CaN Aβ MRNA Gene Silencing Inhibit Cardiac Hypertrophy Induced By Aldosterone

Background and Objective: Cardiac hypertrophy serves as a common pathway in various cardiovascular diseases. It is one of the most important pathological foundations resulting in cardiogenic death. Recently, cardiac hypertrophy caused by the maladjustment of neural and humoral factors, especially renin-angiotensin-aldosterone system (RAAS) has been paid much attention. It has been detected that aldosterone is an important factor which can induce cardiac hypertrophy. It has been showed that CaN-dependent signal passway may play an important role in the development of cardiac hypertrophy. Now some scientists used RNAi technique to study the hypertrophy interrelated gene on cultured cardiac cells, to detect the mechanism of cardiac hypertrophy. But nobody used RNAi technique to study CaN-dependent signal pathway in cardiac hypertrophy. We think it is important to lucubrate the key function of CaN-dependent signal pathway in cardiac hypertrophy.In the present study, we used aldosterone to stimulate cultured neonatal rat cardiomyocytes, at the same time, transfected calcineurin Aβ mRNA target siRNA and it's control. In order to detect the role of CaN in the mechanism of cardiac hypertrophy induced by aldosterone, we observed the effects on hypertrophy by interfered with CsA or spironolactone (spiro) including cell size , changes of gene expression related to cardiac hypertrophy-atrial natriuretic factor (ANF) andp-myosin heavy chain(P-MHC), CaN gene and it's protein expression in myocardium cell. To detect the effect of RNA interfering, we analyzed the expressing levels of the CaN mRNA and CaN protein. Then we candetermine the functional segment of CaN Ap.Method: Cardiomyocyte cultures were isolated by enzymatic disassociation of 1-to-3-day-old neonatal rat hearts and were divided into six groups randomly. Ald group: cells incubated with aldosterone 10^-9mol/L per well; control group: incubated only with medium; Spiron group: incubated with aldosterone 10^-9mol/L and spironolactone 3 × 10^-6mol/L per well; CsA group: incubated with aldosterone 10^-9mol/L and cyclosporin A 5×10^-3mol/L per well; target siRNA group: incubated with aldosterone 10^-9mol/L and 3ul/well of siLentGene^TM transfection reagent and 2ul/well siRNA DNA cassette; target siRNA control group: incubated with aldosterone 10^-9mol/L and 3ul/well of siLentGene^TM transfection reagent and 2ul/well siRNA control DNA cassette. Cell size was quantified by myocyte surface area. Half quantitative PCR was employed to determine the levels of ANF ,p-MHC and CaN Ap mRNA in cardium cells. The expression of CaN and a-actin in cardiac myocytes were observed by immunohistochemical and immunofluorescence staining.Result: The results were that aldosterone can increase the cell size obviously. Aldosterone can increase the gene expression of ANF and P-MHC, which were related to cardiac hypertrophy,. The higher expressions of CaN and a-actin in cardium cells by immunohistochemical staining were also showed. CsA and spironolactone can inhibit the cell sizes induced by aldosterone, and the the gene expressions of ANF and P-MHC as well as CaN Aβ mRNA were also inhibited. They also can down-regulate the expressions of CaN Aβ and a-actin. It suggests that aldosterone may play an important role in the development of cardiac hypertrophy. We used siRNA to interfer CaN AβmRNA expression. The cell sizes were inhibited and the gene expressions of ANF and P-MHC which were related to cardiac hypertrophy were down-regulated, as well...