Tatsuya Morimoto

The dietary compound curcumin inhibits p300 histone acetyltransferase activity and prevents heart failure in rats

Hemodynamic overload in the heart can trigger maladaptive hypertrophy of cardiomyocytes. A key signaling event in this process is nuclear acetylation by histone deacetylases and p300, an intrinsic histone acetyltransferase (HAT). It has been previously shown that curcumin, a polyphenol responsible for the yellow color of the spice turmeric, possesses HAT inhibitory activity with specificity for the p300/CREB-binding protein. We found that curcumin inhibited the hypertrophy-induced acetylation and DNA-binding abilities of GATA4, a hypertrophy-responsive transcription factor, in rat cardiomyocytes. Curcumin also disrupted the p300/GATA4 complex and repressed agonist- and p300-induced hypertrophic responses in these cells. Both the acetylated form of GATA4 and the relative levels of the p300/GATA4 complex markedly increased in rat hypertensive hearts in vivo. The effects of curcumin were examined in vivo in 2 different heart failure models: hypertensive heart disease in salt-sensitive Dahl rats and surgically induced myocardial infarction in rats. In both models, curcumin prevented deterioration of systolic function and heart failure-induced increases in both myocardial wall thickness and diameter. From these results, we conclude that inhibition of p300 HAT activity by the nontoxic dietary compound curcumin may provide a novel therapeutic strategy for heart failure in humans.

α- and β-Adrenergic Pathways Differentially Regulate Cell Type–Specific Apoptosis in Rat Cardiac Myocytes

Background—The apoptosis of cardiac myocytes may play a role in the development of heart failure. Norepinephrine is one of the factors activated in heart failure and can induce myocardial cell apoptosis in culture. However, it is unknown if α- and β-adrenergic pathways coordinately or differentially regulate apoptosis and if this apoptotic pathway uses common or cell type–specific apoptotic signals. Methods and Results—We stimulated cultured neonatal rat cardiac myocytes with an α1-adrenergic agonist (PE, phenylephrine), a β-adrenergic agonist (isoproterenol [Iso]) or a membrane-permeable cAMP analogue (8-Br-cAMP) in serum-free conditions for 48 hours. Iso and 8-Br-cAMP markedly increased the number of TUNEL-positive cells (%TUNEL-positive nuclei >40%) compared with saline stimulation (<10%). DNA fragmentation was also confirmed by ladder formation in agarose gels. Apoptotic myocytes were characterized by cell shrinkage and nuclear condensation, consistent with morphological features of apoptosis. The Iso...

Cardiac p300 is involved in myocyte growth with decompensated heart failure

ABSTRACT A variety of stresses on the heart initiate a number of subcellular signaling pathways, which finally reach the nuclei of cardiac myocytes and cause myocyte hypertrophy with heart failure. However, common nuclear pathways that lead to this state are unknown. A zinc finger protein, GATA-4, is one of the transcription factors that mediate changes in gene expression during myocardial-cell hypertrophy. p300 not only acts as a transcriptional coactivator of GATA-4, but also possesses an intrinsic histone acetyltransferase activity. In primary cardiac myocytes derived from neonatal rats, we show that stimulation with phenylephrine increased an acetylated form of GATA-4 and its DNA-binding activity, as well as expression of p300. A dominant-negative mutant of p300 suppressed phenylephrine-induced nuclear acetylation, activation of GATA-4-dependent endothelin-1 promoters, and hypertrophic responses, such as increase in cell size and sarcomere organization. In sharp contrast to the activation of cardiac MEK-1, which phosphorylates GATA-4 and causes compensated hypertrophy in vivo, p300-mediated acetylation of mouse cardiac nuclear proteins, including GATA-4, results in marked eccentric dilatation and systolic dysfunction. These findings suggest that p300-mediated nuclear acetylation plays a critical role in the development of myocyte hypertrophy and represents a pathway that leads to decompensated heart failure.

Calcineurin-GATA-6 pathway is involved in smooth muscle-specific transcription.

Intracellular calcium is one of the important signals that initiates the myogenic program. The calcium-activated phosphatase calcineurin is necessary for the nuclear import of the nuclear factor of activated T cell (NFAT) family members, which interact with zinc finger GATA transcription factors. Whereas GATA-6 plays a role in the maintenance of the differentiated phenotype in vascular smooth muscle cells (VSMCs), it is unknown whether the calcineurin pathway is associated with GATA-6 and plays a role in the differentiation of VSMCs. The smooth muscle–myosin heavy chain (Sm-MHC) gene is a downstream target of GATA-6, and provides a highly specific marker for differentiated VSMCs. Using immunoprecipitation Western blotting, we showed that NFATc1 interacted with GATA-6. Consistent with this, NFATc1 further potentiated GATA-6–activated Sm-MHC transcription. Induction of VSMCs to the quiescent phenotype caused nuclear translocation of NFATc1. In differentiated VSMCs, blockage of calcineurin down-regulated the amount of GATA-6-DNA binding as well as the expression of Sm-MHC and its transcriptional activity. These findings demonstrate that the calcineurin pathway is associated with GATA-6 and is required for the maintenance of the differentiated phenotype in VSMCs.

p300 Protein as a Coactivator of GATA-5 in the Transcription of Cardiac-restricted Atrial Natriuretic Factor Gene

A cellular target of adenovirus E1A oncoprotein, p300 is a transcriptional coactivator and a negative regulator of cellular proliferation. A previous study suggests that the p300 family is also involved in cell type-specific transcription in cardiac myocytes. However, nothing is known about which cardiac transcription factor(s) interact with and transactivate through these proteins. The transcription factors GATA-4/5/6 have been implicated as key regulators of cardiogenesis, and they participate in the transcription of many cardiac-specific genes. Here we show that E1A represses the GATA-5-dependent transactivation of a promoter derived from the cardiac-restricted atrial natriuretic factor gene. This repression is correlated with the interaction of E1A with p300, indicating that p300 participates in GATA-5-dependent transactivation. E1A markedly down-regulates endogenous atrial natriuretic factor expression, as well as disrupts the interaction between p300 and GATA-5. A small fragment of p300 containing the carboxyl-terminal cysteine/histidine-rich domain, sufficient to interact with GATA-5, prevents transcriptional activation by GATA-5 as a dominant-negative mutant. Consistent with its role as a coactivator, p300 markedly potentiates GATA-5-activated transcription. These results implicate p300 as an important component of myocardial cell differentiation and provide an insight into the relationship between mechanisms that mediate cell type-specific transcription and cell cycle regulation during cardiogenesis.

Histone Acetyltransferase Activity of p300 Is Required for the Promotion of Left Ventricular Remodeling After Myocardial Infarction in Adult Mice In Vivo

Background— Left ventricular (LV) remodeling after myocardial infarction is associated with hypertrophy of surviving myocytes and represents a major process that leads to heart failure. One of the intrinsic histone acetyltransferases, p300, serves as a coactivator of hypertrophy-responsive transcriptional factors such as a cardiac zinc finger protein GATA-4 and is involved in its hypertrophic stimulus-induced acetylation and DNA binding. However, the role of p300-histone acetyltransferase activity in LV remodeling after myocardial infarction in vivo is unknown. Methods and Results— To solve this problem, we have generated transgenic mice overexpressing intact p300 or mutant p300 in the heart. As the result of its 2–amino acid substitution in the p300-histone acetyltransferase domain, this mutant lost its histone acetyltransferase activity and was unable to activate GATA-4–dependent transcription. The two kinds of transgenic mice and the wild-type mice were subjected to myocardial infarction or sham operation at the age of 12 weeks. Intact p300 transgenic mice showed significantly more progressive LV dilation and diminished systolic function after myocardial infarction than wild-type mice, whereas mutant p300 transgenic mice did not show this. Conclusions— These findings demonstrate that cardiac overexpression of p300 promotes LV remodeling after myocardial infarction in adult mice in vivo and that histone acetyltransferase activity of p300 is required for these processes.

A p300 Protein as a Coactivator of GATA-6 in the Transcription of the Smooth Muscle-Myosin Heavy Chain Gene

The mechanisms that regulate smooth muscle development and differentiation are poorly understood. Although recent studies have suggested the possible role of a zinc finger transcription factor, GATA-6, in the differentiation of vascular smooth muscle cells (VSMCs), the downstream gene targeted by GATA-6 is unknown. The expression of smooth muscle-myosin heavy chain (Sm-MHC) provides a highly specific marker for the differentiated phenotype of VSMCs as well as the smooth muscle cell lineage. Here, we show that GATA-6 bound to a GATA-like motif (−810/−805) within the rat Sm-MHC promoter in a sequence-specific manner and activated this promoter through this site. In addition, we show that the transcriptional coactivator p300 associated with GATA-6 during the transcription of the Sm-MHC gene. A p300/GATA-6 complex in VSMCs was up-regulated by induction of the quiescent phenotype. A wild-type E1A, which interferes with endogenous p300, but not a mutant E1A defective for p300 binding, markedly down-regulated the expression of endogenous Sm-MHC in quiescent-phenotype VSMCs. These studies provide the first identification of a functionally important GATA-6 binding site within a smooth muscle-specific promoter and suggest a role for p300 in the maintenance of the differentiated phenotype in VSMCs as a coactivator of GATA-6.

Identification of p300-targeted Acetylated Residues in GATA4 during Hypertrophic Responses in Cardiac Myocytes

A zinc finger protein, GATA4, is one of the hypertrophy-responsive transcription factors and increases its DNA binding and transcriptional activities in response to hypertrophic stimuli in cardiac myocytes. Activation of GATA4 during this process is mediated, in part, through acetylation by intrinsic histone acetyltransferases such as a transcriptional coactivator p300. However, p300-targeted acetylated sites of GATA4 during myocardial cell hypertrophy have not been identified. By mutational analysis, we showed that 4 lysine residues located between amino acids 311 and 322 are required for synergistic activation of atrial natriuretic factor and endothelin-1 promoters by GATA4 and p300. A tetra-mutant GATA4, in which these 4 lysine residues were simultaneously mutated, retained the ability to localize in nuclei and to interact with cofactors including FOG-2, GATA6, and p300 but lacked p300-induced acetylation, DNA binding, and transcriptional activities. Furthermore, coexpression of the tetra-mutant GATA4 with wild-type GATA4 impaired the p300-induced acetylation, DNA binding, and transcriptional activities of the wild type. When we expressed the tetra-mutant GATA4 in neonatal rat cardiac myocytes using a lentivirus vector, this mutant suppressed phenylephrine-induced increases in cell size, protein synthesis, and expression of hypertrophy-responsive genes. However, its expression did not affect the basal state. Thus, we have identified the most critical lysine residues acting as p300-mediated acetylation targets in GATA4 during hypertrophic responses in cardiac myocytes. The results also demonstrate that GATA4 with simultaneous mutation of these sites specifically suppresses hypertrophic responses as a dominant-negative form, providing further evidence for the acetylation of GATA4 as one of critical nuclear events in myocardial cell hypertrophy.

Cell line-dependent differentiation of induced pluripotent stem cells into cardiomyocytes in mice

AIMS Mouse and human fibroblasts can be directly reprogrammed to pluripotency by the ectopic expression of four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc) to yield induced pluripotent stem (iPS) cells. iPS cells can be generated even without the expression of c-Myc. The present study examined patterns of differentiation of mouse iPS cells into cardiomyocytes in three different cell lines reprogrammed by three or four factors. METHODS AND RESULTS During the induction of differentiation on feeder-free gelatinized dishes, genes involved in cardiogenesis were expressed as in embryonic stem cells and myogenic contraction occurred in two iPS cell lines. However, in one iPS cell line (20D17) generated by four factors, the expression of cardiac-specific genes and the beating activity were extremely low. Treating iPS cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased Nkx2.5 expression in all iPS cell lines. While the basal Nkx2.5 expression was very low in 20D17, the TSA-induced increase was the greatest. TSA also induced the expression of contractile proteins in 20D17. Furthermore, we demonstrated the increased mRNA level of Oct3/4 and nuclear protein level of HDAC4 in 20D17 compared with the other two iPS cell lines. DNA microarray analysis identified genes whose expression is up- or down-regulated in 20D17. CONCLUSIONS Mouse iPS cells differentiate into cardiomyocytes in a cell line-dependent manner. TSA induces myocardial differentiation in mouse iPS cells and might be useful to overcome cell line variation in the differentiation efficiency.

Endothelin-1–Dependent Nuclear Factor of Activated T Lymphocyte Signaling Associates With Transcriptional Coactivator p300 in the Activation of the B Cell Leukemia-2 Promoter in Cardiac Myocytes

Endothelin-1 (ET-1) is a potent survival factor that protects cardiac myocytes from apoptosis. ET-1 induces cardiac gene transcription and protein expression of antiapoptotic B cell leukemia-2 (bcl-2) in a calcineurin-dependent manner. A cellular target of adenovirus early region 1A (E1A) oncoprotein, p300 also activates bcl-2 transcription in cardiac myocytes and is required for their survival. p300 acts as a calcineurin-regulated nuclear factors of activated T lymphocytes (NFATc), downstream targets of calcineurin. In addition, the bcl-2 promoter contains multiple NFAT consensus sequences. These findings prompted us to investigate the role of NFATc in ET-1–dependent and p300-dependent bcl-2 transcription in cardiac myocytes. In primary cardiac myocytes prepared from neonatal rats, mutation of 2 NFAT sites within the bcl-2 promoter completely abolished the ET-1– and p300-induced increases in the activity of this promoter. We show here that p300 markedly potentiates the binding of NFATc1 to the bcl-2 NFAT element by interacting with NFATc1 in an E1A-dependent manner. On the other hand, stimulation of cardiac myocytes with ET-1 causes nuclear translocation of NFATc1, which interacts with p300 and increases DNA binding. Expression of E1A did not change the cardiac nuclear localization of NFATc1 but blocked its interaction with p300, DNA binding, and bcl-2 promoter activation. These findings suggest that ET-1–dependent NFATc signaling associates with p300 in the transactivation of bcl-2 gene in cardiac myocytes.