| Objective: Cardiac hypertrophy is one of adaptive responses of the heart to a variety of pathological stimuli, including hypertension, myocardial infarction, congenital heart disease, heart valvula disease, and perturbations in sarcomeric function, and is the main reason of the refractory heart failure. But the mechanism of cardiac hypertrophy is still not very clear.LIM proteins contain one, two or multiple cysteine-rich zinc-finger motifs and have been discovered to play important roles in a variety of fundamental biological processes including cytoskeleton organization, cell lineage specification and organ development. hhLIM, also named hLIM3 (GenBank AF121260), was cloned by three-element subtraction method from the embryo heart cDNA library. It is one of the members of LIM family since it has the typical LIM domain. Our previous study confirmed that hhLIM can shuttle between the nucleus and the cytoplasm. In differentiated cells, hhLIM mainly distributes in cytoplasm and enhances the stability of the actin cytoskeleton and promotes actin bundling, and then maintains the contractile function of myocardium. In response to stimuli inducing hypertrophy, hhLIM protein shuttled from cytoplasm to nucleus. In the nucleus, hhLIM interacted with Nkx2.5 and synergistically activated the expression of BNP andα-actin.Krüppel-like factors (KLFs) belong to the transcription factor family containing zinc-finger structures, mainly involved in regulating cell growth, differentiation, proliferation and apoptosis. As one of the members of this family, KLF5 (BTEB2) is an important transcription factor in cardiovascular remodeling and plays an important role in cardiac hypertrophy.It has been demonstrated that both KLF5 and hhLIM participated in the process of cardiac hypertrophy, but little is known about their relationship and function in the process of cardiac hypertrophy. In the present study, we investigated the interaction between KLF5 and hhLIM in the process of cardiomyocyte hypertrophy, and provided new evidence to reveal the mechanism of cardiomyocyte hypertrophy.Methods: Primary cardiomyocytes were isolated from the ventricles of 1-3-day-old Sprague-Dawley rats. MTT analysis was used to detect the effect of PE on cell proliferation. Flow cytometric/cell cycle analysis was used to reveal the distribution of H9C2 cells in various phases of the cell cycle. The expression of KLF5, hhLIM, Nkx2.5 and ANF were examined by Western blotting. GST pull-down and co-immunoprecipitation assay were done to examine the interaction between KLF5 and hhLIM. Reporter gene assay was done to detect the effect of KLF5 and hhLIM on the expression of ANF.Results:1 PE induces cardiomyocyte hypertrophyPE (20μmol/L) significantly increased the size in both the long and short cellular axes of cardiomyocytes. RT-PCR and Western blotting showed that the expression of the hypertrophic marker genes ANF and Nkx2.5 was markedly elevated after the cells were treated with PE, indicating that PE-induced cardiomyocyte hypertrophy model is established successively.2 PE induces the expression of KLF5 and hhLIMAfter the cells were treated by different concentrations of PE for the same time or the same concentration of PE for different times, the expression of hhLIM and KLF5 increased in dose- and time-dependent manners in cardiomyocytes, suggesting that KLF5 and hhLIM participate in the process of cardiomyocyte hypertrophy.3 KLF5 and hhLIM cooperatively activate the expression of ANF293A cells were transfected with ANF promoter-reporter plasmids and pRL-TK, pGL3-Basic, pMT-KLF5 or/and pEGFP-hhLIM expression plasmids. Luciferase activity was determined using the dual-luciferase reporter assay system. The results showed that ANF promoter activity was enhanced after transfected with hhLIM or KLF5 expression plasmids, respectively. While co-transfection of KLF5 and hhLIM significantly increased the ANF promoter activity to 2.48-fold compared with respective transfection of KLF5 or hhLIM, suggesting that KLF5 and hhLIM synergistically activate the ANF expression.We further constructed the adenovirus expression vector of KLF5 and hhLIM and infected cardiomyocytes, and then detected the expression of the hypertrophic marker gene ANF using Western blot analysis. Overexpression of KLF5 and hhLIM resulted in a significant increase in the expression of ANF, indicating that KLF5 and hhLIM cooperatively induce cardiomyocyte hypertrophy.4 PE increases the interaction between KLF5 and hhLIMThe above results showed that KLF5 and hhLIM cooperatively activated the expression of ANF. In order to further reveal the molecular mechanism of the interaction between KLF5 and hhLIM, GST pull-down and co-immunoprecipitation assay were performed. The results showed that KLF5 and hhLIM interacted with each other both in primary cardiomyocytes and H9C2 cells before and after treated with PE. Their interaction significantly increased after treated by PE, suggesting that KLF5 and hhLIM interact physically and PE promotes the interaction between KLF5 and hhLIM.5 Silencing of KLF5 inhibits PE-induced expression of ANFOur previous study confirmed that hhLIM was a cotranscription factor. So, we supposed that hhLIM activated the expression of ANF by interacting with KLF5. In order to identify this, H9C2 cells were transfected with rat KLF5-specific small interfering RNA (KLF5-siRNA) to silence the expression of endogenous KLF5. The expression of ANF was detected by Western blot analysis. The results showed that the level of ANF protein was reduced 59.3% in the cells transfected with KLF5-specific siRNA compared with those transfected with NS-siRNA, suggesting that KLF5 mediates the expression of ANF induced by PE.Conclusions:1 PE induces the expression of cardiac hypertrophic marker gene ANF and cardiomyocyte hypertrophy, but does not affect the proliferation of cardiac myocytes and the progress of cell cycle. 2 KLF5 and hhLIM mediate the expression of ANF induced by PE.3 KLF5 and hhLIM cooperatively activate the expression of ANF by interacting with each other.
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