Atrial Natriuretic Factor Transcription Research Articles

Protective effect of histone methyltransferase NSD3 on ISO-induced cardiac hypertrophy.

Nuclear receptor-binding SET domain 3 (NSD3) is a lysine methyltransferase that plays important roles in multiple biological activities; however; its potential roles in the cardiovascular system remain unknown. In this study, we found that NSD3 expression is reduced by isoproterenol (ISO) stimuli both invitro and invivo. Overexpression of NSD3 attenuates ISO-induced cardiomyocyte hypertrophy. Mechanistically, ISO treatment decreases H3K27me2/3 modifications on the atrial natriuretic factor (ANF) promoter by suppressing NSD3 and inhibits the association between NSD3 and bromodomain-containing protein 4 (BRD4), thus suppressing the BRD4-mediated H3K27ac modifications, which ultimately promote ANF transcription and cardiomyocyte hypertrophy. In conclusion, NSD3 decreases ANF expression and, thereby, attenuates ISO-induced cardiomyocyte hypertrophy.

Cardiac Myocyte-specific Ablation of Follistatin-like 3 Attenuates Stress-induced Myocardial Hypertrophy

Transforming growth factor-β family cytokines have diverse actions in the maintenance of cardiac homeostasis. Follistatin-like 3 (Fstl3) is an extracellular regulator of certain TGF-β family members, including activin A. The aim of this study was to examine the role of Fstl3 in cardiac hypertrophy. Cardiac myocyte-specific Fstl3 knock-out (KO) mice and control mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Cardiac hypertrophy was assessed by echocardiography and histological and biochemical methods. KO mice showed reduced cardiac hypertrophy, pulmonary congestion, concentric LV wall thickness, LV dilatation, and LV systolic dysfunction after TAC compared with control mice. KO mice displayed attenuated increases in cardiomyocyte cell surface area and interstitial fibrosis following pressure overload. Although activin A was similarly up-regulated in KO and control mice after TAC, a significant increase in Smad2 phosphorylation only occurred in KO mice. Knockdown of Fstl3 in cultured cardiomyocytes inhibited PE-induced cardiac hypertrophy. Conversely, adenovirus-mediated Fstl3 overexpression blocked the inhibitory action of activin A on hypertrophy and Smad2 activation. Transduction with Smad7, a negative regulator of Smad2 signaling, blocked the antihypertrophic actions of activin A stimulation or Fstl3 ablation. These findings identify Fstl3 as a stress-induced regulator of hypertrophy that controls myocyte size via regulation of Smad signaling.

ZFP260 Is an Inducer of Cardiac Hypertrophy and a Nuclear Mediator of Endothelin-1 Signaling

Pressure and volume overload induce hypertrophic growth of postnatal cardiomyocytes and genetic reprogramming characterized by reactivation of a subset of fetal genes. Despite intense efforts, the nuclear effectors of cardiomyocyte hypertrophy remain incompletely defined. Endothelin-1 (ET-1) plays an important role in cardiomyocyte growth and is involved in mediating the neurohormonal effects of mechanical stress. Here, we show that the phenylephrine-induced complex-1 (PEX1), also known as zinc finger transcription factor ZFP260, is essential for cardiomyocyte response to ET-1 as evidenced in cardiomyocytes with PEX1 knockdown. We found that ET-1 enhances PEX1 transcriptional activity via a PKC-dependent pathway which phosphorylates the protein and further potentiates its synergy with GATA4. Consistent with a role for PEX1 in cardiomyocyte hypertrophy, overexpression of PEX1 is sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. Importantly, transgenic mice with inducible PEX1 expression in the adult heart develop cardiac hypertrophy with preserved heart function. Together, the results identify a novel nuclear effector of ET-1 signaling and suggest that PEX1 may be a regulator of the early stages of cardiac hypertrophy.

The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling

Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-induced MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal α-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.

Abstract 5270: Crystal Structure of Nkx2.5 Homeodomain in Complex with Anf-242 Motif

NKX2.5 is a homeodomain (HD) containing transcription factor preferentially expressed in cardiomyocytes. Mutations in the NKX2.5 gene have been identified in patients with congenital heart disease, suggesting its critical roles in cardiac development and maturation. NKX2.5 is reported to regulate transcription of atrial natriuretic factor (ANF), potentially through direct binding to -242 bp proximal promoter region (ANF-242). ANF-242 contains two palidromic NKX2.5 binding sites, and occupation of both sites by NKX2.5 has been reported. In addition, physical interaction between NKX2.5 proteins has been suggested to facilitate DNA binding through domains outside of the HD. However, detailed crystal structure of NKX2.5 protein is currently not available. The HD of human NKX2.5 (aa 138–197; C193S) with the oxidizable Cys193 substituted with Ser was co-crystallized with 19bp double stranded DNA including ANF-242 region. This mutant was reported to have the same DNA binding characteristics as the wild type. The crystal structure was determined at 1.8 Å resolution with final R-working and R-free values of 0.234 and 0.278, respectively. Consistent with previous studies, two molecules of NKX2.5 HD are bound to ANF-242 region without physical interaction between the two proteins. Column purification demonstrated that NKX2.5 behaves as dimers in solution in a concentration dependent manner. These results suggest that dimeric NKX2.5 HD in solution may change conformation upon DNA binding. Among various NK class proteins, the three-dimensional structure has only been determined in the HD of Drosophila vnd/NK2 (ventral nervous system defective proteins) using NMR. When the two HDs are compared, NKX2.5 HD shows extended DNA contacts to a total of 8 base pairs. Most of the residues of HD implicated in pathology of mutants are involved in DNA binding and structural stabilization. The structure of NKX2.5 HD with ANF-242 shows that the HD occupies two binding sites of ANF-242 without physical interaction between themselves. This high resolution crystal structure of NKX2.5 protein provides a detailed picture of protein and DNA interactions, which allows us to predict DNA binding of mutants identified in human patients. This research has received full or partial funding support from the American Heart Association, AHA Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).

Induction by left ventricular overload and left ventricular failure of the human Jumonji gene (JARID2) encoding a protein that regulates transcription and reexpression of a protective fetal program

We identified changes in Jumonji (JARID2) expression in failing human hearts and determined its effects on expressions of atrial natriuretic factor (ANF), myosin light chain 2a (MLC2A), and alpha myosin heavy chain (MHCA), genes associated with both human heart failure and the fetal gene program. Left ventricular outflow tract cardiac biopsy samples were taken from 31 patients with aortic valvular stenosis. Hearts were grouped according to left ventricular size and function: nonfailing hearts (undilated with good function) and failing hearts (dilated with poor function). Protein levels were determined by Western blotting, and messenger RNA transcript levels by ratiometric reverse transcriptase-polymerase chain reaction. Luciferase assays in HL-2 cells were used to assess effects of Jarid2 on Anf, Mlc2a, and Mhca transcriptions. Chromatin immunoprecipitation was used to detect interaction of JARID2 with specific target-gene promoters. JARID2 and MHCA expressions were reduced in failing hearts, whereas MLC2A and ANF were increased. In HL-2 cell culture, Jarid2 suppressed Anf and Mlc2a but enhanced Mhca. Jarid2 expression was reduced by cyclic mechanical stress, with concomitant increased Anf and Mlc2a and decreased Mhca expressions, reproducing the expression profile found in decompensated human pressure overload. Jumonji expression is reduced by mechanical stress in human heart failure from aortic stenosis. JARID2 regulates ANF, MLC2A, and MHCA transcription and contributes to reexpression of the fetal gene program in decompensated aortic stenosis. JARID2 appears important in transcriptional regulation of fetal genes and may emerge as a diagnostic marker for left ventricular decompensation in aortic stenosis.

Endocytosis machinery is required for β1-adrenergic receptor-induced hypertrophy in neonatal rat cardiac myocytes

Cardiac hypertrophy by activation of the beta-adrenergic receptor (beta AR) is mediated more efficiently by the beta1-AR than by the beta2-AR. We investigated the signalling mechanism by which the beta1-AR mediates cardiac hypertrophy. Experiments were performed in cultured neonatal rat cardiomyocytes. Hypertrophy was determined by the protein/DNA content and atrial natriuretic factor transcription. Phosphorylation of Akt and Src was assessed by immunoblotting. Isoproterenol (ISO, 10 microM), a non-selective beta-AR agonist, caused selective downregulation of the beta1-AR (control beta1 vs. beta2: 35 vs. 65%, Bmax 78 +/- 4 fmol/mg; 4 h, 10 vs. 90%, 61 +/- 5 fmol/mg). Concanavalin A (Con A, 0.5 microg/mL), an inhibitor of endocytosis, prevented downregulation of beta1-ARs by ISO treatment (4 h, 35 vs. 65%, 73 +/- 8 fmol/mg), suggesting that beta1-ARs selectively undergo endocytosis. Interference with beta1-AR endocytosis by Con A, carboxyl terminal peptide of beta-AR kinase-1, dominant negative (DN) beta-arrestin-1, or DN dynamin inhibited beta-adrenergic hypertrophy, suggesting that the endocytosis machinery plays a key role in mediating beta-adrenergic hypertrophy. Activation of Akt by the beta1-AR was blocked by inhibition of the endocytosis machinery, suggesting that endocytosis mediates activation of Akt. Akt plays a critical role in beta-adrenergic hypertrophy, since DN Akt blocked ISO-induced hypertrophy. beta-Adrenergic activation of Akt is mediated by Src, which associates with the endocytosis machinery and is necessary and sufficient to mediate beta-adrenergic hypertrophy. Activation of the endocytosis machinery is required for activation of Akt, which, in turn, critically mediates beta1-AR-induced cardiac hypertrophy.

Phenylephrine hypertrophy, Ca2+-ATPase (SERCA2), and Ca2+signaling in neonatal rat cardiac myocytes

We endeavored to use a basic and well-controlled experimental system to characterize the extent and time sequence of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) involvement in the development of cardiac hypertrophy, including transcription, protein expression, Ca(2+) transport, and cytoplasmic Ca(2+) signaling. To this end, hypertrophy of neonatal rat cardiac myocytes in culture was obtained after adrenergic activation with phenylephrine (PE). Micrographic assessment of myocyte size, rise of [(14)C]phenylalanine incorporation and total protein expression, and increased transcription of atrial natriuretic factor demonstrated unambiguously the occurrence of hypertrophy. An early and prominent feature of hypertrophy was a reduction of the SERCA2 transcript, as determined by RT-PCR with reference to a stable marker such as glyceraldehyde-3-phosphate dehydrogenase. Reduction of Ca(2+)-ATPase protein levels and Ca(2+) transport activity to approximately 50% of control values followed with some delay, evidently as a consequence of a primary effect on transcription. Cytosolic Ca(2+) signaling kinetics, measured with a Ca(2+)-sensitive dye after electrical stimuli, were significantly altered in hypertrophic myocytes. However, the effect of PE hypertrophy on cytosolic Ca(2+) signaling kinetics was less prominent than observed in myocytes subjected to drastic SERCA2 downregulation with small interfering RNA or inhibition with thapsigargin (10 nM). We conclude that SERCA2 undergoes significant downregulation after hypertrophic stimuli, possibly due to lack of SERCA gene involvement by the hypertrophy transcriptional program. The consequence of SERCA2 downregulation on Ca(2+) signaling is partially compensated by alternate Ca(2+) transport mechanisms. These alterations may contribute to a gradual onset of functional failure in long-term hypertrophy.

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.

Glycogen Synthase Kinase 3β Inhibits Myocardin-Dependent Transcription and Hypertrophy Induction Through Site-Specific Phosphorylation

Cardiomyocyte hypertrophy is transcriptionally controlled and inhibited by glycogen synthase kinase 3beta (GSK3beta). Myocardin is a muscle-specific transcription factor with yet unknown relation to hypertrophy. Therefore, we investigated whether myocardin is sufficient to induce cardiomyocyte hypertrophy and whether myocardin is regulated by GSK3beta through site-specific phosphorylation. Adenoviral myocardin overexpression induced cardiomyocyte hypertrophy in neonatal rat cardiomyocytes, with increased cell size, total protein amount, and transcription of atrial natriuretic factor (ANF). In vitro and in vivo (HEK 293 cells) kinase assays with synthetic peptides and full-length myocardin demonstrated that myocardin was a "primed" GSK3beta substrate, with serines 455 to 467 and 624 to 636 being the major GSK3beta phosphorylation sites. Myocardin-induced ANF transcription and increase in total protein amount were enhanced by GSK3beta blockade (10 mmol/L LiCl), indicating that GSK3beta inhibits myocardin. A GSK3beta phosphorylation-resistant myocardin mutant (8xA) activated ANF transcription twice as potently as wildtype myocardin under basal conditions with GSK3beta being active. Conversely, a GSK3beta phospho-mimetic myocardin mutant (8xD) was transcriptionally repressed after GSK3beta blockade, indicating that GSK3beta phosphorylation at the sites identified inhibits myocardin transcriptional activity. GAL4-myocardin fusion constructs demonstrated that GSK3beta phosphorylation reduced the intrinsic myocardin transcriptional activity. A cell-permeable (Antennapedia protein transduction tag) peptide containing the mapped myocardin GSK3beta motifs 624 to 636 induced hypertrophy of cultured cardiomyocytes, suggesting that the peptide acted as substrate-based GSK3beta inhibitor in cardiomyocytes. Therefore, we conclude that the GSK3beta-myocardin interaction constitutes a novel molecular control of cardiomyocyte hypertrophy. Phosphorylation by GSK3beta comprises a novel post-transcriptional regulatory mechanism of myocardin.

Immunolocalization of type 2 inositol 1,4,5-trisphosphate receptors in cardiac myocytes from newborn mice.

The precise localization and role of inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) in cardiac muscle cells are largely unknown. It is believed that waves and oscillations in cytosolic free calcium triggered by activation of InsP(3)Rs underlie modifications of cellular responses that lead to changes in gene expression in other cells. However, how changes in cytosolic calcium alter gene expression in cardiac cells is unknown. Moreover, it is unclear how changes in cytosolic calcium that alter gene expression do so independently of effects of calcium on other cellular functions, such as contraction. Here we show that InsP(3)R type 2 is the only isoform present in cardiac myocytes isolated from neonatal mouse ventricles. We also show that type 2 InsP(3)Rs are associated with the nucleus and that activation of type 2 InsP(3)Rs with endothelin-1 or phenylephrine selectively increases transcription of atrial natriuretic factor and skeletal alpha-actin. Type 2 InsP(3)Rs are also in striations. Activation of InsP(3)Rs with adenophostin A in permeabilized cells induced calcium release in the nuclear domain and other regions of the cell away from the nucleus. Agonist-induced increase in gene expression and calcium release were blocked by the InsP(3)R inhibitors 2-aminoethoxydiphenyl borate and xestospongin C. The spatial separation of type 2 InsP(3)Rs provides support for the concept that microdomains of calcium discretely alter various cell processes. Our experiments suggest that calcium released by InsP(3)Rs in the nuclear domain provides a direct mechanism for the control of gene expression, whereas release of calcium in the cytoplasm may modulate other processes, such as contraction.

Regional Heterogeneity in Gene Expression Profiles: A Transcript Analysis in Human and Rat Heart

Background: Investigations involving biopsies of human cardiac tissue often assume that myocardial samples from a specific location are representative of the entire heart. Hypothesis: There are significant regional differences in gene expression in the heart. Methods: We used two models. In the first model, seven whole human hearts were cut in 1-cm slices from apex to base and 11 distinct regions were sampled. Full thickness left ventricular tissue was further subdivided equally into an inner, outer, and middle region. In the second model, hearts were removed from adult Sprague-Dawley rats and were divided into 4 regions. Using species-specific quantitative reverse transcriptase-polymerase chain reaction, we measured transcript levels of myosin heavy chain β (MHC-β), glucose transporter 1 (GLUT 1), and atrial natriuretic factor (ANF) in tissue samples from both models. Results: In human heart, there were significant differences in transcript levels between regions. The following patterns could be recognized among the seven hearts. ANF expression was highest in the subendocardial region. MHC-β and GLUT 1 transcript levels were higher in the right ventricle than the left ventricle. As expected, ANF transcript levels were highest in the atria, where MHC-β and GLUT 1 expression was low. Analogous to the human studies, MHC-β and GLUT 1 transcript levels were low in rat atria as compared to ventricles. In rat heart, MHC-β expression was higher in the left ventricle than the right ventricle while GLUT 1 expression was not significantly different between ventricles. Conclusion: Despite the large variability in transcript levels among different regions in human hearts, certain patterns in gene expression emerged suggesting that different anatomical regions of the heart also differ in respect to gene expression.

Upregulation of GLUT1 expression is necessary for hypertrophy and survival of neonatal rat cardiomyocytes

During hypertrophy the heart increases its utilization of glucose and decreases that of fatty acids, resuming a fetal pattern of substrate metabolism. As demonstrated here, GLUT1 protein expression is increased in association with in vivo pressure-overload-induced hypertrophy. The relationship of changes in GLUT1 to enhanced glucose uptake and to cardiomyocyte hypertrophy and survival is not known. To explore this question we first examined the effect of prostaglandin F2α (PGF2α), an established hypertrophic agonist, on GLUT1 expression and glucose uptake in neonatal rat ventricular myocytes (NRVMs). PGF2α treatment for 24 h led to a fivefold increase in GLUT1 expression and a sixfold increase in glucose uptake. However, NRVMs cultured in the absence of glucose or with 3– O–methyl glucose, a competitive inhibitor of glucose uptake, still exhibited PGF2α-induced hypertrophic growth. In addition, we determined that overexpression of GLUT1 using adenovirus was insufficient to cause an increase in cell size, myofibrillar organization, or atrial natriuretic factor (ANF) expression. On the other hand, adenoviral overexpression of antisense GLUT1 (which blocked PGF2α-induced increases in GLUT1 protein) prevented PGF2α-stimulated cell enlargement and increases in ANF transcription. Overexpression of GLUT1 or addition of PGF2α also protected cells against serum deprivation-induced apoptosis; this effect was blocked by antisense GLUT1 but, surprisingly, was not dependent on glucose. Together, these data suggest that upregulation of GLUT1 serves a role in agonist-induced hypertrophy and survival which can be dissociated from its role in glucose transport.

Hypertrophic phenotype of cardiac calcium/calmodulin-dependent protein kinase II is reversed by angiotensin converting enzyme inhibition.

Calcium-dependent mechanisms and the renin angiotensin system (RAS) are critically involved in the hypertrophic growth of the myocardium. The calcium/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous mediator in calcium signaling and modulates calcium handling and growth mechanisms in cardiomyocytes. Here we present data on expression of cardiac isoforms of CaMKIIdelta, the dominant form in the myocardium, in compensatory hypertrophy of stroke-prone spontaneously hypertensive rats (SHRSP) compared to the normotensive Wistar-Kyoto (WKY) control strain. Cardiac hypertrophy in SHRSP was documented by an increased heart weight/body weight ratio (HW/BW) of 31% (p < 0.05) and a more than six-fold elevated atrial natriuretic factor (ANF) transcript level (p < 0.05). Compensatory hypertrophic growth in SHRSP produced a specific phenotype of CaMKIIdelta isoforms characterized by increased transcript levels of the embryonic/neonatal isoform delta4 (48%, p < 0.05) and the isoform delta9 (31%, p < 0.05) with no changes in delta2 and delta3. Inhibition of angiotensin converting enzyme (ACE) by cilazapril completely regressed myocardial hypertrophy, normalized ANF transcript levels, and restored the normal phenotype of CaMKIIdelta by reducing transcripts for delta4 and delta9 to levels present in WKY controls. Our data suggest the importance of specific changes in the CaMKII isoform composition for growth processes in the myocardium.

Glycogen Synthase Kinase 3β Regulates GATA4 in Cardiac Myocytes

Inactivation of glycogen synthase kinase 3beta (GSK3beta) is critical for transcription of atrial natriuretic factor (ANF) by beta-adrenergic receptors in cardiac myocytes. We examined the mechanism by which GSK3beta regulates ANF transcription. Stimulation of beta-adrenergic receptors induced nuclear accumulation of GATA4, whereas beta-adrenergic ANF transcription was suppressed by dominant negative GATA4, suggesting that GATA4 plays an important role in beta-adrenergic ANF transcription. Interestingly, GATA4-mediated transcription was markedly attenuated by GSK3beta. GSK3beta physically associates with GATA4 and phosphorylates GATA4 in vitro. Overexpression of GSK3beta suppressed both basal and beta-adrenergic increases in nuclear expression of GATA4, whereas inhibition of GSK3beta by LiCl caused nuclear accumulation of GATA4, suggesting that GSK3beta negatively regulates nuclear expression of GATA4. The nuclear exportin Crm1 reduced nuclear expression of GATA4, and the reduction was enhanced by GSK3beta but not by kinase-inactive GSK3beta. Leptomycin B, an inhibitor for Crm1, increased basal nuclear GATA4 and suppressed GSK3beta-induced decreases in nuclear GATA4. These results suggest that GSK3beta negatively regulates nuclear expression of GATA4 by stimulating Crm1-dependent nuclear export. Inhibition of GSK3beta by beta-adrenergic stimulation abrogates GSK3beta-induced nuclear export of GATA4, causing nuclear accumulation of GATA4, which may represent an important signaling mechanism mediating cardiac hypertrophy.

Akt Like a Woman

The oncogenic murine AKT8 retrovirus was identified almost 25 years ago, and the presence of the causative oncogenic agent v- akt was subsequently demonstrated in transformed cells or tumors from both mice and humans.1 2 The cellular homolog of v- akt was cloned in the early 1990s and was termed c-Akt (or simply Akt). Largely on the basis of its sequence similarity to both protein kinases A and C, it was concluded that Akt (alternatively known as protein kinase B [PKB]) represented a Ser-/Thr- protein kinase. Three mammalian genes have been identified, with transcripts of akt1 and akt2 being highly expressed in heart. The explosion of interest in Akt was the direct consequence of the realization that it is an effector of the lipid signaling molecule phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] (Figure⇓). Figure 1. Possible actions of Akt in the cardiovascular system. Arrows indicate activation; bars indicate inhibition. Phosphorylation of Bad inhibits its proapoptic activity by reducing its inhibition of the antiapoptotic actions of Bcl-2 and Bcl-XL. The overall consequences of activation of Akt are shown in boxes. For additional explanation, see the text. PDK indicates PtdIns(3,4,5)P3-dependent kinase; eNOS, endothelial NO synthase. By binding to their transmembrane receptor protein tyrosine kinases, a variety of growth factors, including insulin and insulin-like growth factor 1 (IGF1), activate the lipid kinase phosphatidylinositol 3-kinase (PI3K) to phosphorylate the membrane phospholipid PtdIns(4,5)P2, producing PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 remains in the plane of the membrane and may serve to recruit Akt to this compartment from its normal cytoplasmic location by binding to the Akt pleckstrin-homology domain.1 2 In addition, PtdIns(3,4,5)P3 activates PtdIns(3,4,5)P3-dependent protein kinase, which phosphorylates Akt on a Thr- residue (Thr308 in Akt1/PKBα, Thr309 in Akt2/PKBβ, and Thr305 in PKBγ). …

β -Adrenergic Cardiac Hypertrophy is Mediated Primarily by the β1-Subtype in the Rat Heart

Myocardial β -adrenergic receptors (β -ARs) consist ofβ1 - and β2-subtypes, which mediate distinct signaling mechanisms. We examined whichβ -AR subtype mediates cardiac hypertrophy. The β2-subtype is predominant in neonatal rat cardiac myocytes (β1, 36%vβ2, 64%), while theβ1 -subtype predominates in the adult rat heart (59%v 41%). Stimulation of cultured cardiac myocytes in vitro with isoproterenol (ISO), an agonist for β1- andβ2 -ARs, caused hypertrophy of myocytes along with increases in transcription of atrial natriuretic factor (ANF) and actin reorganization. All of these ISO-mediated myocyte responses in vitro were inhibited by aβ1 -AR antagonist, betaxolol, but not by a β2-AR antagonist, ICI 118551. Pertussis toxin failed to affect ISO-induced increases in total protein/DNA content and ANF transcription in vitro. ISO increased LV weight/body weight and ANF transcription in the adult rat in vivo, which were also inhibited by betaxolol but not by ICI 118551. These results suggest that β -AR stimulated hypertrophy is mediated by theβ1 -subtype and by a pertussis toxin-insensitive mechanism

Molecular mechanisms of cardiac hypertrophy induced by toxicants.

Cardiac hypertrophy is an end point of chronic cardiac toxicity from a number of toxicants. Doxorubicin, cocaine, acetaldehyde, monocrotaline, and azide are examples of these toxicants, which may induce hypertrophy by increasing oxidants, circulating levels of catecholamines, and hemodynamic load or by inducing hypoxia. We summarize here the major signal transduction pathways and common changes in gene expression found with the classical hypertrophy inducers angiotensin II, endothelin 1, and catecholamines. Activation of G-proteins, calcium signaling, phosphoinositide 3-kinase (PI3K), certain family members of protein kinase Cs (PKCs), and three branches of mitogenactivated protein kinases (MAPKs), i.e. extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinases (JNKs), are important for developing a hypertrophic phenotype in cardiomyocytes. Characteristic changes of gene expression in hypertrophy include the elevated transcription of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta MHC), skeletal alpha-actin (SkA), certain variants of integrins and perhaps tubulin genes, and reduced expression of the sarcoplasmic reticulum proteins phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPase 2 alpha (SERCA2 alpha), and of the ryanodine receptors. Although which toxicants induce these molecular changes remains to be tested, increasing lines of evidence support that oxidants play a central role in cardiac hypertrophy. Oxidants activate small G-proteins, calcium signaling, PI3K, PKCs, and MAPKs. Oxidants cause cardiomyocytes to enlarge in vitro. Recent developments in transgenic, genomic, and proteomic technologies will provide needed tools to reveal the mechanism of chronic cardiac toxicity at the cellular and molecular levels.

Insulin-like growth factor-binding protein-3 induces fetalization in neonatal rat cardiomyocytes.

We employed cDNA microarrays representing 4000 distinct sequences to profile changes in gene expression in a rodent model of heart disease, namely, progression to heart failure after myocardial infarction. Differential gene expression in the left ventricle was examined at 4-week intervals over a 12-week period after coronary artery ligation in rats. Over this time course, insulin-like growth factor-binding protein-3 (IGFBP-3) was found to have a greater expression than in nondiseased tissues. We then employed quantitative real-time PCR to analyze gene expression in neonatal rat cardiac myocytes that had been treated with recombinantly expressed IGFBP-3 to examine a number of transcriptional responses designed to reflect the heart failure phenotype. The IGFBP-3 protein was shown to induce transcription of atrial natriuretic factor (ANF) and beta-myosin heavy chain (B-MHC). Analysis of conditioned media taken from IGFBP-3-treated cardiac myocyte cultures demonstrated an increase in ANF protein as well as in protein synthesis, as determined by metabolic incorporation of a radiolabeled amino acid. However, transcriptional changes of troponin-1, endothelin-1, or angiotensin-II by IGFBP-3 were not observed.

The Akt-Glycogen Synthase Kinase 3β Pathway Regulates Transcription of Atrial Natriuretic Factor Induced by β-Adrenergic Receptor Stimulation in Cardiac Myocytes

We examined the mechanism of atrial natriuretic factor (ANF) transcription by isoproterenol (ISO), an agonist for the beta-adrenergic receptor (betaAR), in cardiac myocytes. ISO only modestly activated members of the mitogen-activated protein kinase family. ISO-induced ANF transcription was not affected by inhibition of mitogen-activated protein kinases, whereas it was significantly inhibited by KN93, an inhibitor of Ca(2+)/calmodulin-dependent kinase (CaM kinase II). Production of 3'-phosphorylated phosphatidylinositides (3 phosphoinositides) was also required for ISO-induced ANF transcription. ISO caused phosphorylation (Ser-473) and activation of Akt through CaM kinase II- and 3 phosphoinositides-dependent mechanisms. Constitutively active Akt increased myocyte surface area, total protein content, and ANF expression, whereas dominant negative Akt blocked ISO-stimulated ANF transcription. ISO caused Ser-9 phosphorylation and decreased activities of GSK3beta. Overexpression of GSK3beta inhibited ANF transcription, which was reversed by ISO. ISO failed to reverse the inhibitory effect of GSK3beta(S9A), an Akt-insensitive mutant. Kinase-inactive GSK3beta increased ANF transcription. Cyclosporin A partially inhibited ISO-stimulated ANF transcription, indicating that calcineurin only partially mediates ANF transcription. These results suggest that both CaM kinase II and 3 phosphoinositides mediate betaAR-induced Akt activation and ANF transcription in cardiac myocytes. Furthermore, betaAR-stimulated ANF transcription is predominantly mediated by activation of Akt and subsequent phosphorylation/inhibition of GSK3beta.