’ was reduced,E’/A’ ratio was abnormal,E/E’ ratio was increased(P ? 0.05),and EF and shortening rate(FS)were normal in RCM mice.(2)No significant changes of systolic and diastolic functions in WT mice with 0.3mg/kg M-compound(P > 0.05).IVRT,E’,and E/E’ were partially or completely reversed in TG mice(P ? 0.05),with no significant changes of EF and FS.However,LVEDD,left ventricular end-diastolic volume(LVEDV),left ventricular end-systolic diameter(LVESD),and left ventricular end-systolic volume(LVESV)were significantly reduced(P ? 0.05).(3)Diastolic function of WT mice was not changed significantly after1mg/kg M-compound treatment(P > 0.05),and LVESD and LVESV were increased significantly However,EF and FS were decreased significantly(P ? 0.05).IVRT,E’,and E/E’ were partially or completely reversed in TG mice,with increased LVESD and LVESV(P ? 0.05).But EF and FS were also decreased significantly.(4)After 3 months of intraperitoneal injection of 50 mg/kg/day T-compound,the systolic and diastolic function of WT mice were not changed.IVRT was completely reversed,and LVEDD,LVESD,LVEDV,and LVESV were increased(P ? 0.05),along with remained EF and FS in each group.(5)Mice were treated with 4 times dosage of T-compound,and there was no abnormal performance and death.Conclusion:(1)M-compound,interacted with myosin,could improve cardiac diastolic function in RCM mice,but also has adverse effects on systolic function.High doses of M-compound could seriously impair systolic function,and the effect is in a dose-dependent behaver.(2)Long-term low-dose T-compound,interacted with myosin,could improve cardiac diastolic function in RCM mice,and the systolic function is not affected,which suggests T-compound is better than M-compound to use in vivo.PART Ⅳ CATECHIN IMPROVES DIASTOLIC FUNCTION IN CHILDREN WITH CARDIOMYOPATHYObjective: Previous parts showed,HCM and RCM are characterized by diastolic dysfunction,severe conditions in the middle and late stage,limited treatments and poor prognosis.Catechin EGCG with good safety could improve the diastolic function in cardiomyopathy mice with diastolic dysfunction without effect on systolic function.This study was aimed to assess the efficacy and safety of catechin EGCG in the treatment of cardiomyopathy children with diastolic dysfunction.Methods: Twelve cardiomyopathy children(HCM and RCM)with diastolic dysfunction,aged 0.8 to 14.2 years,were included in this study.The initial dose of catechin was 15mg/kg/day and gradually increased to 50mg/kg/day for 12 months.They also received routine treatments.All children underwent echocardiography,laboratory and electrocardiogram before and after catechin treatment.Results: After treatment for 6 months,LVEDV(40±28 vs 53±28 ml,P = 0.028)was increased by 17.1%,stroke volume(SV,25 ± 16 vs 32 ± 17 ml,P = 0.022)increased by 14.9%,and IVRT significantly decreased(P = 0.047).After 12 months,LVEDV(40±28 vs 48±33 ml,P = 0.011)was increased by 17.0%,SV(25 ± 16 vs 30 ± 17 ml,P = 0.022)increased by 23%,and the E/A ratio was improved(P = 0.018).IVRT was significantly decreased(P = 0.033).No significant changes were observed in LVESD,LVEDD and LVESV before and after treatment(P > 0.05).(2)There was no change in EF,ventricular wall thickness and atrial size(P > 0.05).(3)There were no significant changes in heart rate and blood pressure(P > 0.05).(4)No significant side effects were observed in the patients tested.Conclusion: This study indicates that catechin EGCG,is helpful and safe in correcting the impaired relaxation in pediatric cardiomyopathy patients with diastolic dysfunction.