GRP78 (Glucose-Regulated Protein of 78 kDa) Promotes Cardiomyocyte Growth Through Activation of GATA4 (GATA-Binding Protein 4).
ABSTRACT: The heart manifests hypertrophic growth in response to elevation of afterload pressure. Cardiac myocyte growth involves new protein synthesis and membrane expansion, of which a number of cellular quality control machineries are stimulated to maintain function and homeostasis. The unfolded protein response is potently induced during cardiac hypertrophy to enhance protein-folding capacity and eliminate terminally misfolded proteins. However, whether the unfolded protein response directly regulates cardiac myocyte growth remains to be fully determined. Here, we show that GRP78 (glucose-regulated protein of 78 kDa)-an endoplasmic reticulum-resident chaperone and a critical unfolded protein response regulator-is induced by cardiac hypertrophy. Importantly, overexpression of GRP78 in cardiomyocytes is sufficient to potentiate hypertrophic stimulus-triggered growth. At the in vivo level, TG (transgenic) hearts overexpressing GRP78 mount elevated hypertrophic growth in response to pressure overload. We went further to show that GRP78 increases GATA4 (GATA-binding protein 4) level, which may stimulate Anf (atrial natriuretic factor) expression and promote cardiac hypertrophic growth. Silencing of GATA4 in cultured neonatal rat ventricular myocytes significantly diminishes GRP78-mediated growth response. Our results, therefore, reveal that protein-folding chaperone GRP78 may directly enhance cardiomyocyte growth by stimulating cardiac-specific transcriptional factor GATA4.
Project description:Cardiac hypertrophy is a common response to injury and hemodynamic stress and an important harbinger of heart failure and death. Herein, we identify the Kruppel-like factor 15 (KLF15) as an inhibitor of cardiac hypertrophy. Myocardial expression of KLF15 is reduced in rodent models of hypertrophy and in biopsy samples from patients with pressure-overload induced by chronic valvular aortic stenosis. Overexpression of KLF15 in neonatal rat ventricular cardiomyocytes inhibits cell size, protein synthesis and hypertrophic gene expression. KLF15-null mice are viable but, in response to pressure overload, develop an eccentric form of cardiac hypertrophy characterized by increased heart weight, exaggerated expression of hypertrophic genes, left ventricular cavity dilatation with increased myocyte size, and reduced left ventricular systolic function. Mechanistically, a combination of promoter analyses and gel-shift studies suggest that KLF15 can inhibit GATA4 and myocyte enhancer factor 2 function. These studies identify KLF15 as part of a heretofore unrecognized pathway regulating the cardiac response to hemodynamic stress.
Project description:GATA4 is a transcription factor that is up-regulated during cardiac hypertrophy and plays a fundamental role in myocyte growth and survival. In this study, we investigate the transcriptional vs. post-transcriptional mechanisms that are involved in regulating GATA4 in the heart during neonatal and pressure overload-induced hypertrophic growth.GATA4 protein is significantly higher during pressure overload-induced (2.9 ± 0.4-fold) and neonatal (6.8 ± 1-fold) hypertrophic growth vs. the normal adult mouse heart. Using RNA polymerase II immunoprecipitation combined with deep sequencing, we confirmed that active transcription of the Gata4 gene remained unchanged during hypertrophy, whereas it was two-fold higher in the neonatal vs. adult heart, commensurate with the mRNA levels. These results suggested a post-transcriptional mode of regulation of its expression, which prompted the identification of a conserved sequence in its 3'-untranslated region that was responsible for reduced translation via miR-26b. Overexpression of miR-26b reduced GATA4-dependent transcription, endothelin-induced hypertrophy, and sensitized the cells to apoptotic insults. Additionally, miR-26b targeted phospholipase C-?1, which, in turn, inhibited miR-26b expression, creating a double-negative feedback loop. Accordingly, overexpression of miR-26b in the heart inhibited up-regulation of its targets and the development of hypertrophy. However, knockdown of miR-26b is not sufficient for inducing hypertrophy.Down-regulation of miR-26b in the heart is required for the up-regulation of GATA4 and the induction of pressure-induced cardiac hypertrophy. The results also underscore the functional relevance of miRNAs in regulating gene expression during cardiac hypertrophy.
Project description:Cardiac hypertrophy is an adaptive growth process that occurs in response to stress stimulation or injury wherein multiple signal transduction pathways are induced, culminating in transcription factor activation and the reprogramming of gene expression. GATA4 is a critical transcription factor in the heart that is known to induce/regulate the hypertrophic program, in part, by receiving signals from MAPKs. Here we generated knock-in mice in which a known MAPK phosphorylation site at serine 105 (S105) in Gata4 that augments activity was mutated to alanine. Homozygous Gata4-S105A mutant mice were viable as adults, although they showed a compromised stress response of the myocardium. For example, cardiac hypertrophy in response to phenylephrine agonist infusion for 2 wk was largely blunted in Gata4-S105A mice, as was the hypertrophic response to pressure overload at 1 and 2 wk of applied stimulation. Gata4-S105A mice were also more susceptible to heart failure and cardiac dilation after 2 wk of pressure overload. With respect to the upstream pathway, hearts from Gata4-S105A mice did not efficiently hypertrophy following direct ERK1/2 activation using an activated MEK1 transgene in vivo. Mechanistically, GATA4 mutant protein from these hearts failed to show enhanced DNA binding in response to hypertrophic stimulation. Moreover, hearts from Gata4-S105A mice had significant changes in the expression of hypertrophy-inducible, fetal, and remodeling-related genes.
Project description:Acetyltransferase p300 is essential for cardiac development and is thought to be involved in cardiac myocyte growth through MEF2- and GATA4-dependent transcription. However, the importance of p300 in the modulation of cardiac growth in vivo is unknown.Pressure overload induced by transverse aortic coarctation, postnatal physiological growth, and human heart failure were associated with large increases in p300. Minimal transgenic overexpression of p300 (1.5- to 3.5-fold) induced striking myocyte and cardiac hypertrophy. Both mortality and cardiac mass were directly related to p300 protein dosage. Heterozygous loss of a single p300 allele reduced pressure overload-induced hypertrophy by approximately 50% and rescued the hypertrophic phenotype of p300 overexpressers. Increased p300 expression had no effect on total histone deacetylase activity but was associated with proportional increases in p300 acetyltransferase activity and acetylation of the p300 substrates histone 3 and GATA-4. Remarkably, a doubling of p300 levels was associated with the de novo acetylation of MEF2. Consistent with this, genes specifically upregulated in p300 transgenic hearts were highly enriched for MEF2 binding sites.Small increments in p300 are necessary and sufficient to drive myocardial hypertrophy, possibly through acetylation of MEF2 and upstream of signals promoting phosphorylation or nuclear export of histone deacetylases. We propose that induction of myocardial p300 content is a primary rate-limiting event in the response to hemodynamic loading in vivo and that p300 availability drives and constrains adaptive myocardial growth. Specific reduction of p300 content or activity may diminish stress-induced hypertrophy and forestall the development of heart failure.
Project description:The transcriptional code that programs maladaptive cardiac hypertrophy involves the zinc finger-containing DNA binding factor GATA-4. The highly related transcription factor GATA-6 is also expressed in the adult heart, although its role in controlling the hypertrophic program is unknown.To determine the role of GATA-6 in cardiac hypertrophy and homeostasis.Here, we performed a cardiomyocyte-specific conditional gene targeting approach for Gata6, as well as a transgenic approach to overexpress GATA-6 in the mouse heart. Deletion of Gata6-loxP with Nkx2.5-cre produced late embryonic lethality with heart defects, whereas deletion with ?-myosin heavy chain-cre (?MHC-cre) produced viable adults with >95% loss of GATA-6 protein in the heart. These latter mice were subjected to pressure overload-induced hypertrophy for 2 and 6 weeks, which showed a significant reduction in cardiac hypertrophy similar to that observed Gata4 heart-specific deleted mice. Gata6-deleted mice subjected to pressure overload also developed heart failure, whereas control mice maintained proper cardiac function. Gata6-deleted mice also developed less cardiac hypertrophy following 2 weeks of angiotensin II/phenylephrine infusion. Controlled GATA-6 overexpression in the heart induced hypertrophy with aging and predisposed to greater hypertrophy with pressure overload stimulation. Combinatorial deletion of Gata4 and Gata6 from the adult heart resulted in dilated cardiomyopathy and lethality by 16 weeks of age. Mechanistically, deletion of Gata6 from the heart resulted in fundamental changes in the levels of key regulatory genes and myocyte differentiation-specific genes.These results indicate that GATA-6 is both necessary and sufficient for regulating the cardiac hypertrophic response and differentiated gene expression, both alone and in coordination with GATA-4.
Project description:Fetal cardiac gene reactivation is a hallmark of pathological cardiac hypertrophy (PCH) driven by cardiac transcription factors (TFs) such as nuclear factor of activated T-cells (NFATs). Nuclear import of dephosphorylated NFATs catalyzed by calcineurin (CaN) is a well-established hypertrophic mechanism. Here we report that NFATc4 expression is also up-regulated by newly expressed protein kinase D3 (PKD3) to induce PCH. In both in vitro and in vivo cardiac hypertrophic models, the normally undetectable PKD3 was profoundly up-regulated by isoproterenol followed by overt expression of cardiac TFs including NFATc4, NK family of transcription factor 2.5 (Nkx2.5), GATA4 and myocyte enhancer factor 2 (MEF2). Using gene silencing approaches, we demonstrate PKD3 is required for increasing the expression of NFATc4, Nkx2.5, and GATA4 while PKD1 is required for the increase in MEF2D expression. Upstream induction of PKD3 is driven by nuclear entry of CaN-activated NFATc1 and c3 but not c4. Therefore, PKD3 is a pivotal mediator of the CaN-NFATc1/c3-PKD3-NFATc4 hypertrophic signaling cascade and a potential new drug target for the PCH.
Project description:Cardiac hypertrophy is controlled by a complex signal transduction and gene regulatory network, containing multiple layers of crosstalk and feedback. While numerous individual components of this network have been identified, understanding how these elements are coordinated to regulate heart growth remains a challenge. Past approaches to measure cardiac myocyte hypertrophy have been manual and often qualitative, hindering the ability to systematically characterize the network's higher-order control structure and identify therapeutic targets. Here, we develop and validate an automated image analysis approach for objectively quantifying multiple hypertrophic phenotypes from immunofluorescence images. This approach incorporates cardiac myocyte-specific optimizations and provides quantitative measures of myocyte size, elongation, circularity, sarcomeric organization, and cell-cell contact. As a proof-of-concept, we examined the hypertrophic response to ?-adrenergic, ?-adrenergic, tumor necrosis factor (TNF?), insulin-like growth factor-1 (IGF-1), and fetal bovine serum pathways. While all five hypertrophic pathways increased myocyte size, other hypertrophic metrics were differentially regulated, forming a distinct phenotype signature for each pathway. Sarcomeric organization was uniquely enhanced by ?-adrenergic signaling. TNF? and ?-adrenergic pathways markedly decreased cell circularity due to increased myocyte protrusion. Surprisingly, adrenergic and IGF-1 pathways differentially regulated myocyte-myocyte contact, potentially forming a feed-forward loop that regulates hypertrophy. Automated image analysis unlocks a range of new quantitative phenotypic data, aiding dissection of the complex hypertrophic signaling network and enabling myocyte-based high-content drug screening.
Project description:Cardiac hypertrophy is regulated by the zinc finger-containing DNA binding factors Gata4 and Gata6, both of which are required to mount a productive growth response of the adult heart. To determine if Gata4 and Gata6 are redundant or have non-overlapping roles, we performed cardiomyocyte-specific conditional gene deletions for Gata4 and Gata6 in conjunction with reciprocal replacement with a transgene encoding either Gata4 or Gata6, during the pressure overload response. We determined that Gata4 and Gata6 play a redundant and dosage-sensitive role in programming the hypertrophic growth response itself following pressure overload stimulation. However, non-redundant functions were identified as functional decompensation induced by either Gata4 or Gata6 deletion was not rescued by the reciprocal transgene, and only Gata4 heart-specific deletion produced a reduction in capillary density after pressure overload. Gene expression profiling from hearts of these gene-deleted mice showed both overlapping and unique transcriptional codes, with Gata4 exhibiting the strongest impact. These results indicate that Gata4 and Gata6 play a dosage-dependent and semi-redundant role in programming cardiac hypertrophy, but that each has a unique role in maintaining cardiac homeostasis and adaptation to injury that cannot be compensated by the other. Microarray-bassed gene expression profiling identified overlapping, distinct, and quantitatively/differentially regulated classes of Gata4 or Gata6 regulated genes. To determine if Gata4 and Gata6 are redundant or have non-overlapping roles in programming cardiac hypertrophic responses and adaptation to stress or injury, we performed cardiomyocyte-specific conditional gene deletions for Gata4 and Gata6 in conjunction with reciprocal replacement with a transgene encoding either Gata4 or Gata6, during the pressure overload response.
Project description:Pressure overload and prolonged angiotensin II (Ang II) infusion elicit cardiac hypertrophy in Ang II receptor 1 (AT(1)) null mouse, whereas Ang II receptor 2 (AT(2)) gene deletion abolishes the hypertrophic response. The roles and signals of the cardiac AT(2) receptor still remain unsettled. Promyelocytic leukemia zinc finger protein (PLZF) was shown to bind to the AT(2) receptor and transmit the hypertrophic signal. Using PLZF knockout mice we directed our studies on the function of PLZF concerning the cardiac specific transcription factor GATA4, and GATA4 targets.PLZF knockout and age-matched wild-type (WT) mice were treated with Ang II, infused at a rate of 4.2 ng·kg(-1)·min(-1) for 3 weeks. Ang II elevated systolic blood pressure to comparable levels in PLZF knockout and WT mice (140 mmHg). WT mice developed prominent cardiac hypertrophy and fibrosis after Ang II infusion. In contrast, there was no obvious cardiac hypertrophy or fibrosis in PLZF knockout mice. An AT(2) receptor blocker given to Ang II-infused wild type mice prevented hypertrophy, verifying the role of AT(2) receptor for cardiac hypertrophy. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that PLZF bound to the GATA4 gene regulatory region. A Luciferase assay verified that PLZF up-regulated GATA4 gene expression and the absence of PLZF expression in vivo produced a corresponding repression of GATA4 protein.PLZF is an important AT(2) receptor binding protein in mediating Ang II induced cardiac hypertrophy through an AT(2) receptor-dependent signal pathway. The angiotensin II-AT(2)-PLZF-GATA4 signal may further augment Ang II induced pathological effects on cardiomyocytes.
Project description:RATIONALE:Endoplasmic reticulum (ER) stress dysregulates ER proteostasis, which activates the transcription factor, ATF6 (activating transcription factor 6?), an inducer of genes that enhance protein folding and restore ER proteostasis. Because of increased protein synthesis, it is possible that protein folding and ER proteostasis are challenged during cardiac myocyte growth. However, it is not known whether ATF6 is activated, and if so, what its function is during hypertrophic growth of cardiac myocytes. OBJECTIVE:To examine the activity and function of ATF6 during cardiac hypertrophy. METHODS AND RESULTS:We found that ER stress and ATF6 were activated and ATF6 target genes were induced in mice subjected to an acute model of transverse aortic constriction, or to free-wheel exercise, both of which promote adaptive cardiac myocyte hypertrophy with preserved cardiac function. Cardiac myocyte-specific deletion of Atf6 (ATF6 cKO [conditional knockout]) blunted transverse aortic constriction and exercise-induced cardiac myocyte hypertrophy and impaired cardiac function, demonstrating a role for ATF6 in compensatory myocyte growth. Transcript profiling and chromatin immunoprecipitation identified RHEB (Ras homologue enriched in brain) as an ATF6 target gene in the heart. RHEB is an activator of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), a major inducer of protein synthesis and subsequent cell growth. Both transverse aortic constriction and exercise upregulated RHEB, activated mTORC1, and induced cardiac hypertrophy in wild type mouse hearts but not in ATF6 cKO hearts. Mechanistically, knockdown of ATF6 in neonatal rat ventricular myocytes blocked phenylephrine- and IGF1 (insulin-like growth factor 1)-mediated RHEB induction, mTORC1 activation, and myocyte growth, all of which were restored by ectopic RHEB expression. Moreover, adeno-associated virus 9- RHEB restored cardiac growth to ATF6 cKO mice subjected to transverse aortic constriction. Finally, ATF6 induced RHEB in response to growth factors, but not in response to other activators of ATF6 that do not induce growth, indicating that ATF6 target gene induction is stress specific. CONCLUSIONS:Compensatory cardiac hypertrophy activates ER stress and ATF6, which induces RHEB and activates mTORC1. Thus, ATF6 is a previously unrecognized link between growth stimuli and mTORC1-mediated cardiac growth.