Regulating Cardiomyocyte Growth Research Articles

Mst4, a novel cardiac STRIPAK complex–associated kinase, regulates cardiomyocyte growth and survival and is upregulated in human cardiomyopathy

Myocardial failure is associated with adverse remodeling which includes apoptotic loss of cardiomyocytes, hypertrophy as well as alterations in cell-cell contacts. Striatin-interacting phosphatase and kinase (STRIPAK) complexes and their kinase Mst4 have been linked to the development of different diseases. The role and targets of Mst4 in cardiomyocytes have not been investigated, yet.Multi tissue immunoblot experiments show highly enriched Mst4-expression in rodent hearts. Analyses of human biopsy samples from patients suffering from dilated cardiomyopathy revealed that Mst4 is upregulated (5,8-fold p<0.001) compared with non-failing controls. Increased abundance of Mst4 could also be detected in mouse models of cardiomyopathy. We confirmed that Mst4 interacts with STRIPAK components in neonatal rat ventricular cardiomyocytes, indicating that STRIPAK is present in the heart. Immunofluorescence stainings and molecular interaction studies revealed that Mst4 is localized to the intercalated disc and interacts with several intercalated disc proteins. Overexpression of Mst4 in neonatal rat cardiomyocytes results in hypertrophy compared with controls. In adult rat cardiomyocytes, Mst4-overexpression increases cellular and sarcomeric fractional shortening (p<0.05), indicating enhanced contractility. Overexpression of Mst4 also inhibits apoptotic cell death shown by reduction of cleaved Caspase3 (-69%, p<0.0001) and Caspase7 (-80%, p<0.0001) as well as cleaved Parp1 (-27%, p<0.001). To elucidate potential Mst4 targets, we performed phosphoproteomics analyses in neonatal rat cardiomyocytes after Mst4 overexpression and inhibition. The results revealed several target candidates of Mst4 at the intercalated disc and sarcolemma. ConclusionWe identified Mst4 as a novel cardiac kinase that is upregulated in human cardiomyopathy and regulates cardiomyocyte growth and survival.

Cpeb4 regulates cardiomyocyte growth and function by the modulating the expression of Zeb1

Cpeb4 regulates cardiomyocyte growth and function by the modulating the expression of Zeb1

METTL14 is required for exercise-induced cardiac hypertrophy and protects against myocardial ischemia-reperfusion injury

RNA m6A modification is the most widely distributed RNA methylation and is closely related to various pathophysiological processes. Although the benefit of regular exercise on the heart has been well recognized, the role of RNA m6A in exercise training and exercise-induced physiological cardiac hypertrophy remains largely unknown. Here, we show that endurance exercise training leads to reduced cardiac mRNA m6A levels. METTL14 is downregulated by exercise, both at the level of RNA m6A and at the protein level. In vivo, wild-type METTL14 overexpression, but not MTase inactive mutant METTL14, blocks exercise-induced physiological cardiac hypertrophy. Cardiac-specific METTL14 knockdown attenuates acute ischemia-reperfusion injury as well as cardiac dysfunction in ischemia-reperfusion remodeling. Mechanistically, silencing METTL14 suppresses Phlpp2 mRNA m6A modifications and activates Akt-S473, in turn regulating cardiomyocyte growth and apoptosis. Our data indicates that METTL14 plays an important role in maintaining cardiac homeostasis. METTL14 downregulation represents a promising therapeutic strategy to attenuate cardiac remodeling.

Abstract 378: Physiologic Role of Rpl13a SnoRNAs in Cell Size Regulation

Objectives: Box C/D small nucleolar RNAs (snoRNA) are a multifunctional family of ncRNAs that play a critical role in guiding 2'- O -methylation (Nm) of ribosomal RNA (rRNA). My work has recently revealed that box C/D snoRNAs can also direct methylation of messenger RNA (mRNA), and that this methylation can regulate mRNA translation in the heart. In studies of genetically-engineered mice lacking four box C/D snoRNAs, I have observed that the knockout animals have relatively small hearts compared to wild-type. The objective of my current study is to define the specific mechanisms by which box C/D snoRNAs regulate heart size. Methods: I investigated the hearts of genetically-engineered mice lacking four box C/D snoRNAs from the Rpl13a locus (snoRNAs U32a, U33, U34 and U35a ). I also used antisense oligonucleotides to knock down these snoRNAs in H9c2 rat cardiomyoblasts. Changes in gene and protein expression were assessed by qPCR and immunoblot. Relative Nm modification of mRNA was determined by reverse transcription at low dNTP concentrations, followed by real-time PCR (RTL-P). Results: Germline knockout of the four box C/D Rpl13a snoRNAs ( U32a, U33, U34 and U35a ) in adolescent mice produces developmentally smaller hearts. In H9c2 rat cardiomyoblasts, knockdown of Rpl13a snoRNAs by antisense oligonucleotides significantly reduces H9c2 cell size. These concordant effects on organ and cell size suggest that the Rpl13a snoRNAs might be regulating critical pathways that determine cell growth. Using a candidate gene approach, I found that Mtor mRNA and protein expression is significantly reduced in hearts from Rpl13a snoRNA knockout mice. Preliminary results using the RTL-P method suggest that Mtor is subject to snoRNA-guided Nm modification, which is decreased in Rpl13a snoRNA knockout mouse hearts. Conclusion: These results suggest that Rpl13a snoRNAs regulate cardiomyocyte growth, at least in part, by guiding Nm modification of Mtor mRNA.

Abstract 248: Microfibrillar-Associated Protein 4 Regulates Maladaptive Cardiac Remodeling

Cardiac hypertrophy is a well-known risk factor for heart failure. Cardiomyocytes are often considered the ultimate culprit of cardiac remodeling following stress, despite comprising less than half of the heart’s total cells. Recent work has begun to recognize the importance of non-myocyte populations in the heart, yet how signaling between these and cardiomyocytes can dictate cardiac remodeling is still largely unknown. We have found that Microfibrillar-associated protein 4 (MFAP4) is a novel mediator of intercellular communication in the heart, and is specifically required for cardiac adaptation to stress. MFAP4 is an extracellular matrix protein that has been previously shown as a necessary component of arterial remodeling following injury, but its role in cardiac hypertrophy has not been explored. Our data revealed that, in the heart, MFAP4 is selectively secreted by vascular smooth muscle cells and endothelial cells in response to transforming growth factor β (TGFβ) activation. To determine the role of MFAP4 in cardiac hypertrophy, we subjected MFAP4-deficient mice to two forms of stress: chronic pressure overload or 1 week administration of the pro-fibrotic agent Angiotensin II (AngII). MFAP4-deficient mice developed accelerated cardiac hypertrophy, fibrotic remodeling, and marked cardiac functional defects with pressure overload when compared to wild-type animals. Also, AngII administration was sufficient to exacerbate cardiac hypertrophy prior to marked cardiac functional decline in MFAP4-deficient mice, suggesting a primary role for MFAP4 in regulating cardiomyocyte growth. Furthermore, aging-induced cardiac stress led to left ventricular dilation and spontaneous development of functional defects in otherwise unstressed MFAP4-deficient mice. Mechanistically, MFAP4 possess an integrin-binding domain and can affect cardiac homeostasis downstream of cardiomyocyte integrins via altered signaling through the FAK-ERK cascade, well-known to dictate cardiomyocyte geometric remodeling. Overall, our study demonstrates a critical role for MFAP4 on cardiac hypertrophy and function post-injury, underscoring the importance of non-myocyte-derived factors on cardiomyocyte pathophysiology.

The Heparan Sulfate Proteoglycan Glypican-6 Is Upregulated in the Failing Heart, and Regulates Cardiomyocyte Growth through ERK1/2 Signaling.

Pressure overload is a frequent cause of heart failure. Heart failure affects millions of patients worldwide and is a major cause of morbidity and mortality. Cell surface proteoglycans are emerging as molecular players in cardiac remodeling, and increased knowledge about their regulation and function is needed for improved understanding of cardiac pathogenesis. Here we investigated glypicans (GPC1-6), a family of evolutionary conserved heparan sulfate proteoglycans anchored to the extracellular leaflet of the cell membrane, in experimental and clinical heart failure, and explored the function of glypican-6 in cardiac cells in vitro. In mice subjected to pressure overload by aortic banding (AB), we observed elevated glypican-6 levels during hypertrophic remodeling and dilated, end-stage heart failure. Consistently, glypican-6 mRNA was elevated in left ventricular myocardium from explanted hearts of patients with end-stage, dilated heart failure with reduced ejection fraction. Glypican-6 levels correlated negatively with left ventricular ejection fraction in patients, and positively with lung weight after AB in mice. Glypican-6 mRNA was expressed in both cardiac fibroblasts and cardiomyocytes, and the corresponding protein displayed different sizes in the two cell types due to tissue-specific glycanation. Importantly, adenoviral overexpression of glypican-6 in cultured cardiomyocytes increased protein synthesis and induced mRNA levels of the pro-hypertrophic signature gene ACTA1 and the hypertrophy and heart failure signature genes encoding natriuretic peptides, NPPA and NPPB. Overexpression of GPC6 induced ERK1/2 phosphorylation, and co-treatment with the ERK inhibitor U0126 attenuated the GPC6-induced increase in NPPA, NPPB and protein synthesis. In conclusion, our data suggests that glypican-6 plays a role in clinical and experimental heart failure progression by regulating cardiomyocyte growth through ERK signaling.

(436) - The Ubiquitin Ligase Mule Is Required for the Maintenance of Normal Cardiac Function

Heart failure (HF) is the leading cause of morbidity and mortality worldwide. When exposed to injury, the heart compensates for the increased workload by cardiomyocyte (CM) hypertrophy, ultimately leading to maladaptive remodelling and HF. Defining the mechanisms underlying HF and understanding the factors regulating CM growth has immense clinical consequence. Protein ubiquitination is a key mechanism for controlling biological signalling pathways. The X-chromosome linked Mcl-1 ubiquitin ligase E3 (Mule) gene mediates the polyubiquitination and degradation of several proteins including Arf, Mcl-1, c-Myc and p53, all known to be involved in the control of cell growth and apoptosis.

Angiotensin II and the JNK pathway mediate urotensin II expression in response to hypoxia in rat cardiomyocytes

Cardiomyocyte hypoxia causes cardiac hypertrophy through cardiac-restricted gene expression. Urotensin II (UII) cooperates with activating protein 1 (AP1) to regulate cardiomyocyte growth in response to myocardial injuries. Angiotensin II (AngII) stimulates UII expression, reactive oxygen species (ROS) production, and cardiac hypertrophy. This study aimed to evaluate the expression of UII, ROS, and AngII as well as their genetic transcription after hypoxia treatment in neonatal cardiomyocytes. Cultured neonatal rat cardiomyocytes were subjected to hypoxia for different time periods. UII (Uts2) protein levels increased after 2.5% hypoxia for 4 h with earlier expression of AngII and ROS. Both hypoxia and exogenously added AngII or Dp44mT under normoxia stimulated UII expression, whereas AngII receptor blockers, JNK inhibitors (SP600125), JNK siRNA, or N-acetyl-l-cysteine (NAC) suppressed UII expression. The gel shift assay indicated that hypoxia induced an increase in DNA-protein binding between UII and AP1. The luciferase assay confirmed an increase in transcription activity of AP1 to the UII promoter under hypoxia. After hypoxia, an increase in (3)H-proline incorporation in the cardiomyocytes and expression of myosin heavy chain protein, indicative of cardiomyocyte hypertrophy, were observed. In addition, hypoxia increased collagen I expression, which was inhibited by SP600125, NAC, and UII siRNA. In summary, hypoxia in cardiomyocytes increases UII and collagen I expression through the induction of AngII, ROS, and the JNK pathway causing cardiomyocyte hypertrophy and fibrosis.

DYRK2 negatively regulates cardiomyocyte growth by mediating repressor function of GSK-3β on eIF2Bε.

BackgroundA prerequisite of hypertrophic response of the myocardium is an increase in protein synthesis. A central regulator of translation initiation is Eukaryotic initiation factor 2B (eIF2B). Here we assessed the hypothesis that regulation of protein synthesis via eIF2Bε is essential to cardiac hypertrophic response in vivo.MethodsTwo transgenic mouse lines were generated with cardiac restricted overexpression of eIF2Bε or its mutant eIF2Bε-eIFS535A, which cannot be inactivated by phosphorylation through GSK-3β.Results(1) Under baseline conditions eIF2Bε transgenic mice showed no difference in cardiac phenotype compared to wild type, whereas in the mutant eIF2Bε-S535A an increase in LV/tibia length (7.5±0.4 mg/mm vs. 6.2±0.2 mg/mm, p<0.001) and cardiomyocyte cross sectional area (13004±570 vs. 10843±347 RU, p<0.01) was observed. (2) Cardiac overexpression of eIF2Bε did not change the response of the heart to pathologic stress induced by chronic isoproterenol treatment. (3) Cardiac overexpression of the eIF2Bε transgene was followed by overexpression of DYRK2 which is known to prime the inhibitory action of GSK-3β on eIF2Bε, while DYRK1A and GSK-3β itself were not increased. (4) In C57BL/6 mice after 48 h of isoproterenol-stimulation or aortic banding, eIF2Bε was increased and DYRK2 was concomitantly decreased. (5) In line with these in vivo findings, siRNA knockdown of DYRK2 in cultured cardiomyocytes resulted in decreased levels of p(S535)- eIF2Bε, (6) whereas adenoviral induced overexpression of DYRK2 was accompanied by clearly increased phosphorylation of eIF2Bε, indicating a coordinated response pattern (7) Adenoviral induced overexpression of DYRK2 leads to significantly reduced cardiomyocyte size and diminishes hypertrophic response to adrenergic stimulation.ConclusionsThe interaction of GSK-3β and its priming kinase DYRK2 regulate the activity of eIF2Bε in cardiac myocytes. DYRK2 is a novel negative regulator of cardiomyocyte growth. DYRK2 could serve as a therapeutic option to regulate myocardial growth.

Abstract 19199: Cardiomyocyte Enriched MicroRNA, Mir-378, Regulates Hypertrophy Agonists Stimulated Growth of Cardiac Myocytes

MicroRNAs are small non-coding RNAs that have emerged as important negative regulators of gene expression. We recently described miR-378 as a cardiomyocyte enriched miRNA that targets IGF1/IGF1R signaling and regulates post-natal cardiac remodeling ( JBC 287(16):12913-26, 2012 ). In this study we tested the hypothesis that miR-378 regulates cardiomyocyte growth induced by hypertrophy stimuli. To test miR-378 involvement in cardiac hypertrophy, pressure overload was induced in adult mice by transverse aortic constriction (TAC) for 2 wks or 8 wks. TAC produced about 40% increase in HW / tibia length ratio with no overt cardiac dysfunction at 2 wks (hypertrophy, H group) and 30% reduction in %FS at 8 wks (heart failure, HF group). Expression of miR-378 was reduced by about 50% in H group but it was induced by 1.2-fold in HF group. Deficiency of miR-378 (30% to 60%) was also noted when neonatal rat ventricular cardiomyocytes (NRVM) were exposed to hypertrophy agonists, phenylephrine (PE, 20uM), AngII (100 nM) or isoproterenol (10 uM) for 48 h. Inhibition of miR-378 in NRVM by 378-antimiR, resulted in enhanced PE stimulated cardiomyocyte growth as reflected by 25% increase in cardiomyocyte size by WGA staining. We also noted significatly more induction of ANF in response to PE treatment in 378-antimiR transfected NRVM as compared to scramble control. Conversely, over-expression of miR-378 by 378-mimic in NRVM led to significant inhibition of PE induced [3] Leu incorporation and ANF induction. It also counteracted PE-stimulated activation of Ras and downstream signaling cascade such as pERK1/2, p70S6K and pAKT. One of the well accepted markers of pathological remodeling is nuclear translocation of NFATC which activates hypertrophy gene program in response to hypertrophy stimulus. By confocal imaging we found 378-mimic totally obliterated nuclear occupancy of NFATC 48 h after PE treatment. Data indicate that deficiency of miR-378 contributes to the development of cardiac hypertrophy, and by inhibiting Ras-signaling, miR-378 acts as an anti-hypertrophy microRNA. Our findings implicate that over-expression of miR-378 will counteract pathological remodeling and protect the heart from maladaptive hypertrophy and heart failure.

Abstract P279: YAP, a Transcriptional Cofactor of the Hippo Pathway, Regulates Cardiomyocyte Growth, Survival, and Proliferation

Mst1 and Lats2, components of the mammalian Hippo pathway, stimulate apoptosis and inhibit hypertrophy of cardiomyocytes (CMs), thereby mediating reperfusion injury and heart failure. YAP, a transcription factor co-factor, is negatively regulated by the Hippo pathway, and controls cell survival, proliferation and tissue regeneration. The role of YAP in regulating growth and death of CMs is poorly understood. YAP overexpression in CMs induced cardiac hypertrophy, as indicated by increases in cell size (+1.2 fold, p&lt;0.01), protein content (+1.1 fold, p&lt;0.01) and ANF (luciferase reporter activity +1.7 fold, mRNA +2.2 fold, and staining +2.7 fold, p&lt;0.01). Lats2 phosphorylates YAP at Serine 127, which induces cytoplasmic translocation of YAP, whereas YAP(S127A) is localized constitutively in the nucleus. Expression of YAP(S127A) enhanced hypertrophy in cultured CMs compared to that of wild type YAP (+1.87 fold ANF staining, p&lt;0.05), suggesting that the Mst1/Hippo pathway negatively regulates cardiac hypertrophy through YAP. YAP inhibited cell death induced by H2O2 treatment, as evaluated with TUNEL staining (-65%, p&lt;0.05) and CellTiter Blue assays (+34.9%, p&lt;0.01), indicating that YAP plays an essential role in mediating CM survival. Interestingly, YAP also significantly increased Ki67 positive cells in cultured CMs compared to LacZ (+2.65 fold, p&lt;0.05). We used a mouse model of chronic myocardial infarction (MI) to evaluate the function of YAP in the heart in vivo. Although YAP is diffusely localized both in the nucleus and cytosol in CMs in control hearts, CMs in the border zone of MI exhibited nuclear localization of YAP whereas YAP was excluded from the nucleus in CMs in the remodeling area four days after MI (+6.52 fold and +1.28 fold). Some of the YAP positive CMs in the border zone exhibited positive co-staining with Ki67, suggesting that YAP potentially induces CM proliferation. A significant increase in nuclear YAP and Ki67 positive CMs (+2.95 fold, p&lt;0.01 and +2.18 fold, p&lt;0.05) was also observed in neonatal rat hearts whose apex was surgically resected three days before euthanasia. These results suggest that YAP plays an important role in mediating not only hypertrophy and survival, but also proliferation of CMs in response to myocardial injury.

Role of miR-1 in regulating cardiomyocyte growth

Objective To construct and identify miR-1 adenovirus vector,and to analyze its effect on cardiac hypertrophy.Methods The primers of miR-1 precursor were designed for PCR amplification,and the PCR products were cloned into adenovirus shuttle plasmid pAdTrack and linearized by enzyme PmeⅠ;the resultant plasmid was co-transfected into E.coli BJ5183 cells with adenovirus backbone plasmid pAdEasy-1 for homologous recombination.Then the recombinant plasmid was identified,linearized and packaged into QBI-293A cells to amplify the recombinant adenovirus Ad-miR-1,which was then used to infect cardiomyocytes.Real-time quantitative PCR was used to observe the expression of miR-1 and two hypertrophic markers,the atrial natriuretic peptide(Nppa) and β-myosin heavy chain(myh7),in cultured primary cardiomyocytes.Cell surface area was analyzed using software AxioVision 4.7.1(Carl Zeiss).Results Sequencing and enzyme digestion showed that the miR-1 recombinant plasmid was successfully constructed.Real-time quantitative PCR confirmed that adenovirus Ad-miR-1 significantly enhanced intracellular miR-1 expression in cardiomyocytes and reduced cell surface area and the expression of Nppa and myh7.Conclusion The adenovirus expressing miR-1 has been successfully constructed and it can be transfected into cardiomyocytes to increase the expression of miR-1,thus inhibiting cardiomyocyte growth.

Crystal Structure of Cardiac-specific Histone Methyltransferase SmyD1 Reveals Unusual Active Site Architecture

SmyD1 is a cardiac- and muscle-specific histone methyltransferase that methylates histone H3 at lysine 4 and regulates gene transcription in early heart development. The unique domain structure characterized by a "split" SET domain, a conserved MYND zinc finger, and a novel C-terminal domain (CTD) distinguishes SmyD1 from other SET domain containing methyltransferases. Here we report the crystal structure of full-length SmyD1 in complex with the cofactor analog sinefungin at 2.3 Å. The structure reveals that SmyD1 folds into a wrench-shaped structure with two thick "grips" separated by a large, deep concave opening. Importantly, our structural and functional analysis suggests that SmyD1 appears to be regulated by an autoinhibition mechanism, and that unusually spacious target lysine-access channel and the presence of the CTD domain both negatively contribute to the regulation of this cardiovascularly relevant methyltransferase. Furthermore, our structure also provides a structural basis for the interaction between SmyD1 and cardiac transcription factor skNAC, and suggests that the MYND domain may primarily serve as a protein interaction module and cooperate SmyD1 with skNAC to regulate cardiomyocyte growth and maturation. Overall, our data provide novel insights into the mechanism of SmyD1 regulation, which would be helpful in further understanding the role of this protein in heart development and cardiovascular diseases.

Myostatin is upregulated following stress in an Erk-dependent manner and negatively regulates cardiomyocyte growth in culture and in a mouse model.

Myostatin is well established as a negative regulator of skeletal muscle growth, but its role in the heart is controversial. Our goal in this study was to characterize myostatin regulation following cardiomyocyte stress and to examine the role of myostatin in the regulation of cardiomyocyte size. Neonatal cardiomyocytes were cultured and stressed with phenylephrine. Adenovirus was used to overexpress myostatin or dominant negative myostatin in culture. Adeno-associated virus was used to overexpress myostatin or dominant negative myostatin in mice. Myostatin is upregulated following cardiomyocyte stress in an Erk-dependent manner that is associated with increased nuclear translocation and DNA binding activity of MEF-2. Myostatin overexpression leads to decreased and myostatin inhibition to increased cardiac growth both in vitro and in vivo due to modulation of Akt and NFAT3 pathways. Myostatin is a negative regulator of cardiac growth, and further studies are warranted to investigate the role of myostatin in the healthy and failing heart.

Wnt Signaling Molecules in Left Ventricular Remodeling

Under acute or chronic stresses, the adult heart undergoes a remodeling process that involves cardiomyocyte hypertrophy accompanied by apoptosis, necrosis, and fibrosis that lead to impaired cardiac contractility. The role of endogenous regeneration in this process is currently under investigation. Sustained deleterious stimuli will lead to a decompensated form of hypertrophy often culminating in heart failure.1 This form of hypertrophy is often referred to as “maladaptive.” When dealing with hypertrophy, it appears important to distinguish between the term being used on the cellular and molecular level (enlargement of individual cardiomyocytes and re-expression of fetal/embryonic genes) and the organ level (increased heart weight, left ventricular wall thickness, and functional diastolic and systolic impairment). In our view, these processes are certainly linked but not identical. Hypertrophy on the organ levels summarizes several independent cellular and molecular processes (see below), where cardiomyocyte growth is not necessarily the most important. Independent of its origin, cardiac hypertrophy is associated with alterations in cardiac geometry, mass, architecture, and function controlled by a complex network of interconnected and abundant signal-transduction pathways.2 New signaling molecules are emerging as possible targets to specifically attenuate maladaptive hypertrophy. Pathological, stress-induced growth of cardiomyocytes was shown to depend on Wnt/β-catenin nuclear signaling rather than its adhesive function in cell adhesion. However, the specificity of the cell type and the molecular mechanisms governing the Wnt signaling–dependent changes are currently unknown.3 In this issue of the Hypertension , the study by Malekar et al4 provides new evidences concerning the …

MicroRNA-1 Negatively Regulates Expression of the Hypertrophy-Associated Calmodulin and Mef2a Genes

Calcium signaling is a central regulator of cardiomyocyte growth and function. Calmodulin is a critical mediator of calcium signals. Because the amount of calmodulin within cardiomyocytes is limiting, the precise control of calmodulin expression is important for the regulation of calcium signaling. In this study, we show for the first time that calmodulin levels are regulated posttranscriptionally in heart failure. The cardiomyocyte-restricted microRNA miR-1 inhibited the translation of calmodulin-encoding mRNAs via highly conserved target sites within their 3' untranslated regions. In keeping with its effect on calmodulin expression, miR-1 downregulated calcium-calmodulin signaling through calcineurin to NFAT. miR-1 also negatively regulated the expression of Mef2a and Gata4, key transcription factors that mediate calcium-dependent changes in gene expression. Consistent with the downregulation of these hypertrophy-associated genes, miR-1 attenuated cardiomyocyte hypertrophy in cultured neonatal rat cardiomyocytes and in the intact adult heart. Our data indicate that miR-1 regulates cardiomyocyte growth responses by negatively regulating the calcium signaling components calmodulin, Mef2a, and Gata4.

Liver X Receptor activator inhibits AngII induced hypertrophic responses in rat neonatal cardiomyocytes

The progression of cardiac hypertrophy and heart failure involves a maladaptation in energy homeostasis. Proteins involved in this key energy metabolic pathway aretranscriptionally regulated by members of the nuclear receptorsuperfamily, such as PPARs and ERRs. The Liver X Receptors (LXRα and LXRβ) are members ofthe nuclear hormone receptor superfamily. These sterol‐responsivetranscription factors regulate the expression of a number ofgenes involved in lipid and glucose metabolism. We hypothesize that LXRs play important roles in regulating the adaptive substrate switch in cardiac hypertrophy and heart failure. We investigated how a LXR synthetic ligand T1317 influences AngII induced hypertrophic responses in cultured rat neonatal cardiomyocytes. We found AngII can induce upregulation of LXRalpha transcript levels in cultured cardiomyocytes (n=4, P&lt;0.05). Pretreatment of a LXR T1317 (1, 5, 10 uM) in cultured cardiomyocytes 2 hours prior to the 24 hour AngII treatment prevented hypertrophic responses. T1317 pretreatment suppressed an AngII induced elevation of protein synthesis in cardiomyocytes (n=6, P&lt;0.05). Furthermore, T1317 pretreatment prevented an AngII induced increase of cell surface size (n=25, P&lt;0.05). T1317 pretreatment also suppressed the expression of hypertorphic marker ANF induced by AngII treatment. Therefore, we conclude that LXRs play important roles in regulating cardiomyocyte growth, possibly by regulating intracellular energy balance.

Not All Hypertrophy Is Created Equal

See related article, pages 1164–1174 Cardiac hypertrophy is the natural response of myocardium to various stressors, including neurohormonal stimuli, hemodynamic overload, and injury. In the face of continued stress, pathological hypertrophy progresses to a loss of cardiomyocytes, the development of fibrosis, and, ultimately, heart failure. Emerging evidence has shown that glycogen synthase kinase-3β (GSK-3β) is an important negative regulator of cardiomyocyte hypertrophy, yet inhibition of GSK-3β has been shown to reduce cell death after ischemia reperfusion. Therefore, it has been difficult to predict what the consequences of chronic inhibition of GSK-3 in the heart would be because it might be a balance between the potential for the aggravation of cardiac hypertrophy versus antiapoptotic effects. In this issue of Circulation Research , Hirotani and colleagues report that sustained inhibition of GSK-3β in postneonatal hearts results in well-compensated “physiologic” cardiac hypertrophy and, most intriguingly, exerts protective effects against the development of “pathological” hypertrophy and heart failure with pressure overload.1 Although there are a number of concerns that need to be addressed before targeting GSK-3 for the treatment of heart failure, this “best of both worlds” outcome provides an interesting and favorable rationale for such intervention. The GSK-3 family of protein kinases is encoded by 2 genes, α and β. They are highly conserved throughout evolution and are critical to the regulation of diverse biological processes ranging from organ development to cell death, including cell growth, cell cycling, cytoskeletal organization, and metabolism.2,3 Furthermore, dysregulation of GSK-3β has been implicated in the pathogenesis of many human diseases including Alzheimer disease, diabetes, and tumorgenesis.4–8 GSK-3α (51 kDa) is the larger of the two and has a glycine-rich N terminus of unknown function. GSK-3β (47 kDa) and α share significant sequence homology (97%) in their kinase domains, however the homology between the …

Abstract 594: Identification of Cell Cycle Regulatory and Inflammatory Genes as Predominant Targets of p38 MAPK in the Heart

Mitogen-activated protein kinases (MAPKs) regulate cardiomyocyte growth and apoptosis in response to extracellular stimulation, but the downstream effectors that mediate their pathophysiological effects remain poorly understood. We determined the targets and role of p38 MAPK in the heart in vivo by using local adenovirus-mediated gene transfer of constitutively active upstream kinase mitogen-activated protein kinase kinase 3b (MKK3bE) and wild-type p38α in rats. DNA microarray analysis of animals with cardiac-specific overexpression of p38 MAPK revealed that 264 genes were upregulated and 2 genes were down-regulated more than 2-fold. A large number of previously unknown p38 target genes were found, such as cell cycle regulators cyclins A2, and B1 as well as transcription factors Runx1 and Krox20. Many fundamental inflammation-associated genes like interleukin-6 (IL-6) and toll-like receptor-2 as well as cell adhesion genes selectin E and osteopontin were also elevated in p38 MAPK overexpressing hearts. The complete list of 266 differentially expressed genes was loaded into PathwayAssist to build the interaction map showing potential connections among genes regulated by p38 MAPK overexpression in the heart. A central signaling molecule in this pathway was IL-6, which has been implicated in the regulation of 20 genes found to be affected by p38 MAPK. Further, using gel mobility shift assays we identified several cardiac transcription factors including serum response factor (SRF), activator protein-1 (AP-1), nuclear factor κB (NF-κB) that were directly activated by p38 MAPK overexpression. Finally, we determined the functional significance of the altered cardiac gene expression profile by histological analysis and echocardiographic measurements, which indicated that p38 MAPK overexpression induced gene expression results in myocardial cell proliferation, inflammation and fibrosis. In conclusion, we defined the novel target genes and transcription factors as well as the functional effects of p38 MAPK in the heart. Our results demonstrate that the pathophysiological consequence of p38 MAPK overexpression is myocardial cell proliferation and inflammation associated with fibrosis in the adult heart.

Identification of Cell Cycle Regulatory and Inflammatory Genes As Predominant Targets of p38 Mitogen-Activated Protein Kinase in the Heart

Mitogen-activated protein kinases (MAPKs) regulate cardiomyocyte growth and apoptosis in response to extracellular stimulation, but the downstream effectors that mediate their pathophysiological effects remain poorly understood. We determined the targets and role of p38 MAPK in the heart in vivo by using local adenovirus-mediated gene transfer of constitutively active upstream kinase mitogen-activated protein kinase kinase 3b (MKK3bE) and wild-type p38alpha in rats. DNA microarray analysis of animals with cardiac-specific overexpression of p38 MAPK revealed that 264 genes were upregulated more than 2-fold including multiple genes controlling cell division, cell signaling, inflammation, adhesion, and transcription. A large number of previously unknown p38 target genes were found. Using gel mobility-shift assays we identified several cardiac transcription factors that were directly activated by p38 MAPK. Finally, we determined the functional significance of the altered cardiac gene-expression profile by histological analysis and echocardiographic measurements, which indicated that p38 MAPK overexpression-induced gene expression results in myocardial cell proliferation, inflammation, and fibrosis. In conclusion, we defined the novel target genes and transcription factors as well as the functional effects of p38 MAPK in the heart. Expression profiling of p38 MAPK overexpression identified cell cycle regulatory and inflammatory genes critical for pathological processes in the adult heart.