Expression Of Fetal Cardiac Genes Research Articles

Genome Editing and Diabetic Cardiomyopathy.

Differential gene expression is associated with diabetic cardiomyopathy (DMCM) and culminates in adverse remodeling in the diabetic heart. Genome editing is a technology utilized to alter endogenous genes. Genome editing also provides an option to induce cardioprotective genes or inhibit genes linked to adverse cardiac remodeling and thus has promise in ameliorating DMCM. Non-coding genes have emerged as novel regulators of cellular signaling and may serve as potential therapeutic targets for DMCM. Specifically, there is a widespread change in the gene expression of fetal cardiac genes and microRNAs, termed genetic reprogramming, that promotes pathological remodeling and contributes to heart failure in diabetes. This genetic reprogramming of both coding and non-coding genes varies with the progression and severity of DMCM. Thus, genetic editing provides a promising option to investigate the role of specific genes/non-coding RNAs in DMCM initiation and progression as well as developing therapeutics to mitigate cardiac remodeling and ameliorate DMCM. This chapter will summarize the research progress in genome editing and DMCM and provide future directions for utilizing genome editing as an approach to prevent and/or treat DMCM.

Maresin-1 induces cardiomyocyte hypertrophy through IGF-1 paracrine pathway.

The resolution of inflammation is closely linked with tissue repair. Recent studies have revealed that macrophages suppress inflammatory reactions by producing lipid mediators, called specialized proresolving mediators (SPMs); however, the biological significance of SPMs in tissue repair remains to be fully elucidated in the heart. In this study, we focused on maresin-1 (MaR1) and examined the reparative effects of MaR1 in cardiomyocytes. The treatment with MaR1 increased cell size in cultured neonatal rat cardiomyocytes. Since the expression of fetal cardiac genes was unchanged by MaR1, physiological hypertrophy was induced by MaR1. SR3335, an inhibitor of retinoic acid-related orphan receptor α (RORα), mitigated MaR1-induced cardiomyocyte hypertrophy, consistent with the recent report that RORα is one of MaR1 receptors. Importantly, in response to MaR1, cardiomyocytes produced IGF-1 via RORα. Moreover, MaR1 activated phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and wortmannin, a PI3K inhibitor, or triciribine, an Akt inhibitor, abrogated MaR1-induced cardiomyocyte hypertrophy. Finally, the blockade of IGF-1 receptor by NVP-AEW541 inhibited MaR-1-induced cardiomyocyte hypertrophy as well as the activation of PI3K/Akt pathway. These data indicate that MaR1 induces cardiomyocyte hypertrophy through RORα/IGF-1/PI3K/Akt pathway. Considering that MaR1 is a potent resolving factor, MaR1 could be a key mediator that orchestrates the resolution of inflammation with myocardial repair.

Effects of chronically increased VEGF-A on the aging heart.

Whether approaches to chronically increase VEGF-A in the heart may have beneficial effects and prevent the development of heart failure, in part by improving cardiac perfusion, or whether this increase could have detrimental effects on cardiac performance in the aging heart, has not been tested yet. In this study, a genetic mouse model with a chronic increase in VEGF-A in the heart is shown to have increased cardiac angiogenesis and develop cardiac hypertrophy with enhanced basal cardiac performance with age progression. However, in aged hearts, this increase in VEGF-A was associated with higher expression of fetal cardiac genes and reduced cardiac performance after β-agonistic stress, features consistent with pathologic cardiac hypertrophy. Expression of Nod-like receptor protein (NLRP)-3 was increased in the hearts of the mice, and its genetic inactivation prevented increased fetal cardiac gene expression and partially rescued the impaired cardiac performance after β-agonistic stimulation in aged hearts without reducing cardiac angiogenesis or hypertrophy. Thus, although a chronic increase in cardiac VEGF-A may improve cardiac perfusion, long-term upregulation of VEGF-A leads to reduced cardiac performance under stress, an effect that can be partially inhibited by NLRP3 inactivation. Targeting NLRP3 shifts the VEGF-A-induced cardiac hypertrophy from a pathologic toward a more physiologic hypertrophy.-Marneros, A. G. Effects of chronically increased VEGF-A on the aging heart.

The actin-organizing formin protein Fhod3 is required for postnatal development and functional maintenance of the adult heart in mice

Cardiac development and function require actin-myosin interactions in the sarcomere, a highly organized contractile structure. Sarcomere assembly mediated by formin homology 2 domain-containing 3 (Fhod3), a member of formins that directs formation of straight actin filaments, is essential for embryonic cardiogenesis. However, the role of Fhod3 in the neonatal and adult stages has remained unknown. Here, we generated floxed Fhod3 mice to bypass the embryonic lethality of an Fhod3 knockout (KO). Perinatal KO of Fhod3 in the heart caused juvenile lethality at around day 10 after birth with enlarged hearts composed of severely impaired myofibrils, indicating that Fhod3 is crucial for postnatal heart development. Tamoxifen-induced conditional KO of Fhod3 in the adult heart neither led to lethal effects nor did it affect sarcomere structure and localization of sarcomere components. However, adult Fhod3-deleted mice exhibited a slight cardiomegaly and mild impairment of cardiac function, conditions that were sustained over 1 year without compensation during aging. In addition to these age-related changes, systemic stimulation with the α1-adrenergic receptor agonist phenylephrine, which induces sustained hypertension and hypertrophy development, induced expression of fetal cardiac genes that was more pronounced in adult Fhod3-deleted mice than in the control mice, suggesting that Fhod3 modulates hypertrophic changes in the adult heart. We conclude that Fhod3 plays a crucial role in both postnatal cardiac development and functional maintenance of the adult heart.

Midkine Deteriorates Cardiac Remodeling via Epidermal Growth Factor Receptor Signaling in Chronic Kidney Disease.

In chronic kidney disease, activation of the epidermal growth factor receptor (EGFR) leads to cardiac hypertrophy, which affects morbidity and mortality. In patients with renal insufficiency and heart failure, the expression of midkine, a heparin-binding growth factor, is increased. Therefore, we investigated the association between midkine and EGFR in the induction of cardiac hypertrophy and dysfunction in chronic kidney disease. We performed subtotal nephrectomies in midkine-knockout mice and wild-type mice. We found that subtotal nephrectomy-induced cardiac hypertrophy and phosphorylation of extracellular signal-regulated kinase 1/2 and AKT were attenuated in midkine-knockout mice compared with wild-type mice. An antiphosphotyrosine receptor antibody array was used to demonstrate that EGFR phosphorylation in the heart was also lower in midkine-knockout mice than in wild-type mice. Midkine induced EGFR, extracellular signal-regulated kinase 1/2, and AKT phosphorylation and led to hypertrophy in neonatal rat cardiomyocytes. Pretreatment with EGFR inhibitors or EGFR silencing suppressed midkine-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 and AKT, induction of fetal cardiac gene expression, and hypertrophy in cardiomyocytes. To confirm the association between midkine and EGFR in vivo, mice subjected to subtotal nephrectomy were treated with the EGFR inhibitor gefitinib. Gefitinib treatment attenuated subtotal nephrectomy-induced cardiac hypertrophy. These results indicate that midkine might be a key mediator of cardiorenal interactions through EGFR activation, which plays a crucial role in cardiac hypertrophy in chronic kidney disease.

Role of Histone Demethylases in Cardiomyocytes Induced to Hypertrophy.

Epigenetic changes induced by histone demethylases play an important role in differentiation and pathological changes in cardiac cells. However, the role of the jumonji family of demethylases in the development of cardiac hypertrophy remains elusive. In this study, the presence of different histone demethylases in cardiac cells was evaluated after hypertrophy was induced with neurohormones. A cell line from rat cardiomyocytes was used as a biological model. The phenotypic profiles of the cells, as well as the expression of histone demethylases, were studied through immunofluorescence, transient transfection, western blot, and qRT-PCR analysis after inducing hypertrophy by angiotensin II and endothelin-1. An increase in fetal gene expression (ANP, BNP, and β-MHC) was observed in cardiomyocytes after treatment with angiotensin II and endothelin-1. A significant increase in JMJD2A expression, but not in UTX or JMJD2C expression, was observed. When JMJD2A was overexpressed in cardiomyocytes through transient transfection, the effect of neurohormones on fetal cardiac gene expression was increased. We conclude that JMJD2A plays a principal role in the regulation of fetal cardiac genes, which increase in expression during the pathological hypertrophic process.

Abstract 15317: Deletion of Low Molecular Weight Protein Tyrosine Phosphatase (Acp1) Protects Against Stress-induced Cardiomyopathy

Background: The low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene, is a ubiquitously expressed phosphatase whose in vivo function remains poorly understood. Genetic studies revealed an association between ACP1 and a variety of human cardiovascular disorders. However, evidence of a direct involvement of LMPTP in cardiac diseases is still lacking. Methodology: We generated the first mouse model of Lmptp deficiency and studied the response of the mice under basal condition or to long-term pressure overload using comprehensive experimental approaches such as q-PCR, siRNA technology, fluorescent microscopy and immuneprecipitation technique. Results: Acp1 -/- mice develop normally, and aging mice do not show any obvious signs of pathology in major tissues under basal condition. However, Acp1 -/- mice are strikingly resistant to pressure overload hypertrophy and heart failure. Lmptp expression is high in the embryonic mouse heart, decreased in the post-natal stage, and increased in the adult mouse and human failing heart. Consistent with their protected phenotype, Acp1 -/- mice subjected to pressure overload hypertrophy have attenuated fibrosis and decreased expression of hypertrophic markers. Transcriptional profiling and analysis of molecular signaling shows that the resistance of Acp1 -/- mice to pathological cardiac stress correlates with marginal expression of fetal cardiac genes, increased insulin receptor beta phosphorylation and inactivation of CaMKIIδ pathway. Conclusion: Our data show that ablation of Lmptp inhibits pathological cardiac remodeling and suggest that inhibition of LMPTP may be of therapeutic relevance for the treatment of heart failure in human.

Growth Factor Midkine Deteriorates Cadiac Hypertrophy Via Epidermal Growth Factor Receptor Signaling: A Novel Mediator of Cardio-renal Interaction

Background: Midkine (MK), a 13-kDa heparin-binding growth factor, has shown to be up-regulated in injured renal tubule, and released into circulation. We recently reported that cardiac-specific overexpression of MK exacerbates cardiac hypertrophy in mice. However, it remains unknown by which mechanisms MK affects on heart. Although receptors for MK are thought to be receptor-type tyrosine kinase, specific receptor for MK has not been determined. Here we show that MK acts by activating the epidermal growth factor receptor (EGFR) and its downstream signaling and leads to cardiac hypertrophy. Methods and Results: MK phosphorylated EGFR (Tyr1068) on neonatal rat cardiomyocytes. MK also promoted phosphorylation of ERK1/2 and Akt, induced fetal cardiac gene expression, and led to cardiomyocyte hypertrophy. Pre-treatment with EGFR inhibitors attenuated MK-induced ERK1/2 and Akt phosphorylation, and induction of fetal cardiac gene, as well as cardiomyocyte hypertrophy in neonatal rat cardiomyocytes. EGFR silencing by siRNA also attenuated MK-induced cardiomyocyte hypertrophy. Next, we examine whether the action of MK mediates EGFR transactivation using TAPI-2, a metalloproteinase inhibitor, to inhibit EGFR transactivation. Pre-treatment with TAPI-2 did not affect MK-induced elevated cardiac fetal gene activity and activation of EGFR, suggesting that MK may directly phosphorylated EGFR rather than mediating EGFR transactivation. For in vivo study, subtotal nephrectomy was created with MK systemic knockout (MK-KO) mice and littermate wild-type (WT) mice. Subtotal nephrectomy induced cardiac hypertrophy and poor survival rate in WT mice, whereas cardiac hypertrophy and survival rate was improved in MK-KO mice compared with WT mice. Conclusions: We demonstrated for the first time that EGFR signaling plays a crucial role in MK-induced cardiac hypertrophy, and MK is thought to be one of key molecule mediating cardio-renal interaction.

Kruppel-like Factor 4 Protein Regulates Isoproterenol-induced Cardiac Hypertrophy by Modulating Myocardin Expression and Activity

Kruppel-like factor 4 (KLF4) plays an important role in vascular diseases, including atherosclerosis and vascular injury. Although KLF4 is expressed in the heart in addition to vascular cells, the role of KLF4 in cardiac disease has not been fully determined. The goals of this study were to investigate the role of KLF4 in cardiac hypertrophy and to determine the underlying mechanisms. Cardiomyocyte-specific Klf4 knockout (CM Klf4 KO) mice were generated by the Cre/LoxP technique. Cardiac hypertrophy was induced by chronic infusion of the β-adrenoreceptor agonist isoproterenol (ISO). Results showed that ISO-induced cardiac hypertrophy was enhanced in CM Klf4 KO mice compared with control mice. Accelerated cardiac hypertrophy in CM Klf4 KO mice was accompanied by the augmented cellular enlargement of cardiomyocytes as well as the exaggerated expression of fetal cardiac genes, including atrial natriuretic factor (Nppa). Additionally, induction of myocardin, a transcriptional cofactor regulating fetal cardiac genes, was enhanced in CM Klf4 KO mice. Interestingly, KLF4 regulated Nppa expression by modulating the expression and activity of myocardin, providing a mechanical basis for accelerated cardiac hypertrophy in CM Klf4 KO mice. Moreover, we showed that KLF4 mediated the antihypertrophic effect of trichostatin A, a histone deacetylase inhibitor, because ISO-induced cardiac hypertrophy in CM Klf4 KO mice was attenuated by olmesartan, an angiotensin II type 1 antagonist, but not by trichostatin A. These results provide novel evidence that KLF4 is a regulator of cardiac hypertrophy by modulating the expression and the activity of myocardin.

Transcriptional Regulation of the Fetal Cardiac Gene Program

Reactivation of the fetal cardiac gene program in adults is a reliable marker of cardiac hypertrophy and heart failure. Normally, genes within this group are expressed in the fetal ventricles during development, but are silent after birth. However, their expression is re-induced in the ventricular myocardium in response to various cardiovascular diseases, and potentially plays an important role in the pathological process of cardiac remodeling. Thus, analysis of the molecular mechanisms that govern the expression of fetal cardiac genes could lead to the discovery of transcriptional regulators and signaling pathways involved in both cardiac differentiation and cardiac disease. In this review we will summarize what is currently known about the transcriptional regulation of the fetal cardiac gene program.

Self-beating Atypically Shaped Cardiomyocytes Survive a Long-term Postnatal Development While Preserving the Expression of Fetal Cardiac Genes in Mice

The present study was designed to examine the postnatal developmental changes of atypically shaped cardiomyocytes (ACMs) prepared from the heart of newborn [postnatal day 1 (day-1)] through aged (12-month-old) mice. ACMs were identified as a novel type of self-beating cardiomyocyte with a peculiar morphology in mouse cardiac ventricles. The cell length of ACMs significantly increased during the first three postnatal months and further increased over the following 9 months. In contrast, the population of ACMs was significantly decreased within the first 5 weeks and reached a plateau in the adult stage. ACMs obtained from newborn and adult mice exhibited similar spontaneous action potentials. The expression of the fetal cardiac gene products atrial natriuretic peptide and voltage-gated T-type Ca(2+) channel Ca(V)3.2 was confirmed by immunostaining in ACMs obtained from both newborn and aged mice. These observations provide evidence that ACMs that exhibit spontaneous beating survive the long-term postnatal development of cardiac ventricles while preserving the expression of fetal cardiac genes. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Abstract 5658: Immune Reconstitution Prevents Autoimmune Inflammation and Right Ventricular Remodeling in Immunodeficiency-Associated Pulmonary Hypertension

Pulmonary arterial hypertension (PH) is a frequently lethal condition commonly associated with autoimmune conditions. Because autoimmunity is closely linked to immunodeficiency, we utilize athymic (AT) nude rats lacking normal T cells to demonstrate how PH is accentuated by a lack of normal regulatory T cell activity. The purpose of this study was to determine whether immune reconstitution (IR) prevents autoimmune inflammation (AI), right ventricular (RV) remodeling and PH using inbred AT rats reconstituted with euthymic spleen cells. Methods and Results: Inbred WAG AT nude rats were given a single injection of vascular endothelial growth factor receptor (VEGFR) antagonist SU5416 (20mg/kg sc), AT+SU group. Control AT was treated with vehicle only (DMSO). In adoptive transfer IR experiments, AT rats received unfractionated 20×10 6 euthymic rat spleen cells, CD4+T cells, CD8+T cells, CD4+CD25+T cells isolated with magnetic cell sorting before or after SU5416 administration. 2D-Echo, pulmonary hemodynamics and RV/LV+S ratio were assessed at 10d and 21d. Echo revealed decreased RV fractional shortening (p<0.05) and PA acceleration time (p<0.005) in AT+SU compared with IR and controls. IR prevented increase of RVSP compared to AT+SU (P<0.05) and of RV/LV+S (P<0.001). RV fetal cardiac gene expression (a-MHC, b-MHC, SERCA, BNP, SkACT, ANP) was consistent with pathologic stress pattern injury pattern and was significantly attenuated by IR. Furthermore, IR reduced expression of Th1, TH2 cytokines and pulmonary infiltration of macrophages, mast cells, B cells, IgG deposition and up-regulated lung BMPR2, FOXP3+, c-kit+, HOX11+ expression with increased c-kit+ and BMPR2 expression being noted in the pulmonary vascular endothelium. Conclusions: This study demonstrates that T cell deficiency is permissive for PH development and that IR prevents hemodynamic RV remodeling, attenuates AI and limits PH. Interestingly, that PH prevention is associated with increased regenerative HOX11+, C-kit stem cell populations.

Class II HDACs mediate CaMK-dependent signaling to NRSF in ventricular myocytes

We recently reported that a transcriptional repressor, neuron-restrictive silencer factor (NRSF), represses expression of fetal cardiac genes, including atrial and brain natriuretic peptide (ANP and BNP), by recruiting class I histone deacetylase (HDAC) and that attenuation of NRSF-mediated repression contributes to the reactivation of fetal gene expression during cardiac hypertrophy. The molecular mechanism by which the activity of the NRSF–HDAC complex is inhibited in cardiac hypertrophy remains unresolved, however. In the present study, we show that class II HDACs (HDAC4 and 5), which are Ca/calmodulin-dependent kinase (CaMK)-responsive repressors of hypertrophic signaling, associate with NRSF and participate in NRSF-mediated repression. Blockade of the CaMK-class II HDAC signaling pathway using a CaMK-resistant HDAC5 mutant, a CaMK inhibitor (KN62) or a dominant-negative CaMK mutant inhibited ET-1-inducible ANP and BNP promoter activity, but that inhibitory effect was abolished by mutation of the neuron-restrictive silencer element (NRSE) within the ANP and BNP promoter. In addition, adenovirus-mediated expression of a dominant-negative NRSF mutant abolished the inhibitory effect of KN62 on ET-1-inducible endogenous ANP gene expression in ventricular myocytes. Finally, the interaction between NRSF and class II HDACs was decreased in both in vitro and in vivo models of cardiac hypertrophy. These findings show that ET-1-induced CaMK signaling disrupts class II HDAC–NRSF repressor complexes, thereby enabling activation of ANP and BNP gene transcription in ventricular myocytes, and shed light on a novel mechanism by which the fetal cardiac gene program is reactivated.

Toxicogenomic Profile of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin in the Murine Fetal Heart: Modulation of Cell Cycle and Extracellular Matrix Genes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and similar environmental contaminants have been demonstrated to be potent cardiovascular teratogens in developing piscine and avian species. In the present study, we investigated the effects of TCDD on gene expression during murine cardiovascular development. C57Bl6N pregnant mice were dosed with 1.5, 3.0, or 6.0 microg TCDD/kg on gestational day (GD) 14.5, and microarray analysis was used to characterize the global changes in fetal cardiac gene expression on GD 17.5. TCDD significantly altered expression of a number of genes involved in xenobiotic metabolism, cardiac homeostasis, extracellular matrix production/remodeling, and cell cycle regulation. Interestingly, while the AhR-responsive genes Cyp1A1, Cyp1B1, Ugt1a6, and Ahrr, were all induced by TCDD in the fetal murine heart, other AhR-responsive genes, Cyp1a2, Nqo1, and Gsta1, were not. Quantitative real-time polymerase chain reactions confirmed the changes in expression of several G1/S-type cyclins and extracellular matrix-related genes. These results demonstrate the global changes in cardiac gene expression that result from TCDD exposure of the fetal murine heart and implicate genes involved in cell cycle and extracellular matrix regulation in TCDD-induced cardiac teratogenicity and functional deficits.

NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function.

Reactivation of the fetal cardiac gene program is a characteristic feature of hypertrophied and failing hearts that correlates with impaired cardiac function and poor prognosis. However, the mechanism governing the reversible expression of fetal cardiac genes remains unresolved. Here we show that neuron-restrictive silencer factor (NRSF), a transcriptional repressor, selectively regulates expression of multiple fetal cardiac genes, including those for atrial natriuretic peptide, brain natriuretic peptide and alpha-skeletal actin, and plays a role in molecular pathways leading to the re-expression of those genes in ventricular myocytes. Moreover, transgenic mice expressing a dominant-negative mutant of NRSF in their hearts exhibit dilated cardiomyopathy, high susceptibility to arrhythmias and sudden death. We demonstrate that genes encoding two ion channels that carry the fetal cardiac currents I(f) and I(Ca,T), which are induced in these mice and are potentially responsible for both the cardiac dysfunction and the arrhythmogenesis, are regulated by NRSF. Our results indicate NRSF to be a key transcriptional regulator of the fetal cardiac gene program and suggest an important role for NRSF in maintaining normal cardiac structure and function.

Ischemic protection and myofibrillar cardiomyopathy: dose-dependent effects of in vivo deltaPKC inhibition.

To delineate the in vivo cardiac functions requiring normal delta protein kinase C (PKC) activity, we pursued loss-of-function through transgenic expression of a deltaPKC-specific translocation inhibitor protein fragment, deltaV1, in mouse hearts. Initial results using the mouse alpha-myosin heavy chain (alphaMHC) promoter resulted in a lethal heart failure phenotype. Viable deltaV1 mice were therefore obtained using novel attenuated mutant alphaMHC promoters lacking one or the other thyroid response element (TRE-1 and -2). In transgenic mouse hearts, deltaV1 decorated cytoskeletal elements and inhibited ischemia-induced deltaPKC translocation. At high levels, deltaV1 expression was uniformly lethal, with depressed cardiac contractile function, increased expression of fetal cardiac genes, and formation of intracardiomyocyte protein aggregates. Ultrastructural and immunoconfocal analyses of these aggregates revealed focal cytoskeletal disruptions and localized concentrations of desmin and alphaB-crystallin. In individual cardiomyocytes, cytoskeletal abnormalities correlated with impaired contractile function. Whereas desmin and alphaB-crystallin protein were increased approximately 4-fold in deltaV1 hearts, combined overexpression of these proteins at these levels was not sufficient to cause any detectable cardiac pathology. At low levels, deltaV1 expression conferred striking resistance to postischemic dysfunction, with no measurable effects on basal cardiac structure, function, or gene expression. Intermediate expression of deltaV1 conferred modest basal contractile depression with less ischemic protection, associated with abnormal cardiac gene expression, and a histological picture of infrequent cardiomyocyte cytoskeletal deformities. These results validate an approach of deltaPKC inhibition to protect against myocardial ischemia, but indicate that there is a threshold level of deltaPKC activation that is necessary to maintain normal cardiomyocyte cytoskeletal integrity.

Activated MEK5 induces serial assembly of sarcomeres and eccentric cardiac hypertrophy.

Mitogen-activated protein kinase (MAPK) pathways couple intrinsic and extrinsic signals to hypertrophic growth of cardiomyocytes. The MAPK kinase MEK5 activates the MAPK ERK5. To investigate the potential involvement of MEK5-ERK5 in cardiac hypertrophy, we expressed constitutively active and dominant-negative forms of MEK5 in cardiomyocytes in vitro. MEK5 induced a form of hypertrophy in which cardiomyocytes acquired an elongated morphology and sarcomeres were assembled in a serial manner. The cytokine leukemia inhibitory factor (LIF), which stimulates MEK5 activity, evoked a similar response. Moreover, a dominant-negative MEK5 mutant specifically blocked LIF-induced elongation of cardiomyocytes and reduced expression of fetal cardiac genes without blocking other aspects of LIF-induced hypertrophy. Consistent with the ability of MEK5 to induce serial assembly of sarcomeres in vitro, cardiac-specific expression of activated MEK5 in transgenic mice resulted in eccentric cardiac hypertrophy that progressed to dilated cardiomyopathy and sudden death. These findings reveal a specific role for MEK5-ERK5 in the induction of eccentric cardiac hypertrophy and in transduction of cytokine signals that regulate serial sarcomere assembly.

Decompensation of pressure-overload hypertrophy in G alpha q-overexpressing mice.

Receptor-mediated activation of myocardial Gq signaling is postulated as a biochemical mechanism transducing pressure-overload hypertrophy. The specific effects of Gq activation on the functional and morphological adaptations to pressure overload are not known. To determine the effects of intrinsic myocyte G alpha q signaling on the left ventricular hypertrophic response to experimental pressure overload, transgenic mice overexpressing G alpha q specifically in the heart (G alpha q-25) and nontransgenic siblings underwent microsurgical creation of transverse aortic coarctation and the morphometric, functional, and molecular characteristics of these pressure-overloaded hearts were compared at increasing times after surgery. Before aortic banding, isolated G alpha q-25 ventricular myocytes exhibited contractile depression (depressed +dl/dt and -dl/dt) and G alpha q-25 hearts showed a pattern of fetal gene expression similar to the known characteristics of nontransgenic pressure-overloaded mice. Three weeks after transverse aortic banding, G alpha q-25 left ventricles hypertrophied to a similar extent (approximately 30% increase) as nontransgenic mice. However, whereas nontransgenic mice exhibited concentric left ventricular remodeling with maintained ejection performance (compensated hypertrophy), G alpha q-25 left ventricles developed eccentric hypertrophy and ejection performance deteriorated, ultimately resulting in left heart failure (decompensated hypertrophy). The signature hypertrophy-associated progress of fetal cardiac gene expression observed at baseline in G alpha q-25 developed after aortic banding of nontransgenic mice but did not significantly change in aortic-banded G alpha q-25 mice. Intrinsic cardiac myocyte G alpha q activation stimulates fetal gene expression and depresses cardiac myocyte contractility. Superimposition of the hemodynamic stress of pressure overload on G alpha q overexpression stimulates a maladaptive form of eccentric hypertrophy that leads to rapid functional decompensation. Therefore G alpha q-stimulated cardiac hypertrophy is functionally deleterious and compromises the ability of the heart to adapt to increased mechanical load. This finding supports a reevaluation of accepted concepts regarding the mechanisms for compensation and decompensation in pressure-overload hypertrophy.

996-10 A Novel Approach to Identifying mRNAs Differentially Expressed Under Hemodynamic Pressure Overload During Sheep Fetal Heart Development

The adaptive response to pressure overload is different in neonatal hearts with congenital lesions than in adult hearts with acquired lesions. In adult hearts this response is characterized by an altered pattern of gene expression. Little is known about the changes in gene expression that occur in neonates. We have used a novel approach to examine these changes in expression of messenger RNAs in an in vivo model of fetal lambs heart after induced acute pressure overload. In ewes (which normally carry twin gestationsl the main pulmonary artery of one fetal lamb was banded in utero for one hour to produce an acute pressure overload on the right ventricle (RV) while the twin underwent a sham operation. Total RNA was isolated and reverse transcribed from both the banded and control RV The resulting cDNAs were amplified by the polymerase chain reaction technique using generalized oligonucleotide primers and the products displayed on sequencing gels. Comparative analysis of the differentially expressed cDNA bands from each condition revealed both up-regulation and down-regulation of several cardiac genes in the banded fetal hearts compared with the control lamb hearts. No morphological changes in the cardiac tissues were observed during this same time period. Eighty (300–500bp) differentially expressed cDNAs were identified. Twenty-three of these were analyzed by Northem blot and four were confirmed as being differentially expressed. Two of these were sequenced, identifying the 3’-untranslated regions which had no identifiable homology to the sequences in GENEBANK. This study reports on using an in vivo fetal lamb model to study changes in gene expression induced by hemodynamic pressure overload using a technique that identifies all differentially expressed messages. This is the first study to examine alterations in fetal cardiac gene expression to hemodynamic overload in a large mammalian model.

Norepinephrine in Neonatal Rat Ventricular Myocytes: Association with the Cell Nucleus and Binding to Nuclear α1- and β-Adrenergic Receptors

Chronic exposure of neonatal rat ventricular myocytes to norepinephrine (NE) has been demonstrated to induce fetal cardiac gene expression and hypertrophy. The precise signaling mechanism of NE induction, as well as the long delay for the onset of NE effect, are not well understood. To examine the possibility that the hormone may be transported into the cell and exerts its effect through an intracellular site, ventricular myocytes from neonatal rats were incubated with [3H]-labeled NE and the cytosolic and nuclear fractions of the cell were measured for radioactivity. The presence of intracellular adrenergic binding sites was also explored.Following incubation of neonatal rat ventricular myocytes with [3H]NE for different time intervals (from 30 min to 22 h), the highest proportion (more than 80%) of NE taken up by the cell was recovered in the nuclear fraction. The nuclear accumulation was slow and time-dependent, being non-detectable in the first 60 min. Furthermore, isolated nuclei from the ventricular myocytes contain binding sites for [3H] prazosin and dihydroalprenolol, suggesting the presence of α1, and β1, adrenergic receptors. The apparent KD and Bmax were 0.6 nM and 0.6 fmol/mg protein for α1-adrenergic receptors, while β-adrenergic nuclear receptors exhibited an apparent KD of 12 nM and a Bmax of 61 fmol/mg protein. Thus, neonatal rat ventricular myocytes to NE accumulate the hormone in the cell nucleus where it can bind to high affinity α1- and β-adrenergic receptors.