Atrial Natriuretic Factor Gene Research Articles

The gp130/STAT3 signaling pathway mediates β‐adrenergic receptor‐induced atrial natriuretic factor expression in cardiomyocytes

beta-Adrenergic receptor (beta-AR)-induced cardiac remodeling is closely linked with the re-expression of the atrial natriuretic factor (ANF) gene. However, the exact molecular mechanism of this response remains elusive. Here, we demonstrate that the beta-AR agonist isoproterenol potently evokes the tyrosine phosphorylation of STAT3 and increases its transcriptional activity in an extracellularly regulated kinase 1/2 and glycoprotein (gp)130 signaling-dependent manner in rat cardiomyocytes. Interestingly, both specific silencing of signal transducers and activators of transcription 3 (STAT3) expression by lentivirus-mediated RNA interference and antagonism of gp130 signaling lead to significant inhibition of isoproterenol-stimulated ANF expression. Together, these results indicate that gp130/STAT3 signaling has an essential role in ANF expression by beta-AR stimulation.

Human heart LIM protein activates atrial-natriuretic-factor gene expression by interacting with the cardiac-restricted transcription factor Nkx2.5

hhLIM [human heart LIM (Lin-11/IsI-1/Mec-3) protein] is a muscle-specific LIM-only protein that consists of two LIM motifs. hhLIM functions as a positive regulator for cardiac hypertrophy. Here we report that hhLIM serves as a cofactor regulating the expression of the ANF (atrial natriuretic factor) gene in H9c2 rat cardiomyoblast cells. We found that hhLIM promoted the expression of the ANF gene in H9c2 cells, but not in A293 human embryonic kidney cells. Furthermore, we showed that hhLIM interacted with Nkx2.5 (a cardiac-restricted transcription factor) in vivo and in vitro using its N-terminal LIM domain and enhanced the binding ability of Nkx2.5 to the NKE (Nkx2.5-binding element) boxes in the ANF promoter. These results suggest that hhLIM promotes the specific expression of the ANF gene by co-operating with Nkx2.5.

Metabolic and signaling alterations in dystrophin-deficient hearts precede overt cardiomyopathy

The cytoskeletal protein dystrophin has been implicated in hereditary and acquired forms of cardiomyopathy. However, much remains to be learned about the role of dystrophin in the heart. We hypothesized that the dystrophin-deficient heart displays early alterations in energy metabolism that precede overt cardiomyopathy. We evaluated the metabolic and functional phenotype of dystrophin-deficient mdx mouse hearts at 10–12 weeks, when no major histological or echocardiographic abnormalities are reported. Ex vivo working mdx heart perfusions with stable isotopes revealed a marked shift in substrate fuel selection from fatty acids to carbohydrates associated with enhanced oxygen consumption. They also unmasked in the mdx heart: (i) compromised cardiac contractile function and efficiency, (ii) reduced cellular integrity, and (iii) exacerbated alterations in mitochondrial citric acid cycle-related parameters and in nutrient signaling pathways related to Akt. The observed shift in substrate selection cannot be explained by metabolic gene remodeling. However, mdx mice hearts showed an increased expression of the atrial natriuretic factor ( anf) gene, an activator of the nitric oxide (NO)/cGMP signaling pathway and marker of cardiac remodeling, and, only as the cardiomyopathy progresses (at 25 weeks of age), an increased expression of the α 1 subunit of soluble guanylate cyclase, which is known to negatively correlate with the activity NO/cGMP pathway. Collectively, our results highlight early metabolic and signaling alterations in the dystrophin-deficient heart, which may predispose these hearts to contractile dysfunction and sarcolemmal fragility. They also suggest the presence of a “sub-clinical” defect in the NO/cGMP pathway, which in vivo, at an early age, may be compensated by enhanced anf gene expression.

A Possible Anti-Inflammatory Role of Angiotensin II Type 2 Receptor in Immune-Mediated Glomerulonephritis during Type 1 Receptor Blockade

We previously reported that angiotensin II type 1 receptor (AT1R) blockade attenuates renal inflammation/fibrogenesis in immune-mediated glomerulonephritis via angiotensin II type 2 receptor (AT2R). In the present study, further in vivo experiments revealed that AT2R was expressed in tubular epithelial cells of nephritic kidneys in mice, and feedback activation of the renin-angiotensin system during AT1R blockade significantly reduced p-ERK, but not intranuclear nuclear factor-kappaB, levels via AT2R. This led to reduction in mRNA levels of the proinflammatory mediator monocyte chemoattractant protein-1 and overall interstitial inflammation and subsequent fibrogenesis. Specific blockade of ERK expression in tubular epithelium by anti-sense oligodeoxynucleotides also attenuated interstitial inflammation, mimicking the anti-inflammatory action of AT2R in nephritic kidneys. Alternatively, we succeeded in confirming such an AT(2)R function by demonstrating that AT1R blockade did not confer renoprotection in nephritic, AT2R gene-deficient mice. Additional in vitro experiments revealed that AT2R activation did not affect nuclear factor-kappaB activation by tumor necrosis factor-alpha in cultured tubular epithelial cells, although it inhibited ERK phosphorylation, which reduced monocyte chemoattractant protein-1 mRNA levels. These results suggest that feedback activation of AT2Rs in tubular epithelium of nephritic kidneys plays an important role in attenuating interstitial inflammation.

Role of inositol 1,4,5-trisphosphate receptors in alpha1-adrenergic receptor-induced cardiomyocyte hypertrophy1

Aim:Intracellular Ca2+ plays pivotal roles in diverse cellular functions, including gene transcription that underlies cardiac remodeling during stress responses. However, the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the mediation of cardiac intracellular Ca2+ and hypertrophic growth remains elusive. Prior work with neonatal rat ventricular myocytes suggests that activation of IP3Rs may be linked to α1 adrenergic receptor (α1 AR) increased stereotyped Ca2+ spark occurrence and global Ca2+ oscillations. Thus, we hypothesized that Ca2+ release through IP3Rs was necessary for α1 AR-stimulated cardiac hypertrophy.Methods:We used myoinositol 1,4,5-trisphosphate hexakis (butyryloxymethyl) ester (IP3BM), a membrane-permeant ester of IP3, to activate IP3Rs directly, and Fluo 4/AM to measure intracellular Ca2+ signaling.Results:IP3BM (10 μmol-L−1) mimicked the effects of phenylephrine, a selective agonist of α1AR, in increments in local Ca2+ spark release (especially in the perinuclear area) and global Ca2+ transient frequencies. More importantly, IP3R inhibitors, 2-aminoethoxydiphenyl borate and Xestospongin C, abolished the IP3BM-induced Ca2+ responses, and significantly suppressed α1AR-induced cardiomyocyte hypertrophy assayed by cell size, [3H] leucine incorporation and atrial natriuretic factor gene expression, during sustained (48 h) phenylephrine stimulation.Conclusion:These results, therefore, provide cellular mechanisms that link IP3R signaling to α1 AR-stimulated gene expression and cardiomyocyte hypertrophy.

The Transcriptional Coactivator CAMTA2 Stimulates Cardiac Growth by Opposing Class II Histone Deacetylases

Postnatal cardiac myocytes respond to diverse signals by hypertrophic growth and activation of a fetal gene program. In an effort to discover regulators of cardiac hypertrophy, we performed a eukaryotic expression screen for activators of the atrial natriuretic factor (ANF) gene, a cardiac-specific marker of hypertrophic signaling. We discovered that a family of transcriptional coactivators, called CAMTAs, promotes cardiomyocyte hypertrophy and activates the ANF gene, at least in part, by associating with the cardiac homeodomain protein Nkx2-5. The transcriptional activity of CAMTAs is governed by association with class II histone deacetylases (HDACs), which negatively regulate cardiac growth. Mice homozygous for a mutation in a CAMTA gene are defective in cardiac growth in response to pressure overload and neurohumoral signaling, whereas mice lacking HDAC5, a class II HDAC, are sensitized to the prohypertrophic actions of CAMTA. These findings reveal a transcriptional regulatory mechanism that modulates cardiac growth and gene expression by linking hypertrophic signals to the cardiac genome.

High Fat/Low Carbohydrate Feeding Prevents Cardiac Hypertrophy, LV Remodeling and ANF Gene Induction, Independent of Hypertension

Chronic hypertension (HTN) causes left ventricular hypertrophy (LVH) and dilation, which leads to heart failure. Diets high in fat and low in carbohydrate are recommended to patients at risk, but their impact on LV adaptations to HTN are unknown. Male Dahl salt sensitive rats were fed for 12 wk with either 1) low fat (10% kcal fat), 2) low fat/high salt (6% NaCl), or 3) high fat/high salt (60% kcal fat, 67% saturated, 33% unsaturated; 6% NaCl) (n=10/group). Tail cuff blood pressure (BP) measurements and echocardiographic assessment of LV dimensions were performed before & after 11wks on diets. There were no differences in these parameters between groups prior to feeding, and body mass was not different among groups. BP increased with salt feeding, with no differences between low fat & high fat groups (P<0.05, 126±2, 201±3 and 193±3 mmHg respectively). Compared to the low fat/low salt group, HTN lead to an increase in LV mass (3.38±0.19 vs 2.13±0.05 g/kg) and end diastolic area (EDA; 0.80±0.03 vs 0.66±0.02 cm2), which corresponded with an 11-fold induction of atrial natriuretic factor (ANF) mRNA expression (10.7±0.2 vs 1.0±0.1%) in the low fat/high salt group. In contrast, despite similar elevations in BP, the high fat/high salt group had significantly reduced LV mass (2.74±0.11 g/kg), LV EDA (0.66±0.03), and ANF expression (3.8±0.1%) compared to the low fat/high salt group. Conclusion High fat low carbohydrate feeding prevented HTN induced LVH and LV remodeling. We speculate that the mechanism for this dramatic effect may be reduced cardiac protein synthesis secondary to a reduction in plasma insulin associated with chronic low carbohydrate/high fat diets.

Cardiac-Specific Overexpression of Diacylglycerol Kinase ζ Prevents Gq Protein-Coupled Receptor Agonist-Induced Cardiac Hypertrophy in Transgenic Mice

Diacylglycerol is a lipid second messenger that accumulates in cardiomyocytes when stimulated by Gqalpha protein-coupled receptor (GPCR) agonists such as angiotensin II, phenylephrine, and others. Diacylglycerol functions as a potent activator of protein kinase C (PKC) and is catalyzed by diacylglycerol kinase (DGK) to form phosphatidic acid and inactivated. However, the functional roles of DGK have not been previously examined in the heart. We hypothesized that DGK might prevent GPCR agonist-induced activation of diacylglycerol downstream signaling cascades and subsequent cardiac hypertrophy. To test this hypothesis, we generated transgenic (DGKzeta-TG) mice with cardiac-specific overexpression of DGKzeta. There were no differences in heart size and heart weight between DGKzeta-TG and wild-type littermate mice. The left ventricular function was normal in DGKzeta-TG mice. Continuous administration of subpressor doses of angiotensin II and phenylephrine caused PKC translocation, gene induction of atrial natriuretic factor, and subsequent cardiac hypertrophy in WT mice. However, in DGKzeta-TG mice, neither translocation of PKC nor upregulation of atrial natriuretic factor gene expression was observed after angiotensin II and phenylephrine infusion. Furthermore, in DGKzeta-TG mice, angiotensin II and phenylephrine failed to increase cross-sectional cardiomyocyte areas and heart to body weight ratios. Phenylephrine-induced increases in myocardial diacylglycerol levels were completely blocked in DGKzeta-TG mouse hearts, suggesting that DGKzeta regulated PKC activity by controlling cellular diacylglycerol levels. These results demonstrated the first evidence that DGKzeta negatively regulated the hypertrophic signaling cascade and resultant cardiac hypertrophy in response to GPCR agonists without detectable adverse effects in in vivo hearts.

Convergence of Protein Kinase C and JAK-STAT Signaling on Transcription Factor GATA-4

Angiotensin II (AII), a potent vasoactive hormone, acts on numerous organs via G-protein-coupled receptors and elicits cell-specific responses. At the level of the heart, AII stimulation alters gene transcription and leads to cardiomyocyte hypertrophy. Numerous intracellular signaling pathways are activated in this process; however, which of these directly link receptor activation to transcriptional regulation remains undefined. We used the atrial natriuretic factor (ANF) gene (NPPA) as a marker to elucidate the signaling cascades involved in AII transcriptional responses. We show that ANF transcription is activated directly by the AII type 1 receptor and precedes the development of myocyte hypertrophy. This response maps to STAT and GATA binding sites, and the two elements transcriptionally cooperate to mediate signaling through the JAK-STAT and protein kinase C (PKC)-GATA-4 pathways. PKC phosphorylation enhances GATA-4 DNA binding activity, and STAT-1 functionally and physically interacts with GATA-4 to synergistically activate AII and other growth factor-inducible promoters. Moreover, GATA factors are able to recruit STAT proteins to target promoters via GATA binding sites, which are sufficient to support synergy. Thus, STAT proteins can act as growth factor-inducible coactivators of tissue-specific transcription factors. Interactions between STAT and GATA proteins may provide a general paradigm for understanding cell specificity of cytokine and growth factor signaling.

Overexpression of calmodulin induces cardiac hypertrophy by a calcineurin-dependent pathway

The possible role of calcineurin in cardiac hypertrophy induced by calmodulin (CaM) overexpression in the heart was investigated. CaM transgenic (CaM-TG) mice developed marked cardiac hypertrophy and exhibited up-regulation of atrial natriuretic factor (ANF) and β-myosin heavy chain gene expression in the heart during the first 2 weeks after birth. The activity of calcineurin in the heart was also significantly increased in CaM-TG mice compared with wild-type littermates. Treatment of CaM-TG mice with the calcineurin inhibitor FK506 (1 mg/kg per day) prevented the increase in the heart-to-body weight ratio as well as that in cardiomyocyte width. FK506 also inhibited the induction of fetal-type cardiac gene expression in CaM-TG mice. Overexpression of CaM in cultured rat cardiomyocytes activated the ANF gene promoter in a manner sensitive to FK506. Activation of a calcineurin-dependent pathway thus contributes to the development of cardiac hypertrophy induced by CaM overexpression in the heart.

Dissecting Hypertension by Obesity Identifies a Locus at 1p36

The elevated arterial pressure that defines essential hypertension is regarded as the manifestation of a diverse array of interacting genetic and environmental causes. As such, hypertension is a heterogeneous disorder in which multiple contributing factors are responsible for the overarching phenotype of high blood pressure that is the primary clinical manifestation observed. Such heterogeneity has undoubtedly hampered efforts to elucidate the genetic basis of essential hypertension. Overweight and obesity are well known to increase the risk of essential hypertension. Yet, there are many hypertensive individuals whose weight is normal. So does obesity hypertension have a different underlying genetic cause than lean hypertension? In the current issue, Pausova et al report finding a locus with suggestive linkage to hypertension and then reanalyzed their data after dividing their families into lean and obese.1 Of considerable interest, the significance of the linkage peak increased for the obese families but disappeared in the case of the families with hypertension who were not obese. The striking contrast in results for each category of hypertension lends strong support to the idea that hypertension of obesity has a different genetic basis than other categories of hypertension. Not only is this finding important in discovery of the genetic basis of obesity hypertension, but it has obvious implications for approaches that might be used to identify the basis for other “intermediate phenotypes” of hypertension. The study by Pausova et al involved 55 extended families from the geographically remote French-Canadian Saguenay/Lac-St-Jean region of Quebec. This relatively small, isolated population has been spared the level of genetic “noise” present in populations elsewhere. As a result, the degree of genetic homogeneity should be elevated and likely contributed to the …

The Zinc Finger-Only Protein Zfp260 Is a Novel Cardiac Regulator and a Nuclear Effector of α1-Adrenergic Signaling

alpha1-Adrenergic receptors mediate several biological effects of catecholamines, including the regulation of myocyte growth and contractility and transcriptional regulation of the atrial natriuretic factor (ANF) gene whose promoter contains an alpha1-adrenergic response element. The nuclear pathways and effectors that link receptor activation to genetic changes remain poorly understood. Here, we describe the isolation by the yeast one-hybrid system of a cardiac cDNA encoding a novel nuclear zinc finger protein, Zfp260, belonging to the Krüppel family of transcriptional regulators. Zfp260 is highly expressed in the embryonic heart but is downregulated during postnatal development. Functional studies indicate that Zfp260 is a transcriptional activator of ANF and a cofactor for GATA-4, a key cardiac regulator. Knockdown of Zfp260 in cardiac cells decreases endogenous ANF gene expression and abrogates its response to alpha1-adrenergic stimulation. Interestingly, Zfp260 transcripts are induced by alpha1-adrenergic agonists and are elevated in genetic models of hypertension and cardiac hypertrophy. The data identify Zfp260 as a novel transcriptional regulator in normal and pathological heart development and a nuclear effector of alpha1-adrenergic signaling.

Structural Determinants of the Agonist-independent Association of Human Peroxisome Proliferator-activated Receptors with Coactivators

Lipid homeostasis is controlled by various nuclear receptors (NRs), including the peroxisome proliferator-activated receptors (PPARalpha, delta, and gamma), which sense lipid levels and regulate their metabolism. Here we demonstrate that human PPARs have a high basal activity and show ligand-independent coactivator (CoA) association comparable with the NR constitutive androstane receptor. Using PPARgamma as an example, we found that four different amino acid groups contribute to the ligand-independent stabilization of helix 12 of the PPAR ligand-binding domain. These are: (i) Lys329 and Glu499, mediating a charge clamp-type stabilization of helix 12 via a CoA bridge; (ii) Glu352, Arg425, and Tyr505, directly stabilizing the helix via salt bridges and hydrogen bonds; (iii) Lys347 and Asp503, interacting with each other as well as contacting the CoA; and (iv) His351, Tyr(355), His477, and Tyr501, forming a hydrogen bond network. These amino acids are highly conserved within the PPAR subfamily, suggesting that the same mechanism may apply for all three PPARs. Phylogenetic trees of helix 12 amino acid and nucleotide sequences of all crystallized NRs and all human NRs, respectively, indicated a close relationship of PPARs with constitutive androstane receptor and other constitutive active members of the NR superfamily. Taking together, the ligand-independent tight control of the position of the PPAR helix 12 provides an effective alternative for establishing an interaction with CoA proteins. This leads to high basal activity of PPARs and provides an additional view on PPAR signaling.

Adenovirus-Mediated Overexpression of Diacylglycerol Kinase-ζ Inhibits Endothelin-1–Induced Cardiomyocyte Hypertrophy

Diacylglycerol (DAG) is a lipid second messenger that transiently accumulates in cells stimulated by endothelin-1 (ET-1) and other Galphaq protein-coupled receptor agonists. Diacylglycerol kinase (DGK) is thought to be an enzyme that controls the cellular levels of DAG by converting it to phosphatidic acid; however, the functional role of DGK has not been examined in cardiomyocytes. Because DGK inactivates DAG, a strong activator of protein kinase C (PKC), we hypothesized that DGK inhibited ET-1-induced activation of a DAG-PKC signaling cascade and subsequent cardiomyocyte hypertrophy. Real-time reverse transcription-polymerase chain reaction demonstrated a significant increase of DGK-zeta mRNA by ET-1 in cardiomyocytes. To determine the functional role of DGK-zeta, we overexpressed DGK-zeta in cardiomyocytes using a recombinant adenovirus encoding rat DGK-zeta (Ad-DGKzeta). ET-1-induced translocation of PKC-epsilon was blocked by Ad-DGKzeta (P<0.01). Ad-DGKzeta also inhibited ET-1-induced activation of extracellular signal-regulated kinase (P<0.01). Luciferase reporter assay revealed that ET-1-mediated increase of activator protein-1 (AP1) DNA-binding activity was significantly inhibited by DGK-zeta (P<0.01). In cardiomyocytes transfected with DGK-zeta, ET-1 failed to cause gene induction of atrial natriuretic factor, increases in [3H]-leucine uptake, and increases in cardiomyocyte surface area. We demonstrated for the first time that DGK-zeta blocked ET-1-induced activation of the PKC-epsilon-ERK-AP1 signaling pathway, atrial natriuretic factor gene induction, and resultant cardiomyocyte hypertrophy. DGK-zeta might act as a negative regulator of hypertrophic program in response to ET-1, possibly by controlling cellular DAG levels.

Interleukin-18 Is a Pro-hypertrophic Cytokine That Acts through a Phosphatidylinositol 3-Kinase-Phosphoinositide-dependent Kinase-1-Akt-GATA4 Signaling Pathway in Cardiomyocytes

In patients with congestive heart failure, high serum levels of the proinflammatory cytokine interleukin (IL)-18 were reported. A positive correlation was described between serum IL-18 levels and the disease severity. IL-18 has also been shown to induce atrial natriuretic factor (ANF) gene expression in adult cardiomyocytes. Because re-expression of the fetal gene ANF is mostly associated with hypertrophy, a hallmark of heart failure, we hypothesized that IL-18 induces cardiomyocyte hypertrophy. Treatment of the cardiomyocyte cell line HL-1 with IL-18 induced hypertrophy as characterized by increases in protein synthesis, phosphorylated p70 S6 kinase, and ribosomal S6 protein levels as well as cell surface area. Furthermore, IL-18 induced ANF gene transcription in a time-dependent manner as evidenced by increased ANF secretion and ANF promoter-driven reporter gene activity. Investigation into possible signal transduction pathways mediating IL-18 effects revealed that IL-18 activates phosphoinositide 3-kinase (PI3K), an effect that was blocked by wortmannin and LY-294002. IL-18 induced Akt phosphorylation and stimulated its activity, effects that were abolished by Akt inhibitor or knockdown. IL-18 stimulated GATA4 DNA binding activity and increased transcription of a reporter gene driven by multimerized GATA4-binding DNA elements. Pharmacological inhibition or knockdown studies revealed that IL-18 induced cardiomyocyte hypertrophy and ANF gene transcription via PI3K, PDK1, Akt, and GATA4. Most importantly, IL-18 induced ANF gene transcription and hypertrophy of neonatal rat ventricular myocytes via PI3K-, Akt-, and GATA4-dependent signaling. Together these data provide the first evidence that IL-18 induces cardiomyocyte hypertrophy via PI3K-dependent signaling, defines a mechanism of IL-18-mediated ANF gene transcription, and further supports a role for IL-18 in inflammatory heart diseases including heart failure.

Determinants of inducible brain natriuretic peptide promoter activity

Atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are polypeptide hormones belonging to the cardiac-derived mammalian natriuretic peptide system. These hormones share the same biological properties and receptors and both play important roles in the maintenance of fluid and electrolyte balance and in cardiovascular growth. Most hemodynamic and neurohumoral stimuli can coordinately increase ANF and BNP gene expression. However, instances of discoordinated ANF and BNP gene expression have been described, providing an opportunity for investigating the mechanisms that differentially regulate the expression of the natriuretic peptide genes. For example, exposure of cardiocytes in culture to certain pro-inflammatory cytokines and conditioned medium from mixed lymphocyte cultures upregulate BNP but not ANF gene expression. BNP promoter activity is also upregulated under these conditions but the cis-acting elements involved in this phenomenon are not known. In comparison to the ANF gene, less is known about BNP promoter consensus elements that regulate gene expression by mechanical or neurohumoral agonists. A number of cis-acting elements for GATA, Nkx2.5, NF-κB and TEF transcription factors have recently been identified within the BNP promoter that regulate BNP expression in response to specific agonists. This review focuses on the information available regarding cis-acting determinants responsible for inducible BNP transcription.

Hairy-related Transcription Factors Inhibit GATA-dependent Cardiac Gene Expression through a Signal-responsive Mechanism

Combinatorial actions of transcription factors in multiprotein complexes dictate gene expression profiles in cardiac development and disease. The Hairy-related transcription factor (HRT) family of basic helix-loop-helix proteins is composed of transcriptional repressors highly expressed in the cardiovascular system. However, it has remained unclear whether HRT proteins modulate gene expression driven by cardiac transcriptional activators. Here, we have shown that HRT proteins inhibit cardiac gene transcription by interfering with GATA transcription factors that are implicated in cardiac development and hypertrophy. HRT proteins inhibited GATA-dependent transcriptional activation of cardiac gene promoters such as the atrial natriuretic factor (ANF) promoter. Adenovirus-mediated expression of Hrt2 suppressed mRNA expression of ANF and other cardiac-specific genes in cultured cardiomyocytes. Among various signaling molecules implicated in cardiomyocyte growth, constitutively active Akt1/protein kinase B alpha relieved Hrt2-mediated inhibition of GATA-dependent transcription. HRT proteins physically interacted with GATA proteins, and the basic domain of HRT was critical for physical association as well as transcriptional inhibition. These results suggest that HRT proteins may regulate specific sets of cardiac genes by modulating the function of GATA proteins and other cardiac transcriptional activators in a signal-dependent manner.

Jumonji Represses Atrial Natriuretic Factor Gene Expression by Inhibiting Transcriptional Activities of Cardiac Transcription Factors

Mice with a homozygous knockout of the jumonji (jmj) gene showed abnormal heart development and defective regulation of cardiac-specific genes, including the atrial natriuretic factor (ANF). ANF is one of the earliest markers of cardiac differentiation and a hallmark for cardiac hypertrophy. Here, we show that JMJ represses ANF gene expression by inhibiting transcriptional activities of Nkx2.5 and GATA4. JMJ represses the Nkx2.5- or GATA4-dependent activation of the reporter genes containing the ANF promoter-enhancer or containing the Nkx2.5 or GATA4-binding consensus sequence. JMJ physically associates with Nkx2.5 and GATA4 in vitro and in vivo as determined by glutathione S-transferase pull-down and immunoprecipitation assays. Using mutational analyses, we mapped the protein-protein interaction domains in JMJ, Nkx2.5, and GATA4. We identified two DNA-binding sites of JMJ in the ANF enhancer by gel mobility shift assays. However, these JMJ-binding sites do not seem to mediate ANF repression by JMJ. Mutational analysis of JMJ indicates that the protein-protein interaction domain of JMJ mediates the repression of ANF gene expression. Therefore, JMJ may play important roles in the down-regulation of ANF gene expression and in heart development.

Cardiac-specific overexpression of diacylglycerol kinaseζ prevents angiotensin II-induced cardiac hypertrophy in vivo

Background: Gq protein-coupled receptor (GPCR) agonists such as angiotensin II (AII), endothelin-1, and phenylephrine increase cellular levels of diacylglycerol (DAG). DAG, a potent activator of protein kinase C (PKC), is catalyzed and inactivated by diacylglycerol kinase (DGK). Therefore, DGK may act as a negative regulator for GPCR signaling pathway. Previous studies showed that DGKζ was the predominant isozyme in the heart. Methods and Results: To characterize the functional role of DGKζ in vivo, we generated transgenic (TG) mice with cardiac-specific overexpression of DGKζ using an α-myosin heavy chain promoter. Protein level of DGKζ was 21-fold and kinase activity was 19-fold in TG hearts compared to wild-type (WT) mice. There were no differences in heart size, heart weight, and left ventricular function determined by echocardiography between TG and WT mice. Continuous administration of AII caused the increase of blood pressure, PKCα translocation, atrial natriuretic factor (ANF) gene induction, and cardiac hypertrophy in WT mice. However in TG mice, AII did not induce translocation of PKCα and up-regulation of ANF. Furthermore, increases in cross-sectional cardiomyocyte area and heart/body weight ratio by AII were abolished in TG mice. Conclusion: These results demonstrated the first evidence that DGKζ blocked hypertrophic signaling pathway and resultant cardiac hypertrophy by AII in vivo. DGKζ may be one of therapeutic targets to prevent cardiac hypertrophy.

Protein kinase C-alpha-induced hypertrophy of neonatal rat ventricular myocytes.

Protein kinase C (PKC) isoenzymes play a critical role in cardiomyocyte hypertrophy. At least three different phorbol ester-sensitive PKC isoenzymes are expressed in neonatal rat ventricular myocytes (NRVMs): PKC-alpha, -delta, and -epsilon. Using replication-defective adenoviruses (AdVs) that express wild-type (WT) and dominant-negative (DN) PKC-alpha together with phorbol myristate acetate (PMA), which is a hypertrophic agonist and activator of all three PKC isoenzymes, we studied the role of PKC-alpha in signaling-specific aspects of the hypertrophic phenotype. PMA induced nuclear translocation of endogenous and AdV-WT PKC-alpha in NRVMs. WT PKC-alpha overexpression increased protein synthesis and the protein-to-DNA (P/D) ratio but did not affect cell surface area (CSA) or cell shape compared with uninfected or control AdV beta-galactosidase (AdV betagal)-infected cells. PMA-treated uninfected cells displayed increased protein synthesis, P/D ratio, and CSA and elongated morphology. PMA did not further enhance protein synthesis or P/D ratio in AdV-WT PKC-alpha-infected cells. To assess the requirement of PKC-alpha for these PMA-induced changes, AdV-DN PKC-alpha or AdV betagal-infected NRVMs were stimulated with PMA. Without PMA, AdV-DN PKC-alpha had no effects on protein synthesis, P/D ratio, CSA, or shape vs. AdV betagal-infected NRVMs. PMA increased protein synthesis, P/D ratio, and CSA in AdV betagal-infected cells, but these parameters were significantly reduced in PMA-stimulated AdV-DN PKC-alpha-infected NRVMs. Overexpression of DN PKC-alpha enhanced PMA-induced cell elongation. Neither WT PKC-alpha nor DN PKC-alpha affected atrial natriuretic factor gene expression. Insulin-like growth factor-1 also induced nuclear translocation of endogenous PKC-alpha. PMA but not WT PKC-alpha overexpression induced ERK1/2 activation. However, AdV-DN PKC-alpha partially blocked PMA-induced ERK activation. Thus PKC-alpha is necessary for certain aspects of PMA-induced NRVM hypertrophy.