Study On Implementing Calcineurin Aβ Gene Silencing In Hypertrophic Myocardium In Vivo

Background and objectives Myocardial hypertrophy is recognized as an independent risk factor of morbidity and mortality of cardiovascular disease. Calcineurin signal pathway plays an important role. CsA and FK506 are often used to inhibit calcineurin to reduce hypertrophy. But the side effects limit their clinical application. It is very important to find a safe and effective method to inhibit CaN pathway. In the study, we select effective RNAi target site of CaN Aβ in cultured myocardial cells. Then, report gene is transfected to myocardium in vivo to compare transfection efficiency. At last, we induce CaNAβ gene silencing of hypertrophic myocardium in vivo. We aim to provide a new feasible method to improve transfection efficiency and obtain extensive transfection of myocardium comparably. And we mean to find a new effective and safe way to inhibit CaNAβ gene expressing in vivo to prevent and treat myocardial hypertrophy. Methods 1. Selecting effective target sites. Constructing shRNA expression plasmid targeting 3 different sites of CaN Aβ(si1280, si1539, si1053) and transfecting cultured myocardial cells. Cells are devided into 6 groups, Control group; Ald group; Ald+ si1280 group, Ald+ si1539 group, Ald+si1053 group and Ald+siGFP group (negative control). Diameter is measured and mRNA levels of CaN Aβ, ANF and β-MHC are detected 48h after transfection. 2. Effective method to transfect myocardium in vivo. PLacZ is used as a report gene. Adult rats are divided into control group, intrapericardial injection group and negative group, sublingual vein injection group and negative group. 6ds after transfection, issues of heart, liver, lung and kidney are stained with X-gal to observe transfection to myocardium and non-target transfection. 3. CaN Aβ gene silencing in vivo. Adult rats are divided into Control group, hypertrophy model group, intrapericardial injection group and negative group, sublingual vein injection group and negative group. 6ds after transfection, the concentration of CK, CK-MB, LDH, GPT, GOT,Cr, UN and Ua in plasma is detected. Cardiac muscle was collected to detect CaN Aβ mRNA levels and CaN Aβ protein expression by immunofluorescence. Results 1. The level of CaN Aβ, ANF, β-MHC mRNA and diameters increased inducing by Ald (p<0.05). There was no significant difference between Ald group and siGFP group (P>0.05). Compared with siGFP group, the diameters and CaN Aβ, ANF mRNA level of si1280,si1539 and si1053 groups decreased (P<0.05). β-MHC mRNA level of si1280, si1053 were also decreased (P<0.05). CaN Aβ mRNA level of three intervention group has no difference between each other (P>0.05). The diameter of sil280 group is smaller than sil539 and sil053 group (P<0.05). ANF mRNA of sil280 group is lower than sil539 and sil053 group (P<0.05). β-MHC mRNA level is lower than si1539 group and have no difference with sil053 (P>0.05). 2. After PLacZ transfection of myocardial in vivo, only intrapericardial injection group have been stained in some myocytes under epicardium. Staining of liver, kidney, and lung of all of groups show negative results. 3. All the surgical groups have no significant difference for CaN Aβ mRNA level (P>0.05). Immunofluorescence staining shows that, the CaN Aβ protein expression of intrapericardial injection group decreased in some myocytes under epicardium, while other groups show no decreased fluorescence. Conclusions CaN Aβ gene silencing can reduce myocardial hypertrophy in cultured cells, si1280 (21bp) of CaN Aβ gene is the most effective target site for siRNA. The method of intrapericardial injection of plasmid, microbubbles and erzymes can improve transfection efficiency of non-viral plasmid with satisfying targeted transfection. But the scope of transfected myocytes is still limited. CaN Aβ shRNA expressing plasmid transfection in vivo by pericardial injection results in decreased CaN Aβ protein expression of small part of myocytes, and CaN Aβ mRNA only shows decreased trend. The dosage of non-viral vector and the parameters of ultrasound energy should be optimized in further study.