Xupei Huang

Correcting diastolic dysfunction by Ca2+ desensitizing troponin in a transgenic mouse model of restrictive cardiomyopathy.

Several cardiac troponin I (cTnI) mutations are associated with restrictive cardiomyopathy (RCM) in humans. We have created transgenic mice (cTnI(193His) mice) that express the corresponding human RCM R192H mutation. Phenotype of this RCM animal model includes restrictive ventricles, biatrial enlargement and sudden cardiac death, which are similar to those observed in RCM patients carrying the same cTnI mutation. In the present study, we modified the overall cTnI in cardiac muscle by crossing cTnI(193His) mice with transgenic mice expressing an N-terminal truncated cTnI (cTnI-ND) that enhances relaxation. Protein analyses determined that wild type cTnI was replaced by cTnI-ND in the heart of double transgenic mice (Double TG), which express only cTnI-ND and cTnI R193H in cardiac myocytes. The presence of cTnI-ND effectively rescued the lethal phenotype of RCM mice by reducing the mortality rate. Cardiac function was significantly improved in Double TG mice when measured by echocardiography. The hypersensitivity to Ca(2+) and the prolonged relaxation of RCM cTnI(193His) cardiac myocytes were completely reversed by the presence of cTnI-ND in RCM hearts. The results demonstrate that myofibril hypersensitivity to Ca(2+) is a key mechanism that causes impaired relaxation in RCM cTnI mutant hearts and Ca(2+) desensitization by cTnI-ND can correct diastolic dysfunction and rescue the RCM phenotypes, suggesting that Ca(2+) desensitization in myofibrils is a therapeutic option for treatment of diastolic dysfunction without interventions directed at the systemic beta-adrenergic-PKA pathways.

Myofilament anchoring of protein kinase C-epsilon in cardiac myocytes

Regulatory proteins on muscle filaments are substrates for protein kinase C (PKC) but mechanisms underlying activation and translocation of PKC to this non-membrane compartment are poorly understood. Here we demonstrate that the epsilon isoform of PKC (ϵ-PKC) activated by arachidonic acid (AA) binds reversibly to cardiac myofibrils with an EC50 of 86 nM. Binding occurred near the Z-lines giving rise to a striated staining pattern. The delta isoform of PKC (δ-PKC) did not bind to cardiac myofibrils regardless of the activator used, and the alpha isoform (α-PKC) bound only under strong activating conditions. Three established PKC anchoring proteins, filamentous actin (F-actin), the LIM domain protein Cypher-1, and the coatamer protein β′-COP were each tested for their involvement in cytoskeletal anchoring. F-actin bound ϵ-PKC selectively over δ-PKC and α-PKC, but this interaction was readily distinguishable from cardiac myofilament binding in two ways. First, the F-actin/ϵ-PKC interaction was independent of PKC activation, and second, the synthetic hexapeptide LKKQET derived from the C1 region of ϵ-PKC effectively blocked ϵ-PKC binding to F-actin, but was without effect on its binding to cardiac myofilaments. Involvement of Cypher-1 was ruled out on the basis of its absence from detergent-skinned myofibrils that bound ϵ-PKC, despite its presence in intact cardiac myocytes. The ϵ-PKC translocation inhibitor peptide EAVSLKPT reduced activated ϵ-PKC binding to cardiac myofibrils in a concentration dependent manner, suggesting that a RACK2 or a similar protein plays a role in ϵ-PKC anchoring in cardiac myofilaments.

Inhibition of p300-HAT results in a reduced histone acetylation and down-regulation of gene expression in cardiac myocytes.

AIMS Histone acetylation plays an important role in cardiogenesis, but the underlying mechanism is unclear. In this study, we investigated the relationship between histone hypo-acetylation and the expression of cardiac-specific genes to explore the underlying mechanisms. MAIN METHODS Cardiac-specific genes that physically interacted with p300 protein in mouse hearts were analyzed using chromatin immunoprecipitation (ChIP) assays. The cultured mouse neonatal cardiac myocytes were treated with curcumin with different concentrations and durations. The changes of histone acetyltransferase (HAT) activities, histone acetylation, cardiac-specific genes expression, and structure of chromatin were assessed by ELISA, Western blotting, quantitative RT-PCR, and ChIP assays, respectively. KEY FINDINGS Results from the ChIP assay showed that GATA4, Nkx2.5, and Mef2c physically interacted with p300 protein. After treatment with 30 μM curcumin for 24h, the HAT activities of cardiac myocytes were inhibited significantly. And the acetylation of whole histone H3 was reduced by 0.3983-fold compared to control groups (P<0.05). Accordingly, the expression of cardiac-specific genes, GATA4, Nkx2.5, and Mef2c, were significantly down-regulated. Acetylation of histone H3 bound with promoter regions of these genes was significantly reduced. SIGNIFICANCE p300 interacts with cardiac-specific genes, GATA4, Nkx2.5 and Mef2c, and inhibition of p300-HAT by curcumin down-regulates their expression through the inhibition of histone H3 acetylation in the promoter regions. This finding indicates that p300-HAT mediated histone H3 acetylation plays an important role in the regulation of cardiac gene expression, which is a novel mechanism of epigenetic regulation in the heart during the development and in case of some congenital heart diseases.

Roles of planar cell polarity pathways in the development of neutral tube defects

Neural tube defects (NTDs) are the second most common birth defect in humans. Despite many advances in the understanding of NTDs and the identification of many genes related to NTDs, the fundamental etiology for the majority of cases of NTDs remains unclear. Planar cell polarity (PCP) signaling pathway, which is important for polarized cell movement (such as cell migration) and organ morphogenesis through the activation of cytoskeletal pathways, has been shown to play multiple roles during neural tube closure. The disrupted function of PCP pathway is connected with some NTDs. Here, we summarize our current understanding of how PCP factors affect the pathogenesis of NTDs.

Sodium valproate-induced congenital cardiac abnormalities in mice are associated with the inhibition of histone deacetylase

BackgroundValproic acid, a widely used anticonvulsant drug, is a potent teratogen resulting in various congenital abnormalities. However, the mechanisms underlying valproic acid induced teratogenesis are nor clear. Recent studies indicate that histone deacetylase is a direct target of valproic acid.MethodsIn the present study, we have used histological analysis and RT-PCR assays to examine the cardiac abnormalities in mice treated with sodium valproate (NaVP) and determined the effects of NaVP on histone deacetylase activity and the expression of heart development-related genes in mouse myocardial cells.ResultsThe experimental data show that NaVP can induce cardiac abnormalities in fetal mice in a dose-dependent manner. NaVP causes a dose-dependent inhibition of hitone deacetylase (HDAC) activity in mouse myocardial cells. However, the expression levels of HDAC (both HDAC1 and HDAC2) are not significantly changed in fetal mouse hearts after administration of NaVP in pregnant mice. The transcriptional levels of other heart development-related genes, such as CHF1, Tbx5 and MEF2, are significantly increased in fetal mouse hearts treated with NaVP.ConclusionsThe study indicates that administration of NaVP in pregnant mice can result in various cardiac abnormalities in fetal hearts, which is associated with an inhibition of histone deacetylase without altering the transcription of this enzyme.

Methionine sulfoxide reductase A (MsrA) protects cultured mouse embryonic stem cells from H2O2‐mediated oxidative stress

Methionine sulfoxide reductase A (MsrA), a member of the Msr gene family, can reduce methionine sulfoxide residues in proteins formed by oxidation of methionine by reactive oxygen species (ROS). Msr is an important protein repair system which can also function to scavenge ROS. Our studies have confirmed the expression of MsrA in mouse embryonic stem cells (ESCs) in culture conditions. A cytosol‐located and mitochondria‐enriched expression pattern has been observed in these cells. To confirm the protective function of MsrA in ESCs against oxidative stress, a siRNA approach has been used to knockdown MsrA expression in ES cells which showed less resistance than control cells to hydrogen peroxide treatment. Overexpression of MsrA gene products in ES cells showed improved survivability of these cells to hydrogen peroxide treatment. Our results indicate that MsrA plays an important role in cellular defenses against oxidative stress in ESCs. Msr genes may provide a new target in stem cells to increase their survivability during the therapeutic applications. J. Cell. Biochem. 111: 94–103, 2010.

Deficiency of methionine sulfoxide reductase A causes cellular dysfunction and mitochondrial damage in cardiac myocytes under physical and oxidative stresses

Methionine sulfoxide reductase A (MsrA) is an enzyme that reverses oxidation of methionine in proteins. Using a MsrA gene knockout (MsrA(-/-)) mouse model, we have investigated the role of MsrA in the heart. Our data indicate that cellular contractility and cardiac function are not significantly changed in MsrA(-/-) mice if the hearts are not stressed. However, the cellular contractility, when stressed using a higher stimulation frequency (2Hz), is significantly reduced in MsrA(-/-) cardiac myocytes. MsrA(-/-) cardiac myocytes also show a significant decrease in contractility after oxidative stress using H(2)O(2). Corresponding changes in Ca(2+) transients are observed in MsrA(-/-) cardiomyocytes treated with 2Hz stimulation or with H(2)O(2). Electron microscope analyses reveal a dramatic morphological change of mitochondria in MsrA(-/-) mouse hearts. Further biochemical measurements indicate that protein oxidation levels in MsrA(-/-) mouse hearts are significantly higher than those in wild type controls. Our study demonstrates that the lack of MsrA in cardiac myocytes reduces myocardial cells capability against stress stimulations resulting in a cellular dysfunction in the heart.

Spatiotemporal expression of histone acetyltransferases, p300 and CBP, in developing embryonic hearts

Histone acetyltransferases (HATs), p300 and cAMP response element binding protein (CREB)-binding protein (CBP) are two structurally related transcriptional co-activators that activate expression of many eukaryotic genes involved in cellular growth and signaling, muscle differentiation and embryogenesis. However, whether these proteins play important and different roles in mouse cardiogenesis is not clear. Here, we investigate the protein distributions and mRNA expression of the two HATs in embryonic and adult mouse heart during normal heart development by using immunohistochemical and RT-PCR techniques. The data from immunohistochemical experiments revealed that p300 was extensively present in nearly every region of the hearts from embryonic stages to the adulthood. However, no CBP expression was detected in embryonic hearts at day E7.5. CBP expression appeared at the later stages, and the distribution of CBP was less than that of p300. In the developmental hearts after E10.5, both for p300 and CBP, the mRNA expression levels reached a peak on day E10.5, and then were gradually decreased afterwards. These results reveal that both p300 and CBP are related to embryonic heart development. The dynamic expression patterns of these two enzymes during mouse heart development indicate that they may play an important role on heart development. However, there is a difference in spatiotemporal expression patterns between these two enzymes during heart development. The expression of p300 is earlier and more predominate, suggesting that p300 may play a more important role in embryonic heart development especially during cardiac precursor cell induction and interventricular septum formation.

Protective effects of taurine against oxidative stress in the heart of MsrA knockout mice

Taurine has been shown to have potent anti‐oxidant properties under various pathophysiological conditions. We reported previously a cellular dysfunction and mitochondrial damage in cardiac myocytes of methionine sulfoxide reductase A (MsrA) gene knockout mice (MsrA−/−). In the present study, we have explored the protective effects of taurine against oxidative stress in the heart of MsrA−/− mice with or without taurine treatment. Cardiac cell contractility and Ca2+ dynamics were measured using cell‐based assays and in vivo cardiac function was monitored using high‐resolution echocardiography in the tested animals. Our data have shown that MsrA−/− mice exhibited a progressive cardiac dysfunction with a significant decrease of ejection fraction (EF) and fraction shortening (FS) at age of 8 months compared to the wild type controls at the same age. However, the dysfunction was corrected in MsrA−/− mice treated with taurine supplement in the diet for 5 months. We further investigated the cellular mechanism underlying the protective effect of taurine in the heart. Our data indicated that cardiac myocytes from MsrA−/− mice treated with taurine exhibited an improved cell contraction and could tolerate oxidative stress better. Furthermore, taurine treatment reduced significantly the protein oxidation levels in mitochondria of MsrA−/− hearts, suggesting an anti‐oxidant effect of taurine in cardiac mitochondria. Our study demonstrates that long‐term treatment of taurine as a diet supplement is beneficial to a heart that is vulnerable to environmental oxidative stresses. J. Cell. Biochem. 113: 3559–3566, 2012.

Alcohol Consumption During Gestation Causes Histone3 Lysine9 Hyperacetylation and an Alternation of Expression of Heart Development-Related Genes in Mice

BACKGROUND Alcohol abuse during gestation may cause congenital heart diseases (CHDs). The underlying mechanisms of alcohol-induced cardiac deformities are still not clear. Recent studies suggest that histone modification may play a crucial role in this pathological process. Moreover, our previous studies reported that ethanol could induce histone3 lysine9 (H3K9) hyperacetylation and overexpression of heart development-related genes in vitro. The aim of this study was to investigate the effect of alcohol consumption during gestation on the imbalance of H3K9 acetylation and the alternation of the expression of heart development-related genes during cardiogenesis. METHODS Pregnant mice were exposed to a single dose of alcohol (10 μl/g/d, 56% alcohol) by gavage every day in the morning from embryo day 7.5 (E7.5) to E15.5. Hematoxylin and eosin (H&E) staining was applied for observing the structure of the embryonic hearts. Western blotting and quantitative real-time polymerase chain reaction were used for detecting the level of H3K9 acetylation and gene expression. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities were detected by colorimetric assay and fluorometric assay. RESULTS H&E staining of cardiac tissue showed abnormalities of embryonic hearts at E17.5. The level of H3K9 acetylation reached peak at E17.5 and decreased sharply to a low level at birth and maintained at low level afterward. Alcohol exposure increased H3K9 acetylation at E11.5, E14.5, E17.5, and E18.5, respectively (p < 0.05), and enhanced the expression of Gata4 in the embryonic hearts at E14.5 and E17.5, Mef2c at E14.5, and Nkx2.5 at E14.5 and E17.5, (p < 0.05) but not for Tbx5 (p > 0.05). On embryonic day 17.5, HAT activities of embryonic hearts increased significantly, however alcohol exposure did not alter HDAC activities. CONCLUSIONS These data indicate a time course of H3K9 acetylation change during heart development and demonstrate that alcohol exposure in utero may induce an increase of HAT activities, which results in H3K9 hyperacetylation and an increase of the expression of heart development-related genes. These findings reveal a novel epigenetic mechanism that connects the alcohol consumption during the pregnancy and the development of CHD in the fetus.