Down regulation of myocardial β1-adrenoceptor signal transduction system in pacing-induced failure in dogs with aortic stenosis-induced left ventricular hypertrophy

We recently demonstrated that rapid ventricular pacing caused cardiac failure (Failure) in dogs with aortic stenosis-induced left ventricular hypertrophy (Hypertrophy) and isoproterenol caused no significant increases in function, O₂ consumption and intracellular cyclic AMP level in the failing hypertrophied hearts. We tested the hypothesis that alterations in the ₁-adrenoceptor-signal transduction pathway would correlate with the reduced functional and metabolic responses to -adrenergic stimulation during the transition from the compensated hypertrophy to failure. Pressure overload-induced left ventricular hypertrophy was created using aortic valve plication in 10 dogs over a 6-month period. Five months after aortic valve plication, congestive heart failure was induced in 5 dogs by rapid ventricular pacing at 240 bpm for 4 weeks. The density of myocardial ₁-adrenoceptors (fmoles/mg membrane protein; fmoles/g wet tissue) was significantly reduced in the Failure dogs (176 ± 19; 755 ± 136) when compared to those of the Control (344 ± 51; 1,551 ± 203) and the Hypertrophy (298 ± 33; 1,721 ± 162) dogs. The receptor affinities were not significantly different among all groups. There was a small but significant decrease in the percentage of ₁-adrenoceptors of the failing hypertrophied hearts (62 ± 3%) when compared to that of the hypertrophied hearts (77 ± 5%). The basal myocardial adenylyl cyclase activity (rmoles/mg protein/min) was significantly lower in the Failure dogs (45 ± 4) than in the Control (116 ± 14) and Hypertrophy (86 ± 6) dogs. The forskolin (0.1 mM)-stimulated adenylyl cyclase activity was also significantly lower in the Failure dogs (158 ± 17) than in the Control dogs (296 ± 35) and slightly lower than in the Hypertrophy dogs (215 ± 10). There were no significant differences in low Km cyclic AMP-phosphodiesterase activities among all groups. We conclude that down regulation of ₁-adrenoceptors and reduced adenylyl cyclase activities contribute to the decreases in myocardial functions and -adrenergic responses in the failing hypertrophied hearts induced by rapid ventricular pacing.     

Energy metabolism and mechanical recovery after cardioplegia in moderately hypertrophiedrats

It is well established that severe hypertrophy induces metabolic and structural changes in the heart which result in enhanced susceptibility to ischemic damage during cardioplegic arrest while much less is known about the effect of cardioplegic arrest on moderately hypertrophied hearts. The aim of this study was to elucidate the differences in myocardial high energy phosphate metabolism and in functional recovery after cardioplegic arrest and ischemia in mildly hypertrophied hearts, before any metabolic alterations could be shown under baseline conditions.Cardiac hypertrophy was induced in rats by constriction of the abdominal aorta resulting in 20% increase in heart weight/body weight ratio (hypertrophy group) while sham operated animals served as control. In both groups, isolated hearts were perfused under normoxic conditions for 40 min followed by infusion of St.Thomas' Hospital No. 1 cardioplegia and 90 min ischemia at 25øC with infusions of cardioplegia every 30 min. The changes in ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) were followed by³¹ P nuclear magnetic resonance (NMR) spectroscopy. Systolic and diastolic function was assessed with an intraventricular balloon before and after ischemia.Baseline concentrations of PCr, ATP and Pi as well as coronary flow and cardiac function were not different between the two groups. However, after cardioplegic arrest PCr concentration increased to 61.8 ± 4.9 mol/g dry wt in the control group and to 46.3 ± 2.8 mol/g in hypertrophied hearts. Subsequently PCr, pH and ATP decreased gradually, concomitant with an accumulation of Pi in both groups. PCr was transiently restored during each infusion of cardioplegic solution while Pi decreased. PCr decreased faster after cardioplegic infusions in hypertrophied hearts. The most significant difference was observed during reperfusion: PCr recovered to its pre-ischemic levels within 2 min following restoration of coronary flow in the control group while similar recovery was observed after 4 min in the hypertrophied hearts. A greater deterioration of diastolic function was observed in hypertrophied hearts.Moderate hypertrophy, despite absence of metabolic changes under baseline conditions could lead to enhanced functional deterioration after cardioplegic arrest and ischemia. Impaired energy metabolism resulting in accelerated high energy phosphate depletion during ischemia and delayed recovery of energy equilibrium after cardioplegic arrest observed in hypertrophied hearts could be one of the underlying mechanisms.     

Down-regulation of mitofusin-2 expression in cardiac hypertrophy in vitro and in vivo

Mitofusin-2 (Mfn2) suppresses smooth muscle cell proliferation through inhibition of the Ras-extracellular signal-regulated kinases (ERK1/2) pathway. Since the ERK1/2 pathway is implicated in mediating hypertrophic signaling, we studied the changes in Mfn2 in cardiac hypertrophy using in vitro and in vivo models. Phenylephrine was used to induce hypertrophy in neonatal rat ventricular myocytes (NRVMs). In vivo hypertrophy models included spontaneously hypertensive rats (SHR), pressure-overload hypertrophy by transverse aortic constriction (TAC), hypertrophy of non-infarcted myocardium following myocardial infarction (MI), and cardiomyopathy due to cardiac-restricted overexpression of beta(2)-adrenergic receptors (beta(2)-TG). We determined hypertrophic parameters and analysed expression of atrial natriuretic peptide (ANP) and Mfn2 by real-time PCR. Phosphorylated-ERK1/2 (phospho-ERK) was measured by Western blot. Mfn2 was downregulated in phenylephrine treated NRCMs (by approximately 40%), hypertrophied hearts from SHR (by approximately 80%), mice with TAC (at 1 and 3 weeks, by approximately 50%), and beta(2)-TG mice (by approximately 20%). However, Mfn2 was not downregulated in hypertrophied hearts with 15 weeks of TAC, nor in hypertrophied non-infarcted myocardium following MI. phospho-ERK1/2 was increased in hypertrophied myocardium at 1 week post-TAC, but not in non-infarcted myocardium after MI, indicating that downregulated Mfn2 may be accompanied by an increase of phospho-ERK1/2. This study shows, for the first time, downregulated Mfn2 expression in hypertrophied hearts, which depends on the etiology and time course of hypertrophy. Further study is required to examine the causal relationship between Mfn2 and cardiac hypertrophy.     

Reduced cGMP signaling activates NF-kappaB in hypertrophied hearts of mice lacking natriuretic peptide receptor-A

Mice lacking natriuretic peptide receptor-A (NPRA) develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for cardiac hypertrophic growth in the absence of NPRA signaling are not yet known. We sought to determine the activation of nuclear factor-kappaB (NF-kappaB) in Npr1 (coding for NPRA) gene-knockout (Npr1-/-) mice exhibiting cardiac hypertrophy and fibrosis. NF-kappaB binding activity was 4-fold greater in the nuclear extract of Npr1-/- mutant mice hearts as compared with wild-type (Npr1+/+) mice hearts. In parallel, inhibitory kappaB kinase-beta activity and IkappaB-alpha protein phosphorylation were also increased 3- and 4-fold, respectively, in hypertrophied hearts of mutant mice. cGMP levels were significantly reduced 5-fold in plasma and 10-fold in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates NF-kappaB binding activity associated with hypertrophic growth of mutant mice hearts.     

GPR39 promotes cardiac hypertrophy by regulating the AMPK–mTOR pathway and protein synthesis

Hypertrophic growth of the cardiomyocytes is one of the core mechanisms underlying cardiac hypertrophy. However, the mechanism underlying cardiac hypertrophy remains not fully understood. Here we provided evidence that G protein-coupled receptor 39 (GPR39) promotes cardiac hypertrophy via inhibiting AMP-activated protein kinase (AMPK) signaling. GRP39 expression is overexpressed in hypertrophic hearts of humans and transverse aortic constriction (TAC)-induced cardiac hypertrophy in mice. In neonatal cardiomyocytes, adenovirus-mediated overexpression of GPR39 promoted angiotensin II-induced cardiac hypertrophy, while GPR39 knockdown repressed hypertrophic response. Adeno-associated virus 9-mediated knockdown of GPR39 suppressed TAC-induced decline in fraction shortening and ejection fraction, increase in heart weight and cardiomyocyte size, as well as overexpression of hypertrophic fetal genes. A mechanism study demonstrated that GPR39 repressed the activation of AMPK to activate the mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase β-1 (S6K1), subsequently promoted de novo protein synthesis. Inhibition of mTOR with rapamycin blocked the effects of GPR39 overexpression on protein synthesis and repressed cardiac hypertrophy. Collectively, our findings demonstrated that GPR39 promoted cardiac hypertrophy via regulating the AMPK–mTOR–S6K1 signaling pathway, and GRP39 can be targeted for the treatment of cardiac hypertrophy.     

Insulin Receptor Substrates Are Essential for the Bioenergetic and Hypertrophic Response of the Heart to Exercise Training

Insulin and insulin-like growth factor 1 (IGF-1) receptor signaling pathways differentially modulate cardiac growth under resting conditions and following exercise training. These effects are mediated by insulin receptor substrate 1 (IRS1) and IRS2, which also differentially regulate resting cardiac mass. To determine the role of IRS isoforms in mediating the hypertrophic and metabolic adaptations of the heart to exercise training, we subjected mice with cardiomyocyte-specific deletion of either IRS1 (CIRS1 knockout [CIRS1KO] mice) or IRS2 (CIRS2KO mice) to swim training. CIRS1KO hearts were reduced in size under basal conditions, whereas CIRS2KO hearts exhibited hypertrophy. Following exercise swim training in CIRS1KO and CIRS2KO hearts, the hypertrophic response was equivalently attenuated, phosphoinositol 3-kinase (PI3K) activation was blunted, and prohypertrophic signaling intermediates, such as Akt and glycogen synthase kinase 3β (GSK3β), were dephosphorylated potentially on the basis of reduced Janus kinase-mediated inhibition of protein phosphatase 2a (PP2A). Exercise training increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein content, mitochondrial capacity, fatty acid oxidation, and glycogen synthesis in wild-type (WT) controls but not in IRS1- and IRS2-deficient hearts. PGC-1α protein content remained unchanged in CIRS1KO but decreased in CIRS2KO hearts. These results indicate that although IRS isoforms play divergent roles in the developmental regulation of cardiac size, these isoforms exhibit nonredundant roles in mediating the hypertrophic and metabolic response of the heart to exercise.     

Chronoinotropic reaction of the hypertrophied myocardium using an electromechanical coupling mathematical model.]

Static and dynamic chrono-inotropic responses were recorded from both normal and hypertrophic rat auricular myocardium. The slope of the static force-frequency relation from hypertrophied heart was steeper than in the control hearts. The cellular mechanisms underlying changes in the force frequency response associated with hypertrophy of the heart were studied by means of a mathematical model of excitation-contraction coupling. The characteristic features of hypertrophied heart force-frequency relations are shown to be due to the enhanced volume of the intracellular Ca-stores in contrast to the total volume of the cardiomyocyte.     

Cardiac remodeling associated with protein increase and lipid accumulation in early-stage chronic kidney disease in rats

Chronic kidney disease (CKD) is associated with increased risks of cardiovascular morbidity and mortality. Cardiac remodeling including myocardial fibrosis and hypertrophy is frequently observed in CKD patients. In this study, we investigate the mechanism involved in cardiac hypertrophy associated with CKD using a rat model, by morphological and chemical component changes of the hypertrophic and non-hypertrophic hearts. Sprague–Dawley rats were 4/5 nephrectomized (Nx) at 11 weeks of age and assigned to no treatment and treatment with AST-120, which was reported to affect the cardiac damage, at 18 weeks of age. At 26 weeks of age, the rats were euthanized under anesthesia, and biochemical tests as well as analysis of cardiac condition were performed by histological and spectrophotometric methods. Cardiac hypertrophy and CKD were observed in 4/5 Nx rats even though vascular calcification and myocardial fibrosis were not detected. The increasing myocardial protein was confirmed in hypertrophic hearts by infrared spectroscopy. The absorption of amide I and other protein bands in hypertrophic hearts increased at the same position as in normal cardiac absorption. Infrared spectra also showed that lipid accumulation was also detected in hypertrophic heart. Conversely, the absorptions of protein were obviously reduced in the myocardium of non-hypertrophic heart with CKD compared to that of hypertrophic heart. The lipid associated absorption was also decreased in non-hypertrophic heart. Our results suggest that cardiac remodeling associated with relatively early-stage CKD may be suppressed by reducing increased myocardial protein and ameliorating cardiac lipid load.     

The Profile of Cardiac Cytochrome c Oxidase (COX) Expression in an Accelerated Cardiac-Hypertrophy Model

Summary The contribution of the mitochondrial components, the main source of energy for the cardiac hypertrophic growth induced by pressure overload, is not well understood. In the present study, complete coarctation of abdominal aorta was used to induce the rapid development of cardiac hypertrophy in rats. One to two days after surgery, we observed significantly higher blood pressure and cardiac hypertrophy, which remained constantly high afterwards. We found an early increased level of cytochrome c oxidase (COX) mRNA determined by in-situ hybridization and dot blotting assays in the hypertrophied hearts, and a drop to the baseline 20 days after surgery. Similarly, mitochondrial COX protein level and enzyme activity increased and, however, dropped even lower than baseline 20 days following surgery. In addition, in natural hypertension-induced hypertrophic hearts in genetically hypertensive rats, the COX protein was significantly lower than in normotensive rats. Taken together, the lower efficiency of mitochondrial activity in the enlarged hearts of long-term complete coarcted rats or genetically hypertensive rats could be, at least partially, the cause of hypertensive cardiac disease. Additionally, the rapid complete coarctation-induced cardiac hypertrophy was accompanied by a disproportionate COX activity increase, which was suggested to maintain the cardiac energy-producing capacity in overloaded hearts.     

Insulin-like growth factor I receptor signaling is required for exercise-induced cardiac hypertrophy

The receptors for IGF-I (IGF-IR) and insulin (IR) have been implicated in physiological cardiac growth, but it is unknown whether IGF-IR or IR signaling are critically required. We generated mice with cardiomyocyte-specific knockout of IGF-IR (CIGF1RKO) and compared them with cardiomyocyte-specific insulin receptor knockout (CIRKO) mice in response to 5 wk exercise swim training. Cardiac development was normal in CIGF1RKO mice, but the hypertrophic response to exercise was prevented. In contrast, despite reduced baseline heart size, the hypertrophic response of CIRKO hearts to exercise was preserved. Exercise increased IGF-IR content in control and CIRKO hearts. Akt phosphorylation increased in exercise-trained control and CIRKO hearts and, surprisingly, in CIGF1RKO hearts as well. In exercise-trained control and CIRKO mice, expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and glycogen content were both increased but were unchanged in trained CIGF1RKO mice. Activation of AMP-activated protein kinase (AMPK) and its downstream target eukaryotic elongation factor-2 was increased in exercise-trained CIGF1RKO but not in CIRKO or control hearts. In cultured neonatal rat cardiomyocytes, activation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) prevented IGF-I/insulin-induced cardiomyocyte hypertrophy. These studies identify an essential role for IGF-IR in mediating physiological cardiomyocyte hypertrophy. IGF-IR deficiency promotes energetic stress in response to exercise, thereby activating AMPK, which leads to phosphorylation of eukaryotic elongation factor-2. These signaling events antagonize Akt signaling, which although necessary for mediating physiological cardiac hypertrophy, is insufficient to promote cardiac hypertrophy in the absence of myocardial IGF-I signaling.     

Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth

Postnatal cardiac hypertrophies have traditionally been classified into physiological or pathological hypertrophies. Both of them are induced by hemodynamic load. Cardiac postnatal hypertrophic growth is regarded as a part of the cardiac maturation process that is independent of the cardiac working load. However, the functional significance of this biological event has not been determined, mainly because of the difficulty in creating an experimental condition for testing the growth potential of functioning heart in the absence of hemodynamic load. Recently, we generated a novel transgenic mouse model (alphaMHC-BMP10) in which the cardiac-specific growth factor bone morphogenetic protein 10 (BMP10) is overexpressed in postnatal myocardium. These alphaMHC-BMP10 mice appear to have normal cardiogenesis throughout embryogenesis, but develop to smaller hearts within 6 weeks after birth. alphaMHC-BMP10 hearts are about half the normal size with 100% penetrance. Detailed morphometric analysis of cardiomyocytes clearly indicated that the compromised cardiac growth in alphaMHC-BMP10 mice was solely because of defect in cardiomyocyte postnatal hypertrophic growth. Physiological analysis further demonstrated that the responses of these hearts to both physiological (e.g. exercise-induced hypertrophy) and pathological hypertrophic stimuli remain normal. In addition, the alphaMHC-BMP10 mice develop subaortic narrowing and concentric myocardial thickening without obstruction by four weeks of age. Systematic analysis of potential intracellular pathways further suggested a novel genetic pathway regulating this previously undefined cardiac postnatal hypertrophic growth event. This is the first demonstration that cardiac postnatal hypertrophic growth can be specifically modified genetically and dissected out from physiological and pathological hypertrophies.     

Src Homology 2 Domain-containing Phosphatase 2 (Shp2) Is a Component of the A-kinase-anchoring Protein (AKAP)-Lbc Complex and Is Inhibited by Protein Kinase A (PKA) under Pathological Hypertrophic Conditions in the Heart

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Our results identify a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that the tyrosine phosphatase, Shp2, is a component of the A-kinase-anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits its protein-tyrosine phosphatase activity. Given the important cardiac roles of both AKAP-Lbc and Shp2, we investigated the AKAP-Lbc-Shp2 interaction in the heart. AKAP-Lbc-tethered PKA is implicated in cardiac hypertrophic signaling; however, mechanism of PKA action is unknown. Mutations resulting in loss of Shp2 catalytic activity are also associated with cardiac hypertrophy and congenital heart defects. Our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Thus, while induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote compensatory cardiac hypertrophy.     

The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro

Cardiac hypertrophy is a major risk factor for heart failure and associated patient morbidity and mortality. Research investigating the aberrant molecular processes that occur during cardiac hypertrophy uses primary cardiomyocytes from neonatal rat hearts as the standard experimental in vitro system. In addition, some studies make use of the H9C2 rat cardiomyoblast cell line, which has the advantage of being an animal-free alternative; however, the extent to which H9C2 cells can accurately mimic the hypertrophic responses of primary cardiac myocytes has not yet been fully established. To address this limitation, we have directly compared the hypertrophic responses of H9C2 cells with those of primary rat neonatal cardiomyocytes following stimulation with hypertrophic factors. Primary rat neonatal cardiomyocytes and H9C2 cells were cultured in vitro and treated with angiotensin II and endothelin-1 to promote hypertrophic responses. An increase in cellular footprint combined with rearrangement of cytoskeleton and induction of foetal heart genes were directly compared in both cell types using microscopy and real-time rtPCR. H9C2 cells showed almost identical hypertrophic responses to those observed in primary cardiomyocytes. This finding validates the importance of H9C2 cells as a model for in vitro studies of cardiac hypertrophy and supports current work with human cardiomyocyte cell lines for prospective molecular studies in heart development and disease.     

Subcellular distribution of the enzymes of the malate-aspartate shuttle in rat heart and effect of experimental cardiac hypertrophy

The effect of experimental cardiac hypertrophy on the enzymes of the malate - aspartate shuttle aspartate aminotransferase (AAT) and malate dehydrogenase (MDH) was studied. ( l ) Aortic constriction in adult rats resulted in 25% cardiac hypertrophy in 2 1/2-3 weeks. Total DNA (mg per heart) did not change. ( 2 ) The proportions of mitochondrial and cytosolic isozymes of AAT and MDH did not change as a result of cardiac h y p e r t r o p h y . About two-thirds of each enzyme occurred in the mitochondrial form and one-third in the cytosolic form. ( 3 ) Total AAT in hypertrophic hearts, in enzyme units per mg DNA, increased by 24% compared to AAT content in the hearts of sham-operated animals . Total MDH did not change. SoIubilized protein increased by 20%. Normal hearts contained 10 times more enzyme units of MDH than of AAT. (4) Cardiac growth stimulation induced in newborn rats did not result in specific changes of either enzyme. It is suggested that true cardiac hypertrophy acts as a specific stimulus for the possibly rate-limiting enzyme AAT of the shuttle.     

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.     

Cytoskeletal networks and the regulation of cardiac contractility: microtubules, hypertrophy, and cardiac dysfunction

The cytoskeleton as classically defined for eukaryotic cells consists of three systems of protein filaments: the microtubules, the intermediate filaments, and the microfilaments. In mature striated muscle such as the heart of the adult mammal, these three types of cytoskeletal filaments are superimposed spatially on the myofilaments, a specialized system of contractile protein filaments. Each of these systems of protein filaments has the potential to respond in an adaptive or maladaptive manner during load-induced hypertrophic cardiac growth. However, the extent to which such hypertrophy is compensatory is also critically dependent on the type of hemodynamic overload that serves as the hypertrophic stimulus. Thus cardiac hypertrophy is not intrinsically maladaptive; rather, it is the nature of the inducing load rather than hypertrophy itself that is responsible, through effects on structural and/or regulatory proteins, for the frequent deterioration of initially compensatory hypertrophy into the congestive heart failure state. As one example reviewed here of this load specificity of maladaptation, increased microtubule network density is a persistent feature of severely pressure-overloaded, hypertrophied, and failing myocardium that imposes a primarily viscous load on active myofilaments during contraction.     

CIP4 is required for the hypertrophic growth of neonatal cardiac myocytes

Background

CIP4 is a scaffold protein that regulates membrane deformation and tubulation, organization of the actin cytoskeleton, endocytosis of growth factor receptors, and vesicle trafficking. Although expressed in the heart, CIP4 has not been studied with regards to its potential function in cardiac myocytes.

Results

We now show using RNA interference that CIP4 expression in neonatal rat ventricular myocytes is required for the induction of non-mitotic, hypertrophic growth by the α-adrenergic agonist phenylephrine, the IL-6 cytokine leukemia inhibitor factor, and fetal bovine serum, as assayed using morphometry, immunocytochemistry for the hypertrophic marker atrial natriuretic factor and [³H]leucine incorporation for de novo protein synthesis. This requirement was consistent with the induction of CIP4 expression by hypertrophic stimulation. The inhibition of myocyte hypertrophy by CIP4 small interfering oligonucleotides (siRNA) was rescued by expression of a recombinant CIP4 protein, but not by a mutant lacking the N-terminal FCH domain responsible for CIP4 intracellular localization.

Conclusions

These results imply that CIP4 plays a significant role in the intracellular hypertrophic signal transduction network that controls the growth of cardiac myocytes in heart disease.