The Study Of Recombinant Adeno-associated Virus Vectors Serotype 1 Gene Transfer Of SERCA2a On Chronic Heart Failure Beagles Induced By Rapid-pacing

[Background] Heart failure is a major cause of morbidity and mortality in the world, and as the population ages, the disease burden will continue to increase. Even though new treatments for heart failure have had a significant impact on mortality and the course of the disease, they do not reverse or cure the underlying pathological state of the heart. With the advent of novel intracellular targets involving cell contractility and survival, and increasingly efficient gene transfer methodologies, gene-based therapies are emerging as promising therapeutic strategies in patients afflicted heart failure. A body of scientific evidence points that several key regulatory molecules on plasma membrane or sarcoplasmic reticulum (SR) in heart failure have distorted functions, intrinsic defects intracellular calcium handling arised in cardiomyocytes.Sarco-endoplasmic reticulum calcium ATPase (SERCA2a) plays a pivotal role in intracellular Ca²⁺ handling in cardiac myocytes. Previous studies have proved that adeno-associated virual gene transfer of SERCA2a can effectively function in chronic heart failure rats. We will study the efficiency of recombinant adeno-associated virus vectors serotype 1 gene transfer of SERCA2a ( rAAV1- SERCA2a) in a large animal model of chronic heart failure, and the possible mechanisms of the therapy effects. To understand the pathophysiological mechanisms of chronic heart failure further.Part I : To make animal model of chronic heart failure (CHF) induced by rapid right ventricular pacing. The beagles received the rapid right ventricular (230 beats/min) for 30 days. A reduced rate (180 beats/min) was continued for another 30 days . The beagles were divided two group:(a) control group (n=4);(b) pacing group (n=16).The high frequency echocardiographic, the hemodynamicstudy , the pathologic analysis of hearts were undergone. All beagles had clinical conditions of CHF after rapid right ventricular (230 beats/min) for 30 days. Left ventricular systolic function was significantly reduced at day 30 and remained low at day 60, including the ejection fraction, LVSP, LV + dp/dtmax - dp/dtmax (P < 0.05) . LVEDP was significantly increased (P < 0.05) . Histologic examination of ventricles revealed changes characterized by cardiac cell edema, fatty degeneration, focal cardiacmyofiber necrosis, interstitial edema, neutrophils and lymphocytes infiltration and vascular congestion.Rapid right ventricular pacing provided a consistent model of CHF. This model can be used to study a variety of new interventions for heart failure.Part II: To determine the utility of recombinant adeno-associated virus vectors serotype 1, 2 (rAAV1, rAAV2) in the transduction of mice heart. Recombinant adeno-associated virus serotype 1, 2 carrying either enhanced green fluorescent protein(EGFP) or luciferase(LUC) reporter gene were respectively injected into pericardium in mice. After 30 days, cryosection was analyzed by Confocal microscopy to examine the expression of EGFP, gene expressions of LUC in myocardium were respectively in 14 and 45 days monitored using a cooled CCD camera. Green fluorescence was detected in cryosection of the hearts in all mice . The expression of rAAV1-EGFP was seen in cardiac myocyte of epicardium to endocardium. The expression of rAAV2-EGFP was limited in some cardiac myocyte of epicardium. The intensity and spread of rAAV1-EGFP expression was higher than rAAV2. The expression intenisty and duration of rAAV1-LUC reporter gene were better than of rAAV2-LUC reporter gene in cardiac myocyte.As gene transducing vector in mice heart, rAAV1 is superior to rAAV2.Part III: To study the efficiency of SERCA2a gene therapy on chronic heart failure (CHF) beagles, and the possible mechanisms of the therapy effects. Beagles were used to make an animal model with heart failure after rapid right ventricular pacing. The beagles were divided into four group: (a) control group(n=4), (b) HF group (n=4), (c) HF+EGFP group (n=4), (d) HF+SERCA2a group (n=8), and group d was divided into two sub-groups: 30 days and 60 days after gene transfer. rAAV1-EGFP ( 1×10¹² vg ) and rAAV1- SERCA2a ( 1×10¹² vg ) were respectively delivered intramyocardially. After 30 days, cryosection was analyzed by Confocal microscopy to examine the expression of EGFP. SERCA2a expression was assessed by RT-PCR Western-Blot and immunohistochemistry after gene transfer 30 days and 60 days. SERCA2a is regulated by phospholamban( PLN ), so PLN was also assessed by RT-PCR.30 days after transfection, the expression of rAAV1-EGFP was seen in cardiac myocyte, prominently in the anterior wall of left ventricle.The positive cells were distributed separately, scatterly or focally. After gene transfer of SERCA2a in CHF beagles for 30 days, the ratio of SERCA2a mRNA/ GAPDHmRNA and SERCA2a/ β -actin was significantly higher than the HF group (P < 0.05).By immunohistochemistry, the expression of SERCA2a in the myocardial tissue was higher in the HF+SERCA2a group (gene transfer for 30 days) than the HF group (P < 0.05). After gene SERCA2a transfer 60 days, the mRNA level and protein expression of SERCA2a in the myocardial tissue were kept high levels. The heart function was also improved along with the increase of SERCA2a expression. However, the PLN mRNA level was unchanged.It shows that intramyocardial injection of rAAV1- SERCA2a can improve the contractile function in beagles chronic heart failure. It may be helpful for next investigations with rAAV1- SERCA2a on treatment of human heart failure.Part IV: To test the effects of gene transfer of SERCA2a on left ventricular (LV) remodeling and apoptosis. Experiment group is the same to part III. Apoptosis cardiomyocyte was examined with electron microscopy, The apoptosis index of cardiomyocyte was evaluated by TUNEL. matrix metalloproteinase-9(MMP-9) and Bax expression were assessed by immunohistochemistry .The nucleus of apoptosis cardiomyocyte was observed concentrated andfragmented by electron microscope. The apoptosis index of cardiomyocyte , the protein expression of MMP-9 and Bax in beagles with HF and HF+EGFP group were much higher than control group (P < 0.05). Moreover, the apoptosis index , MMP-9, Bax were much lower in HF+ SERCA2a group than in HF group(P < 0.05)In this study, gene transfer of SERCA2a is beneficial to improve cardiac function and reduce LV remodeling with chronic heart failure.