Hypertrophic Responses In Cardiac Myocytes Research Articles

Disruption of the cyclic AMP phosphodiesterase-4 (PDE4)–HSP20 complex attenuates the β-agonist induced hypertrophic response in cardiac myocytes

The small heat shock protein HSP20 is known to be cardioprotective during times of stress and the mechanism underlying its protective abilities depends on its phosphorylation on Ser16 by PKA (protein kinase A). Although the external stimuli that trigger Ser16 phosphorylation have been well studied, the events that modulate spatial and temporal control of this modification remain to be clarified. Here, we report that inhibition of cAMP phosphodiesterase-4 (PDE4) induces the phosphorylation of HSP20 in resting cardiac myocytes and augments its phosphorylation by PKA following β-adrenergic stimulation. Moreover, using peptide array technology, in vitro binding studies, co-immunoprecipitation techniques and immunocytochemistry, we show that HSP20 binds directly to PDE4 within a region of the conserved catalytic domain. We also show that FRET-based, genetically-encoded cAMP reporters anchored to HSP20 exhibit a larger response to PDE4 inhibition compared to free cytosolic cAMP reporters, suggesting that the interaction with PDE4 is crucial in modulating the highly localised pool of cAMP to which HSP20 is exposed. Using information gleaned from peptide array analyses, we developed a cell-permeable peptide that serves to inhibit the interaction of PDE4 with HSP20. Disruption of the HSP20–PDE4 complex, using this peptide, suffices to induce phosphorylation of HSP20 by PKA and to protect against the hypertrophic response measured in neonatal cardiac myocytes following chronic β-adrenergic stimulation.

Myocardin-Related Transcription Factor A Is a Common Mediator of Mechanical Stress- and Neurohumoral Stimulation-Induced Cardiac Hypertrophic Signaling Leading to Activation of Brain Natriuretic Peptide Gene Expression

Subjecting cardiomyocytes to mechanical stress or neurohumoral stimulation causes cardiac hypertrophy characterized in part by reactivation of the fetal cardiac gene program. Here we demonstrate a new common mechanism by which these stimuli are transduced to a signal activating the hypertrophic gene program. Mechanically stretching cardiomyocytes induced nuclear accumulation of myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), in a Rho- and actin dynamics-dependent manner. Expression of brain natriuretic peptide (BNP) and other SRF-dependent fetal cardiac genes in response to acute mechanical stress was blunted in mice lacking MRTF-A. Hypertrophic responses to chronic pressure overload were also significantly attenuated in mice lacking MRTF-A. Mutation of a newly identified, conserved and functional SRF-binding site within the BNP promoter, or knockdown of MRTF-A, reduced the responsiveness of the BNP promoter to mechanical stretch. Nuclear translocation of MRTF-A was also involved in endothelin-1- and angiotensin-II-induced activation of the BNP promoter. Moreover, mice lacking MRTF-A showed significantly weaker hypertrophic responses to chronic angiotensin II infusion than wild-type mice. Collectively, these findings point to nuclear translocation of MRTF-A as a novel signaling mechanism mediating both mechanical stretch- and neurohumoral stimulation-induced BNP gene expression and hypertrophic responses in cardiac myocytes.

RbAp48/46 are Novel Components of p300/GATA4 Complex Required for Hypertrophic Responses in Cardiac Myocytes

RbAp48/46 are Novel Components of p300/GATA4 Complex Required for Hypertrophic Responses in Cardiac Myocytes

Differential effects of Pyk2 and FAK on the hypertrophic response of cardiac myocytes

The related cytoplasmic non-receptor tyrosine kinases Pyk2 (proline-rich tyrosine kinase 2) and FAK (focal adhesion kinase) have been implicated in phenylephrine-induced G-protein-coupled receptor-mediated signaling mechanisms leading to cardiomyocyte hypertrophy. We report that, in phenylephrine-stimulated neonatal rat ventricular myocytes (NRVM), Pyk2 augments expression of the hypertrophic marker atrial natriuretic factor (ANF) but reduces cytoskeletal organization and cell spreading. In contrast, FAK attenuates ANF production but does not alter cytoskeletal organization and cell spreading. Pyk2 and FAK exhibit differential localization in both unstimulated and phenylephrine-stimulated myocytes. Pyk2 catalytic activity is required for Pyk2 to augment ANF secretion but is not necessary to reduce cell spreading. Pyk2 autophosphorylation is required but not sufficient for Pyk2 to augment ANF secretion. Expression of the Pyk2 FERM domain as an autonomous fragment inhibits phenylephrine-mediated ANF secretion and reduces cell spreading. In addition, expression of the Pyk2 FERM domain inhibits the ability of Pyk2 to augment ANF secretion; this is correlated with reduced Pyk2 autophosphorylation. These data indicate that Pyk2 and FAK have different roles and occupy different positions in signaling pathways leading to the development of cardiomyocyte hypertrophy.

MyD88-dependent nuclear factor-κB activation is involved in fibrinogen-induced hypertrophic response of cardiomyocytes

Plasma fibrinogen has been defined as a risk factor of cardiovascular disease and may play a role in the development of cardiac hypertrophy. We have previously demonstrated that the Toll-like receptor 4 (TLR4)-mediated myeloid differentiation primary response protein 88 (MyD88)-dependent nuclear factor-kappaB (NF-kappaB) pathway is involved in cardiac hypertrophy. The present study aimed to investigate whether fibrinogen will stimulate the hypertrophic response of cardiac myocytes and to examine the role of the TLR4/MyD88/NF-kappaB pathway in fibrinogen-induced cardiac hypertrophy. Cardiac hypertrophy was induced by transverse aortic banding for 5 weeks in Sprague-Dawley rats. The deposition of fibrinogen in the left ventricle, as determined by immunohistochemistry and immunoblotting, was increased. Aortic banding also significantly enhanced the association of TLR4 with MyD88 and increased NF-kappaB activity. In-vitro studies showed that fibrinogen induced a dose-dependent, hypertrophic response of neonatal cardiomyocytes. Fibrinogen stimulation significantly increased myocyte size, 3H-leucine incorporation and mRNA levels of atrial natriuretic peptide (ANP); fibrinogen challenge also significantly increased associations of TLR4 with MyD88 and NF-kappaB binding activity. Transient transfection of cardiomyocytes with a dominant-negative MyD88 plasmid significantly attenuated the fibrinogen-induced hypertrophic response of neonatal cardiac myocytes and blunted fibrinogen-increased activation of the TLR4/MyD88/NF-kappaB signaling pathway. Our results suggest that fibrinogen induces hypertrophic response of cardiomyocytes partially through a TLR4-mediated, MyD88-dependent NF-kappaB pathway.

Tollip attenuated the hypertrophic response of cardiomyocytes induced by IL-1beta

We examined the role of Tollip in the hypertrophic response of cardiomyocytes. C57BL/6 mice were subjected to transverse aortic constriction (TAC) for 2 weeks and age-matched sham surgical operated mice served as control. TAC significantly reduced the association of Tollip with IRAK-1 by 66.4 percent and increased NF-kappaB binding activity by 86.5 percent and the levels of phospho-p38 by 114.6 percent in the myocardium compared with sham control, respectively. In vitro experiments showed that IL-1beta stimulation also significantly reduced the association of Tollip with IRAK-1 and increased NF-kappaB binding activity in neonatal cardiomyocytes. Tollip overexpression by transfection of cardiac myocytes significantly attenuated the IL-1beta-induced hypertrophic response of cardiac myocytes as evidenced by reduced cell size (16.4 percent) and decreased ANP expression (33.3 percent). Overexpression of Tollip also reduced NF-kappaB binding activity by 30.7 percent and phospho-p38 by 47.1 percent, respectively. The results suggest that Tollip could be a negative regulator during the development of cardiac hypertrophy. The negative regulation of cardiac hypertrophy by Tollip may involve downregulation of the MyD88-dependent NF-kappaB activation pathway.

The kinase activity of Cdk9 is required for phosphorylation of p300 and acetylation of GATA4 as well as cardiomyocyte hypertrophy

A zinc finger protein GATA-4 is one of the factors involved in transcriptional regulation during myocardial cell hypertrophy. In response to hypertrophic stimuli, GATA-4 forms a complex with an intrinsic histone acetyltransferase (HAT), p300, and increases its transcriptional activity by acetylation. By tandem affinity purification and mass spectrometric analyses, we identified cyclin-dependent kinase-9 (Cdk9), a component of positive transcription elongation factor b (P-TEFb), as a novel GATA4-binding partner. Cdk9 also forms a complex with p300 as well as GATA-4. However, precise functional relationship between p300/GATA-4 and P-TEFb is unknown. Intact p300 induced not only the acetylation of GATA-4 but also the interaction of GATA-4 with P-TEFb. Furthermore, p300 induced the hyperphosphorylation of RNA Pol II, suggesting that p300 is involved in regulating the kinase activity of Cdk9. All of these effects were inhibited by a dominant-negative form of (DN-) p300. Meanwhile, DN-Cdk9, which loses its kinase activity by a single amino acid substitution, inhibited p300-induced hyperphophorylation of RNA Pol II. Notably, DN-Cdk9 inhibited phosphorylation of p300 as well as p300-induced acetylation and DNA binding of GATA-4, indicating a requirement of Cdk9 in the HAT activity of p300. Finally, both DN-p300 and DN-Cdk9 inhibited phenylephrine-induced hypertrophic responses in cardiac myocytes. These findings demonstrate that Cdk9 forms a functional complex with the p300/GATA4 and is required for p300/GATA4-transcriptional pathway during cardiomyocyte hypertrophy.

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.

Abstract 535: Proteomics Analysis Identifies Positive Transcription Elongation Factor b as a Novel GATA-4-binding Partner Involved in Hypertrophic Responses in Cardiomyocytes

Introduction: A zinc finger protein GATA-4 is one of the factors involved in transcriptional regulation during myocardial cell hypertrophy. In response to hypertrophic stimuli, GATA-4 forms a large complex with ERK, NFATc and an intrinsic histone acetyltransferase, p300. Disruption of this complex results in the inhibition of hypertrophic responses in cardiomyocytes. Hypothesis: We assessed the hypothesis that proteomics analysis may identify novel GATA-4-binding partners required for myocardial cell hypertrophy. Methods: GATA-4 was stably expressed as FLAG-HA-epitope fusion in HeLa cells by retroviral transduction. GATA-4 complex was purified from nuclear extracts of these cells by sequential immunoprecipitation with an anti-FLAG antibody followed by an anti-HA antibody (Tandem Affinity Purification). Results: By mass spectrometric analyses, we identified 73 GATA-4 binding proteins, including histone modifying factors, methyltransferase, HDAC and unknown proteins. One was cyclin-dependent kinases-9 (cdk9), a component of Positive Transcription Elongation Factor b (P-TEFb). Immunoblotting analyses demonstrated that GATA-4 formed a complex with p300, cdk9 and cyclin T1, another component of P-TEFb. A dominant-negative form of p300 disrupted the interaction of GATA-4 with cdk9 or cyclin T1. Conversely, a dominant-negative form of cdk9 and a cdk9 kinase inhibitor, 5,6-dichloro-1-h-ribofuranosyl-benzimidazole (DRB) inhibited p300-induced activation of GATA-4-dependent transcription as well as the acetylation of GATA-4. Stimulation of cardiomyocytes with phenylephrine (PE) increased the binding of GATA-4/p300 with cdk9 or cyclin T1. DN-cdk9 and DRB not only disrupted the complex of GATA-4/p300 with cdk9 or cyclin T1, but also repressed PE-induced hypertrophic responses such as myofibrillar organization, increase in cell size and promoter activation of the endothelin-1 and atrial natriuretic factors in cardiomyocytes. Conclusion: These findings demonstrate that cdk9 and cyclin T1 form a functional protein complex with GATA-4/p300 and that this large complex is a prerequisite for hypertrophic responses in cardiac myocytes.

Gα12/13-mediated Up-regulation of TRPC6 Negatively Regulates Endothelin-1-induced Cardiac Myofibroblast Formation and Collagen Synthesis through Nuclear Factor of Activated T Cells Activation

Sustained elevation of [Ca(2+)](i) has been implicated in many cellular events. We previously reported that alpha subunits of G(12) family G proteins (Galpha(12/13)) participate in sustained Ca(2+) influx required for the activation of nuclear factor of activated T cells (NFAT), a Ca(2+)-responsive transcriptional factor, in rat neonatal cardiac fibroblasts. Here, we demonstrate that Galpha(12/13)-mediated up-regulation of canonical transient receptor potential 6 (TRPC6) channels participates in sustained Ca(2+) influx and NFAT activation by endothelin (ET)-1 treatment. Expression of constitutively active Galpha(12) or Galpha(13) increased the expression of TRPC6 proteins and basal Ca(2+) influx activity. The treatment with ET-1 increased TRPC6 protein levels through Galpha(12/13), reactive oxygen species, and c-Jun N-terminal kinase (JNK)-dependent pathways. NFAT is activated by sustained increase in [Ca(2+)](i) through up-regulated TRPC6. A Galpha(12/13)-inhibitory polypeptide derived from the regulator of the G-protein signaling domain of p115-Rho guanine nucleotide exchange factor and a JNK inhibitor, SP600125, suppressed the ET-1-induced increase in expression of marker proteins of myofibroblast formation through a Galpha(12/13)-reactive oxygen species-JNK pathway. The ET-1-induced myofibroblast formation was suppressed by overexpression of TRPC6 and CA NFAT, whereas it was enhanced by TRPC6 small interfering RNAs and cyclosporine A. These results suggest two opposite roles of Galpha(12/13) in cardiac fibroblasts. First, Galpha(12/13) mediate ET-1-induced myofibroblast formation. Second, Galpha(12/13) mediate TRPC6 up-regulation and NFAT activation that negatively regulates ET-1-induced myofibroblast formation. Furthermore, TRPC6 mediates hypertrophic responses in cardiac myocytes but suppresses fibrotic responses in cardiac fibroblasts. Thus, TRPC6 mediates opposite responses in cardiac myocytes and fibroblasts.

CDK9 is a Novel Component of p300/GATA-4 Complex Required for Hypertrophic Responses in Cardiac Myocytes

CDK9 is a Novel Component of p300/GATA-4 Complex Required for Hypertrophic Responses in Cardiac Myocytes

Identification of p300-targeted Acetylated Sites in GATA-4 During Hypertrophic Responses in Cardiac Myocytes

Identification of p300-targeted Acetylated Sites in GATA-4 During Hypertrophic Responses in Cardiac Myocytes

Carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy

Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of the plasma membrane Na+-H+ exchanger (NHE1) and Cl- -HCO3- exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H+ and HCO3-, their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37 +/- 2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA, twofold in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane-anchored isoform, CAIV. The membrane-permeant CA inhibitor, 6-ethoxyzolamide (ETZ), both prevented and reversed PE-induced hypertrophy in a concentration-dependent manner in neonate cardiomyocytes (IC50=18 microm). ETZ and the related CA inhibitor methazolamide prevented hypertrophy in adult cardiomyocytes. In addition, ETZ inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC50 values of 1.2 +/- 0.3 microm and 2.7 +/- 0.3 microm. PE significantly increased neonatal cardiomyocyte Ca2+ transient frequency from 0.33 +/- 0.4 Hz to 0.77 +/- 0.04 Hz following 24 h treatment; these Ca2+ -handling abnormalities were completely prevented by ETZ (0.28 +/- 0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype.

Induction of c-fos mRNA Expression by Pure Pressure Overload in Cultured Cardiac Myocytes

Mechanical stress by pressure overload due to hypertension or valvular heart disease such as aortic valve stenosis induces cardiac hypertrophy. It has been well established that the mechanical stretch of cardiac myocytes in vitro induces hypertrophic responses such as the expression of immediate early response genes including c-fos. However, it remains uncertain whether the mechanical forces due to pure atmospheric pressure can induce similar responses in cardiac myocytes. We thus cultured rat neonatal cardiac myocytes in an atmospheric pressure chamber apparatus and determined the effects of pure pressure stress on c-fos gene expression. Pressures greater than 80 mmHg enhanced c-fos mRNA after 30 minutes. These results suggest that pure atmospheric pressure overload can also induce early hypertrophic responses in cardiac myocytes.

A Transgenic Mouse Model of Heart Failure Using Inducible Gαq

Receptors coupled to Galpha q play a key role in the development of heart failure. Studies using genetically modified mice suggest that Galpha q mediates a hypertrophic response in cardiac myocytes. Galpha q signaling in these models is modified during early growth and development, whereas most heart failure in humans occurs after cardiac damage sustained during adulthood. To determine the phenotype of animals that express increased Galpha q signaling only as adults, we generated transgenic mice that express a silent Galpha q protein (Galpha qQ209L-hbER) in cardiac myocytes that can be activated by tamoxifen. Following drug treatment to activate Galpha q Q209L-hbER, these mice rapidly develop a dilated cardiomyopathy and heart failure. This phenotype does not appear to involve myocyte hypertrophy but is associated with dephosphorylation of phospholamban (PLB), decreased sarcoplasmic reticulum Ca2+-ATPase activity, and a decrease in L-type Ca2+ current density. Changes in Ca2+ handling and decreased cardiac contractility are apparent 1 week after Galpha qQ209L-hbER activation. In contrast, transgenic mice that express an inducible Galpha q mutant that cannot activate phospholipase Cbeta (PLCbeta) do not develop heart failure or changes in PLB phosphorylation, but do show decreased L-type Ca2+ current density. These results demonstrate that activation of Galpha q in cardiac myocytes of adult mice causes a dilated cardiomyopathy that requires the activation of PLCbeta. However, increased PLCbeta signaling is not required for all of the Galpha q-induced cardiac abnormalities.

FOG-2 Competes with GATA-4 for Transcriptional Coactivator p300 and Represses Hypertrophic Responses in Cardiac Myocytes

A multizinc finger protein, FOG-2, associates with a cardiac transcription factor, GATA-4, and represses GATA-4-dependent transcription. GATA-4 is required not only for normal heart development but is also involved in hypertrophic responses in cardiac myocytes; however, the effects of FOG-2 on these responses are unknown. The interaction of GATA-4 with a transcriptional coactivator p300 is required for its full transcriptional activity and the activation of the embryonic program during myocardial cell hypertrophy. We show here that exogenous FOG-2 represses phenylephrine-induced hypertrophic responses such as myofibrillar organization, increases in cell size, and hypertrophy-associated gene transcription. Using immunoprecipitation Western blotting, we demonstrate that FOG-2 physically interacted with p300 and reduced the binding of GATA-4 to p300. In addition, in COS7 cells, in which the function of endogenous p300 is disrupted, FOG-2 is unable to repress the GATA-4-dependent transcriptional activities; however, FOG-2 markedly repressed the p300-mediated increase in the DNA-binding and transcriptional activities of GATA-4 in these cells. Similarly, FOG-2 inhibited a phenylephrine-induced increase in the p300/GATA-4 interaction, the GATA-4/DNA-binding, and transcriptional activities of GATA-4-dependent promoters in cardiac myocytes as well. These findings demonstrate that FOG-2 represses hypertrophic responses in cardiac myocytes and that p300 is involved in these repressive effects.

Heme oxygenase-1 inhibition of MAP kinases, calcineurin/NFAT signaling, and hypertrophy in cardiac myocytes.

Heme oxygenases (HO) are the rate-limiting enzymes in heme degradation, catalyzing the breakdown of heme to equimolar quantities of biliverdin (BV), carbon monoxide (CO), and ferrous iron. The inducible HO isoform, HO-1, confers protection against ischemia/reperfusion (I/R)-injury in the heart. We hypothesized that HO-1 and its catalytic by-products constitute an antihypertrophic signaling module in cardiac myocytes. The G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) (30 nmol/l) stimulated a robust hypertrophic response in cardiac myocytes isolated from 1- to 3-day-old Sprague-Dawley rats, with increases in cell surface area (planimetry), sarcomere assembly (confocal laser scanning microscopy), and prepro-atrial natriuretic peptide (ANP) mRNA expression. Adenoviral overexpression of HO-1, but not beta-galactosidase, significantly inhibited ET-1 induced cardiac myocyte hypertrophy. The antihypertrophic effects of HO-1 were mimicked by BV (10 micromol/l) and the CO-releasing molecule [Ru(CO)3Cl2]2 (10 micromol/l), strongly suggesting a critical involvement of BV and CO in the antihypertrophic effects of HO-1. Both BV and CO suppressed extracellular signal-regulated kinases (ERK1/ERK2) and p38 mitogen-activated protein kinase (MAPK) activation by ET-1 stimulation. Moreover, BV and CO inhibited the prohypertrophic calcineurin/NFAT pathway. This inhibition occurred upstream from calcineurin because BV and CO inhibited NFAT activation in response to ET-1 stimulation but not in response to adenoviral expression of a constitutively active calcineurin mutant. Upstream-inhibition of the calcineurin/NFAT pathway by CO occurred independent from cGMP and cGMP-dependent protein kinase type I (PKG I). Heme oxygenase-1 and its catalytic by-products, BV and CO, constitute a novel antihypertrophic signaling pathway in cardiac myocytes. Biliverdin and CO inhibition of MAPKs and calcineurin/NFAT signaling provides a mechanistic framework how heme degradation products may promote their antihypertrophic effects.

Csm, a Cardiac-specific Isoform of the RNA Helicase Mov10l1, Is Regulated by Nkx2.5 in Embryonic Heart

Nkx2.5 (also called Csx) is an evolutionarily conserved cardiac transcription factor of the homeobox gene family. Nkx2.5 is required for early heart development, because Nkx2.5 null mice die before completion of cardiac looping. To identify genes regulated by Nkx2.5 in the developing heart, we performed differential screening in combination with suppression subtractive hybridization using RNA isolated from wild-type and Nkx2.5 null hearts at embryonic day 8.5. One gene that we found to be markedly down-regulated in the hearts from Nkx2.5 null embryos is an isoform of Mov10 like-1 (Mov10l1), a putative RNA helicase expressed in testis. We named this novel isoform as Csm (cardiac-specific isoform of Mov10l1). Csm is identical with the 3' region of the Mov10l1 gene, but its transcript starts from the exon 16 of Mov10l1. The conceptual protein encoded by Csm cDNA contains a helicase motif as well as ATPase and RNA interaction motifs. Csm is expressed specifically in the heart, and its expression in the heart is restricted to cardiac myocytes. Csm potentiated phenylephrine-induced hypertrophic response in cardiac myocytes. Furthermore, transient cotransfection analysis showed that Nkx2.5 transactivates the Csm promoter, suggesting that Nkx2.5 is essential for embryonic Csm expression.

Chronic activation of extracellular-signal-regulated protein kinases by phenylephrine is required to elicit a hypertrophic response in cardiac myocytes.

Extracellular-signal-regulated protein kinases (ERKs) are activated rapidly and transiently in response to phenylephrine (PE) and endothelin-1 (ET-1) in cardiac myocytes, but whether this is linked to the subsequent development of the hypertrophic phenotype remains equivocal. To investigate this, we examined the dependence of the hypertrophic response on the length of exposure to PE in neonatal myocyte cultures. In addition to the initial transient activation of ERKs (maximum at 5-10 min), PE (10 microM) induced a second, more prolonged peak of activity several hours later. The activity of a transfected atrial natriuretic factor-luciferase reporter gene was increased 10- to 24-fold by PE. This response was inhibited by the alpha(1)-antagonist prazosin (100 nM) and by U0126 (10 microM) and PD184352 (1 microM), inhibitors of ERK activation, irrespective of whether these were added before or up to 24 h after the addition of PE. Prazosin had no effect on ET-1 (50 nM)-stimulated atrial natriuretic factor-luciferase activity. Protein synthesis was enhanced by 35+/-6% by PE, and this was blocked by prazosin added 1 h after the addition of PE, but decreased only by half when added 8 h after PE. Similarly, PE (48 h) increased myocyte area by 49% and this was prevented by prazosin added 1 h after PE, but decreased only by half when added at 24 h. These results demonstrate that prolonged exposure to PE is required to elicit alterations in gene expression, protein synthesis and cell size, characteristic of hypertrophied myocytes, and they confirm that the initial peak of ERK activity is insufficient to trigger hypertrophic responses.

Signalling components involved in the coupling of alpha 1-adrenoceptors to phospholipase D in neonatal rat cardiac myocytes.

Activation of phospholipase D (PLD) is assumed to be one major pathway by which alpha(1)-adrenoceptors (alpha(1)ARs) induce hypertrophic responses in cardiac myocytes. Heterotrimeric G proteins, protein kinase C (PKC) isoforms, protein tyrosine kinases, monomeric GTPases of the ADP-ribosylation factor (ARF) and Rho families, and as important cofactor phosphatidylinositol 4,5-bisphosphate (PIP(2)) seem to participate in the G protein-coupled receptor dependent regulation of PLD. We therefore studied the role of these components in the coupling of alpha(1)ARs to PLD in neonatal rat cardiac myocytes (NRCM). Stimulation of alpha(1)ARs, most likely of the alpha(1A) subtype, by noradrenaline increased PLD activity three- to fourfold concomitant with the stimulation of phospholipase C (PLC). In contrast, the partial agonist phenylephrine stimulated PLC, but failed to increase PLD activity. The PLC and PLD responses were pertussis toxin insensitive and treatment of the cells with the G(q)-activating toxin of Pasteurella multocida stimulated both phospholipases about fourfold. Over-expression of the G(q)-and G(i)-type-specific regulator of G protein signalling RGS4 blunted alpha(1)AR-induced PLC and PLD stimulation. Ro 31-8220, known to inhibit Ca(2+)-dependent and -independent PKC isoforms, strongly inhibited PLD activity, whereas Gö 6976, known to inhibit preferentially Ca(2+)-dependent PKC isozymes, was without effect. The ARF signalling inhibitor brefeldin A, protein tyrosine kinase inhibitors and the Rho-inactivating toxin B of Clostridium difficile blunted alpha(1)AR-induced PLD stimulation and largely reduced the cellular PIP(2) content. In membranes of toxin B-treated NCRM, PLD activity was similarly reduced, but was fully restored by addition of exogenous PIP(2). We conclude, that alpha(1A)ARs stimulate PLD activity via a G(q/11)-PLCbeta-novel PKC isoform-dependent pathway in NRCM. ARF and Rho GTPases as well as protein tyrosine kinases contribute to PLD stimulation in NRCM, most likely by regulating the supply of PIP(2).