Adenovirus-Mediated Overexpression Of TNNI3K Promotes Cardiomyocyte Hypertrophy

Aims:Cardiac troponin I interacting kinase (TNNI3K), a novel cardiac-specific kinase gene, shares similar structure with integrin-linked kinase(ILK). It has been demonstrated that ILK play an important role in the regulation of cardiac development and hypertrophy. Thus, TNNI3K might take part in the progress of cardiac hypertrophy. The aim of the present study was to investigate the effects of TNNI3K on cardiac hypertrophy.Methods:To investigate whether TNNI3K take part in the hypertrophy, cardiomyocytes were induced with endothelin-1 (ET-1) (100 nnol/L) and the TNNI3K mRNA expression was determined using quantitative real-time reverse transcript polymerase chain reaction (RT-PCR). To obtain enough adenovirus carrying human TNNI3K (Ad-TNNI3K), was amplified in HEK293A cells and purified in the CsCl using density gradient centrifuge. To determine a reasonable adenovirus infection dose of adenoviruses, cardiomyocytes were infected with an adenovirus carrying human TNNI3K at varying multiplicity of infection (MOI) for 48 hours, the efficiency of Ad-TNNI3K was detected with green fluorescence protein (GFP) under the fluorescence microscopy and the expression of TNNI3K was analyzed by western blot. To discovery the function role of TNNI3K in the cardiomyocytes, cardiomyocytes treated or without treated with ET-1 were infected with a control adenovirus (Ad-GFP), or Ad-TNNI3K. In each group, actin was stained with phalloidine-TRICT to demonstrate the sarcomere organization; cell surface area was determined with confocal microscopy software; the rate of protein synthesis was determined by the incorporation of 3H-leucine; changes of fetal-type genes such as atrial natriuretic factor (ANF) and a-myosin heavy chain (a-MHC) were measured using quantitative RT-PCR. Phosphorylation of Akt was detected by western blot. Finally, the expression of TNNI3K in cardiomyopathy (normal, n=3; dilated cardiomyopathy, n=6; hypertrophic cardiomyopathy, n=4) was analyzed using immunohistochemisty.Results:Quantitative real-time PCR analysis demonstrated a significant increase in TNNI3K mRNA expression in hypertrophic cardiomyocytes induced by endothelin-1 (ET-1). Adenoviruses were successfully amplified in 293A cells and purified in CsCl density gradient centrifuge. A reasonable adenovirus infection dose of adenoviruses was considered as MOI 100. Hypertrophic effect of TNNI3K on cardiomyocytes:Compared to Ad-GFP, the Ad-TNNI3K induced an increases in sarcomere organization, cell surface area,3H-leucine incorporation, and?-MHC re-expression. This type of hypertrophic phenomenon is similar to that observed in Ad-GFP-infected hypertrophic cardiomyocytes induced by ET-1; In hypertrophic cardiomyocytes, Ad-TNNI3K induced an increase in sarcomere organization, cell surface area and 3H-leucine incorporation compared to Ad-GFP. The expression of TNNI3K in dilated cardiomyopathy and hypertrophic cardiomyopathy is higher than that in normal hearts.Conclusion:These results suggest that TNNI3K overexpression induces cardiomyocytes hypertrophy and accelerates hypertrophy in hypertrophic cardiomyocytes. Therefore, TNNI3K may be an interesting target for the clinical treatment of hypertrophy. adenovirus-mediated TNNI3K overexpression enhance Ca2+ sensitivity in adult rat cardiac myocytesObjective To provide a useful model for studying the function of TNNI3K during the heart contraction, hypertrophy, heart failure and other heart diseases, adult rat cardiac myocytes were cultured and the effect of TNNI3K overexpression on Ca2+sensitivity was determined.Methods Sprague-Dawley male rats were anesthetized and the heart was quickly removed from the chest. Then the heart was retroperfused with an enzyme solution containing collegenase II. After digestion was terminated, left ventricular tissues were split into single myocyte and stored in K-B buffer for 2 h. Before myocytes were seeded in improved M199 medium, myocytes were treated with grade Ca2+medium. Then the Ca2+sensitivity of myocytes infected with Ad-GFP or Ad-TNNI3K were determined using IonOptix system.Results Successfully isolated and cultured adult rat cardiac myocytes. TNNI3K overexpression enhanced the Ca2+sensitivity of myocytes.Conclusion The methods established in our experiment were effective and provided a useful tool to investigate the contraction function of TNNI3K. Aims:Sarcomere organization could be regulated by cross-linking protein and is a character of cardiac hypertrophy. Nexilin, a actin filamine binding protein, might play a role in the regulation of sarcomere organization and contractility. The purpose of this study was to investigate the function role of nexilin in cardiomyocyte.Methods:(1) To determine the expression of nexilin in cardiac hypertrophy, cardiomyocytes were induced hypertrophy with endothelin-1 (ET-1). nexilin mRNA expression was analyzed by real-time reverse transcription-polymerase chain reaction at different time; the expression of nexilin in hypertrophic cardiomyopathy and dilated cardiomyopathy were measured using immunohistochemisty. (2) To obtain nexilin cDNA fragment, total RNA was extracted from adult rat heart, reverse transcribed into cDNA. nexilin cDNA fragment was amplified using polymerase chain reaction, cloned into pcDNA3 vector, and proofed by sequencing. (3) nexilin knockdown adenovirus recombination:nexilin cDNA fragment was subcloned into pEGFP-N1 vector; Four shRNAs were synthesized and inserted in pGCSIL-U6 vector; HEK293A cells were co-transfected with these two vectors for 48 h. To evaluated the effect of knockdown in vitro,total protein was extracted and EGFP-Flag-nexilin fusion protein was detected with Flag antibody by western blot; To recombinant adenovirus, HEK293A cells were co-transfected with shuttle vectors constructed with effective shRNA and adenovirus skeleton vectors. (4) To evaluated over-expression and knock down effects of adenovirus, cardiomyocytes were infected with adenovirus carrying full-length nexilin cDNA or effective shRNA at indicated MOI dose. Nexilin expression was detected with nexilin antibody by western blot. (5) To get the location of nexilin in cardiomyocyte, neonatal rat cardiomyocytes were transfected with pEGFP-N1-nexilin. The cellular location of nexilin was determined under the confocal microscopy. (6) To determine the effect of nexilin on cardiomyocyte cytoskeleton, Cardiomyocytes were seeded on glass. Then cardiomyocytes treated or untreated with ET-1 were infected with control adenovirus (Ad-GFP) or adenovirus overexpressing nexilin (Ad-nexilin) or adenovirus carrying shRNA that knocking down nexilin (Ad-nexilinRNAi or Ad-shRNA) for 24 h. Actin was stained with phalloidine-TRICT to demonstrate the sarcomere organization in each group. (7) cardiomyocytes were treated as above description and the contractility was measured using Ion Optix system. Results:(1) nexilin mRNA expression was upregulated in ET-1-induced hypertrophy; nexilin protein expression also was upregulated and the expreeson was higher in dilated cardiomyopathy than that in hypertrophic cardiomyopathy. (2) nexilin cDNA fragment was obtained. High effective shRNA was screened and constructed into adenovirus. (3) The effects of nexilin knock down was the best on MOI 40; Effects of nexilin overexpression mediated by adenovirus was better on MOI 40 and best on MOI 100. (4) nexilin-EGFP fusion protein incorporated and located in sarcomere. (5) nexilin overexpression maintained and increased the sarcomere organization, enhanced cell shortening, departure velocity and relaxation velocity; On the contrary, nexilin knock down dissipated sarcomere organization, decreased the shortening, departure velocity and relaxation velocity; compared with normal cardiomyocytes, ET-1-induced hypertrophy increased sarcomere organization and cell shortening.Conclusion:Nexilin regulated cardiomyocytes contractility possibly via controlling sarcomere organization. It plays a vital role in maintaining cardiac myocyte morphology and contractile function and is involved in cardiac hypertrophy formation.