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Cardiac Contractility Modulation Effect on Cardiac Function of Chronic Heart Failure Rabbits and Its Mechanism

Author: LiuHuiLiang
Tutor: QiXiaoYong
School: Hebei Medical University
Course: Internal Medicine
Keywords: Cardiac contractility modulation Absolute refractory periodelectrical stimulation Chronic heart failure Oxidative stress Myosin heavychain Cardiomyocyte apoptosis Sarcoplasmic endoplasmic reticulumcalcium ATPase
CLC: R541.6
Type: PhD thesis
Year: 2014
Downloads: 3
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Abstract


Chronic heart failure (CHF) is a common complication of cardiovasculardisease; its incidence is increasing year by year and threatening the health ofus. Recently, along with the related basic study of heart failure andevidence-based development, we have more information about pathogenesis,pathophysiology and clinical prevention and treatment of heart failure.Successful establishment of heart failure animal model can provide objectivesupport and positive contributions to basic and clinical research.The animal models of CHF mainly include myocardial ischemia model,pressure load model, capacity load and cardiomyopathy. Pressure overloadheart failure model can be used to study the compensatory hypertrophy todecompensated heart failure pathophysiology changes. At present, coarctationof the aorta model is the most optimum model in all left ventricular pressureoverload heart failure model. Pig, dog, rabbit, rat, guinea pigs and monkeys,baboons are often used to establish the animal model of heart failure and dog,pig and rabbit are most common used. The advantage of rabbit is relativelyclose to human in some cell electrophysiological characteristics. The earlystage of pressure overload is mainly through compensatory hypertrophy,which by hypertrophy of myocardial cell, to overcome the load increased,guarantee the normal ejection fraction. Lately, suffering from heart failure iscombined results of various factors including changes in gene expression,hormones, apoptosis, energy metabolism, oxidative stress, vasculardysfunction, arrhythmia, collagen deposition and so on.Cardiac contraction modulation is a non-excitatory current (NEC) electricstimulation applied during absolute refractory period (ARPES), which isabsolute refractory period electrical stimulation (ARPES). It does not inducemyocardial cell action potentials but can boost the myocardial contractility and improve the heart function. Animal experiments and clinical trials showedthat treatment of CCM can improve left ventricular systolic function; increaseleft ventricular ejection fraction, and no extra consumption of oxygen, whichcan be used for the treatment of heart failure.Superoxide dismutase (SOD), an important free radical scavenger, caneffectively remove free radical in the body and protect cells from damage.Malondialdehyde (MDA), the final product of free radical damage tobiological membranes, has strong toxicity to the biological organisms.Glutathione peroxidase (GSH-Px) is a significant catalytic decomposition ofhydrogen peroxide enzyme, can protect the structure and function of cellmembrane function. Studies have shown that: oxygen free radicals areinvolved in the pathophysiological process of CHF. MDA levels in plasmaincreased with the degree of CHF while SOD activity decreased, which canreflect the degree and prognosis of CHF. Nevertheless, the effect of CCM onoxidative stress in patients with heart failure has not been reported and needfurther study.Renin angiotensin-aldosterone system (RAAS) plays an important role inthe occurrence and development process of CHF. Myocardial fibrosis (MF) isan important manifestation of ventricular remodeling. Angiotensin Ⅱ(Ang II)is a main medium induced MF and myocardial hypertrophy, which increasedsignificantly in CHF patients and the elevated level in CHF, was significantlyassociated with mortality. Ang II overexpression resulted in myocardialmetabolism and abnormal function, promoted the pressure overload inducedcardiac dysfunction, caused the vascular and myocardial remodeling, thesepathological changes can further activate RAAS, which is a vicious spiral.Myocardial contractile protein composed by myosin, actin, tropomyosin.Cardiac myosin accounted for60%of the protein, which is the majorstructural protein of myocardium. Heart only expressed myosin heavy chain(MHC) α-MHC and β-MHC, formed alpha-alpha and beta-beta with the twomer and alpha-beta ISO two dimers, which formed respectively the isoenzymeV1, V2and V3. V1has the highest activity of Ca2+and actin-activated ATPase, followed by V2, V3is the lowest. Ventricular muscle reexpressed of embryonicβ-MHC gene instead of α-MHC, increase proportion in total MHC expression,when left ventricular pressure overload and stimulated by mechanical stretchor neurohumoral factors. β-MHC has lower affinity to actin than α-MHC,consequently myocardial contractility is weak, and therefore slow down thespeed of contraction caused myocardial contractile dysfunction. WhetherCCM enhanced myocardial contractility is by changing the expression ofmyosin heavy chain still need further study.Cell apoptosis is an active cell death. Synthesis of related proteins andregulation genes was involved in apoptosis. The Caspase family and Bcl-2family are the most important apoptosis regulatory protein. Apoptosis iscontrolled by complex signal network system. As we all known, there arethree major signaling pathways: the mitochondrial pathway; the death receptorpathway and the endoplasmic reticulum pathway. These signal transductionpathways can activate apoptosis performer, cysteine aspartic acid protease(Caspases-3), it can hydrolysis of various cell components and cell apoptosis.The expression of aldehyde dehydrogenase2(ALDH2) is aboundant in themitochondrion provide energy dependent cardiac and its expressionsignificantly decreases in heart failure. ALDH2can inhibit the apoptosis ofmyocardial cells. Studies have found that the degree of the apoptosis ofmyocardial cells was related to the ratio of myocardial cells expressingBcl-2/Bax, the ratio of Bcl-2/Bax increased can inhibit myocardial cellapoptosis, and the ratio of Bcl-2/Bax decreased to promote apoptosis ofmyocardial cells.Sarcoplasmic reticulum, an important intracellular calcium storage device,plays an important role in the regulation of calcium concentration in thecytoplasm. Ca2+is a key factor in myocardial systolic and diastolic activity.Sarcoplasmic reticulum Ca2+-ATP enzyme (SERCA) plays a key role in Ca2+uptake, storage and release. SERCA2is composed by SERCA2a, SERCA2band SERCA2c, SERCA2a seems to be the mainly enzyme in the heart. Ca2+uptake of sarcoplasmic reticulum is mainly affected by SERCA and phospholamban (PLN). PLN through phosphorylation and dephosphorylationregulates Ca2+uptake of SERCA2a. In human and experimental animal modelsof heart failure was found the expression and activity of SERCA2a proteindecreased obviously compared with the normal myocardium. In chronic heartfailure, the expression of the activity and mRNA of SERCA2a decreased is thefeatures of the changes of myocardial dysfunction and increased theexpression of SERCA2a can promote the recovery of myocardial contractilityand cardiac function improvement. There some reports have found that theexpression of SERCA2amRNA in myocardium is not positive correlation withthe SERCA2a protein level in heart failure, in different period thedevelopment of heart failure, the SERCA2a activity of myocardial is not same.Whether there is relationship between CCM enhance myocardial contractilityand regulation of SERCA2a and PLN on Ca2+uptake, storage and release alsoneed further study.The aim of the present study is to observe the effects and mechanism ofCCM on cardiac function in rabbits with heart failure induced by ascendingaorta ring tied up.Part1Effects of cardiac contractility modulation on cardiac function inrabbits with chronic heart failureObjective: The aim of present study was to observe the effects of cardiaccontractility modulation on cardiac function in rabbits with chronic heartfailure induced by ligating ascending aortic.Methods:6months of32healthy New Zealand rabbits were randomlydivided into three groups: sham operation group (SHAM, n=8);heart failuremodel group(HF, n=12);CCM therapy group (CCM, n=12). In the HF groupand CCM group, we made cerclage constriction in the ascending aorta rootdistal1cm after thoracotomy. In the CCM group, we present a pediatrictemporary pacemaker electrode in the anterior left ventricular wall near apexduring the operation. In the SHAM group, we made a thoracotomy withoutaortic cerclage. Three groups respectively in the preoperative andpostoperative4weeks,8weeks and12weeks did electrocardiogram examinations. In SHAM group and HF group, the changes of ANP andNT-proBNP in serum were observed at12weeks after the operation. In theCCM group, we gave an absolute refractory period electrical stimulation at12weeks, lasting6hours everyday for7days, blood samples after ARPES.Results:1The general situation of32New Zealand rabbits: no death in SHAMgroup and after12weeks. In HF group,2rabbits died with pneumothorax and1rabbit died with heart failure after operation, at12weeks for9rabbits. InCCM group,1rabbits died during operation,2rabbits died after operation and1rabbit bitted off the electrode wire, no CCM stimulations was delivered andruled out,8effective models at12weeks. The successful rate of model was70.8%. Pre-operation weight (Weight), respiratory rate (R) and heart rate (HR)had no significant difference between the three groups of New Zealand rabbits.After12weeks HF group and CCM group rabbits showed spiritless, move less,cyanosis, respiratory. Compared with the control group, heart rate wasincreased, but body weight decreased.2Echocardiogram: Conventional measured the echocardiographicindexes, LVEF<50%could meet the standard of heart failure. Preoperativethe indexes (for example LVEF) of the three groups had no significantdifference. The indexes(for example LVEF)between the three groups haddifference after4weeks of the operation, but the difference is not statisticallysignificant, some rabbits of the HF and CCM groups had meet the standard ofheart failure at8weeks and all rabbits of HF and CCM groups meet thestandard of heart failure at12weeks. Compared with SHAM group, IVS,IVPW, LVESD and LVEDD of HF and CCM group significantly increased,LVEF, LVFS and E/A were significantly decreased.Compared with HFgroup, the heart function had improved after ARPES electrostimulation inCCM group.3The level of serum marker in heart failure rabbits: Compared withcontrol group, serum ANP and NT-proBNP in HF and CCM groups significantly increased at12weeks. Compared with HF group, CCM groupsignificantly decreased.Conclutions: Ascending aortic root ligated allows effective establishmentof a rabbit model of chronic congestive heart failure. CCM signal treatmentcan improve cardiac function in rabbits with heart failure. The cardiaccontractility enhancement, which is the chang of LVESD, LVEF and E/A,implied the cardiac funtion improved.Part2Effects of cardiac contractility modulation in response to oxidativestress on chronic heart failure in rabbitsObjective: To observe the antioxidant capacity in chronic heart failure inrabbit and clearing oxygen free radical changes, study the effects of CCM onoxidative stress in rabbits with chronic heart failure and its mechanism.Methods:8rabbits in CCM group only give ARPES electricalstimulation6hours a day, to stimulate a week in a row. At the end of theARPES blood specimens, using colorimetry to detect the plasma SOD, MDA,GSH-Px levels. SHAM and HF groups at12weeks in pursuance of rabbit testthe corresponding indicators.Results:1CCM on rabbit the levels of SOD, GSH-Px in serum: The influence ofHF group compared with the SHAM group, the plasma SOD, GSH-Px levelsdrop, statistically significant. CCM group compared with the SHAM group,the levels of SOD, GSH-Px had also decreased. After the ARPES electricalstimulation, SOD and GSH-Px of CCM group are obviously higher than theHF group.2CCM on the level of MDA in plasma in chronic heart failure: theinfluence of HF group compared with the SHAM group, the level of MDA inplasma significantly increases. CCM group compared with the SHAM group,the MDA level has also significantly increased. CCM group are significantlylower than the HF group, the MDA level was statistically difference.3rabbit chronic heart failure of oxidative stress and degree of heartfailure relationship analysis: linear correlation analysis showed that the NT- proBNP and negatively correlated with SOD, GSH-Px correlation coefficient(r=0.809), respectively (r=0.895); NT-proBNP and MDA were positivelycorrelated (r=0.848).ANP was positively correlation with the NT-proBNP (r=0.914).Conclusions: heart failure in rabbit body exist obvious peroxidationdamage, CCM can increase levels of SOD and GSH-Px, decrease MDA. Thismay be related to CCM stimulation can increase myocardial contraction andimprove cardiac systolic and diastolic function. Plasma MDA, SOD, GSH-Pxchanges can objectively reflect the degree of CHF and prognosis.Part3Effect of cardiac contractile modulation on the expression ofmyosin heavy chain in chronic heart failure rabbitObjective: To investigate the molecular mechanism of CCM improve theexpression of myosin heavy chain in heart failure induced by pressure loadincreaseMethods: The expression of protein and mRNA of α-MHC, β-MHC wasrespectively detected by real-time fluorescent quantitative reversetranscription PCR and Western-blot. The content of Ang II in serum was testedby ELISA. Separation of left ventricular mass weighed to calculate leftventricular mass index.Results:1The levels of AngⅡ in plasma of HF and CCM were significantlyhigher than SHAM group. The levels of Ang Ⅱin CCM group than HF aredecreased, but no statistical significance.2Ang Ⅱ and ANP, NT-proBNP were positively correlated, thecorrelation coefficient respectively (r=0.946, r=0.875, P=0.000).3Effect of CCM stimulation on the expression of α-MHC and β-MHCmRNA: The expression of α-MHC mRNA is obvious differences in the threegroups. HF group significantly decreased compared with the SHAM group.CCM group compared with the SHAM group is markedly reduced, but nostatistical difference was found between two groups. After ARPES electricalstimulation α-MHC mRNA in CCM is obviously higher than HF group. The expression of β-MHC mRNA is obvious difference in the three groups. HFgroup increased significantly compared with the SHAM group. CCM groupcompared with the SHAM group, the expression of β-MHC mRNA has alsoincreased significantly. CCM group after ARPES electrical stimulation β-MHC mRNA expression significantly decreased, compared with HF groupwas statistically significant.4CCM stimulation expression of α-MHC and β-MHC protein: theexpression of α-MHC protein in the SHAM group compared with beta actionreference expressed at a higher level, and expression of β-MHC protein atlower levels. HF and CCM group of expression of α-MHC protein decreasedobviously and expression of β-MHC increased significantly. CCM groupcompared with HF group, ARPES electrical stimulation after the expression ofα-MHC protein raised and β-MHC protein decreased.5Left ventricular mass indexes (LVMI) in HF and CCM group increasedcompared with SHAM group. There are differences between the HF and CCMgroups, but no statistical difference.Conclusions: The LVMI and Ang Ⅱwas heighter in heart failure;ARPES electrical stimulation had no significant effect on LVMI and Ang Ⅱinsuch a short time. The expression of α-MHC mRNA and protein decreased inmyocardial of heart failure, the expression of β-MHC mRNA and proteinincreased. After treatment of CCM, the expression of α-MHC mRNA andprotein increased the expression of β-MHC mRNA and protein decreased.CCM enhanced cardiac contractility through the improvement ofreconstruction of myocardial myosin heavy chain in heart failure rabbit.Part4Cardiac contractile modulation effects on regulation of calciumcontraction in rabbit cardiomyocytes with chronic heart failureObjective:To observed the effects and mechanisms of CCM on themRNA and protein expression of PLN and SERCA2a in chronic heart failurerabbit myocardial.Methods: The mRNA and protein expression level of SERCA2a andPLN in myocardial tissue was detected by RT-PCR and Western-blot method. Results:1Effect of CCM stimulating on mRNA expression of SERCA2a andPLN: the mRNA expression of SERCA2a in HF group was obviously lowerthan that in SHAM group. There were no significant differences betweenCCM group and SHAM group. The expression of SERCA2a mRNA geneincreased significantly higher in CCM group after ARPES electricalstimulation than that in HF group. The mRNA expression of PLN in both HFgroup and CCM group were much higher than that in SHAM group. ThemRNA expression of PLN in CCM group after ARPES electrical stimulationdecreased significantly compared with HF group. SERCA2a/PLN (S/P) ratiosin SHAM group, HF group and the CCM group respectively were0.86±0.07,0.057±0.001,0.38±0.07, and there was statistics different in the threegroups (P <0.01). After ARPES electrical stimulation in CCM group, the ratioof S/P increased significantly in CCM group than that in HF group.2Effect of CCM stimulation on protein expression of SERCA2a andPLN: β-action as internal reference, SERCA2a protein expression at a highlevel that was similar to the reference level in SHAM group, while PLNprotein at the lower expression level that was similar to the reference level.SERCA2a protein expressions were significantly decreased in HF group andthe CCM group and expression of PLN protein was significantly increased.After ARPES electrical stimulation in CCM group compared with HF groupthat SERCA2a protein expression increased and the PLN protein expressiondecreased, while the change of PLN was more obviously.3Correlation analysis showed that NT-proBNP was negatively correlatedwith SERCA2a, and the correlation coefficient was obviously (r=-0.856P=0.000); NT-proBNP is positive correlation with PLN(r=0.918P=0.000);SERCA2a was negatively correlated with PLN (r=0.918P=0.000).Conclusions: Protein and mRNA of SERCA2a were lower expression inheart failure myocardium of rabbits, while mRNA and protein of PLN werehigher expression. Protein and mRNA expression of SERCA2a increased afterCCM stimulated while PLN decreased. CCM improved cardiac systolic and diastolic function through influencing of calcium regulating protein mRNAand protein expression. ARPES had greater influence on mRNA and proteinexpression of PLN. PLN could be observation index that more sensitive thanSERCA2a, and the ratio of S/P may be more likely to determine and reflectthe improvement of heart function.Part5Cardiac contractility modulation effects on the apoptosis ofchronic heart failure in rabbit cardiomyocytesObjective: To observe the apoptosis in the development of heart failurein cardiomyocytes and investigate the mechanism. To observe theantiapoptotic mechanism of CCM improving cardiac function.Methods: After electrical stimulation in CCM group, SHAM group andHF group was killed animal model during12weeks then directly took leftventricular myocardial specimens, then mRNA and protein gene expression ofBcl-2, Bax, ALDH2and Caspases-3in myocardial tissue was detected byRT-PCR and Western-blot. Using flow cytometry to test the rate ofcardiomyocytes apoptosis. Cardiomyocytes apoptosis was observed byTUNEL methodResults:1GAPDH as internal reference, there was a marked difference about themRNA expression of Bcl-2、Bax in three groups. The mRNA expression ofBcl-2in CCM group decreased significantly comparied with SHAM group,while Bax increased significantly. The mRNA expression of Bcl-2increasedsignificantly after ARPES stimulation in CCM comparied with HF group,while mRNA expression of Bax decreased. Bcl-2/Bax ratios in three grouprespectively were0.92±0.22、0.05±0.14、0.17±0.04and there were statisticsdifferent. Bcl-2/Bax ratios after ARPES stimulation increased significantly inCCM group comparied with HF group.2With β-action as internal reference, Bcl-2protein at a higher expressionlevel in SHAM group, while Bax protein at the lower expression level. Theexpression of Bcl-2protein decreased significantly in HF group and CCMgroup, while the expression of Bax protein increased significantly. After ARPES stimulation, CCM group compared with HF group, there wasappeared that Bcl-2protein expression increased and Bax protein expressiondecreased.3The mRNA expression of ALDH2, Caspase-3in three groups wasremarkable difference. HF group compared with SHAM group, the expressionof ALDH2decreased significantly and mRNA expression of Caspase-3increased significantly. CCM group compared with SHAM group, the mRNAexpression of ALDH2decreased, but there was no significantly differencebetween two groups, while Caspase-3increased and there was statisticsdifference. After ARPES stimulation in CCM group compared with HF group,the mRNA of ALDH2increased significantly while Caspase-3decreasedsignificantly.4SHAM group was similar to β-action the reference levels and ALDH2protein at high expression level while Caspase-3was expressed at lower level.ALDH2protein expression decreased significantly in the HF group and CCMgroup while Caspase-3protein expression levels increased significantly. AfterARPES stimulation, CCM group compared with HF group showed thatALDH2protein expression increased and expression of Caspase-3proteindecreased significantly.5Flow cytometry was used to observe the changes of cardiomyocytesapoptosis: There were obvious differences between the three groups ofcardiomyocytes apoptosis. The percentage of cardiomyocytes apoptosis in HFgroup and CCM group increased obviously compared with SHAM group.There were significant differences among three groups. The number ofcardiomyocytes after ARPES stimulation durning one week of CCM groupdecreased significantly than that in HF group.6Cardiomyocyte apoptosis by TUNEL observed: SHAM group and thenormal reference seen similar, HF and SHAM group than the CCM groupincreased apoptotic bodies, but apoptotic bodies were lesser in the CCM groupthan HF group.Conclusions: The mRNA and protein expression of antiapoptosis gene Bcl-2, ALDH2in rabbits with heart failure reduced, while pro apoptotic geneBax and Caspase-3increased. The percentage of cardiomyocytes increased,which showed that obvious apoptosis in rabbits with chronic heart failure. ThemRNA and protein expression of Bcl-2, ALDH2increased after treatment withCCM, while Bax and Caspase-3decreased, and the Ratio of Bcl-2/Baxincreased, CCM group reduced myocardial apoptosis than HF group. Itsuggested that CCM stimulation not only enhanced myocardial contractilityand improved heart function but also reversal the occurrence and developmentof cardiomyocytes apoptosis in reverse. This might be able to enhancemyocardial contractility.

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CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Heart disease > Blood circulation failure
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