Role Of Galectin-3Pathways In The Pathogenesis Of Cardiac Remodeling In The Pressure Overload Mice Model With Heart Failure

Objective:Galectin-3, as a kind of inflammatory reaction medium, plays animportant role in mediating ventricular remodeling. However, ventricularremodeling is the basis of development of heart failure, which determinesclinical outcomes and is associated with progression and poor prognosisof heart failure. We establish the pressure overload mice model oftransverse aortic constriction with a modified method in a minimallyinvasive way. When the model of heart failure is set up, we measure thelevel of expression of galectin-3in cardiac muscle and investigate therelationship between galectin-3and pathogenesis of ventricularremodeling in heart failure.Method:1. Preparation of animal models: Male Kunming mice weighted 18-24g are anesthetized by intraperitoneal injection of pentobarbital25mg.kg-1, then open the cavity of chest and detach the aortic arch. One6-0no damage silk is placed between the innominate artery and the leftcommon carotid artery, then ligature together with25G syringe needle(D=0.5mm).The constriction is established successfully when the needleis pulled out. In sham-operated group, the aortic arch is just detached andthe silk is placed without ligation.2. Echocardiographic studies: To evaluate the cardiac structuralresponses to aortic arch banding,2-dimension (2D) and M modetransthoracic echocardiography (TEE) are deployed to monitor the leftventricular function and left ventricular remodeling changes induced bypressure overload at the8th week after the surgery. The left ventricularanterior wall thickness at end-diastole (AWd)，the left ventricular anteriorwall thickness at end-systole (AWs)，the left ventricular posterior wallthickness at end-diastole (PWd)，the left ventricular posterior wallthickness at end-systole (PWs)，the left ventricular end-diastolicdimension (LVEDd)，the external left ventricular diastolic diameter(EXLVDd), the left ventricular end-systolic dimension (LVESd) and theleft ventricular fractional shortening (FS) are measured.3. Hemodynamic studies: To evaluate the cardiac functional responsesto aortic arch banding, a microtip catheter transducer is cannulatedthrough the right carotid artery for the pressure measurement, such as the left ventricular systolic pressure (LVSP)，the left ventricular end-diastolicpressure (LVEDP)，the maximum left ventricular developed pressureincrease rate (+dp/dtmax), the maximum left ventricular developedpressure decrease rate (-dp/dtmax) are recorded with continuousphysiological monitors to evaluate the chamber function. After theexperiment, the heart is taken out and cardiac weight is measured, whichis useful to obtain the index of left ventricular hypertrophy (LVHI), thatis LV weight—to—body weight ratio (LW/BW).4. Histopathological studies: To evaluate the cardiac morphologyresponses to aortic arch banding, histopathological characteristics ofcardiac muscle are observed through HE and MASSON dying. At the sametime collagen volume fraction (CVF) is calculated. Besides, theexpression of galectin-3in cardiac muscle is localized by the method ofimmuno-histochemistry.5. The expression level of galectin-3mRNA, collagenous fiberⅠmRNA, collagenous fiber Ⅲ mRNA in myocardium are determined bythe method of reverse transcription polymerase chain reaction (RT-PCR).The expression of galectin-3in myocardium is determined by the methodof Westen-Blot.6. Statistical analysis: SPSS18.0is used for statistical analysis.Continuous variables are expressed as means±SD. Continuous datasbetween2groups are compared with Student’s t-test. A value of P＜0.05 is considered statistical significance.Results:1. Echocardiographic datas: Compared with the sham-operated group,the left ventricular anterior wall thickness at end-diastole (AWd)，the leftventricular anterior wall thickness at end-systole (AWs)，the leftventricular posterior wall thickness at end-diastole (PWd)，the leftventricular posterior wall thickness at end-systole (PWs)，the external leftventricular diastolic diameter (EXLVDd) are higher (p <0.05) in theoperated group. The left ventricular end-diastolic dimension (LVEDd) andthe left ventricula fractional shortening (FS) in the operated group aresignificantly lower compared with the sham-operated group (p <0.05).2. Hemodynamic datas: As summarized in Table2, Fig.4and5, at the8th week after surgery, the pressure overload mice model with heartfailure are established successfully. Compared with the sham-operatedgroup, the left ventricular end-diastolic pressure (LVEDP) increasedsignificantly (6.4±1.8vs4.6±0.8，P<0.05) in the operated group; The leftventricular systolic pressure (LVSP), the maximum left ventriculardeveloped pressure increase rate (+dp/dtmax), the maximum leftventricular developed pressure decrease rate (-dp/dtmax) decreasedsignificantly (LVSP：93.4±4.5vs111.4±2.5；+dp/dtmax：5214.5±464.9vs7400.5±208.2；-dp/dtmax:5120.8±240.6vs6468.5±209.6， P<0.05).3. Histopathological studies: As showed in Table3and Fig.7, the index of left ventricular hypertrophy (LVHI) in the operated group isincreased significantly compared with the sham-operated group (5.2±0.3vs3.1±0.2，P<0.05). Light microscope examination in the operatedgroup, as it shows, hypertrophic cardiomyocytes，thickening of smallvessel wall and hyperplasia of stroma with inflammatory cells infiltrationare seen in the operated group, that is more serious than thesham-operated group. It’s the evidence that CVF is higher in the operatedgroup than in the sham-operated group (12.02±0.71vs2.56±0.42，P<0.05). Furthermore, the level of expression of galectin-3in cardiacmuscle detected by the method of immuno-histochemistry enhancedremarkablely, especially in the region of myocardial fibrosis.4. The expression levels of galectin-3mRNA, collagenous fiber ImRNA, collagenous fiber III mRNA: Table5and Fig.11show theexpression levels of galectin-3mRNA, collagenous fiber I mRNA,collagenous fiber III mRNA in myocardium of each group mice. The ratiolevel of galectin-3mRNA and GAPDH mRNA is significantly high in theoperated group than in the sham-operated group (1.67±0.08vs0.83±0.06，P<0.05). The ratio level of collagenous fiber I mRNA and GAPDH mRNAincreased significantly in the operated group than in the sham-operatedgroup (1.35±0.03vs0.38±0.02，P<0.05). The ratio level of collagenousfiber III mRNA and GAPDH mRNA is significantly high in the operatedgroup than in the sham-operated group (0.46±0.07vs0.24±0.04，P<0.05). Besides, the ratio level of collagenous fiber I mRNA and collagenousfiber III mRNA also increased significantly in the operated group than inthe sham-operated group (2.88±0.76vs1.62±0.56，P<0.05).5. Western-Blot: The protein level of galectin-3expression in theoperated group increased significantly compared with the sham-operatedgroup （1.48±0.07vs0.74±0.05P＜0.05).Conclusions:1. The pressure overload mice model with heart failure of transverseaortic constriction can be established successfully in a modifiedminimally invasive way. The models’ pathophysiological evolvement ismore closed to pressure-overloading CHF, which play a vital role in thestudy of mechanism of CHF.2. The ventricular remodeling plays an important role inpressure-overloading CHF which is characteried with the ventricularfunctional remodeling (systolic and diastolic) and the ventricularstructural remodeling (the left ventricular size, the shape and thehistological components).3. The level expression of galectin-3enhances greatly in cardiacmuscle in pressure-overloading CHF, it’s more remarkable in myocardialfibrosis area, this phenomenon implies that galectin-3plays a vital role inthe evolvement of ventricular remodeling in the pressure-overloading CHF.