Functional Analysis Of Heart High-expressing Genes KLHL31 And Hole In Cardiomyocyte Hypertrophy

Cardiac hypertrophy is a common heart disease, and is an adaptation reaction of myocardial cell to kinds of pathologic stimuli. In order to adapt to the pathological environment, myocardial cells sustain the balance of cardiac output and tissue priming volume through compensatory hypertrophy. If the hypertrophy reaction of cardiac muscle could not be inhibited or be released, ultimately lead to ventricular dilatation, contractibility disfunction, and turn to cardiac failure, which would increase the incidence of a disease and mortality of heart disease. So exploring the etiopathogenisis of cardiac hypertrophy is a hot research field of cardiovascular disease study. In recent years, the research of cardiac hypertrophy has made a great progress, but the molecular mechanism is still unclear. Therefore, researching in molecular mechanism of cardiac hypertrophy and founding effective method is one of the most important subjects in the field of the practice of basic theory and clinical researches.To clarifying the molecular mechanisms of cardiac hypertrophy, human heart high-expressing genes were selected to study their functions in cardiac hypertrophy.At first, pathologic cardiac hypertrophic mouse models were established by injecting isoprenaline (ISO) or Levothyroxine(L-THY), RNA was extracted from the heart of the mouse models for further RT-PCR analyses, and the results show that heart high-expressing genes KLHL31 and hole were up-regulation in cardiac hypertrophic mouse.KLHLS1 was high expressed in human heart and skeletal muscle, and KLHL31 protein contains a conservative BTB domain and repeat Kelch domain. Recently, some researches showed that KLHL31 play an important role in differentiation of skeletal muscle, yet its function in adult heart was unknown.To investigate effects of KLHL31 on cardiac hypertrophy, the primary cardiomyocytes of neonatal rat was isolated. KLHL31 was up-regulated in primary cardiomyocytes stimulated by FBS.H9c2 cells derived from rat cardiac tissue was used for cell model to further study on whether KLHL31 was involve in cardiac hypertrophy. The H9c2 cells line that expressing KLHL31 and KLHL31-RNAi stably was established successfully, hypertrophic marker genes was detected. Obviously, hypertrophic marker genes were up-regulated in over-expressing KLHL31 cells, while were down-regulated in KLHL31-RNAi cells. These results suggested cardiac hypertrophy may be induced partly by KLHL31.Bioinformatics analysis shows that the promoter region of KLHL31 contains the binding site CArG of SRF (serum response factor, SRF). Recently, several reports show that SRF plays a pivotal role in cardiac hypertrophy, and many signal pathways induce cardiac hypertrophy by activating SRF. Whether does KLHL31 regulate cardiac hypertrophy by inactivating SRF? Firstly, we cloned and characterized the human KLHL31 promoter which contains highly conserved serum response factor (SRF) sites. When the recombinant plasmid pGL4.1-KLHL31 and SRF were co-transfected, the activity of luciferase was increased considerably. Then, we demonstrated that SRF binds to the CArG site on KLHL31 promoter region using EMSA. Moreover, over-expressed KLHL31 promoted the transcription activity of endogenetic SRF. These results suggest that KLHL31 maybe regulate the development of cardiac hypertrophy by binding SRF.With the aim of identifying novel genes involved in human heart development and diseases, hole gene was cloned from human embryo cDNA library, hole genes are highly conserved in evolution from lower vertebrates to high vertebrates such as chicken, mouse, hole forms an independent family in evolution, yet its function is unknown. Human hole is located in chromosome 14, and its cDNA is 1.5 kb, coding a protein of 319aa. Human hole was expressed highly in heart. Though hole has no obvious functional domain, it contains a binding site of ERK (D-domain) and proline-rich SH3 binding motifs.To further study on whether was hole involve in cardiac hypertrophy. The H9c2 cells line that expressing hole and hole-RNAi stably was established successfully, hypertrophic marker genes were detected. Obviously, hypertrophic marker genes were down-regulated in over-expressing-hole cells. These results suggested hole maybe act as a inhibitor in development of cardiac hypertrophy.Reporter assay analysis showed that hole protein strongly inhibit the activity of ANF and NFAT which were involve in cardiac hypertrophy. Whether hole regulates cardiac hypertrophy through its binding site of ERK or its proline-rich SH3 binding motifs in its C-terminus? Several mutants of the recombinant plasmid pCMV-tag2B-hole were constructed by restriction digestion and site-directed mutagenesis. The recombinant plasmid pCMV-tag2B-DD and pCMV-tag2B-MD mutants mean that the plasmids contain only the region in binding site of ERK and mutated ERK D domain site; pCMV-tag2B-hole-TD mutant means that the plasmid contain the transmenbrane region and the binding site of ERK; pCMV-tag2B-hole-TD mutant mens the plasmid contain the region in proline-rich SH3 binding motifs; pCMV-tag2B-hole-MD mutant means the region in binding site of ERK has been mutated; pCMV-tag2B-hole-MP mutant means the region in proline-rich SH3 binding motifs has been mutated; pCMV-tag2B-hole-MDP means the regions both in binding site of ERK and proline-rich SH3 binding motifs have been mutated. Then, the several mutants were transfect respectively in HEK293 cell, the report assay showed that the binding site of ERK of hole protein played a crucial role in inhibiting the transcriptional activities of hypertrophic signal molecule.BALB/c mice were intravenously injected with pCMV-tag2B-KLHL31 or pCMV-tag2B-hole. At 1 day after plasmid injection, mice were injected by ISO as we described previously. The heart/body weight (HW/BW) ratios of mice which were be injected plasmid pCMV-tag2B-KLHL31 was significantly increased, as a result of cardiac hypertrophy development, and the expression of marker genes of cardiac hypertrophy were up-regulated, whereas the heart/body weight (HW/BW) significantly lower in the mice given ISO and pCMV-tag2B-hole, and the expression of marker genes were down-regulated. Those results were well consistent with the previous results in cell model.Finally, we use a set of pathological hypertrophic cardiomyopathy patients heart samples to analysis whether KLHL31 and hole were related to cardiac hypertrophy. The results show that KLHL31and hole were also up-regulated in the heart tissue of the patients with hypertrophic cardiomyopathy, which was as the same as those observed in mouse model and cell model. These results indicate that KLHL31 and hole may be involved both in cardiac hypertrophy in mice and human heart.