Effects Of Biventricular Electric Stimulations Applied During Absolute Refractory Period On Cardiac Function Of Rabbits With Heart Failure

Chronic heart failure is the end state of a variety of cardiovasculardisease; its etiology is diverse, with high incidence. The underlyingmechanisms leading to heart failure progression is cardiac remodeling, cardiacremodeling is an adaptive response of cardiac structure and function inirritation occurs, these adaptive changes in the early for the maintenance ofnormal cardiac function, but with the worsening heart failure process, thesechanges lead to the occurrence of pump failure and malignant arrhythmia.Cardiac remodeling includes structural remodeling and electricalremodeling. Structural remodeling increase ventricular volume and weight,change ventricular shape (transverse diameter increased spherical), decreasemyocardial contractility. With the changes of cardiac structure, cardiacelectrical activity changes. Electrical remodeling includes the changes of avariety of ion channels, excitation contraction coupling (e.g. sarcoplasmreticulum calcium cycle) and the gap junctions between cells. These changeslead to reduced cardiac electrical stability, cardiac autonomic elevated,ventricular myocyte resting potential and action potential amplitude decreases,conduction slowing, prolonged the action potential duration; hemodynamicsbased arrhythmia prone to ventricular tachycardia and ventricular fibrillation.The animal model of chronic congestive heart failure mainly includesmyocardial ischemia type, pressure load type, capacity load andcardiomyopathy. This research through the ascending aorta ring tied upinduced chronic congestive heart failure animal models, and observes thechanges of heart mechanical and electrical physiological function. To explorethe methods to establish the animal model of chronic heart failure, whichprovided important data for the change of heart failure, hemodynamics in the development of neuroendocrine system, myocardial cells and subcellularlevels change.In recent years, the treatment of CHF has made great progress, but as thedisease progresses, many severe CHF patients still have obvious symptoms,responds poorly to medication treatment. At the same time, people are activelyseeking a variety of non-drug therapeutic method. The heart transplantation isthe most effective method in the treatment of CHF, but high medical cost,limited donors make its application limited. Cardiac stem cell transplantationis still in the stage of basic research, cardiac resynchronization pacingtechnique in the treatment of CHF can improve the hemodynamics andclinical symptoms of the patients, but the accommodation is limited.Digoxin is a kind of traditional positive inotropic drugs and can reducethe death or rehospitalization rate of heart failure, but did not reduce all-causemortality in patients with heart failure, and also resulted in a slight increase inthe rate of occurrence of sudden death. Other dependent adenosine increasedpositive inotropic drugs such as amrinone, milrinone, due to increasedmortality in patients with chronic heart failure has been abandoned. Thesedrugs can improve cardiac function in the acute phase, accompanied byarrhythmia increased risk, especially in severe left ventricular function decline,acute ischemia or catecholamines increased.Cardiac contraction modulation is a new mechanical device therapie. itdoes not induce myocardial cell action potentials but can boost the myocardialcontractility and improve the heart function though electric stimulationsapplied during absolute refractory period (ARPES).Sarcoplasm reticulum Ca2+ATP enzyme (SERCA2) is also calledsarcoplasm reticulum Ca2+pump, a major protein involved in the regulation ofcalcium inside the cell, can promote the myocardial systolic and diastolicfunction. During every heartbeat, a pulse of calcium is released from thesarcoplasmic reticulum (SR) into the cytoplasm of cardiac muscle cellsthrough a calcium release channel (RyR, ryanodine receptor), thus triggeringmuscle contraction. During relaxation, SERCA2resequesters the calcium back into the internal sarcoplasmic reticulum storage pool, thus priming thenext release of calcium. SERCA2is divided into three kinds of subtypes(SERCA2a, SERCA2b and SERCA2c). The major SERCA2a express in theheart and skeletal muscle of slow muscle fibers, heart is mainly SERCA2a.SERCA2b exist widely in a variety of tissues; can partially replace thefunction of SERCA2a. In chronic heart failure cases, the changes ofmyocardial dysfunction are the decrease of SERCA2a expression, andincreased the expression of SERCA2a can promote the recovery decreasedmyocardial contractility, cardiac function was improved which in animalexperiments and clinical studies have confirmed.MicroRNAs (miRNAs) are a group of newly discovered small RNAs,non-coding, contains approximately19-25nucleotide sequence, whichrepresent one of the most exciting areas of modern medical science as theymodulate the complex regulatory network of gene expression. A large body ofevidence indicates that: miRNAs plays an important role in the pathogenesisof heart failure. MiRNAs133has high expression in heart, have a closerelationship with cardiac hypertrophy, inhibit the cardiomyocyte hypertrophy,and resist apoptosis, participates play an important role in the regulation ofmyocardial remodeling. It has been shown that the level of miRNAs133expression decreased obviously in the heart failure animal model. Anotherstudy showed that miRNAs can bind to the SERCA2a3’non coding region,plays the role of the expression of SERCA2a post transcriptional regulation.Previous experimental and clinical research of cardiac contractionmodulation use the method of single ventricular stimulation, biventricularstimulation is better than single ventricular stimulation in improving heartfunction is unclear, the experiment will be discussed. Biventricular stimulationboost heart failure cardiac function and electrical remodeling, the specificmechanism is not clear. Using the methods of fluorescence quantitativepolymerase chain reaction analysis, we analyze the effect of CCM on theexpression level of rabbit heart failure ventricular muscle SERCA2andmiRNAs133gene, and explore its mechanism of action at the molecular level. Part1: The changes of cardiac mechanical and electrical physiology inrabbits with chronic congestive heart failure.Objective: Rabbits models of choric congestive heart failure were madeby legating ascending aortic root of rabbits, and observed the changes ofcardiac mechanical and electrophysiological.Methods:40New Zealand white rabbits were randomly divided into2groups: heart failure group (n=30); sham operation group (n=10); the changesof Serum BNP, echocardiography and electrophysiology were observed at12weeks after and during the operation.Results:1The general situation of40New Zealand white rabbits:5rats died and2rats did not meet the inclusion criteria of the degree of heart failure after12weeks in operation heart failure group; no death in in sham operation groupand after12weeks. Preoperative heart failure group and sham operation groupweight (Weight), respiratory rate (R) and heart rate (HR) had no significantdifference（P>0.05); After12weeks heart failure rabbits showed spiritless,cyanosis, respiratory. Compared with the sham operation group, heart ratewere increased in operation heart failure group (P<0.05); but body weightdecreased (P<0.05).2The comparisons of diastolic threshold(DT), effective factoryperiod(ERP), ventricular fibrillation threshold(VFT), QT dispersion (QTcd),LVEDD(mm), LVESD(mm), EF(%), FS(%), IVS(mm), LVPW (mm), E/A,SPWMD and IVMD were not different between two groups before theoperation（P>0.05). Compared with sham-operated group, DT, ERP, VFT,EF(%), FS(%) and E/A in heart failure group were significantly decreased(P<0.05), LVEDD(mm), LVESD(mm), IVS(mm), LVPW(mm), sPwMD andIVMD were significantly increased(P<0.05) but QTcd was not different(P>0.05) at12weeks after the operation.Conclusion: ascending aortic root legated allows effective establishmentof a rabbit model of chronic congestive heart failure. Cardiac machinery andelectrophysiology changes accordingly after heart failure. Part2: Effects of biventricular electric stimulations applied duringabsolute refractory period on cardiac function of rabbits with heartfailure.Objective: To investigate the effects of biventricular electric stimulationsduring absolute refractory period on cardiac function and ventricularremolding of rabbits with chronic heart failure，explore the best pattern andthe safety of Cardiac Contractility Modulation(CCM).Methods:23rabbits were divided into three groups：sham-operatedgroup, LV cardiac contractility modulation （LV-CCM） group，biventricularcardiac contractility modulation（BV-CCM）group. Rabbit’s models of choricheart failure were made by legating ascending aortic root of rabbits. Thenelectrical stimulations during the absolute refractory period were delivered onthe anterior wall of left ventricle in LV-CCM group and on the anterior wall ofboth left ventricle and right ventricle in BV-CCM group lasting six hourseveryday for seven days. Changes in ventricular structure，cardiac functionand elctrocardiology were observed before and after CCM stimulation.Results:1Compared with sham-operated group，heart weight，heart weight index，LVESD， LVEDD in LV-CCM and BV-CCM group were significantlydecreased (P<0.05), while their LVEF and FS were significantly increased(P<0.05), especially in BV-CCM; IVS, LVPM,E wave，A wave and E/A ratiowere similar among groups. Plasma BNP levels in three groups was nosignificant difference before CCM delivered (P>0.05); However plasma BNPlevels were highest in the sham-operated group, followed by a LV-CCM group,BV-CCM group were the lowest after CCM-delivered (P<0.05).2Effect of CCM stimulation on plasma BNP level: The plasma level ofBNP in the three groups had no significant difference before CCM stimulated(P>0.05); After CCM stimulation plasma BNP level in three groups:biventricular stimulation group decreased obviously, followed by leftventricular stimulation group (P<0.05), control group did not changesignificantly (P<0.05) respectively. 3Effect of CCM stimulation on ventricular remodeling: Compared withthe control group decreased heart weight and heart weight index decreased inpostoperative left ventricular stimulation group and biventricular stimulationgroup, with the control group decreased, with biventricular group decreasedmore obviously (P<0.05).Conclusion: Biventricular electric currents delivered during the ARPcould significantly enhance the contractility of myocardium and improvecardiac function and reverse ventricular remodeling safely.Part3: Effects of biventricular electric stimulations applied duringabsolute refractory period on cardiac electrophysiological function ofrabbits with heart failure.Objective: To investigate the biventricular electric stimulations appliedduring absolute refractory period lead to what influence on heart failurecardiac electrophysiological remodeling of rabbits with heart failure. We wantto know that this effect is the basis for the improvement of heart function or isindependent of ventricular remodeling, whether will it produce new dangerousarrhythmia.Methods:23rabbits were divided into three groups：control group， LVcardiac contractility modulation （LV-CCM） group，biventricular cardiaccontractility modulation（BV-CCM）group. Rabbit’s models of choric heartfailure were made by legating ascending aortic root of rabbits. Then electricalstimulations during the absolute refractory period were delivered on theanterior wall of left ventricle in LV-CCM group and on the anterior wall ofboth left ventricle and right ventricle in BV-CCM group lasting six hourseveryday for seven days. Changes in electrophysiological and arrhythmiawere observed before and after CCM stimulation.Results:1The changes of rabbits diastolic threshold in the three groups afterCCM stimulation: compared with the control group, diastolic thresholdincrease in LV cardiac contractility modulation group and biventricularcardiac contractility modulation group, especially in BV-CCM group (P<0.05).2The changes of rabbits ERP in the three groups after CCM stimulation:compared with the control group, ERP increase in LV cardiac contractilitymodulation group and biventricular cardiac contractility modulation group,especially in BV-CCM group (P<0.05).3The changes of rabbits VFT in the three groups after CCM stimulation:compared with the control group, VFT increase in LV cardiac contractilitymodulation group and biventricular cardiac contractility modulation group,especially in BV-CCM group (P<0.05).4The changes of rabbits QTcd in the three groups after CCM stimulation:compared with the control group, QTcd has declined in LV cardiaccontractility modulation group and biventricular cardiac contractilitymodulation group, but no statistically significant difference (P>0.05).5Holter monitoring showed that regardless whether or not CCMdelivered the heart rate have no change in heart rate in LV-CCM and BV-CCMgroup. Compared with sham-operated group there is no increase in ventriculararrhythmias.Conclusion: BV-CCM can improve diastolic threshold, ventricularfibrillation threshold and prolong the effective factory period, increase theelectrical stability of the reversal of heart failure, structural remodeling andelectrical remodeling, and does not increase the incidence of ventriculararrhythmia, which is more advantageous to a single ventricular stimulation.Part4: To explore the effect of BV-CCM on the expression level ofrabbit heart failure ventricular muscle SERCA2and miRNAs133geneObjective: To compare the effects of biventricular pacing andconventional single-ventricular pacing for CCM on cardiac contractilefunction and the underlying molecular mechanisms.Methods:23rabbits were divided into three groups: control group (n=7),left ventricular CCM (LV-CCM) group (n=8), and biventricular CCM(BV-CCM) group (n=8). A rabbit model of chronic heart failure wasestablished by legating ascending aortic root of rabbits. Then electrical stimulations during the absolute refractory period were delivered to theanterior wall of left ventricle in the LV-CCM group and on the anterior wall ofboth left and right ventricles in the BV-CCM group lasting six hours per dayfor seven days. After operation, the left ventricular myocardium specimenswere taken. Using method of fluorescence quantitative polymerase chainreaction, we analyze the effect of CCM on the expression level of rabbit heartfailure ventricular muscle SERCA2and miRNAs133gene, and explore itsmechanism of action at the molecular level.mRNA and miRNA Quantification: Total RNA was extracted usingTRIzol (Invitrogen, Carlsbad, CA), and small RNA-enriched RNA fractionswere obtained using the miRNeasy and miRNA cleanup kits. SERCA2amRNA and miR-133levels were analyzed using the TaqMan quantitativereal-time PCR (qRT-PCR) method, and quantified with an ABI7000Real-Time PCR System. Taqman Primer assays for miRNAs and the reagentsfor reverse transcriptase and qRT-PCR reactions were obtained from AppliedBiosystems. Relative expression was calculated using the comparative cyclethreshold (Ct) method (2-ΔΔCt). mRNA and miRNA levels were normalizedto GAPDH.Results: The intracellular Ca2+handling was significantly improved byboth LV-CCM and BV-CCM, as reflected by up regulation ofsarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) protein levels by1.7and2.4fold, and simultaneous up regulation of a cardiac-enriched microRNAmiR-133levels by2.6and3.3fold, in LVP-CCM and BVP-CCM, respectively,compared to control group.Conclusion: CCM stimulation may regulate the expression ofmyocardial miRNAs133, which up regulates the expression of SERCA2andpromotes cardiac function.