The Histone Demethylase PHF8 Represses Cardiac Hypertrophy Upon Pressure Overload

BackgroudMammalian heart will undergo cardiac hypertrophy in the case of press overload; these changes may be compensatory initially to manage the increased workload on the heart. However, sustained hypertrophy results in cardiac decompensation, followed by heart failure. The main pathological represents of myocardial hypertrophy include: cardiomyocyte hypertrophy,extracellular matrix remodeling, myocardial interstitial proliferation etc. Cardiomyocyte protein synthesis, can lead to myocardial cell volume increases, namely cardiomyocyte hypertrophy. Studies confirmed that the formation of cardiomyocyte hypertrophy required four factors:the stimulus signal, signal conduction, activation of nuclear gene transcription, and transformation of cardiac phenotype. The ultimate effect is the transformations of cardiac phenotype from "mature" to "embryonic". There have been many studies about the mechanism of cardiac hypertrophy, but its specific mechanism remains unclear, and there is still no precise and effective way to prevent cardiac hypertrophy. Present studies confirm that in the process of cardiac hypertrophy, a group of genes normally expressed in the fetal heart development is re-expressed, and involved in the regulation of pathological cardiac hypertrophy.Histone is an integral part of the eukaryotic chromatin. The amino terminus of histone subunits is called histone tails free, many residues on histone tails can be covalently modified, different modifications of different residues can produce a variety of different signals, which can be identified by other signaling proteins and regulate the gene expression in eukaryotes, resulting in transcriptional activation or gene silencing, and thus produce a variety of biological effects. Studies have shown that histone modifications are closely related to the pathogenesis of many diseases, such as chronic kidney disease, tumor, nervous system diseases, and endocrine system diseases. In the cardiovascular field, histone modifications also play an important role.Plant Homeo Domain Finger protein 8 (PHF8) is a newly discovered histone demethylase in recent years, which can regulate gene expression through demethylation to histone lysine residues. PHF8 is a member of histone demethylase JmjC family, and the JmjC family plays an important role in development, differentiation, endocrinal regulation and development of a variety of diseases. PHF8 as a member of the JmjC family also plays an important role histone modifications. PHF8 erases repressive histone marks H4K20mel and H3K9me1/2, and binds to H3K4me3, to participate in developmental regulation and the development of many diseases. But the role of PHF8 in the cardiovascular system especially in cardiac hypertrophy is unclear.This research will be designed both in vivo and in vitro to investigate the role of PHF8 in cardiac hypertrophy, and to explore the molecular mechanisms of its action, so as to look for new opportunities and therapeutic targets in prevention and treatment of cardiac hypertrophy.Material and Methods1. Generation of Transgenic Mice:The cardiac-specific human PHF8 transgenic mice were generated.2. Pathological Hypertrophy by Transaortic Constriction (TAC): Pressure overload hypertrophy was induced by TAC of the ascending aorta of mice.3. Echocardiography of Mice: Echocardiography was performed to detect changes of cardiac structure and function of mice.4. Cell Culture and Hypertrophic Growth Cell Model: neonatal rat cardiomyocytes (NRCMs)were isolated and cultured, and phenylephrine (PE,100μM)was used to induce hypertrophic growth of NRCMs.5. PHF8 Interference in NRCMs: Adenoviral vectors were used in our experiments to interference the expression of PHF8 in NRCMs.6. HE Staining, Wheat Germ Agglutinin Staining, Masson Staining: The above staining methods were used to show the gross changes of cardiac hypertrophy, myocardial cell size and myocardial fibrosis respectively.7. Western Blot: To detect the expression of PHF8、ANPand related signaling proteins in protein levels.8. RT-PCR: To detect the expression of PHF8、ANP、β-MPC、BNP and related signaling proteins in gene levels.9.3H-leucine incorporation To detect the level of protein synthesis in cardiomyocytes.10. Luciferase assay To detect promoter activity of embryonic gene in cardiomyocytes.11. Immunofluorescence: To detect the size of cardiomyocytes.Results1. PHF8 level is down-regulated upon pathological hypertrophy. the mRNA and protein levels of PHF8 were significantly down-regulated in hearts from patients with heart failure accomplished with cardiac hypertrophy and cardiac hypertrophy wide-type C57BL/6 mice, we got a similar conclusion in hypertrophic growing NRMCs after PE stimulation.These findings implicate that PHF8 may participate in pathological hypertrophy.2. PHF8 overexpression represses cardiac remodeling upon pressure overload. After 4 weeks of TAC, the cardiac function of PHF8-Tg mice was significantly better than wild-type (WT) mice, and the area of fibrosis in left ventricle of PHF8-Tg mice was significantly reduced compared to that in WT mice, fibrosis markers (COL1A1, COL3A1, CTGF, TGFp1, fibronectin and a-SMA) and hypertrophic fetal genes (ANP, BNP, p-MHC, ACTA1 and IL-6) were markedly down-regulated in PHF8-Tg mice. These results suggest that PHF8 inhibits pathological cardiac remodeling upon pressure overload.3. PHF8 regulates hypertrophic growth of NRCMs. Upon PE treatment in NRCMs, PHF8 overexpression inhibited fetal protein synthesis while PHF8 knockdown promoted fetal protein synthesis, In addition, the PE-induced promoter activities of ANP and β-MHC was down-regulated by PHF8 overexpression and up-regulated by PHF8 knockdown. The findings indicate that PHF8 negatively regulate the hypertrophic response of cardiac myocytes in vitro.4. PHF8 regulates Akt-mTOR pathway. PHF8 overexpression significantly decreased TAC-induced phosphorylation of Akt and mTOR, which indicates that PHF8 overexpression blocked TAC-induced Akt-mTOR pathway. Meanwhile, PHF8 knockdown promoted PE-induced activation of Akt-mTOR pathway.5. Rapamycin rescues the phenotype of PHF8 loss. We found that rapamycin treatment inhibited PE-induced hypertrophic growth of NRCMs and blocked the effect of PHF8 deficiency on hypertrophic growth, rapamycin also inhibited PE-induced protein synthesis and promoter activity of ANP and β-MHC, and PHF8 knockdown induced up-regulation of protein synthesis and completely blocked the hypertrophic gene expression. Therefore, rapamycin rescues the phenotype of PHF8 deficiency.Conclusion1. PHF8 inhibits pathological cardiac remodeling upon pressure overload and hypertrophic growth of cardiac myocytes;2. PHF8 blocks the cardiac hypertrophic response partially through the Akt-mTOR pathway;3. PHF8 may be an alternative of rapamycin in the role of anti-hypertrophic;4. In this study, we elucidated the function of PHF8 in pathological hypertrophy both in vivo and in vitro, and explored the potential mechanisms of its function, in order to provide new ideas and therapeutic targets for prevention and treatment of cardiac hypertrophy.