| BackgroundDilated cardiomyopathy(DCM) is a kind of common cardiomyopathy, involvingdilation of the left, right or both ventricle chambers, with impaired systolic function.Previous epidemiological studies have indicated a prevalence of DCM was36.5per100,000of the population. However, these data reflect older diagnosis modalities andtherefore likely significantly underestimate the disease prevalence. DCM seems tooccur much more commonly. Progressive heart failure and various ventriculararrhythmias are common complications in patients with patients, which are associatedwith a significant mortality.Despite most cases of DCM are sporadic, genetic factors still perform adominant role in the pathogenesis of DCM. It has been reported that geneticinheritance accounts for approximately30%to50%of cases, which mainly includespathologic mutations in approximately50genes encoding for proteins involving incytoskeletal elements, sarcomeric elements, signaling and desmosomal pathways.ARVC is reported to be associated with some specific gene ontologies, withmutations in sarcomeric or desmosomal proteins predominating, respectively. Bycontrast, the genetic basis of DCM is highly diverse. Mutations in SCN5A, encodingfor the α-subunit of cardiac voltage-gated sodium channel, and in ABCC9, encodingfor the cardiac specific SUR2A subunit of the KATPpotassium channel, have beenreported in DCM with arrhythmia. Given that the DCM patients are tend to becomplicated with various ventricular arrhythmias, or even malignant arrhythmiassuch as ventricular tachycardia (VT) or ventricular fibrillation (VF) which may leadto sudden death, researches on the molecular genetic mechanisms of DCM associatedarrhythmia susceptibility will have great significance.Gender difference has been indicated in the prevalence of DCM, while thereasons are unclear. Whether the sex hormones or disruptors are responsible for thisstill needs further investigation. Bisphenol A(BPA), widely recognized as environmental hormone disrupting compound, is one of the highest volume chemicalsused in the synthesis of polycarbonate plastics and epoxy resins. Human exposure ofBPA is continuous and everywhere. Since researchers found BPA can leach fromeveryday plastic containers and be absorbed into mice during consumption ofcontaminated drinks and foods, mice exposure of BPA was shown to be associatedwith reproductive deformity. BPA exposure on human health hazards cannot beignored. A growing number of studies have shown that high exposure to BPA maycause some hormone-sensitive diseases such as reproductive toxicity, precociouspuberty, unexplained spontaneous abortion, breast cancer, prostate cancer and evenlead to brain damage. Recent epidemiological investigations have disclosed theassociations between BPA exposure and some heart diseases. Evidence fromexperimental animal studies suggests that lifelong exposure to BPA can impactcardiac calcium homeostasis as an estrogenizing endocrine disruptor and alter cardiacstructure/function. However, no research on the association between BPA and DCMhas been reported till now. Section1Molecular Genetics and Cellular ElectrophysiologicalStudy on Dilated Cardiomyopathy Patients with VentricularTachycardiaObjectives：Candidate gene approach was designed to identify the novel mutation that wouldbe responsible for dilated cardiomyopathy (DCM) patients with ventriculartachycardia(VT), including genes associated with DCM and potassium channel genes.Electrophysiological analysis, fluorescence confocal microscopy and western blottingwere performed to characterize the functional consequences of the mutation toexplore the pathogenesis of DCM with VT. The present study also analyzed themedical records of the mutation carrier to explore the potential significance ofcandidate genetic testing for diagnosis and treatment.Participants and Methods1. Clinical investigation: The study was approved by the regional institutionalreview board and all subjects gave a written informed consent to participate in thestudy. DCM patients with VT from the cardiovascular department of our hospital ofNanchang university were included. DCM was diagnosed and managed according tothe AHA scientific statement in2006and the recommendations of Chinese experts onthe diagnosis and management of cardiomyopathies. According to the ACC/AHA/ESC2006guidelines for management of patients with ventricular arrhythmiasand the prevention of sudden cardiac death, all participants are required to show atleast one VT episode in ECG recording.2. Genetic screening: Blood samples (5mL) were obtained from all patients.DNA was extracted from peripheral blood leukocytes of patients and controls usingthe method provided in a commercial kit (DP333-01, Tiangen Biotech, Beijing,China). All the exons and exon-intron boundaries of candidate genes, includingLMNA, MYH7, TNNT2, TNNI3, PKP2, DSP, DSG2, SCN5A, KCNQ1, KCNE1,KCNE2and KCNH2, were amplified by polymerase chain reaction (PCR). PCRproducts were purified with SAP-EXON I purification method and sequenced withBigDye Terminator version3.1(Applied Biosystems, Carlsbad, CA, USA).Sequencing reactions were directly sequenced in both directions with an ABI PRISM 3130XL Automatic DNA Sequencer (Applied Biosystems, Carlsbad, CA, USA). Thecontrols were400healthy individuals from the same ethnic background and70DCMpatients without VT.3. Mutagenesis and transfection: Mutated KCNQ1channel cDNA wasconstructed by site-directed mutagenesis system using the method described in theOnline Supplemental Methods. Mutagenic primers were designed according to themutation, and then the mutated plasmid was sequenced to ensure the presence of theR397Q mutation as well as the absence of other substitutions introduced by PCR.Human embryonic kidney293(HEK293) cells were transfected with cDNA clonesencoding Wild-type (WT as a gift from Dr. Chen YH’s laboratory) and R397Q-KCNQ1.4. Cellular Electrophysiology: Whole-cell patch-clamp technique was used toanalysis the electrophysiological characteristics. All relevant parameters such ascurrent density, peak and the activation curve were recorded and analyzed. Allprocedures were done at room temperature. Fitmaster, Sigma Plot, OriginPro7.5software were used for data acquisition and analysis.5. Immunofluorescence was performed to examine the distribution pattern ofthe mutant and WT KCNQ1channel proteins. Triton X-114temperature-inducedphase separation method was used to isolate membrane fraction. Western blotting wasapplied to analyze the expression of the mutant and WT KCNQ1channel proteins.6. All statistics were performed using SPSS18.0software. Values are expressedas means±standard errors of the mean (SEM). Statistical comparisons wereperformed with t-test or one-way ANOVA test. A p value of <0.05was consideredstatistically significant.Results1. Ten unrelated patients with age from16to77years old were included in thisstudy. Three of them are females. The average age was48.7±20.4years old. Twopatients among them were combined with complete right bundle-branch block, andone had complete left bundle-branch block. The echocardiogram showed that therange value of left ventricular end-diastolic dimension (LVEDD) was from53mm to75mm, that of the left ventricular end-systolic dimension (LVESD) was from40mm to67mm and that of left ventricular ejection fraction (LVEF) was from22%to45%.2. Three novel heterozygous missense mutations were found by DNAsequencing, including KCNQ1-p.R397Q, DSG2-p.R824G, SCN5A-p.V1604M, whichwere absent in the400healthy control and70DCM patients without VT.KCNQ1-p.R397Q mutation consisting of G-to-A substitution at nucleotide site1190(c.1190G>A), predicted a substitution of arginine for glutamine at codon site397(p.R397Q). This mutation carrier was a60-year-old male with DCM and VT. Heexperienced radiofrequency ablation and oral amiodarone therapy but had asuboptimal response. Recurrent VT episodes made him receive the secondradiofrequency ablation and implantation of ICD. He was in good condition in thesubsequent follow-up. SCN5A-p.V1604M mutation consisting of G-to-A substitutionat nucleotide site4810(c.4810G>A), predicted a substitution of value for methionineat codon site1604(p.V1604M). This mutation carrier was a61-year-old male withDCM and VT. He had been diagnosed with DCM for ten years. The main symptomswere chest tightness and short of breath. This patient has lost follow-up currently.DSG2-p.R824G mutation consisting of C-to-G substitution at nucleotide site2470(c.2470C>G), predicted a substitution of arginine for glycine at codon site824(p.R824G). This mutation carrier was a65-year-old male with DCM and VT. Theclinical manifestation was mainly described as heart failure and syncope. Despite ofthe severe phenotype, he refused to receive ICD or CRTD implantation. Six monthafter he was discharged, sudden death occurred at home. In addition, five singlenucleotide polymorphisms(SNPs) were identified, including KCNQ1-p.I145I,DSG2-p.R773K, DSG2-p.T835T, MYH7-p.I989I and PKP2-p.P273P. Three of themwere not reported previously, which were DSG2-p.R773K, DSG2-p.T835T, andPKP2-p.P273P.3. WT and mutant R397Q KCNQ1cDNA were respectively transfected intoHEK293cells transiently to examine potassium current density and conduction.R397Q was shown to decrease Ikscurrent density dramatically than WT. However,neither relative activation curves nor time constant of deactivation for R397Q andWT had statistical difference. R397Q didn’t influence the V0.5and slope factor for thevoltage dependence of activation compared with WT. 4. To examine whether the R397Q mutation affects trafficking and membranelocalization in KCNQ1, HEK-293cells transfected with either KCNQ1-WT orKCNQ1-R397Q were analyzed by confocal fluorescence microscopy. Imaging resultsshow that a smaller portion of KCNQ1-R397Q appears to be distributed on thesurface membrane compared with KCNQ1-WT, while KCNQ1-R397Q primarilylocates in the interior of the cells. Western blotting experiments were performed toquantify surface expression of KCNQ1-R397Q versus KCNQ1-WT. Our data showthat KCNQ1-R397Q surface expression levels are significantly lower than that ofKCNQ1-WT (P<0.01).Conclusion1. KCNQ1gene is firstly reported to be associated with patients with DCM andVT. Further functional analysis show that a novel KCNQ1-p.R397Q mutation causesdefective channels through decreasing Iks current density due to traffic proteinproblem and the decreased protein expression on membrane localization, therebyenhancing the arrhythmic susceptibility.2. Two novel heterozygous missense mutations were identified in DCMpatients with VT, DSG2-p.R824G and SCN5A-p.V1604M. It is indicated that DCMpatients prone to VT may be associated with several genes.3. Five SNPs are found in this study, including KCNQ1-p.I145I, DSG2-p.R773K, DSG2-p.T835T, MYH7-p.I989I and PKP2-p.P273P. Three of them,DSG2-p.R773K, DSG2-p.T835T and PKP2-p.P273P, are not reported previously.4. The case of KCNQ1-p.R397Q mutation carrier also indicates that themutation may be responsible for the poor effect of radiofrequency ablation andamiodarone for treating VT. It is suggested that genetic screening could ideallyidentify the DCM patients at high risk of VT and guide the optimal management.5. DCM is considered as a cardiac structural disease, as well as a kind ofchannelopathies. Further study on the underlying molecular mechanism of ionchannel in the pathogenesis of DCM is needed, which will help to find new targets forDCM therapy. Section2Investigation on Serum Bisphenol A and Sex Hormone inDilated Cardiomyopathy PatientsObjectivesTo determine serum BPA concentrations and sex hormonal values in dilatedcardiomyopathy (DCM) patients compared with age-and sex-matched healthycontrols, as well as elucidate the association between BPA and sex hormonal values.Participants and Methods1. A case-control (1:1) study was used to test the hypothesis. Patients diagnosedwith DCM were included from the cardiovascular department in our hospital betweenMarch2012and July2013. The diagnostic criteria of DCM were from the AmericanHeart Association Scientific Statement. The control group was comprised of age-andgender-matched healthy volunteers from the medical center of our hospital during thesame period. Subjects who had taken hormonal drugs in the last3months wereexcluded from the study. The local ethics board of our Hospital approved this studyprotocol. All participants were required to sign a written informed consent beforetheir participation, including collection of blood specimens.2. Six milliliter venous blood was drawn and collected into a general vacuumtube at7:00-10:00from all DCM participants on admission or control population onthe examination day. The samples were centrifuged after retraction of the clot. Thenthe serum was separated and stored at-80℃until assayed for BPA, total testosterone(T), sex hormone-binding globulin (SHBG),17β-estradiol (E2), Estrogen Receptor α(ERα), and Estrogen Receptor β (ERβ). The free androgen index (FAI) was calculatedby the formula: total T in nmol/L×100/SHBG in nmol/L. Serum levels of all valuesmentioned above were determined by using enzyme-linked immunosorbent assay(ELISA) kits according to the manufacturer’s instructions. The BPA ELISA kits, capablefor the quantitative determination BPA in human serum and plasma, were provided byIBL Co., Ltd., Gunma, Japan. The other ELISA kits were provided by Uscn LifeScience Co., Wuhan, China. All serum samples were strictly collected and storedaccording to the manufacturer’s instructions. All measurement procedure was referred tothe operation manual. Duplicate measurement of all test samples and standard wasperformed. The OD values of all samples were examined using multifunctional microplate reader. A standard curve of each assay was created on log-log or semi-loggraph paper, or by using some plot software, for instance, curve expert1.30. Then thesample concentrations were calculated using the OD values and the standard curve.3. Data analysis was performed using the statistical package SPSS version18.0(SPSS, Inc.). Continuous variables were examined for normal distributions andexpressed as mean±SD, which were compared between the two groups usingStudent’s t-test or Mann-Whitney test as a function of the normalcy of the data.Categorical variables were expressed as a percentage, which were compared using theChi-square test. Multiple linear regression analysis was used to investigate theassociation between serum BPA levels and hormonal parameters after controlling andadjusting for potential confounders. All tests are two-sided, and P value less than0.05was considered statistically significant.Results1. Eighty eight DCM patients and eighty eight age-and sex-matched healthycontrols were included in this study. There were59men vs.29women with DCM,and the male: female ratio of DCM incidence rate was2.0:1. The mean age of DCMgroup was59.6±13.2years, while the mean age of healthy controls was59.0±12.7years. No statistical difference between the two groups was noted as a function ofgender.2. Serum BPA levels were significantly higher in the total DCM groupcompared with total healthy controls (6.9±2.7ng/ml vs.3.8±1.9ng/ml, P<0.001).Both DCM patients and controls were divided in two gender subgroups. DCM malesas well as DCM females were comparable for age with their corresponding controlgroups. There were also significant differences in BPA levels between the twosubgroups, male DCM group vs. male healthy control group and female DCM groupvs. female healthy control group. They were (7.0±2.9) ng/ml vs.(3.9±2.0) ng/ml and(6.5±2.2) ng/ml vs.(3.8±1.8) ng/ml, respectively (P<0.001).3. Higher serum SHBG levels were also observed in DCM group comparedwith controls (76.9±30.9vs.41.0±15.6nmol/L, P<0.001), while serum T levels andFAI were significantly lower (488.3±188.2pg/ml vs.555.8±165.8pg/ml,2.9±3.5vs.5.3±2.6, respectively, P<0.001). Both males and females with DCM had significantly higher serum SHBG levels and lower FAI values compared with those in therespective subgroups of controls (P<0.001). Additionally, there was statisticallysignificant difference in serum T levels between DCM males and the correspondingcontrols, but no difference was observed in the female subgroup.4. To assess the association between serum BPA levels and hormone relatedvalues, a linear regression analysis was conducted among all subjects. There was astatistically significant association between serum BPA levels and SHBG levels(β=0.041;95%CI,0.024-0.058, P<0001). No statistical significant association wasnoted between serum BPA level and the independent valuables including T, E2, ERα,ERβ and FAI.Conclusions1. Our findings firstly demonstrated that BPA exposure evident in serum BPAlevels) was higher in those patients with DCM than those healthy controls. In addition,higher serum SHBG and lower FAI was also observed between total DCM group andthe total control group, as well as among the gender subgroups. Serum T levels wassignificantly lower in total DCM patients and male DCM patients than respectivecontrol groups.2. A positive association between BPA and SHBG was found. There is nostatistical association between BPA and other hormonal parameters. However, DCMpatients with higher BPA levels present lower FAI values and T levels. It can beindicated that increased levels of SHBG in DCM patients may be associated withhigher exposure of BPA. |
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