Protein Synthesis In Cardiomyocytes Research Articles

Role of Vasoactive Hormone-Induced Signal Transduction in Cardiac Hypertrophy and Heart Failure.

Heart failure is the common concluding pathway for a majority of cardiovascular diseases and is associated with cardiac dysfunction. Since heart failure is invariably preceded by adaptive or maladaptive cardiac hypertrophy, several biochemical mechanisms have been proposed to explain the development of cardiac hypertrophy and progression to heart failure. One of these includes the activation of different neuroendocrine systems for elevating the circulating levels of different vasoactive hormones such as catecholamines, angiotensin II, vasopressin, serotonin and endothelins. All these hormones are released in the circulation and stimulate different signal transduction systems by acting on their respective receptors on the cell membrane to promote protein synthesis in cardiomyocytes and induce cardiac hypertrophy. The elevated levels of these vasoactive hormones induce hemodynamic overload, increase ventricular wall tension, increase protein synthesis and the occurrence of cardiac remodeling. In addition, there occurs an increase in proinflammatory cytokines and collagen synthesis for the induction of myocardial fibrosis and the transition of adaptive to maladaptive hypertrophy. The prolonged exposure of the hypertrophied heart to these vasoactive hormones has been reported to result in the oxidation of catecholamines and serotonin via monoamine oxidase as well as the activation of NADPH oxidase via angiotensin II and endothelins to promote oxidative stress. The development of oxidative stress produces subcellular defects, Ca2+-handling abnormalities, mitochondrial Ca2+-overload and cardiac dysfunction by activating different proteases and depressing cardiac gene expression, in addition to destabilizing the extracellular matrix upon activating some metalloproteinases. These observations support the view that elevated levels of various vasoactive hormones, by producing hemodynamic overload and activating their respective receptor-mediated signal transduction mechanisms, induce cardiac hypertrophy. Furthermore, the occurrence of oxidative stress due to the prolonged exposure of the hypertrophied heart to these hormones plays a critical role in the progression of heart failure.

Abstract P1166: O-GlcNAcylation Regulates Protein Synthesis In Cardiomyocytes Through Effects On The Translational Regulator Eif2α

The regulated and reversible modification of Ser/Thr residues by O-linked N-Acetylglucosamine (O-GlcNAc) is termed O-GlcNAcylation. O-GlcNAc cycling on and off proteins is regulated by two conserved enzymes, the ‘writer’ O-GlcNAc Transferase (OGT) and the ‘eraser’ O-GlcNAcase (OGA). Crucially, O-GlcNAcylation is implicated in nutrient sensing, cell growth and stress adaptation. Despite these significant roles, the precise pathways impacted by O-GlcNAcylation and what key substrate proteins are involved remain incompletely understood. A hallmark of cardiomyocyte hypertrophy is the rapid production of new proteins that assemble into the contractile machinery of the growing myocyte. eIF2α is a rate-limiting factor in translation initiation. Interestingly, phosphorylation of eIF2α causes the arrest of general mRNA translation and favors the translation of stress-adaptive mRNAs. In the present work we investigated whether altering O-GlcNAcylation affects nascent protein synthesis (NPS) in hypertrophic cardiomyocytes. NPS was significantly elevated by the agonist phenylephrine, and this response was suppressed by OSMI-1 (OGT inhibitor). Moreover, we found that OGT-inhibited cells had increased phosphorylation of eIF2α. Additionally, we found increased protein levels of the stress adaptive factors Atf4 and Chop, whose mRNA translation is favored when eIF2α is phosphorylated. ISRIB, a small molecule designed to suppress the effects of phospho-eIF2α in altering translation, significantly prevented OSMI-1-induced accumulation of Atf4. Furthermore, we used metabolic labeling of glycans in cells, coupled with alkyne-azide ‘click’ chemistry and streptavidin pull-down, to show that eIF2α can be O-GlcNAcylated in cardiomyocytes. Furthermore, protein enrichment followed by mass spectrometry identified potential sites of O-GlcNAcylation on eIF2α. Taken together, these results highlight a novel role of O-GlcNAcylation on cardiomyocyte protein synthesis through the regulation of eIF2α. Future investigations will characterize the upstream mechanisms that promote eIF2α phosphorylation in OGT-inhibited cells and investigate the implications of direct eIF2α O-GlcNAcylation on hypertrophic protein synthesis in cardiomyocytes.

Abstract 2562: O-GlcNAcylation regulates nascent protein synthesis in cardiomyocytes through effects on the translational regulator eIF2α

Abstract 2562: O-GlcNAcylation regulates nascent protein synthesis in cardiomyocytes through effects on the translational regulator eIF2α

The insulin receptor family and protein kinase B (Akt) are activated in the heart by alkaline pH and α1-adrenergic receptors.

Insulin and insulin-like growth factor stimulate protein synthesis and cardioprotection in the heart, acting through their receptors (INSRs, IGF1Rs) and signalling via protein kinase B (PKB, also known as Akt). Protein synthesis is increased in hearts perfused at alkaline pHo to the same extent as with insulin. Moreover, α1-adrenergic receptor (α1-AR) agonists (e.g. phenylephrine) increase protein synthesis in cardiomyocytes, activating PKB/Akt. In both cases, the mechanisms are not understood. Our aim was to determine if insulin receptor-related receptors (INSRRs, activated in kidney by alkaline pH) may account for the effects of alkaline pHo on cardiac protein synthesis, and establish if α1-ARs signal through the insulin receptor family. Alkaline pHo activated PKB/Akt signalling to the same degree as insulin in perfused adult male rat hearts. INSRRs were expressed in rat hearts and, by immunoblotting for phosphorylation (activation) of INSRRs/INSRs/IGF1Rs, we established that INSRRs, together with INSRs/IGF1Rs, are activated by alkaline pHo. The INSRR/INSR/IGF1R kinase inhibitor, linsitinib, prevented PKB/Akt activation by alkaline pHo, indicating that INSRRs/INSRs/IGF1Rs are required. Activation of PKB/Akt in cardiomyocytes by α1-AR agonists was also inhibited by linsitinib. Furthermore, linsitinib inhibited cardiomyocyte hypertrophy induced by α1-ARs in cultured cells, reduced the initial cardiac adaptation (24 h) to phenylephrine in vivo (assessed by echocardiography) and increased cardiac fibrosis over 4 days. We conclude that INSRRs are expressed in the heart and, together with INSRs/IGF1Rs, the insulin receptor family provide a potent system for promoting protein synthesis and cardioprotection. Moreover, this system is required for adaptive hypertrophy induced by α1-ARs.

Sick euthyroid syndrome and its association with cardiogenic shock in acute coronary syndromes

Abstract Introduction Sick euthyroid syndrome (SES) constitutes an acute response to stress, and patients who develop it usually show more severe illness than those who do not. It could be related to disease severity in acute coronary syndrome (ACS), as assessed with Killip-Kimball class (KK), since cardiomyocytes are specifically sensitive to T3 levels. Objective To determine the prevalence of SES and low T3 in patients with ACS, and to assess its association with disease severity. Methods Prospective, observational and single center study in consecutive patients admitted to the CCU with a diagnosis of ACS. Clinical variables were collected from medical records; blood samples were obtained at admission to measure TSH, T3 and free T4 levels. SES was defined as low T3 with normal TSH and free T4. Maximum KK was determined by treating physicians. Categorical variables were compared with the chi-squared test, and categorical variables with Kruskal-Wallis and Wilcoxon tests. Statistical significance was set at p<0.05. Results There were 149 patients with complete data available for analysis. Their age was 67.8±12.4 years, and 64% were male. A total of 16.3% had SES. There were 7.5% patients with SES and KK-A, 34.8% KK-B, 14.3% KK-C and 70% KK-D (p<0.001). Thus, SES was more frequent in patients with some grade of heart failure, particularly cardiogenic shock. Figure 1 shows the difference in T3 values according to Killip-Kimball class. Conclusion Cardiomyocytes lack deiodinase and only possess T3 receptors, which makes them dependent on circulating T3 levels. T3 directly stimulates calcium channel and contractile protein synthesis in cardiomyocytes, and its deficit could affect cardiac contractility. Future studies should determine if thyroid hormone administration in cardiogenic shock can improve contractility and contribute to hemodynamic stability. T3 values according to Killip-Kimball Funding Acknowledgement Type of funding source: None

TIP30 counteracts cardiac hypertrophy and failure by inhibiting translational elongation.

Pathological cardiac overload induces myocardial protein synthesis and hypertrophy, which predisposes to heart failure. To inhibit hypertrophy therapeutically, the identification of negative regulators of cardiomyocyte protein synthesis is needed. Here, we identified the tumor suppressor protein TIP30 as novel inhibitor of cardiac hypertrophy and dysfunction. Reduced TIP30 levels in mice entailed exaggerated cardiac growth during experimental pressure overload, which was associated with cardiomyocyte cellular hypertrophy, increased myocardial protein synthesis, reduced capillary density, and left ventricular dysfunction. Pharmacological inhibition of protein synthesis improved these defects. Our results are relevant for human disease, since we found diminished cardiac TIP30 levels in samples from patients suffering from end‐stage heart failure or hypertrophic cardiomyopathy. Importantly, therapeutic overexpression of TIP30 in mouse hearts inhibited cardiac hypertrophy and improved left ventricular function during pressure overload and in cardiomyopathic mdx mice. Mechanistically, we identified a previously unknown anti‐hypertrophic mechanism, whereby TIP30 binds the eukaryotic elongation factor 1A (eEF1A) to prevent the interaction with its essential co‐factor eEF1B2 and translational elongation. Therefore, TIP30 could be a therapeutic target to counteract cardiac hypertrophy.

Poly(A) tail length regulates PABPC1 expression to tune translation in the heart.

The rate of protein synthesis in the adult heart is one of the lowest in mammalian tissues, but it increases substantially in response to stress and hypertrophic stimuli through largely obscure mechanisms. Here, we demonstrate that regulated expression of cytosolic poly(A)-binding protein 1 (PABPC1) modulates protein synthetic capacity of the mammalian heart. We uncover a poly(A) tail-based regulatory mechanism that dynamically controls PABPC1 protein synthesis in cardiomyocytes and thereby titrates cellular translation in response to developmental and hypertrophic cues. Our findings identify PABPC1 as a direct regulator of cardiac hypertrophy and define a new paradigm of gene regulation in the heart, where controlled changes in poly(A) tail length influence mRNA translation.

Qiliqiangxin Attenuates Phenylephrine-Induced Cardiac Hypertrophy through Downregulation of MiR-199a-5p

Background/Aims: Qiliqiangxin (QL), a traditional Chinese medicine, has long been used to treat chronic heart failure. Previous studies demonstrated that QL could prevent cardiac remodeling and hypertrophy in response to hypertensive or ischemic stress. However, little is known about whether QL could modulate cardiac hypertrophy in vitro, and (if so) whether it is through modulation of specific hypertrophy-related microRNA. Methods: The primary neonatal rat ventricular cardiomyocytes were isolated, cultured, and treated with phenylephrine (PE, 50 µmol/L, 48 h) to induce hypertrophy in vitro, in the presence or absence of pretreatment with QL (0.5 µg/ml, 48 h). The cell surface area was determined by immunofluorescent staining for α-actinin. The mRNA levels of hypertrophic markers including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (MYH7) were assayed by qRT-PCRs. The protein synthesis of cardiomyocytes was determined by the protein/DNA ratio. The miR-199a-5p expression level was quantified in PE-treated cardiomyocytes and heart samples from acute myocardial infarction (AMI) mouse model. MiR-199a-5p overexpression was used to determine its role in the anti-hypertrophic effect of QL on cardiomyocytes. Results: PE induced obvious enlargement of cell surface in cardiomyocytes, paralleling with increased ANP, BNP, and MYH7 mRNA levels and elevated protein/DNA ratio. All these changes were reversed by the treatment with QL. Meanwhile, miR-199a-5p was increased in AMI mouse heart tissues. Of note, the increase of miR-199a-5p in PE-treated cardiomyocytes was reversed by the treatment with QL. Moreover, overexpression of miR-199a-5p abolished the anti-hypertrophic effect of QL on cardiomyocytes. Conclusion: QL prevents PE-induced cardiac hypertrophy. MiR-199a-5p is increased in cardiac hypertrophy, while reduced by treatment with QL. miR-199a-5p suppression is essential for the anti-hypertrophic effect of QL on cardiomyocytes.

Differential ERK1/2 Signaling and Hypertrophic Response to Endothelin-1 in Cardiomyocytes from SHR and Wistar-Kyoto Rats: A Potential Target for Combination Therapy of Hypertension.

Extracellular signal regulated kinase½ (ERK1/2) signaling is critical to endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy. This study was to investigate ERK1/2 signaling and hypertrophic response to ET-1 stimulation in cardiomyocytes (CMs) from spontaneous hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Primary neonatal SHR and WKY CMs were exposed to ET-1 for up to 24 hrs. Minimal basal ERK1/2 phosphorylation was present in WKY CMs, while a significant baseline ERK1/2 phosphorylation was observed in SHR CMs. ET-1 induced a time- and dose-dependent increase in ERK1/2 phosphorylation in both SHR and WKY CMs. However, ET-1-induced ERK1/2 activation occurred much earlier with significantly higher peak phosphorylation level, and stayed elevated for longer duration in SHR CMs than that in WKY CMs. ET-1-induced hypertrophic response was more prominent in SHR CMs than that in WKY CMs as reflected by increased cell surface area, intracellular actin density, and protein synthesis. Pre-treatment with ERK1/2 phosphorylation inhibitor PD98059 completely prevented ET-1-induced ERK1/2 phosphorylation and increases in cell surface area and protein synthesis in SHR and WKY CMs. The specific PI3 kinase inhibitor LY294002 blocked ET-1-induced Akt and ERK1/2 phosphorylation, and protein synthesis in CMs. These data indicated that ERK1/2 signaling was differentially enhanced in CMs, and was associated with increased cardiac hypertrophic response to ET-1 in SHR. ET-1-induced ERK1/2 activation and cardiac hypertrophy appeared to be mediated via PI3 kinase/Akt signaling in SHR and WKY. The differential ERK1/2 activation in SHR CMs by ET-1 might represent a potential target for combination therapy of hypertension.

Testosterone increases GLUT4‐dependent glucose uptake in cardiomyocytes

Testosterone exerts important effects in the heart. Cardiomyocytes are target cells for androgens, and testosterone induces rapid effects via Ca(2+) release and protein kinase activation and long-term effects via cardiomyocyte differentiation and hypertrophy. Furthermore, it stimulates metabolic effects such as increasing glucose uptake in different tissues. Cardiomyocytes preferentially consume fatty acids for ATP production, but under particular circumstances, glucose uptake is increased to optimize energy production. We studied the effects of testosterone on glucose uptake in cardiomyocytes. We found that testosterone increased uptake of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-2-deoxyglucose and [(3) H]2-deoxyglucose, which was blocked by the glucose transporter 4 (GLUT4) inhibitor indinavir. Testosterone stimulation in the presence of cyproterone or albumin-bound testosterone-induced glucose uptake, which suggests an effect that is independent of the intracellular androgen receptor. To determine the degree of GLUT4 cell surface exposure, cardiomyocytes were transfected with the plasmid GLUT4myc-eGFP. Subsequently, testosterone increased GLUT4myc-GFP exposure at the plasma membrane. Inhibition of Akt by the Akt-inhibitor-VIII had no effect. However, inhibition of Ca(2+) /calmodulin protein kinase (CaMKII) (KN-93 and autocamtide-2 related inhibitory peptide II) and AMP-activated protein kinase (AMPK) (compound C and siRNA for AMPK) prevented glucose uptake induced by testosterone. Moreover, GLUT4myc-eGFP exposure at the cell surface caused by testosterone was also abolished after CaMKII and AMPK inhibition. These results suggest that testosterone increases GLUT4-dependent glucose uptake, which is mediated by CaMKII and AMPK in cultured cardiomyocytes. Glucose uptake could represent a mechanism by which testosterone increases energy production and protein synthesis in cardiomyocytes.

Activation of adenosine A1 receptor attenuates tumor necrosis factor-α induced hypertrophy of cardiomyocytes

Tumor necrosis factor (TNF)-α has been implicated in the pathogenesis of cardiac hypertrophy, while the activation of adenosine receptors has been shown to exert antihypertrophic effect on the heart. However, it remains unknown whether adenosine can attenuate hypertrophy induced by TNF-α. This study was aimed to address this issue using transverse aortic constriction (TAC) mouse models and cultured neonatal rat cardiomyocytes. Plasma TNF-α was significantly increased in hypertrophied hearts (Sham vs TAC group: 46.8 ± 2.5 vs 67.0 ± 1.6 pg/ml, P = 0.021), while myocardial TNF-α level, expression of TNF receptor 1 and TNF-α-converting enzyme were positively correlated with heart weight to body weight ratio ( r = 0.930, 0.676 and 0.891, respectively, P < 0.01–0.05). Myocardial adenosine levels were increased significantly at 4 weeks (Sham vs TAC group: 16.15 ± 1.59 vs 86.54 ± 13.49 nmol/mg protein, P < 0.01) and decreased from 6 to 11 weeks after TAC. N6-cyclopentyladenosine, an adenosine A1 receptor agonist inhibited protein synthesis of cardiomyocytes induced by TNF-α in a dose-dependent manner. This antihypertrophic effect could not be mimicked by agonists of A2a, A2b and A3 adenosine receptors. These findings indicate that TNF-α signal system plays important role in the process of cardiac hypertrophy, and activation of adenosine receptor 1 inhibits hypertrophy of cardiomyocytes induced by TNF-α.

Effects of Angiotensin III on Protein, DNA, and Collagen Synthesis of Neonatal Cardiomyocytes and Cardiac Fibroblasts In Vitro

This study compared angiotensin II (Ang II) and angiotensin III (Ang III) for their effects on rat neonatal cardiomyocytes and cardiac fibroblasts in vitro and discussed the possible role of Ang III in the pathogenesis of cardiac remodeling. To do so, protein synthesis, cardiac fibroblast proliferation, collagen synthesis, and secretion in response to treatment with Ang III and Ang II were investigated. Protein synthesis rate was assessed by (3)H-Leucine ((3)H-Leu) incorporation; the content of DNA was defined by (3)H-thymidine ((3)H-TdR) incorporation; and collagen synthesis and secretion were assessed by ( 3)H-proline ((3)H-Pro) incorporation. In neonatal cardiomyocytes, Ang III stimulated protein synthesis in a concentration-dependent manner, whereas in neonatal cardiac fibroblasts, DNA synthesis as well as collagen synthesis and secretion were increased in a concentration-dependent manner. Treatment with captopril, selective aminopeptidase A (APA) inhibitor (EC33), or selective aminopeptidase N inhibitor (PC18) had no effect on these outcomes. Treatment with losartan significantly decreased the effects of Ang III, except for cardiomyocyte protein synthesis. Compared with Ang II, Ang III could stimulate cardiomyocyte protein synthesis, cardiac fibroblast proliferation, and collagen synthesis and secretion. Furthermore, 10(-7) mol/L Ang II but not Ang III significantly increased APA activity in both cardiomyocytes and fibroblasts. All these results show the bioactive effects of Ang III on myocardial cells and suggest that Ang III could be an important independent factor besides Ang II in the regulation of cardiac function and may affect the pathogenesis of cardiac remodeling.

Role of autophagy in heart failure associated with aging

Heart failure is a progressive disease, leading to reduced quality of life and premature death. Adverse ventricular remodeling involves changes in the balance between cardiomyocyte protein synthesis and degradation, forcing these myocytes in equilibrium between life and death. In this context, autophagy has been recognized to play a role in the pathophysiology of heart failure. At basal levels, autophagy performs housekeeping functions, maintaining cardiomyocyte function and ventricular mass. Autophagy also occurs in the failing human heart, and upregulation has been reported in animal models of pressure overload-induced heart failure. Although the factors that determine whether autophagy will be protective or detrimental are not well known, the level and duration of autophagy seem important. Autophagy may antagonize ventricular hypertrophy by increasing protein degradation, which decreases tissue mass. However, the rate of protective autophagy declines with age. The inability to remove damaged structures results in the progressive accumulation of 'garbage', including abnormal intracellular proteins aggregates and undigested materials such as lipofuscin. Eventually, the progress of these changes results in enhanced oxidative stress, decreased ATP production, collapse of the cellular catabolic machinery, and cell death. By contrast, in load-induced heart failure, the extent of autophagic flux can rise to maladaptive levels. Excessive autophagy induction leads to autophagic cell death and loss of cardiomyocytes and may contribute to the worsening of heart failure. Accordingly, the development of therapies that up-regulate the repair qualities of the autophagic process and down-regulate the cell death aspects would be of great value in the treatment of heart failure.

UAP56 is an important regulator of protein synthesis and growth in cardiomyocytes

UAP56, an ATP dependent RNA helicase that also has ATPase activity, is a DExD/H box protein that is phylogenetically grouped with the eukaryotic initiation factor eIF4A, the prototypical member of the DExD/H box family of helicases. UAP56, also known as BAT1, is an essential RNA splicing factor required for spliceosome assembly and mRNA export but its role in protein synthesis is not known. Here we demonstrate that UAP56 regulates protein synthesis and growth in cardiomyocytes. We found that wild-type (WT) UAP56 increased serum induced protein synthesis in HeLa cells. UAP56 mutants lacking ATPase and/or helicase activity inhibited protein synthesis compared with WT UAP56, suggesting that the ATPase and RNA helicase activity of UAP56 is important for protein synthesis. UAP56 siRNA inhibited phenylephrine (PE) induced protein synthesis in cardiomyocytes and inhibited PE induced cardiomyocyte hypertrophy. Our data demonstrate that UAP56 is an important regulator of protein synthesis and plays an important role in the regulation of cardiomyocyte growth.

Blocking eukaryotic initiation factor 4F complex formation does not inhibit the mTORC1-dependent activation of protein synthesis in cardiomyocytes

Activation of the mammalian target of rapamycin complex 1 (mTORC1) causes the dissociation of eukaryotic initiation factor 4E complex (eIF4E)-binding protein 1 (4E-BP1) from eIF4E, leading to increased eIF4F complex formation. mTORC1 positively regulates protein synthesis and is implicated in several diseases including cardiac hypertrophy, a potentially fatal disorder involving increased cardiomyocyte size. The importance of 4E-BP1 in mTORC1-regulated protein synthesis was investigated by overexpressing 4E-BP1, which blocks eIF4F formation in isolated primary cardiomyocytes without affecting other targets for mTORC1 signaling. Interestingly, blocking eIF4F formation did not impair the degree of activation of overall protein synthesis by the hypertrophic agent phenylephrine (PE), which, furthermore, remained dependent on mTORC1. Overexpressing 4E-BP1 also only had a small effect on PE-induced cardiomyocyte growth. Overexpressing 4E-BP1 did diminish the PE-stimulated synthesis of luciferase encoded by structured mRNAs, confirming that such mRNAs do require eIF4F for their translation in cardiomyocytes. These data imply that the substantial inhibition of cardiomyocyte protein synthesis and growth caused by inhibiting mTORC1 cannot be attributed to the activation of 4E-BP1 or loss of eIF4F complexes. Our data indicate that increased eIF4F formation plays, at most, only a minor role in the mTORC1-dependent activation of overall protein synthesis in these primary cells but is required for the translation of structured mRNAs. Therefore, other mTORC1 targets are more important in the inhibition by rapamycin of the rapid activation of protein synthesis and of cell growth.

Liver X Receptor activator inhibits AngII induced hypertrophic responses in rat neonatal cardiomyocytes

The progression of cardiac hypertrophy and heart failure involves a maladaptation in energy homeostasis. Proteins involved in this key energy metabolic pathway aretranscriptionally regulated by members of the nuclear receptorsuperfamily, such as PPARs and ERRs. The Liver X Receptors (LXRα and LXRβ) are members ofthe nuclear hormone receptor superfamily. These sterol‐responsivetranscription factors regulate the expression of a number ofgenes involved in lipid and glucose metabolism. We hypothesize that LXRs play important roles in regulating the adaptive substrate switch in cardiac hypertrophy and heart failure. We investigated how a LXR synthetic ligand T1317 influences AngII induced hypertrophic responses in cultured rat neonatal cardiomyocytes. We found AngII can induce upregulation of LXRalpha transcript levels in cultured cardiomyocytes (n=4, P&lt;0.05). Pretreatment of a LXR T1317 (1, 5, 10 uM) in cultured cardiomyocytes 2 hours prior to the 24 hour AngII treatment prevented hypertrophic responses. T1317 pretreatment suppressed an AngII induced elevation of protein synthesis in cardiomyocytes (n=6, P&lt;0.05). Furthermore, T1317 pretreatment prevented an AngII induced increase of cell surface size (n=25, P&lt;0.05). T1317 pretreatment also suppressed the expression of hypertorphic marker ANF induced by AngII treatment. Therefore, we conclude that LXRs play important roles in regulating cardiomyocyte growth, possibly by regulating intracellular energy balance.

Reduction of protein synthesis and statin-induced cardiomyocyte cell death

The objective of this study was to determine whether an HMG Co A reductase inhibitor (statin) reduces protein synthesis in cardiomyocytes and whether this action maybe an underlying mechanism for statin-induced cell death. Cardiomyocytes from embryonic chick heart were maintained in culture. Cells exposed to lovastatin for 4 h showed a concentration dependent reduction in protein synthesis as assessed by [3H] leucine incorporation and [35S] methionine incorporation. Compared to control, lovastatin 100 microM, which produced a 25% increase in cell death, induced a three-fold reduction in methionine incorporation. [35S] methionine autoradiography showed little (new) protein synthesis at concentrations of lovastatin of 70 microM or higher; an effect that was not limited to specific proteins. Cardiomyocytes treated with lovastatin showed morphologic changes in the nucleoli consistent with insufficient protein synthesis. These cardiomyocytes manifested cell death under conditions of reduced protein synthesis. Interruption of protein synthesis with cycloheximide, a ribosomal RNA transcription inhibitor or reduction in protein substrate availability by lowering the media concentration of fetal calf serum was associated with a concentration-dependent reductions in cell viability. Importantly, stimulation of protein synthesis by higher concentrations of fetal calf serum limited lovastatin-induced cell death. These data suggest that statin-induced inhibition of protein synthesis is an underlying mechanism for statin-induced cell death.

High- but not low-molecular weight FGF-2 causes cardiac hypertrophy in vivo; possible involvement of cardiotrophin-1

The heart expresses high and low molecular weight (hmw, lmw) fibroblast growth factor 2 (FGF-2) isoforms. While the injury-repair-related activities of lmw-FGF-2 have been studied extensively, those of hmw-FGF-2 have not. Thus, we investigated the effects of hmw-FGF-2 on acute as well as chronic responses to myocardial infarction (MI) induced by irreversible coronary occlusion in the rat. Hmw- or lmw-FGF-2 was injected into the ischemic zone during acute evolving MI. Both isoforms were equally effective in reducing infarct size (at 24 h post-MI) and improving heart function up to 6 weeks post-MI, compared to a vehicle-treated infarcted group. Lmw-FGF-2 alone upregulated vascularization in the infarct. Hmw-FGF-2 elicited significant hypertrophy, compared to the vehicle-treated group, at 4–8 weeks post-MI, assessed by ultrasound, heart morphometry and cardiomyocyte cross-sectional area. In addition, hmw- (but not lmw-) FGF-2-treated hearts displayed increased accumulation of the cytokine cardiotrophin-1 and its signal transducer gp130. In culture, hmw- (but not lmw-) FGF-2 increased cardiomyocyte protein synthesis and cell size as well as upregulated cardiotrophin-1 released by cardiac fibroblasts, pointing to similar activities in vivo. Thus, hmw- and lmw-FGF-2 exert isoform-specific effects in the heart and only hmw-FGF-2 triggers cardiomyocyte hypertrophic growth. Direct effects of hmw-FGF-2 on cardiomyocytes, becoming reinforced and sustained by upregulation of cardiotrophin-1 and acting in concert with other factors, are likely to contribute to post-MI hypertrophy.

Thyroid hormone and Na+-K+-ATPase: more than simple transcription

FLUID SECRETION INTO THE DEVELOPING lungs’ air spaces is essential for normal lung development, but this fluid must be cleared at birth so that effective gas exchange can occur. Impaired clearance of this fetal lung liquid, which arises from immaturity of the distal lung epithelium’s active Na transport system (for review, see Ref. 17), causes transient tachypnea of the newborn (2, 12, 13), and, if combined with immaturity of the surfactant system, neonatal respiratory distress syndrome (nRDS) (4, 17). Clearance of air space fluid is driven by active transepithelial Na transport resulting from synchronized activity of apical Na permeant ion channels (e.g., epithelial Na channel, ENaC) and basolateral Na-K-ATPase. The chemical and electrical gradients for Na absorption are, respectively, maintained by Na-K-ATPase and K channels. Perinatal changes in PO2, glucocorticoids, -agonists, and thyroid hormones interact to increase transepithelial Na absorption, to bring about the switch from net fluid secretion to net absorption as lung liquid is cleared at birth. However, despite considerable data from molecular, transgenic, cell culture, and in vivo studies, the fundamental mechanisms of this crucial developmental shift are incompletely understood (5, 19). Barker and coworkers (6) demonstrated that thyroid hormone [triiodothyronine (T3)] played an important role in maturation of the fetal lungs’ ability to respond to an infusion of epinephrine by switching to fluid absorption, and others showed that thyroid hormone in concert with glucocorticoids increased the expression of ENaC (18, 22). Although T3 classically acts as a direct transcriptional activator of target genes and has been shown to potentiate dexamethasone-stimulated transcription of the ENaC promoter (20), T3-induced upregulation of Na-K-ATPase activity in adult rat alveolar epithelial cells was recently shown to be insensitive to actinomycin D (15). This has functional significance. For example, it has been demonstrated that overexpression of Na-KATPase via adenoviral-mediated gene transfer is sufficient to increase alveolar fluid clearance (21). It is known that thyroid hormones have profound effects on the maturation of the fetal lung and that premature infants with nRDS have lower thyroid hormone levels than well preterm infants (9, 10). However, when clinical trials have been conducted wherein augmentation of the thyroid hormonal axis has taken place, there has been no demonstrable benefit. The THORN trial of T3 and hydrocortisone supplementation in preterm infants of less than 30 wk gestation found that low thyroid hormone levels were associated with higher mortality and ventilator dependence (7, 8). However, this trial (8) was unable to demonstrate beneficial effects of T3 and hydrocortisone infusion. Similarly, the provision of thyroid-releasing hormone to infants who had received antenatal glucocorticoid

C-Jun N-terminal Kinase-mediated Stabilization of Microsomal Prostaglandin E2 Synthase-1 mRNA Regulates Delayed Microsomal Prostaglandin E2 Synthase-1 Expression and Prostaglandin E2 Biosynthesis by Cardiomyocytes

Microsomal prostaglandin (PG) E(2) synthase-1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE(2), a key proinflammatory mediator. The purpose of this study was to elucidate the regulation of mPGES-1 mRNA expression in cardiomyocytes, define the role of JNK enzymes in this process, and characterize the role of mPGES-1 in cardiomyocyte PGE(2) biosynthesis. In neonatal cardiomyocytes, interleukin-1beta and lipopolysaccharide (LPS) both stimulated mPGES-1 mRNA expression and increased mPGES-1 mRNA stability and protein synthesis but failed to increase mPGES-1 mRNA transcription. Treatment with the JNK1/2 inhibitor, SP600125, abrogated the increases in mPGES-1 mRNA stability, mPGES-1 protein synthesis, and PGE(2) release induced by interleukin-1beta or LPS. mPGES-1 protein synthesis was observed in LPS-stimulated neonatal cardiomyocytes from jnk1(-/-) or jnk2(-/-) mice. In contrast, infection of jnk1(-/-) cardiomyocytes with an adenovirus encoding phosphorylation-resistant JNK2 (ad-JNK2-DN), or of jnk2(-/-) cardiomyocytes with ad-JNK1-DN, significantly decreased LPS-stimulated mPGES-1 protein synthesis. Similarly, co-infection with ad-JNK1-DN and ad-JNK2-DN attenuated LPS-stimulated mPGES-1 protein synthesis in cardiomyocytes from wild type mice. Targeted deletion of the gene encoding mPGES-1 led to a 3.2-fold decrease in LPS-stimulated PGE(2) release by cardiomyocytes in comparison with wild type cells but had no effect on COX-1, COX-2, mPGES-2, or cytosolic PGES mRNA levels. These studies provide direct evidence that mPGES-1 mRNA levels in cardiomyocytes are augmented by stabilization of mPGES-1 mRNA, that JNK1 or JNK2 can participate in the regulation of mPGES-1 protein synthesis in these cells, and that mPGES-1 catalyzes the majority of LPS-induced PGE(2) biosynthesis by cardiomyocytes.