Cardiac overexpression of myotrophin triggers myocardial hypertrophy and heart failure in transgenic mice

Cardiac hypertrophy and heart failure remain leading causes of death in the United States. Many studies have suggested that, under stress, myocardium releases factors triggering protein synthesis and stimulating myocyte growth. We identified and cloned myotrophin, a 12-kDa protein from hypertrophied human and rat hearts. Myotrophin (whose gene is localized on human chromosome 7q33) stimulates myocyte growth and participates in cellular interaction that initiates cardiac hypertrophy in vitro. In this report, we present data on the pathophysiological significance of myotrophin in vivo, showing the effects of overexpression of cardio-specific myotrophin in transgenic mice in which cardiac hypertrophy occurred by 4 weeks of age and progressed to heart failure by 9-12 months. This hypertrophy was associated with increased expression of proto-oncogenes, hypertrophy marker genes, growth factors, and cytokines, with symptoms that mimicked those of human cardiomyopathy, functionally and morphologically. This model provided a unique opportunity to analyze gene clusters that are differentially up-regulated during initiation of hypertrophy versus transition of hypertrophy to heart failure. Importantly, changes in gene expression observed during initiation of hypertrophy were significantly different from those seen during its transition to heart failure. Our data show that overexpression of myotrophin results in initiation of cardiac hypertrophy that progresses to heart failure, similar to changes in human heart failure. Knowledge of the changes that take place as a result of overexpression of myotrophin at both the cellular and molecular levels will suggest novel strategies for treatment to prevent hypertrophy and its progression to heart failure.     

Myotrophin-kappaB DNA interaction in the initiation process of cardiac hypertrophy

To investigate how cardiac hypertrophy and heart failure develop, we isolated and characterized a candidate initiator, the soluble 12-kDa protein myotrophin, from rat and human hearts. Myotrophin stimulates protein synthesis and myocardial cell growth associated with increased levels of hypertrophy marker genes. Recombinant myotrophin from the cloned gene showed structural/functional motifs, including ankyrin repeats and putative phosphorylation sites for protein kinase C (PKC) and casein kinase II. One repeat, homologous with I kappaB, interacts with rel/NF-kappaB in vitro. We analyzed the interaction of recombinant myotrophin and nuclear extracts prepared from neonatal and adult cardiomyocytes; gel mobility shift assay showed that myotrophin bound to kappaB DNA. To define PKC's role in myotrophin-induced myocyte growth, we incubated neonatal rat myocytes (normal and stretch) with specific inhibitors and found that myotrophin inhibits [3H]leucine incorporation into myocytes and different hypertrophic gene expression in neonatal myocytes. Using confocal microscopy, we observed that a basal level of myotrophin was present in both cytoplasm and nucleus under normal conditions, but under cyclic stretch, myotrophin levels became elevated in the nucleus. Myotrophin gene levels were upregulated when myocytes underwent cyclic stretch or were treated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta and also when excised beating hearts were exposed to high pressure. Our data showed that the myotrophin-kappaB interaction was increased with age in spontaneously hypertensive rats (SHRs) only. Our data provide evidence that myotrophin-kappaB DNA interaction may be an important step in initiating cardiac hypertrophy.     

Nuclear magnetic resonance assignment and secondary structure of an ankyrin-like repeat-bearing protein: myotrophin.

Multidimensional heteronuclear NMR has been applied to the structural analysis of myotrophin, a novel protein identified from spontaneously hypertensive rat hearts and hypertrophic human hearts. Myotrophin has been shown to stimulate protein synthesis in myocytes and likely plays an important role in the initiation of cardiac hypertrophy, a major cause of mortality in humans. Recent cDNA cloning revealed that myotrophin has 11B amino acids containing 2.5 contiguous ANK repeats, a motif known to be involved in a wide range of macromolecular recognition. A series of two- and three-dimensional heteronuclear bond correlation NMR experiments have been performed on uniformly 15N-labeled or uniformly 15N/13C-labeled protein to obtain the 1H, 15N, and 13C chemical shift assignments. The secondary structure of myotrophin has been determined by a combination of NOEs, NH exchange data, 3JHN alpha coupling constants, and chemical shifts of 1H alpha, 13C alpha, and 13 C beta. The protein has been found to consist of seven helices, all connected by turns or loops. Six of the seven helices (all but the C-terminal helix) form three separate helix-turn-helix motifs. The two full ANK repeats in myotrophin are characteristic of multiple turns followed by a helix-turn-helix motif. A hairpin-like turn involving L32-R36 in ANK repeat #1 exhibits slow conformational averaging on the NMR time scale and appears dynamically different from the corresponding region (D65-169) of ANK repeat #2.     

Myotrophin: purification of a novel peptide from spontaneously hypertensive rat heart that influences myocardial growth

Mechanisms involved in the development or the regression of myocardial hypertrophy cannot be fully explained as responses to blood pressure control alone. We had hypothesized that the development of hypertrophy is initiated by a signal (mechanical or humoral) to the myocardium, which in turn produces a soluble factor that triggers protein synthesis and initiates myocardial growth. Using the stimulation of protein synthesis in isolated cardiac myocytes obtained from normal rat hearts as an assay system, we have identified a soluble factor from the hypertrophied myocardium of spontaneously hypertensive rats. This factor, which has been purified to apparent homogeneity, is a protein of 12 kDa. The sequence of three internally liberated peptides containing 7-24 residues was determined. Based on the determined amino acid sequences of these peptides, this factor (designated myotrophin) appears to be a novel protein that shows no homology with any previously described growth factors. Myotrophin is present in human, dog, and rat hypertrophied hearts (28-35% stimulation of protein synthesis over control) and in small amounts in normal hearts (5-6% stimulation). Myotrophin causes two dose-dependent effects in neonatal cardiac myocytes: an increase in the surface area of the myocyte and the appearance of organized myofibrils, which become apparent within 48 h. Myotrophin may play an important role in the pathogenesis of cardiac hypertrophy as well as in the normal development of cardiac myocytes.     

Expression of an active LKB1 complex in cardiac myocytes results in decreased protein synthesis associated with phenylephrine-induced hypertrophy

AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recently, LKB1 was identified as a kinase that regulates AMPK. Using immunoblot analysis, we confirmed high expression of LKB1 in isolated rat cardiac myocytes but show that, under basal conditions, LKB1 is primarily localized to the nucleus, where it is inactive. We examined the role of LKB1 in cardiac myocytes, using adenoviruses that express LKB1, and its binding partners Ste20-related adaptor protein (STRADalpha) and MO25alpha. Infection of neonatal rat cardiac myocytes with all three adenoviruses substantially increased LKB1/STRADalpha/MO25alpha expression, LKB1 activity, and AMPKalpha phosphorylation at its activating phosphorylation site (threonine-172). Since activation of AMPK can inhibit hypertrophic growth and since LKB1 is upstream of AMPK, we hypothesized that expression of an active LKB1 complex would also inhibit protein synthesis associated with hypertrophic growth. Expression of the LKB1/STRADalpha/MO25alpha complex in neonatal rat cardiac myocytes inhibited the increase in protein synthesis observed in cells treated with phenylephrine (measured via [(3)H]phenylalanine incorporation). This was associated with a decreased phosphorylation of p70S6 kinase and its substrate S6 ribosomal protein, key regulators of protein synthesis. In addition, we show that the pathological cardiac hypertrophy in transgenic mice with cardiac-specific expression of activated calcineurin is associated with a significant decrease in LKB1 expression. Together, our data show that increased LKB1 activity in the cardiac myocyte can decrease hypertrophy-induced protein synthesis and suggest that LKB1 activation may be a method for the prevention of pathological cardiac hypertrophy.     

Prevention of cardiac hypertrophy and heart failure by silencing of NF-kappaB

Activation of the nuclear factor (NF)-κB signaling pathway may be associated with the development of cardiac hypertrophy and its transition to heart failure (HF). The transgenic Myo-Tg mouse develops hypertrophy and HF as a result of overexpression of myotrophin in the heart associated with an elevated level of NF-κB activity. Using this mouse model and an NF-κB-targeted gene array, we first determined the components of NF-κB signaling cascade and the NF-κB-linked genes that are expressed during the progression to cardiac hypertrophy and HF. Second, we explored the effects of inhibition of NF-κB signaling events by using a gene knockdown approach: RNA interference through delivery of a short hairpin RNA against NF-κB p65 using a lentiviral vector (L-sh-p65). When the short hairpin RNA was delivered directly into the hearts of 10-week-old Myo-Tg mice, there was a significant regression of cardiac hypertrophy, associated with a significant reduction in NF-κB activation and atrial natriuretic factor expression. Our data suggest, for the first time, that inhibition of NF-κB using direct gene delivery of sh-p65 RNA results in regression of cardiac hypertrophy. These data validate NF-κB as a therapeutic target to prevent hypertrophy/HF.     

The IGF-1-IGF-1 Receptor System Modulates Myocyte Proliferation but Not Myocyte Cellular Hypertrophy in Vitro

In preliminary experiments it was established that the hypertrophic and hyperplastic responses of neonatal cardiac myocytes in culture were associated with enhanced expression of IGF-1 and IGF-1 receptors in these cells. Therefore, to determine the role of IGF-1 receptors on myocyte growth, cells were exposed to antisense oligodeoxynucleotides to IGF-1 receptor mRNA and the effects of this intervention on DNA synthesis, nuclear mitotic division, and changes in the number of myocytes were measured. Moreover, the influence of this procedure on ANF induction and myocyte cell volume was examined. Inhibition of the formation of IGF-1 receptors on myocytes suppressed DNA replication, mitosis, and cell proliferation. In contrast, the antisense treatment did not alter the expression of ANF in myocytes or cellular hypertrophy. Finally, IGF-1 stimulated DNA synthesis in myocytes cultured in serum-free medium, without inducing cellular hypertrophy. In conclusion, ligand activation of IGF-1 receptors on myocytes appears to be coupled with cell proliferation, whereas myocyte cellular hypertrophy seems to be independent from this effector pathway.     

Myotrophin/V-1, a protein up-regulated in the failing human heart and in postnatal cerebellum, converts NFkappa B p50-p65 heterodimers to p50-p50 and p65-p65 homodimers

Myotrophin/V-1 is a cytosolic protein found at elevated levels in failing human hearts and in postnatal cerebellum. We have previously shown that it disrupts nuclear factor of kappaB (NFkappaB)-DNA complexes in vitro. In this study, we demonstrated that in HeLa cells native myotrophin/V-1 is predominantly present in the cytoplasm and translocates to the nucleus during sustained NFkappaB activation. Three-dimensional alignment studies indicate that myotrophin/V-1 resembles a truncated IkappaBalpha without the signal response domain (SRD) and PEST domains. Co-immunoprecipitation studies reveal that myotrophin/V-1 interacts with NFkappaB proteins in vitro; however, it remains physically associated only with p65 and c-Rel proteins in vivo during NFkappaB activation. In vitro studies indicate that myotrophin/V-1 can promote the formation of p50-p50 homodimers from monomeric p50 proteins and can convert the preformed p50-p65 heterodimers into p50-p50 and p65-p65 homodimers. Furthermore, adenovirus-mediated overexpression of myotrophin/V-1 resulted in elevated levels of both p50-p50 and p65-p65 homodimers exceeding the levels of p50-p65 heterodimers compared with Adbetagal-infected cells, where the levels of p50-p65 heterodimers exceeded the levels of p50-p50 and p65-p65 homodimers. Thus, overexpression of myotrophin/V-1 during NFkappaB activation resulted in a qualitative shift by quantitatively reducing the level of transactivating heterodimers while elevating the levels of repressive p50-p50 homodimers. Correspondingly, overexpression of myotrophin/V-1 resulted in significantly reduced kappaB-luciferase reporter activity. Because myotrophin/V-1 is found at elevated levels during NFkappaB activation in postnatal cerebellum and in failing human hearts, this study cumulatively suggests that myotrophin/V-1 is a regulatory protein for modulating the levels of activated NFkappaB dimers during this period.     

Myocardial cell death and regeneration during progression of cardiac hypertrophy to heart failure

Cardiac hypertrophy and ensuing heart failure are among the most common causes of mortality worldwide, yet the triggering mechanisms for progression of hypertrophy to failure are not fully understood. Tissue homeostasis depends on proper relationships between cell proliferation, differentiation, and death and any imbalance between them results in compromised cardiac function. Recently, we developed a transgenic (Tg) mouse model that overexpress myotrophin (a 12-kDa protein that stimulates myocyte growth) in heart resulting in hypertrophy that progresses to heart failure. This provided us an appropriate model to study the disease process at any point from initiation of hypertrophy end-stage heart failure. We studied detailed apoptotic signaling and regenerative pathways and found that the Tg mouse heart undergoes myocyte loss and regeneration, but only at a late stage (during transition to heart failure). Several apoptotic genes were up-regulated in 9-month-old Tg hearts compared with age-matched wild type or 4-week-old Tg hearts. Cardiac cell death during heart failure involved activation of Fas, tumor necrosis factor-alpha, and caspases 9, 8, and 3 and poly(ADP-ribose) polymerase cleavage. Tg mice with hypertrophy associated with compromised function showed significant up-regulation of cyclins,cyclin-dependent kinases (Cdks), and cell regeneration markers in myocytes. Furthermore, in human failing and nonfailing hearts, similar observations were documented including induction of active caspase 3 and Ki-67 proteins in dilated cardiomyopathic myocytes. Taken together, our data suggest that the stress of extensive myocardial damage from longstanding hypertrophy may cause myocytes to reenter the cell cycle. We demonstrate, for the first time in an animal model, that cell death and regeneration occur simultaneously in myocytes during end-stage heart failure, a phenomenon not observed at the onset of the disease process.     

PGE2-induced hypertrophy of cardiac myocytes involves EP4 receptor-dependent activation of p42/44 MAPK and EGFR transactivation

Upon induction of cyclooxygenase-2 (COX-2), neonatal ventricular myocytes (VMs) mainly synthesize prostaglandin E2 (PGE2). The biological effects of PGE2 are mediated through four different G protein-coupled receptor (GPCR) subtypes (EP(1-4)). We have previously shown that PGE2 stimulates cAMP production and induces hypertrophy of VMs. Because the EP4 receptor is coupled to adenylate cyclase and increases in cAMP, we hypothesized that PGE2 induces hypertrophic growth of cardiac myocytes through a signaling cascade that involves EP4-cAMP and activation of protein kinase A (PKA). To test this, we used primary cultures of VMs and measured [3H]leucine incorporation into total protein. An EP4 antagonist was able to partially block PGE2 induction of protein synthesis and prevent PGE2-dependent increases in cell surface area and activity of the atrial natriuretic factor promoter, which are two other indicators of hypertrophic growth. Surprisingly, a PKA inhibitor had no effect. In other cell types, G protein-coupled receptor activation has been shown to transactivate the epidermal growth factor receptor (EGFR) and result in p42/44 mitogen-activated protein kinase (MAPK) activation and cell growth. Immunoprecipitation of myocyte lysates demonstrated that the EGFR was rapidly phosphorylated by PGE2 in VMs, and the EP4 antagonist blocked this. In addition, the selective EGFR inhibitor AG-1478 completely blocked PGE2-induced protein synthesis. We also found that PGE2 rapidly phosphorylated p42/44 MAPK, which was inhibited by the EP4 antagonist and by AG-1478. Finally, the p42/44 MAPK inhibitor PD-98053 (25 micromol/l) blocked PGE2-induced protein synthesis. Altogether, we believe these are the first data to suggest that PGE2 induces protein synthesis in cardiac myocytes in part via activation of the EP4 receptor and subsequent activation of p42/44 MAPK. Activation of p42/44 MAPK is independent of the common cAMP-PKA pathway and involves EP4-dependent transactivation of EGFR.     

The AMPK gamma1 R70Q mutant regulates multiple metabolic and growth pathways in neonatal cardiac myocytes

Although mutations in the gamma-subunit of AMP-activated protein kinase (AMPK) can result in excessive glycogen accumulation and cardiac hypertrophy, the mechanisms by which this occurs have not been well defined. Because >65% of cardiac AMPK activity is associated with the gamma1-subunit of AMPK, we investigated the effects of expression of an AMPK-activating gamma1-subunit mutant (gamma1 R70Q) on regulatory pathways controlling glycogen accumulation and cardiac hypertrophy in neonatal rat cardiac myocytes. Whereas expression of gamma1 R70Q displayed the expected increase in palmitate oxidation rates, rates of glycolysis were significantly depressed. In addition, glycogen synthase activity was increased in cardiac myocytes expressing gamma1 R70Q, due to both increased expression and decreased phosphorylation of glycogen synthase. The inhibition of glycolysis and increased glycogen synthase activity were correlated with elevated glycogen levels in gamma1 R70Q-expressing myocytes. In association with the reduced phosphorylation of glycogen synthase, glycogen synthase kinase (GSK)-3beta protein and mRNA levels were profoundly decreased in the gamma1 R70Q-expressing myocytes. Consistent with GSK-3beta negatively regulating hypertrophy via inhibition of nuclear factor of activated T cells (NFAT), the dramatic downregulation of GSK-3beta was associated with increased nuclear activity of NFAT. Together, these data provide important new information about the mechanisms by which a mutation in the gamma-subunit of AMPK causes altered AMPK signaling and identify multiple pathways involved in regulating both cardiac myocyte metabolism and growth that may contribute to the development of the gamma mutant-associated cardiomyopathy.