Study On The Occurrence Mechanism And Intervention Of Arrhythmia

Commom Variants in TRDN and CALM1 Are Associated with Risk of Sudden Cardiac Death in Chronic Heart Failure Patients in Chinese Han PopulationBackground Chronic heart failure （CHF） represented a major public health problem worldwide owing to high prevalence, poor prognosis and extreme expenditure. Despite of advances in understanding and management of patients with CHF, the morbidity and mortality rates were still high. The 5-year mortality was still>50%, but the current data of long-term prognosis of CHF in China were still missing. More than half of the deaths were due to sustained ventricular tachycardia （VT） or ventricular fibrillation （VF） leading to sudden cardiac death （SCD）. Therefore, prediction and prevention of SCD have been recognized as pivotal step toward improved outcomes. Several indicators have been recognized as potential predictors of SCD, such as hemodynamic status, electrophysiological parameters, biomarkers and inflammatory factors, but the sensitivity and specificity were not satisfied so far.Susceptibility to sudden cardiac death was considered to be a heritable trait in general population. Sudden cardiac death was initiated by lethal arrhythmias, and Ca2+ cycling plays a critical role in heart failure and lethal arrhythmias. Moreover, recent studies suggest that variants （rs3766871, rs790896, and rs17500488, rs3010396, rs7366407） in two calcium handling genes （RyR2 and CASQ2） associated with SCD in subjects with heart failure and coronary artery disease, respectively.ObjectiveThe purpose of this prospective study were 1) to investigate the association between genetic variations of calcium handling genes and prognosis of patients with CHF; 2) to identify calcium handling genes associated with SCD in Chinese Han population.Materials and MethodsWe conducted an ongoing prospective study of patients with CHF from Fu Wai Cardiovascular Hospital from 2005 to 2009. Enrolment criteria included:CHF caused by dilated cardiomyopathy （DCM） or ischemic cardiomyopathy （ICM）; in NYHA （New York Heart Association） functional class II-IV despite optimized medical therapy; and LVEF≤45% in DCM and≤50% in ICM. A standard HF questionnaire was completed during regular outpatient clinics or by telephone contact. Panicipants were genotyped using MALDI-TOF MS genomics platform, MassARRAY system （Sequenom） for twenty variants. For statistical analysis, In addition to the genotype, univariate analysis and multivariate Cox proportionalCommon Variants in TRDN and CALM1 Are Associated with Risk of Sudden Cardiac Death in Chronic Heart Failure Patients in Chinese Han Population hazards models were performed to estimate the effect of genotype on survival. Kaplan-Meier curves with the use of the log-rank test according to the presence or absence of the allele was also analyzed.ResultsWe investigated 20 SNPs representing 10 genes that regulated calcium handling in 1429 patients with CHF, and the genetic association with SCD and all-cause death was analysis. During a median follow-up period of 63 months,538 patients （37.65%） died from CHF, of whom 185 （34.38%） had SCD and the others were NSCD. SNPs that pass a P value cut-off of 0.0025 were considered as significant. We found that patients carrying the CC genotype of rs3814843 on CALM1 gene had greater risks of SCD （HR 5.542,95% CI 2.054-14.948, P=.001） and all cause death （HR 3.484,95% CI 1.651-7.350, P=.001）. After adjusting for other risk factors, significant associations remained. Moreover, patients carrying G allele of rs361508 on TRDN gene also had increased risk of SCD.ConclusionsCommon variant in CALM1 （rs3814843） was associated with increased risk of SCD and all cause death in patients with CHF. Another SNP （rs361508） was also associated with increased risk of SCD. These finding provide further evidence for association of variants in calcium handling regulating proteins and SCD in chronic heart failure.A Novel Lamin A/C Gene Missense Mutation （445 V>E） in Immunoglobulin-like Fold Associated with Left Ventricular NoncompactionBackground Left ventricular noncompaction （LVNC） is a cardiomyopathy and characterized by a meshwork of interwoven myocardial strings lined with endocardium, which constitutes a spongy myocardial layer clearly distinct from the underlying compacted myocardium （two-layered myocardium）. Although the etiological cause of LVNC is largely unknown, the genetic defect is suggested by the familial LVNC. Previous studies have identified mutations in multiple genes （MYH7, ACTC, TNNT2, MYBPC3, LMNA, TAZ, DTNA, and SNTA1 etc.） associated with LVNC. Two mutations （R644C and R190W） of LMNA have also been reported.Lamin A/C （LMNA） gene （GenBank accession no. NC₀00001） maps on the long arm of chromosome 1 （1q21.2-q21.3） and encodes two main proteins, lamin A and lamin C by alternative splicing. They diverge only at their C-termini. Lamin A/C is a type V intermediate filaments protein that contains a N-terminal head, an a-helical rod domain and a long C-terminal tail containing an conserved globular domain exhibiting an immunoglobulin （Ig）-like fold. The Ig-like fold consists of nine p strands, forming two β-sheets of four （2,3,6, and 7） and five （1,4,5,8, and 9） strands, respectively, packed into a classical β sandwich. The targets of Ig-like fold are extremely diverse, including DNA and nuclear proteins, such as lamina-associated protein 2, emerin. Lamin A and lamin C proteins are components of the nuclear envelope, forming the inner nuclear membranes and the nuclear lamina. Stable interactions of lamins with inner nuclear membrane proteins are fundamental for the mechanical integrity of the nuclear envelop.Moreover, mutations of the LMNA gene were associated with diverse phenotypes including Emery-Dreifuss muscular dystrophy （EDMD）, limb girdle muscular dystrophy of type 1B （LGMD）, dilated cardiomyopathy, and familial partial lipodystrophy （FPLD）. Previous studies also demonstrated that LMNA mutation could cause lethal ventricular arrhythmia and sudden cardiac death. Although prophylactic implantable cardioverter-defibrillator （ICD） was implanted to save patients’ lives, the role of LMNA mutation in life-threatening ventricular arrhythmia has still been bleared.ObjectivesPrevious studies demonstrated that two LMNA mutations （R644C and R190W） have associated with familial and sporadic Left ventricular noncompaction （LVNC）. However, the mechanisms underlying these associations have not been elucidated.MethodsGenomic DNA was isolated from peripheral blood leukocytes and analyzed by direct sequencing. Human embryonic kidney 293 cells were transfected with either wild type or mutant LMNA and SCN5A for whole-cell patch clamp experiment and fluorescence microscopy. Point mutation modeling for mutant LMNA was also performed.ResultsOne novel LVNC associated mutation （V445E） in β2 sheet of immunoglobulin-like fold was found in the proband and his father. We also found that the peak current of sodium channel was markedly reduced in mutant LMNA compared with WT while the activation, inactivation and recovery curve was not significantly altered. The mutant lamin A/C were aggregated into multiple highlighted particles. Three β sheets and multiple side chains in immunoglobulin-like fold were altered due to the replacement of a Valine by Glutamic Acid.ConclusionsOur data associated a novel lamin A/C mutation （V445E） with a sudden death form of familial LVNC. The reduced sodium current in mutant LMNA may account for the advent of malignant ventricular arrhythmias. The altered structures of three β sheets and side chains may partially explain the aggregation of lamin A/C protein subjacent to the nuclear envelope.Gene Expression Profile and Proteomics Study of Increased Heart Rate in Shensongyangxin-Treated Bradycardia RabbitsBackgroundBradycardia is a condition in which pulse rate is below 60 beats per minute （bpm）. Coronary artery disease patients and elderly people are at a great risk of developing the abnormally slow heart rate. Currently available drugs （e.g., atropine, dopamine, isoproterenol, and epinephrine） treating bradycardia are temporizing measures only in emergency settings. If the patient does not respond to drugs, temporary or permanent cardiac pacemaker is probably indicated. However, the cost of pacing put a huge financial burden on the family. Consequently, an effective drug aiming at increasing heart rate for a long term is in urgent demand. Traditional Chinese medicines have been used to treat arrhythmia for hundreds of years and ShenSongYangXin （SSYX） is one of such medicines. It is a product consisting of 12 ingredients including Panax ginseng, dwarf lilyturf tuber, and Nardostachys root. Whole-cell patch clamping experiments revealed that SSYX could block multiple ion channels. Preliminary studies also suggested that SSYX was effective in reducing ventricular premature beat and treating bradycardia. In addition, the latest results from a randomized, double-blind, placebo-controlled multicenter trial demonstrated that SSYX is effective for patients with bradycardia. Despite of those results from both basic and clinical research, it has remained a mystery how SSYX plays the positive role in treatment. Especially the affected genes and proteins have not been extensively explored. Therefore, we established an animal model of bradycardia and explored the gene expression and protein outcomes after SSYX treatment.ObjectivesThe present study tries to investigate the molecular mechanisms of SSYX in treating bradycardia using a bradycardia animal model. Both altered genes and proteins of hearts from bradycardia rabbits are extensively explored between groups.MethodsEighteen adult rabbits were randomly assigned in three groups:sham, model, and SSYX treatment groups. Then, heart rate was recorded in rabbits and total RNA was isolated from hearts. Subsequently, gene expression profiling was conducted using gene chip and quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR） was performed to confirm the gene expression results. Patch clamp using human induced pluripotent stem cell-derived cardiomyocytes was also applied to record the calcium current in the presence of SSYX. Finally, differentially expressed proteins were identified using isobaric tags for relative and absolute quantitation （iTRAQ）.ResultsThe mean RR interval was prolonged after six weeks due to the injury of the sinoatrial node in the model group. This effect was partially reversed by 4-week SSYX treatment. cDNA microarray demonstrated that genes related with pacemaker current, calcium ion homeostasis, and signaling were altered by SSYX treatment. Results from patch clamp demonstrated that SSYX reduced the calcium current which is consistent with gene expression results. ITRAQ results also demonstrated increased calcium channels in both sarcoplasmic reticulum （Ryanodine receptor calcium channel 2, calcium ATPase） and mitochondria （voltage-dependent anion channel） and calcium binding proteins after SXSM treatment. While the proteins involved in metabolism were decreased.ConclusionsThe present study shows mRNA remodeling of bradycardia and demonstrates that SSYX is effective in treating bradycardia by reversing altered gene expression in bradycardia atria. Reduced calcium current by SSYX also confirmed the gene expression results. Moreover, increased calcium channels and calcium binding proteins were confirmed again in ventricle at protein level. Therefore, calcium homeostasis plays an essential role in the molecular mechanisms of SSYX.