The Research On Protein Expression And Gene Transcription Of Proteins Influencing The Calcium Homeostasis In Patients With Atrial Fibrillation

Atrial fibrillation(AF)has become the most familiar cardiac arrhythmia.According to the statistics,in the crowd over 60 years of age,AF occurrence rate is up to 1%,and increase with the age.Its clinical significance is what affects hemodynamic and the high incidence of systemic circulation embolism.According to the report,AF decreases cardiac output to 25%or above.The opportunity of brain stroke is 5-7 times higher in non-valvular diseases with AF than non-AF patients,and the incidence of brain embolism in AF with valvular heart diseases fills the occurrence rate higher.But up to the present,it takes place and maintains the mechanism to not yet clarify completely,so no matter is a medicine,radio frequency ablation, double atrium pacing,surgical operation or the implantable atrium defibrillator etc.means,the rhythm recovery and prevention of AF,the sinus rhythm maintenance,the ventricular rate control and even embolism prevention and treatment haven't obtained the breakthrough progress,brought very painful and the heavy burden for sufferer,family,unit,society,nation, already to become the global cardiovascular disease of 21 centuries to learn important challenge that the medical worker face together.Study the enunciation at home and abroad,the natural history of the AF is tended along with change of time in depravation,namely:the occurrence of paroxysmal AF becomes into action the meeting to tend along with change of time in multifarious,continuously time prolong,and often make progress for keep on sex or chronic AF;AF course of illness is more short,medicine or wire reply the success rate of the sinus rhythm more high.This kind of atrium electricity the physiology change that be induct by AF promote the occurrence of the building AF and turn permanently,drive be called the heavy remodeling of the atrium electricity physiology.(The electrophysiological remodeling,EPR)the generally accepted and the most important characteristic in EPR is the atrium action electric potential timing and shouldn't expect to shorten,but the myocardial cell extrovert electric current of clean increase or introverted electric current of clean reduce and all can make act the electric potential and shouldn't expect to shorten.Lack the adaptability reaction along with the high speed rate and cells that the introverted calcium ion flow,the calcium overload and then will attain the degree of threaten the cell vitality,passing the negative feedback mechanism,the function that causes a L type calcium ion to flow(Ica)to show the descend,and induce one L type calcium passage(the L-type Ca2+)egg white expression of bottom adjust,thus cause the variety of the passage structure.Up to the present,the research of abroad to AF mechanism is see in animal more to experiment,the human body research sees still and not much,and fresh see straches the net Ca2 to the same set of case L type calcium passage and muscles+- the protein of ATPase expresses the research of report way;Domestic to AF sufferer the L type calcium passage and muscles strach the net Ca2+- the research of the ATPase expression is then more seldom seen.Purpose:Pass adjusts to control the egg white-cell membrane L type to the main calcium ion(Ca2+)of the sufferer and the sinus rhythm collator myocardial cell of AF of buildingCa2+ the passage and muscles strach the net Ca2+- the ATPase expression of research,inquiry into AF occurrence and the molecular biology mechanism of the maintenances,uncover AF occurrence and maintain the iceberg dime of mechanism,take expect as the follow-up research of the valid prevention and cure AF to lay the foundation.Method:We investigated 63 patients undergoing cardiac surgery(include three groups: chronic AF,paroxysmal AF and sinus rhythm),the right auricle tissues and(or)left Left atrium tissues building specimens were obtaimed in the surgery.Total RNA was extrated from the tissues according to the Tri-zol Reagent Protocol(Gibco).RT-PCR was performed as follows:cDNA was synthesized from 1.5μg of total RNA using M-MLV reverse transcriptase (Promega)and random primers.Single-stranded cDNA was incubated on ice with a specific pair of primers designed to amplify cDNA coding for L type- Ca2+ passage and Ca2+- the content of ATPase.Amplification were performed individely.Amplified products were electrophoresized in 1.5%ethidium bromide-stained agarose gel.The intensities of the amplified cDNA fragment were estimated using a video-densitometer.Western-blot immunoblot Analysis:After boiling for 3 mins,30μg of the sample was subjected to SDS-PAGE in a 10%SDS gel and transferred to PVDF membrane,which was followed by blocking for 1 h with 5%non-fat milk in TBST.The blots were incubated for 12h at 4℃with the primary antibodies(at a 1:1000 dilution for Cx40,Cx43,ERK1,P-ERK1 and GAPDH) followed by incubation for2 h with secondary antibody(horseradish peroxidase conjugated,at a 1:1000 dilution).Immunoreactive bands were visualized with enhanced chemiluminescence reagents.Result:1,The comparison of the clinical data:Preoperative data for the all groups are listed in Table.There were no significant diference in left ventricular(LV)diameter,LV ejection fraction,LV posterior wall thickness and LV septal thickness,left atrial diameters were higher in patients with chronic AF or paroxysmal AF than in patients with NSR with valve disease or (and)NSR without valve disease.2,Alterations in Proteins Levels in Paroxysmal,Persistent AF and sinus group:Changes in protein levels were studied for the genes which showed alterations in mRNA ratios(L-type Ca²⁺and SR Ca²⁺-ATPase).The protein levels of L-type Ca2+ and SR Ca2+ -ATPase were significantly reduced in patients with persistent AF compared to the controls(p＜0.01 &＜0.05).No alterations were found in the protein levels of patients with paroxysmal AF(p＞0.05).3,Alterations in mRNA Levels of L-type Ca²⁺and SR Ca²⁺-ATPase in three groups:Changes in mRNA levels of the gene of interest were determined by comparison of gene-of-interest/GAPDH ratios between patients with persistent AF or with paroxysmal AF, and their matched controls in sinus rhythm.The densities of the amplified GAPDH of the 3 groups of patients were the same for all the genes investigated.Only patients with persistent AF showed a significant reduction of the mRNA ratios of Ltype Ca²⁺⁺/GAPDH and SR Ca²⁺-ATPase/GAPDH(P＜0.01 &＜0.05).No changes were observed in patients with paroxysmal AF compared to the controls in sinus rhythm(p＞0.05).Conclusion:1,Alterations in gene expression of proteins involved in the calcium homeostasis(the SR Ca2+- ATPase and the L- type Ca2+)occur only in patients with long-term persistent AF.2,L-type calcium channel a₁ and sarcoplasmic reticulum Ca 2+ -ATPase mRNA and protein contents were reduced in patients with persistent AF compared to the controls.No changes were observed in patients with paroxysmal AF.