Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2

FGF-2 has been implicated in the cardiac response to hypertrophic stimuli. Angiotensin II (Ang II) contributes to maintain elevated blood pressure in hypertensive individuals and exerts direct trophic effects on cardiac cells. However, the role of FGF-2 in Ang II-induced cardiac hypertrophy has not been established. Therefore, mice deficient in FGF-2 expression were studied using a model of Ang II-dependent hypertension and cardiac hypertrophy. Echocardiographic measurements show the presence of dilated cardiomyopathy in normotensive mice lacking FGF-2. Moreover, hypertensive mice without FGF-2 developed no compensatory cardiac hypertrophy. In wild-type mice, hypertrophy was associated with a stimulation of the c-Jun N-terminal kinase, the extracellular signal regulated kinase, and the p38 kinase pathways. In contrast, mitogen-activated protein kinase (MAPK) activation was markedly attenuated in FGF-2-deficient mice. In vitro, FGF-2 of fibroblast origin was demonstrated to be essential in the paracrine stimulation of MAPK activation in cardiomyocytes. Indeed, fibroblasts lacking FGF-2 expression have a defective capacity for releasing growth factors to induce hypertrophic responses in cardiomyocytes. Therefore, these results identify the cardiac fibroblast population as a primary integrator of hypertrophic stimuli in the heart, and suggest that FGF-2 is a crucial mediator of cardiac hypertrophy via autocrine/paracrine actions on cardiac cells.     

Cdc42 is an antihypertrophic molecular switch in the mouse heart

To improve contractile function, the myocardium undergoes hypertrophic growth without myocyte proliferation in response to both pathologic and physiologic stimulation. Various membrane-bound receptors and intermediate signal transduction pathways regulate the induction of cardiac hypertrophy, but the cardioprotective regulatory pathways or effectors that antagonize cardiac hypertrophy remain poorly understood. Here we identify the small GTPase Cdc42 as a signaling intermediate that restrained the cardiac growth response to physiologic and pathologic stimuli. Cdc42 was specifically activated in the heart after pressure overload and in cultured cardiomyocytes by multiple agonists. Mice with a heart-specific deletion of Cdc42 developed greater cardiac hypertrophy at 2 and 8 weeks of stimulation and transitioned more quickly into heart failure than did wild-type controls. These mice also displayed greater cardiac hypertrophy in response to neuroendocrine agonist infusion for 2 weeks and, more remarkably, enhanced exercise-induced hypertrophy and sudden death. These pathologies were associated with an inability to activate JNK following stimulation through a MEKK1/MKK4/MKK7 pathway, resulting in greater cardiac nuclear factor of activated T cells (NFAT) activity. Restoration of cardiac JNK signaling with an Mkk7 heart-specific transgene reversed the enhanced growth effect. These results identify what we believe to be a novel antihypertrophic and protective cardiac signaling pathway, whereby Cdc42-dependent JNK activation antagonizes calcineurin-NFAT activity to reduce hypertrophy and prevent transition to heart failure.     

Fas receptor signaling inhibits glycogen synthase kinase 3 beta and induces cardiac hypertrophy following pressure overload

Congestive heart failure is a leading cause of mortality in developed countries. Myocardial hypertrophy resulting from hypertension often precedes heart failure. Understanding the signaling underlying cardiac hypertrophy and failure is of major interest. Here, we identified Fas receptor activation, a classical death signal causing apoptosis via activation of the caspase cascade in many cell types, as a novel pathway mediating cardiomyocyte hypertrophy in vitro and in vivo. Fas activation by Fas ligand induced a hypertrophic response in cultured cardiomyocytes, which was dependent on the inactivation of glycogen synthase kinase 3 beta (GSK3 beta) by phosphorylation. In vivo, lpr (lymphoproliferative disease) mice lacking a functional Fas receptor demonstrated rapid-onset left ventricular dilatation and failure, absence of compensatory hypertrophy, and significantly increased mortality in response to pressure overload induction that was accompanied by a failure to inhibit GSK3 beta activity. In contrast, Fas ligand was dispensable for the development of pressure overload hypertrophy in vivo. In vitro, neonatal cardiomyocytes from lpr mice showed a completely abrogated or significantly blunted hypertrophic response after stimulation with Fas ligand or angiotensin II, respectively. These findings indicate that Fas receptor signaling inhibits GSK3 beta activity in cardiomyocytes and is required for compensation of pressure overload in vivo.     

Regulation of cardiac hypertrophy in vivo by the stress-activated protein kinases/c-Jun NH2-terminal kinases

Cardiac hypertrophy often presages the development of heart failure. Numerous cytosolic signaling pathways have been implicated in the hypertrophic response in cardiomyocytes in culture, but their roles in the hypertrophic response to physiologically relevant stimuli in vivo is unclear. We previously reported that adenovirus-mediated gene transfer of SEK-1(KR), a dominant inhibitory mutant of the immediate upstream activator of the stress-activated protein kinases (SAPKs), abrogates the hypertrophic response of neonatal rat cardiomyocytes to endothelin-1 in culture. We now report that gene transfer of SEK-1(KR) to the adult rat heart blocks SAPK activation by pressure overload, demonstrating that the activity of cytosolic signaling pathways can be inhibited by gene transfer of loss-of-function mutants in vivo. Furthermore, gene transfer of SEK-1(KR) inhibited pressure overload-induced cardiac hypertrophy, as determined by echocardiography and several postmortem measures including left ventricular (LV) wall thickness, the ratio of LV weight to body weight, cardiomyocyte diameter, and inhibition of atrial natriuretic factor expression. Our data suggest that the SAPKs are critical regulators of cardiac hypertrophy in vivo, and therefore may serve as novel drug targets in the treatment of hypertrophy and heart failure.     

Molecular aspects of cardiac hypertrophy and their implications in cardiomyopathies

Cardiac hypertrophy or hypertrophy of cardiomyocytes is an adaptive response of the heart against an intrinsic or extrinsic damage in cardiomyocytes. A typical intrinsic defect causing cardiac hypertrophy is the sarcomere mutations found in hypertrophic cardiomyopathy (HCM) and extrinsic defects include cardiac ischemia, pressure- or volume-overload, metabolic diseases and arrhythmias. The hypertrophic response is a compensatory mechanism to augments cardiac output, however, sustained hypertrophy may lead to systolic dysfunction or de-compensation state. It is well known that some patients with HCM develop to dilated-phase or burn-out phase, which resembles dilated cardiomyopathy (DCM). In this review, molecular mechanisms underlying the cardiac hypertrophy in HCM and DCM will be discussed.     

An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation

The cardiac outflow tract develops as a result of a complex interplay among several cell types, including cardiac neural crest cells, endothelial cells, and cardiomyocytes. In both humans and mice, mutations in components of the Notch signaling pathway result in congenital heart disease characterized by cardiac outflow tract defects. However, the specific cell types in which Notch functions during cardiovascular development remain to be defined. In addition, in vitro studies have provided conflicting data regarding the ability of Notch to promote or inhibit smooth muscle differentiation, while the physiological role for Notch in smooth muscle formation during development remains unclear. In this study, we generated mice in which Notch signaling was specifically inactivated in derivatives of the neural crest. These mice exhibited cardiovascular anomalies, including aortic arch patterning defects, pulmonary artery stenosis, and ventricular septal defects. We show that Notch plays a critical, cell-autonomous role in the differentiation of cardiac neural crest precursors into smooth muscle cells both in vitro and in vivo, and we identify specific Notch targets in neural crest that are implicated in this process. These results provide a molecular and cellular framework for understanding the role of Notch signaling in the etiology of congenital heart disease.     

Proapoptotic Rassf1A/Mst1 signaling in cardiac fibroblasts is protective against pressure overload in mice

Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Drosophila Hippo, the master regulator of cell death, proliferation, and organ size in flies. It is the chief component of the mammalian Hippo pathway and promotes apoptosis and inhibits compensatory cardiac hypertrophy, playing a critical role in mediating heart failure. How Mst1 is regulated, however, remains unclear. Using genetically altered mice in which expression of the tumor suppressor Ras-association domain family 1 isoform A (Rassf1A) was modulated in a cell type-specific manner, we demonstrate here that Rassf1A is an endogenous activator of Mst1 in the heart. Although the Rassf1A/Mst1 pathway promoted apoptosis in cardiomyocytes, thereby playing a detrimental role, the same pathway surprisingly inhibited fibroblast proliferation and cardiac hypertrophy through both cell-autonomous and autocrine/paracrine mechanisms, playing a protective role during pressure overload. In cardiac fibroblasts, the Rassf1A/Mst1 pathway negatively regulated TNF-α, a key mediator of hypertrophy, fibrosis, and resulting cardiac dysfunction. These results suggest that the functional consequence of activating the proapoptotic Rassf1A/Mst1 pathway during pressure overload is cell type dependent in the heart and that suppressing this mechanism in cardiac fibroblasts could be detrimental.     

Rat parathyroid hormone 1-34 signals through the MEK/ERK pathway to induce cardiac hypertrophy

This study aimed to characterize the role of the mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in cardiac hypertrophy induced by parathyroid hormone (PTH). Various concentrations of rat PTH1-34 were used to induce hypertrophy in neonatal rat ventricular cardiomyocytes, and the effects were compared with control cells and those treated with PD98059, a selective inhibitor of MEK1. Hypertrophy was assessed in terms of cell diameter, atrial natriuretic peptide (ANP) mRNA expression and protein synthesis; the MEK/ERK pathway was assessed by measuring levels of phosphorylated ERK1/2. Treatment with PTH1-34 at 100 nM for 24 h effectively induced cardiac hypertrophy (increased cell diameter, protein synthesis and ANP mRNA expression) and also increased levels of phosphorylated ERK1/2 compared with normal control cells. Treatment with PTH1-34 plus PD98059 significantly attenuated these changes. These results demonstrate that inhibition of the MEK/ERK pathway blocks PTH1-34-induced cardiac hypertrophy, suggesting that PTH1-34 might signal through the MAPK pathway to induce hypertrophy in cardiomyocytes.     

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.     

NO triggers RGS4 degradation to coordinate angiogenesis and cardiomyocyte growth

Myocardial hypertrophy is an adaptation to increased hemodynamic demands. An increase in heart tissue must be matched by a corresponding expansion of the coronary vasculature to maintain and adequate supply of oxygen and nutrients for the heart. The physiological mechanisms that underlie the coordination of angiogenesis and cardiomyocyte growth are unknown. We report that induction of myocardial angiogenesis promotes cardiomyocyte growth and cardiac hypertrophy through a novel NO-dependent mechanism. We used transgenic, conditional overexpression of placental growth factor (PlGF) in murine cardiac tissues to stimulate myocardial angiogenesis and increase endothelial-derived NO release. NO production, in turn, induced myocardial hypertrophy by promoting proteasomal degradation of regulator of G protein signaling type 4 (RGS4), thus relieving the repression of the Gβγ/PI3Kγ/AKT/mTORC1 pathway that stimulates cardiomyocyte growth. This hypertrophic response was prevented by concomitant transgenic expression of RGS4 in cardiomyocytes. NOS inhibitor L-NAME also significantly attenuated RGS4 degradation, and reduced activation of AKT/mTORC1 signaling and induction of myocardial hypertrophy in PlGF transgenic mice, while conditional cardiac-specific PlGF expression in eNOS knockout mice did not induce myocardial hypertrophy. These findings describe a novel NO/RGS4/Gβγ/PI3Kγ/AKT mechanism that couples cardiac vessel growth with myocyte growth and heart size.     

Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis

The Raf/MEK/extracellular signal-regulated kinase (ERK) signaling pathway regulates diverse cellular processes such as proliferation, differentiation, and apoptosis and is implicated as an important contributor to the pathogenesis of cardiac hypertrophy and heart failure. To examine the in vivo role of Raf-1 in the heart, we generated cardiac muscle-specific Raf-1-knockout (Raf CKO) mice with Cre-loxP-mediated recombination. The mice demonstrated left ventricular systolic dysfunction and heart dilatation without cardiac hypertrophy or lethality. The Raf CKO mice showed a significant increase in the number of apoptotic cardiomyocytes. The expression level and activation of MEK1/2 or ERK showed no difference, but the kinase activity of apoptosis signal-regulating kinase 1 (ASK1), JNK, or p38 increased significantly compared with that in controls. The ablation of ASK1 rescued heart dysfunction and dilatation as well as cardiac fibrosis. These results indicate that Raf-1 promotes cardiomyocyte survival through a MEK/ERK-independent mechanism.     

FGF-2 controls the differentiation of resident cardiac precursors into functional cardiomyocytes

Recent evidence suggests that the heart possesses a greater regeneration capacity than previously thought. In the present study, we isolated undifferentiated precursors from the cardiac nonmyocyte cell population of neonatal hearts, expanded them in culture, and induced them to differentiate into functional cardiomyocytes. These cardiac precursors appear to express stem cell antigen-1 and demonstrate characteristics of multipotent precursors of mesodermal origin. Following infusion into normal recipients, these cells home to the heart and participate in physiological and pathophysiological cardiac remodeling. Cardiogenic differentiation in vitro and in vivo depends on FGF-2. Interestingly, this factor does not control the number of precursors but regulates the differentiation process. These findings suggest that, besides its angiogenic actions, FGF-2 could be used in vivo to facilitate the mobilization and differentiation of resident cardiac precursors in the treatment of cardiac diseases.     

Role of diacylglycerol kinase in cellular regulatory processes: a new regulator for cardiomyocyte hypertrophy

Diacylglycerol (DAG) kinase (DGK) phosphorylates and converts DAG to phosphatidic acid. DGK regulates cellular DAG levels and attenuates DAG signaling. The 10 mammalian DGK isoforms have been identified to date. In cardiac myocytes, DGKalpha, epsilon, and zeta are expressed, and DGKzeta is the predominant isoform. DGKzeta inhibits protein kinase C (PKC) activation and subsequent hypertrophic programs in response to endothelin-1 (ET-1) in neonatal rat cardiomyocytes. DGKzeta blocks cardiac hypertrophy induced by G protein-coupled receptor agonists and pressure overload in vivo. DGKzeta attenuates ventricular remodeling and improves survival after myocardial infarction. These data provide a novel insight for subcellular mechanisms of cardiac hypertrophy and heart failure, and DGKzeta may be a new therapeutic target to prevent cardiac hypertrophy and progression to heart failure.     

Study on the mechanism of HIF1a-SOX9 in glucose-induced cardiomyocyte hypertrophy

A major cause of morbidity and mortality in cardiovascular disease is pathological cardiac hypertrophy. With an increase in the cellular surface area and upregulation of the atrial natriuretic peptide (ANP) gene, cardiac hypertrophy is a prominent feature of diabetic cardiomyopathy. ANP is a hypertrophic marker. Many works have been done to explore how the glucose induces the cardiac hypertrophy. However, it is not enough for us to figure it out. In this study, the influences of different glucose concentrations on cardiomyocytes were examined in vitro. The results showed that cardiomyocytes cultured with 25 mM glucose tended to show a hypertrophic phenotype, while cardiomyocytes cultured with 35 mM glucose tended to undergo apoptosis. An increased expression of SOX9 was observed when cardiomyocytes were cultured with 25 mM glucose, but when the concentration of glucose was increased to 35 mM, the expression of SOX9 decreased. We used the RNAi approach to knockdown SOX9 expression, to assess its effects on cardiomyocyte hypertrophy. The results showed that knockdown of the SOX9 gene suppressed the 25 mM glucose-induced cardiomyocyte hypertrophy. The upregulation of the ANP gene was associated with overexpression of SOX9. Additionally, the results showed that high glucose (HG, 25 mM) treatment increased the expression of hypoxia-inducible factor (HIF)1a. Further study showed that HIF1a participated in regulating SOX9 expression in response to HG. This study revealed a novel regulatory mechanism of HIF1a-SOX9 in high glucose-induced cardiomyocyte hypertrophy, as well as the related molecular mechanisms.     

Altered focal adhesion regulation correlates with cardiomyopathy in mice expressing constitutively active rac1

The ras family of small GTP-binding proteins exerts powerful effects upon cell structure and function. One member of this family, rac, induces actin cytoskeletal reorganization in nonmuscle cells and hypertrophic changes in cultured cardiomyocytes. To examine the effect of rac1 activation upon cardiac structure and function, transgenic mice were created that express constitutively activated rac1 specifically in the myocardium. Transgenic rac1 protein was expressed at levels comparable to endogenous rac levels, with activation of the rac1 signaling pathway resulting in two distinct cardiomyopathic phenotypes: a lethal dilated phenotype associated with neonatal activation of the transgene and a transient cardiac hypertrophy seen among juvenile mice that resolved with age. Neither phenotype showed myofibril disarray and hypertrophic hearts were hypercontractilein working heart analyses. The rac1 target p21-activated kinase translocated from a cytosolic to a cytoskeletal distribution, suggesting that rac1 activation was inducing focal adhesion reorganization. Corroborating results showed altered localizations of src in dilated cardiomyopathy and paxillin in both cardiomyopathic phenotypes. This study, the first examination of rac1-mediated cardiac effects in vivo, demonstrates that dilation and hypertrophy can share a common molecular origin and presents evidence that both timing and concurrent signaling from multiple pathways can influence cardiac remodeling.     

Role of the stress-activated protein kinases in endothelin-induced cardiomyocyte hypertrophy.

The signal transduction pathways governing the hypertrophic response of cardiomyocytes are not well defined. Constitutive activation of the stress-activated protein kinase (SAPK) family of mitogen-activated protein (MAP) kinases or another stress-response MAP kinase, p38, by overexpression of activated mutants of various components of the pathways is sufficient to induce a hypertrophic response in cardiomyocytes, but it is not clear what role these pathways play in the response to physiologically relevant hypertrophic stimuli. To determine the role of the SAPKs in the hypertrophic response, we used adenovirus-mediated gene transfer of SAPK/ERK kinase-1 (KR) [SEK-1(KR)], a dominant inhibitory mutant of SEK-1, the immediate upstream activator of the SAPKs, to block signal transmission down the SAPK pathway in response to the potent hypertrophic agent, endothelin-1 (ET-1). SEK-1(KR) completely inhibited ET-1-induced SAPK activation without affecting activation of the other MAP kinases implicated in the hypertrophic response, p38 and extracellular signal-regulated protein kinases (ERK)-1/ERK-2. Expression of SEK-1(KR) markedly inhibited the ET-1-induced increase in protein synthesis. In contrast, the MAPK/ERK kinase inhibitor, PD98059, which blocks ERK activation, and the p38 inhibitor, SB203580, had no effect on ET-1-induced protein synthesis. ET-1 also induced a significant increase in atrial natriuretic factor mRNA expression as well as in the percentage of cells with highly organized sarcomeres, responses which were also blocked by expression of SEK-1(KR). In summary, inhibiting activation of the SAPK pathway abrogated the hypertrophic response to ET-1. These data are the first demonstration that the SAPKs are necessary for the development of agonist-induced cardiomyocyte hypertrophy, and suggest that in response to ET-1, they transduce critical signals governing the hypertrophic response.