| OBJECTIVE: To construct a recombinant adeno-associated virus vector(rAAV9-AR185-EGFP)with cardiac-specific expression and to prepare two viral preparations,rAAV9-AR185-EGFP and rAAV9-AR117-EGFP,to evaluate the transfection effect in rats.To establish rat heart failure models,to change the phosphorylation level of RyR2 protein in rat heart by intracellular antibodies carried by AAV vector,to study the effect of AR185 antibody on rat heart function and structure,and to explore the possibility of treating heart failure with intracellular antibodies.To study the protective effect of AR185 antibody on calcium homeostasis and contractile function in single rat cardiomyocyte,and to further explore the role and mechanism of hyperphosphorylation of RyR2 protein in the pathogenesis of heart failure.METHODS: The AR185 antibody obtained in the preliminary screening of the research group was used as the target gene.Recombinant type 9 adeno-associated virus(rAAV9)was used as the vector to construct the corresponding rAAV9-AR185-EGFP shuttle plasmid.293 T cells were transfected in vitro to prepare virus preparations through the tail vein.The injection method was used to transfect AAV9-AR185-EGFP into rat and we detected its transfection effect in vivo.Animal models were established and grouped in the experiment.Rats were divided into 4 groups: Sham group(n = 7),saline was injected into the tail vein after suture thoracotomy;HF group(n = 7),myocardial infarction was performed(ligating the left anterior descending coronary artery of rat heart),and saline was injected into the tail vein;AAV-AR117 group(treatment control group,n = 8),myocardial infarction surgery,tail vein injection with rAAV-AR117-EGFP;AAV-AR185 group(treatment group,n = 8),myocardial infarction surgery,tail vein injection with rAAV-AR185-EGFP.After 8 weeks of operation,the cardiac function of the rats was measured using a small animal ultrasound system;the heart weight/body weight ratio of each group were measured;the rat hearts were fixed in 4% PFA solution,after embedding,HE staining and massion staining were performed to compare the microscopic changes of rat hearts between groups.The myocardial microstructural changes were observed in each group.The left ventricle of rat myocardial tissue was obtained,and the conventional tissue samples were prepared.Transmission electron microscopy was used to observe and photograph the electron microscopy images.The changes of myocardial ultrastructure were compared between the groups.The rat cardiomyocytes were isolated by enzymatic hydrolysis,and some of the cardiomyocytes were incubated with Fluo5N-AM intracellular fluorescent calcium probe.The fluorescence intensity in the sarcoplasmic reticulum was detected by laser scanning confocal microscopy(LSCM);Fluo 4-AM intracellular fluorescence calcium probe was used to incubate part of cardiomyocytes.The amplitude of caffeine-induced calcium transient and the frequency of calcium leakage during myocardial cell quiescence were measured using the LSCM system;using the IonOptix single cell contraction detection system to detect the contractility of cardiomyocytes in each group during electrical stimulation.RESULTS: AAV9-AR185-EGFP and AAV9-AR117-EGFP virus preparations were successfully prepared.Four weeks after tail vein injection of the AAV9-AR185-EGFP virus preparation,significant green fluorescent protein expression was observed in rat heart tissue,and no obvious fluorescence signal was detected in other organ tissues.The analysis of cardiac function indexes in each group of rats showed that EF% and FS% in HF group(n=7)were significantly lower than those in Sham group(n=7)(EF%: 32.49±6.46 vs 80.49±6.40,P<0.01;FS%:16.88±4.25 VS 47.65±4.30,P<0.01);LVEDV and LVESV were significantly increased(LVEDV: 498.74±62.97 VS 227.10±33.94,P<0.01;LVESV: 343.75±43.05 VS 55.97 ± 9.75,P < 0.01).At the same time,compared with the AAV-AR117 group(n=8),the ultrasound indexes of the AAV-AR185 group(n=8)were all improved,and the EF% and FS% were significantly higher(EF%: 60.57±7.60).VS 31.30±6.22,P<0.01;FS%:37.72±3.31 VS 16.66±4.60,P<0.01);LVEDV and LVESV were decreased(LVEDV: 432.62±37.02 VS 515.56±59.00,P<0.01;LVESV: 187.22± 44.06 VS 341.63±44.70,P<0.01).A study of microstructure and ultramicrostructure of myocardial tissue in each group showed that compared with the results of the sham group,the ventricular chambers in the HF group expanded,the amplitude of the anterior septum decreased,and the myocardial fibrosis was evident at the operation site.Muscles are loosely arranged and disordered,and mitochondria are extensively damaged.Compared with the AAV-AR117 group,myocardium in the AAV-AR185 group was basically normal and the mitochondrion state was better,and the malignant myocardial remodeling was significantly suppressed.The sarcoplasmic reticulum calcium capacity,calcium transient,calcium leakage,and myocardial cell contractile ability of myocardium in each group were analyzed.Compared to Sham group(n=30)the sarcoplasmic calcium capacity of myocardial cells(n=30)was significantly decreased in HF group(Fluo5N-AM: 26.7±1.4 VS 49.3±3.1,P<0.01);calcium transient amplitude was decreased(Fluo4-AM: 16.7±1.4 VS 34.5±2.5,P<0.01),while the frequency of diastolic calcium release was increased(75.5±7.6 VS 3.7±1.6,P<0.01).In addition,the results of cell shrinkage assessment indicators such as fractional shortening and systolic diastolic velocity have all shown that myocardial cells from failing hearts are significantly impaired in their ability to contract when subjected to electrical stimulation.Compared with the AAV-AR117 group(n=30),the expression of AR185 antibody(n=30)increased the calcium content in the sarcoplasmic reticulum of heart failure rats(Fluo5N-AM: 42.1±2.2 VS 26.5±1.6.(P<0.01);Enhanced calcium transient amplitude(Fluo4-AM: 28.6±2.7 VS 16.8±1.2,P<0.01),and the frequency of diastolic calcium release was significantly decreased(6.8±1.6 VS 73.5±7.3,P<0.01).All indexes of cell contraction function during electrical stimulation have been improved.Conclusion: The rAAV9-AR185 and rAAV9-AR117 constructed and prepared in this experiment have good cardiac transfection effect in vivo,which are consistent with the requirements for carrying out subsequent animal experimental studies.The phosphorylation level of RyR2 is significantly increased during heart failure,and the hyperphosphorylation of RyR2 plays an important role in the pathological process of heart failure.At the entire level of the heart,the AR185 antibody improves the contractile function of failing hearts,protects the heart structure from remodeling impairing.At the cellular level,AR185 antibody can reduce the calcium leakage of myocardial cells in heart failure rats,maintain calcium capacity in sarcoplasmic reticulum,restore calcium homeostasis,and protect myocardial cell contractility.The RyR2 protein is a very promising target in the treatment of heart failure.Intracellular antibody technology is a kind of promising gene therapy method. |
PDF Full Text Request