Cardiac hypertrophy is characterized by an enlargement of individual cardiomyocytes without apparent increase in the cell number. Although it could be a physiologic response, many clinical features such as myocardial infarction, hypertension, or valvu...
Cardiac hypertrophy is characterized by an enlargement of individual cardiomyocytes without apparent increase in the cell number. Although it could be a physiologic response, many clinical features such as myocardial infarction, hypertension, or valvular dysfunctions can cause pathologic hypertrophy. However, since few interventions to prevent cardiac hypertrophy are available yet, it is important to elucidate what molecule is a key factor to develop hypertrophy and how the molecule is regulated. This study was aimed to evaluate the regulation mechanism of histone deacetylase 2 (Hdac2), a class I HDAC, and to find the downstream target gene in various hypertrophy models.
Hdac2 increased the Nppa promoter, most commonly used as molecular markers of hypertrophy, activity. Cardiac hypertrophy, as determined by cell size or by stress fiber formation, was simulated by Hdac2. Heart lysates obtained from isoproterenol (ISP)-treated mice induced phosphorylation of recombinant GST-Hdac2 in vitro, which was completely blocked by treatment with apigenin, a casein kinase (CK) 2 inhibitor. CK2 was activated either in the hearts obtained from ISP-treated mice or in the cardiomyocyte challenged by phenylephrine. Phosphorylation-resistant Hdac2 mutant failed to induce the promoter activity of Nppa and Myh7. However, by immunoprecipitation assay it was observed that alteration of phosphorylation in response to hypertrophic stresses in endogenous Hdac2 was not significant, raising the possible involvement of alternate mechanism. Inducible heat shock protein, HSP70, physically interacted with Hdac2 in cell free systems. Both ATP-binding domain and C-terminal part of HSP70 was responsible for the interaction with Hdac2. The direct in vivo association between HSP70 and Hdac2 was confirmed by protein complementation methods as well as by confocal images. The activity of hexahistidine-tagged recombinant Hdac2 was further potentiated by adding GST-HSP70 in the presence of H9c2 lysates. HSP70ΔABD, a dominant negative HSP70, could not activate Hdac2. Interestingly, transfection of HSP70ΔABD significantly interfered the association between Hdac2 with wild type HSP70. HSP70 induced activation of Nppa promoter in cardiomyocyte, which was completely blocked by siHdac2. Forced expression of HSP70ΔABD blocked the activity of either Hdac2 or Nppa promoter. SK7041, a class I HDAC selective inhibitor, dose-dependently reduced Nppa promoter activity. Nppa promoter mapping study revealed that the region from -130 to -105 was essential for SK7041 action. By searching bioinformatics, KLF4 was predicted for candidate. KLF4 expression was increased in the treatment with SK7041 but downregulated by various hypertrophic stimuli. Transfection of KLF4 reduced either Nppa promoter activity or phenylephrine-triggered increase in the cell size.
In conclusion, these results implicate that HSP70/Hdac2/KLF4 might be targeted as novel molecules for preventing or treating heart disease with hypertrophy.