Hypertrophic Gene Expression Research Articles

Abstract 027: Participation of Cardiac Thyrotroping Releasing Hormone (cTRH) in the Doxorrubicin (D) Induced Cardiotoxicityin in Mice

We have demonstrated TRH hyperactivity in the hypertrophied Left ventricle (LV) of SHR. Its specific inhibition attenuates hypertrophy development in spite of the significant higher pressure observed (Schuman M, Hypertension 2011). LV-TRH over-expression induces several features of the hypertrophied heart, including increases in the apoptotic index Bax/Bcl2, and the activated caspase 3 observed by immunohystochemistry (Schuman M, AJPH&C 2014). In addition, we have found that TRH expression was induced by D in primary cultures of cardiac cells. Based on these results we hypothesized that cTRH could participate in the D cardiotoxicity effects. Indeed, we used C57 adult males (n=10) with a single D or saline injection (200uL,10 mg/kg ip) which previously (24 h, under anesthesia) received an intracardiac injection of a specific TRH-siRNA to inhibit LV-TRH expression or scrambled Con-siRNA. Mice were sacrificed 2,4 and 7 days post D injection and body weight was measured. Genes expression was measured by real time PCR and protein by immunohystochemistry (ANOVA and Tukey test). Body weight showed a mild but not significant decrease in D treated animals. D significantly increased TRH gene expression and TRH protein content reaching the maximum at 7 days post injection (2d: 145%, 4d:190% and 7d: 250%) (p& 0.05), which were not observed in the groups with D+TRH-siRNA indicating the effectiveness of the specific TRH inhibition. Also at this time TRH inhibition attenuates (p&0.05) D-induced increase in the apoptotic index Bax/Bcl2 and the augmented activated caspase 3 content pointing out the participation of the cardiac TRH in the D-induced apoptosis. Similar results were observed with hypertrophic and fibrotic markers gene expression (BNP, BMHC and col III) which showed a significant increase (p& 0.05) only in the groups with D and the intact TRH system (D+Con-siRNA). Fibrosis results were confirmed by Sirius Red and Masson techniques. On the whole, we demonstrated for the first time that LV-TRH system is required for both, Doxorrubicin induction of apoptosis and consecutively hypertrophy and fibrosis in the mouse heart. Even more, we found that cTRH inhibition attenuates doxorrubicin induced damage suggesting a novel mechanisms in the cardiotoxicity injury.

AP-1 family members act with Sox9 to promote chondrocyte hypertrophy.

An analysis of Sox9 binding profiles in developing chondrocytes identified marked enrichment of an AP-1-like motif. Here, we have explored the functional interplay between Sox9 and AP-1 in mammalian chondrocyte development. Among AP-1 family members, Jun and Fosl2 were highly expressed within prehypertrophic and early hypertrophic chondrocytes. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) showed a striking overlap in Jun- and Sox9-bound regions throughout the chondrocyte genome, reflecting direct binding of each factor to the same enhancers and a potential for protein-protein interactions within AP-1- and Sox9-containing complexes. In vitro reporter analysis indicated that direct co-binding of Sox9 and AP-1 at target motifs promoted gene activity. By contrast, where only one factor can engage its DNA target, the presence of the other factor suppresses target activation consistent with protein-protein interactions attenuating transcription. Analysis of prehypertrophic chondrocyte removal of Sox9 confirmed the requirement of Sox9 for hypertrophic chondrocyte development, and in vitro and ex vivo analyses showed that AP-1 promotes chondrocyte hypertrophy. Sox9 and Jun co-bound and co-activated a Col10a1 enhancer in Sox9 and AP-1 motif-dependent manners consistent with their combined action promoting hypertrophic gene expression. Together, the data support a model in which AP-1 family members contribute to Sox9 action in the transition of chondrocytes to the hypertrophic program.

Abstract 240: Differential Impact of RBFox Family Proteins in Adult Murine Heart

Background: The complexity of cardiac transcriptome and proteome is significantly contributed by alternative splicing of mRNA. Alternative splicing is regulated by the cis-regulatory elements located in pre-mRNA together with the trans-activating factors guiding the assembling and function of the spliceosome. In our earlier study, we have observed global changes of alternative splicing events during pressure-overload induced heart failure, and identified RBFox1 as a key regulator for cardiac RNA splicing regulation during postnatal development and pathological remodeling. Both RBFox1 and RBFox2 are highly enriched in cardiomyocytes, and their expression are both significantly repressed in response to pathological stress. Loss-of-function studies for RBFox1 and RBFox2 are achieved using cardiac specific but constitutively active Cre. Therefore, the isoform specific contribution of RBFox1 vs. RBFox2 in maintaining cardiac physiology and homeostasis in adult heart is unknown. Methods and Results: We generated mouse models of cardiac specific and inducible knockout of RBFox1 and RBFox2 individually in adult hearts by breeding the individual floxed alleles with the αMHC-Mer-Cre-Mer mice. At baseline, inactivating RBFox1 in adult heart caused a slight but significant decrease of cardiac function without activating hypertrophy gene expression. However, following myocardial infarction, the RBFox1 deficient hearts showed enhanced global fibrosis in non-infarcted areas comparing to the control animals. In contrast, inactivating RBFox2 in adult mouse heart caused overt heart failure associated with chamber dilation without external stress as early as 2 weeks post tamoxifen administration. Conclusion: We have identified differential impact of RBFox1 and RBFox2 deficiency in adult mouse heart. Our in vivo study illustrate the functional importance of the RBFox family RNA splicing regulators in normal physiology of adult heart, and support the pathogenic contribution of loss of RBFox expression to heart failure. Further analysis focusing on the underlying molecular mechanisms for their differential impact would yield new insights on transcriptome regulation and complexity in cardiac physiology and diseases.

Signalling function of β-catenin is important at early stages of adult heart pathological hypertrophy

The spread of cardiovascular diseases, their significant threat to health and socio-economic burden result in considerable interest of scientists to this problem solution. Lately, not only investigations into new methods of diagnosis and treatment of cardiovascular disease, but also elucidation of the mechanisms underlying their occurrence and course become topical. The aim of our study was to investigate the signaling function of the canonical Wnt-signaling and b-catenin function in the development of pathological hypertrophy of the adult myocardium. Methods. Studies were conducted using transgenic mice BATGIRL and cultures of isolated cardiomyocytes. To induce pathological hypertrophy, lithium chloride and AnglI were used. Changes in the expression of hypertrophic genes and genes involved in the canonical Wnt-signaling were analyzed by real-time PCR and Western-blot analysis. Morphological studies and X-gal staining were performed. Results. Upon the action of hypertrophic stimuli the activation of b-catenin signaling function is shown to occur in the early stages of observation, as evidenced by X-gal staining and changes in gene-targets expression of this signaling (c-Fos, c-Myc, CyclinD1 and TCF-4). There was also observed an increase in the content of activated b-catenin and phosphorylated GSK3b proteins within a day after the action of angiotensin and lithium chloride in the culture of isolated cardiomyocytes. Conclusions. With the development of pathological hypertrophy due to chronic high blood pressure, there occurs the activation of many signal-regulatory mechanisms of cardiomyocytes and one of them is the canonical Wnt-signaling. However, the activation of the canonical Wnt-signaling and β-catenin, in particular, is the early event and obviously essential to run the genetic program of myocardium remodeling.Keywords: β-catenin, hypertrophy, Wnt-signaling, gene expression, myocardium.

Cardiomyocyte Overexpression of FABP4 Aggravates Pressure Overload-Induced Heart Hypertrophy.

Fatty acid binding protein 4 (FABP4) is a member of the intracellular lipid-binding protein family, responsible for the transportation of fatty acids. It is considered to express mainly in adipose tissues, and be strongly associated with inflammation, obesity, diabetes and cardiovasculardiseases. Here we report that FABP4 is also expressed in cardiomyocytes and plays an important role in regulating heart function under pressure overload. We generated heart-specific transgenic FABP4 (FABP4-TG) mice using α myosin-heavy chain (α-MHC) promoter and human FABP4 sequence, resulting in over-expression of FABP4 in cardiomyocytes. The FABP4-TG mice displayed normal cardiac morphology and contractile function. When they were subjected to the transverse aorta constriction (TAC) procedure, the FABP4-TG mice developed more cardiac hypertrophy correlated with significantly increased ERK phosphorylation, compared with wild type controls. FABP4 over-expression in cardiomyocytes activated phosphor-ERK signal and up-regulate the expression of cardiac hypertrophic marker genes. Conversely, FABP4 induced phosphor-ERK signal and hypertrophic gene expressions can be markedly inhibited by an ERK inhibitor PD098059 as well as the FABP4 inhibitor BMS309403. These results suggest that FABP4 over-expression in cardiomyocytes can aggravate the development of cardiac hypertrophy through the activation of ERK signal pathway.

Calcific Aortic Valve Disease: Part 2-Morphomechanical Abnormalities, Gene Reexpression, and Gender Effects on Ventricular Hypertrophy and Its Reversibility.

In part 1, we considered cytomolecular mechanisms underlying calcific aortic valve disease (CAVD), hemodynamics, and adaptive feedbacks controlling pathological left ventricular hypertrophy provoked by ensuing aortic valvular stenosis (AVS). In part 2, we survey diverse signal transduction pathways that precede cellular/molecular mechanisms controlling hypertrophic gene expression by activation of specific transcription factors that induce sarcomere replication in-parallel. Such signaling pathways represent potential targets for therapeutic intervention and prevention of decompensation/failure. Hypertrophy provoking signals, in the form of dynamic stresses and ligand/effector molecules that bind to specific receptors to initiate the hypertrophy, are transcribed across the sarcolemma by several second messengers. They comprise intricate feedback mechanisms involving gene network cascades, specific signaling molecules encompassing G protein-coupled receptors and mechanotransducers, and myocardial stresses. Future multidisciplinary studies will characterize the adaptive/maladaptive nature of the AVS-induced hypertrophy, its gender- and individual patient-dependent peculiarities, and its response to surgical/medical interventions. They will herald more effective, precision medicine treatments.

Skeletal Muscle, but not Cardiovascular Function, Is Altered in a Mouse Model of Autosomal Recessive Hypophosphatemic Rickets.

Autosomal recessive hypophosphatemic rickets (ARHR) is a heritable disorder characterized by hypophosphatemia, osteomalacia, and poor bone development. ARHR results from inactivating mutations in the DMP1 gene with the human phenotype being recapitulated in the Dmp1 null mouse model which displays elevated plasma fibroblast growth factor 23. While the bone phenotype has been well-characterized, it is not known what effects ARHR may also have on skeletal, cardiac, or vascular smooth muscle function, which is critical to understand in order to treat patients suffering from this condition. In this study, the extensor digitorum longus (EDL-fast-twitch muscle), soleus (SOL–slow-twitch muscle), heart, and aorta were removed from Dmp1 null mice and ex-vivo functional tests were simultaneously performed in collaboration by three different laboratories. Dmp1 null EDL and SOL muscles produced less force than wildtype muscles after normalization for physiological cross sectional area of the muscles. Both EDL and SOL muscles from Dmp1 null mice also produced less force after the addition of caffeine (which releases calcium from the sarcoplasmic reticulum) which may indicate problems in excitation contraction coupling in these mice. While the body weights of the Dmp1 null were smaller than wildtype, the heart weight to body weight ratio was higher. However, there were no differences in pathological hypertrophic gene expression compared to wildtype and maximal force of contraction was not different indicating that there may not be cardiac pathology under the tested conditions. We did observe a decrease in the rate of force development generated by cardiac muscle in the Dmp1 null which may be related to some of the deficits observed in skeletal muscle. There were no differences observed in aortic contractions induced by PGF2α or 5-HT or in endothelium-mediated acetylcholine-induced relaxations or endothelium-independent sodium nitroprusside-induced relaxations. In summary, these results indicate that there are deficiencies in both fast twitch and slow twitch muscle fiber type contractions in this model of ARHR, while there was less of a phenotype observed in cardiac muscle, and no differences observed in aortic function. These results may help explain skeletal muscle weakness reported by some patients with osteomalacia and need to be further investigated.

Puerarin Attenuates Cardiac Hypertrophy Partly Through Increasing Mir-15b/195 Expression and Suppressing Non-Canonical Transforming Growth Factor Beta (Tgfβ) Signal Pathway.

BackgroundPrevious studies demonstrated that puerarin has therapeutic effects on cardiac hypertrophy. This study aimed to explore whether the effect of puerarin on attenuating cardiac hypertrophy is related to regulation of microRNAs (miRNAs) and the transforming growth factor beta (TGFβ) signal pathway.Material/MethodsThe therapeutic effect of puerarin was assessed using an angiotensin (Ang) II-induced heart hypertrophy model in mice. The primary cardiomyocytes were used as an in vitro model. MiR-15 family expression was quantified using qRT-PCR analysis. The expression of the genes involved in canonical and non-canonical TGFβ signal pathways was measured using qRT-PCR and Western blot analysis. In vitro cardiac hypertrophic features were assessed by quantifying cardiac hypertrophic genes and measurement of cell surface, protein synthesis, and total protein content.ResultsPuerarin attenuated cardiac hypertrophy and increased miR-15b and miR-195 expression in the mouse cardiac hypertrophy model and in primary cardiomyocytes. It suppressed both canonical and non-canonical TGFβ signal pathways, partially through miR-15b and miR-195. Puerarin reduced mRNA expression of cardiac hypertrophic genes, reduced cell surface area, and lowered the rate of protein synthesis and the total protein content induced by Ang II. Knockdown of endogenous miR-15b and miR-195 partly abrogated these effects. Knockdown of endogenous p38, but not Smad2/3/4, presented similar effects as miR-15b.ConclusionsPuerarin administration enhances miR-15b and miR-195 expression in an Ang II-induced cardiac hypertrophy model, through which it suppresses both canonical and non-canonical TGFβ signal pathways at the same time. However, the effect of puerarin on attenuating cardiac hypertrophy is mainly through the non-canonical TGFβ pathway.

Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads.

Chondrogenic differentiation of mesenchymal stromal cells (MSCs) in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM) was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG) analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05) chondrogenic but lower hypertrophic (p < 0.05) gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis.

Carfilzomib-induced cardiotoxicity mitigated by dexrazoxane through inhibition of hypertrophic gene expression and oxidative stress in rats

Carfilzomib (CFZ) is an inhibitor of proteasome that is generally used in the treatment of multiple myeloma but due to its cardiotoxicity clinical use may be limited. Dexrazoxane (DZR), an inhibitor of topoisomerase-II, prevents cardiac damage by reducing the formation of reactive oxygen species and hypertrophic gene expression. This study evaluated the protective effect of DZR on CFZ-induced cardiotoxicity. Thirty-two male Albino rats were randomly divided into four groups (n = 8). Group I received DMSO, Group II received CFZ (4 mg/kg, intraperitoneally [i.p.]) twice weekly up to day 16, Group III received DZR (20 mg/kg, i.p.) for 16 days and CFZ twice weekly for 16, Group IV received DZR (40 mg/kg, i.p.) for 16 days and CFZ twice weekly for 16. CFZ-induced cardiotoxicity was assessed by hematological, biochemical, mRNA expression, oxidative stress and histopathological studies. CFZ-induced significant changes have been observed in blood parameters including red blood cells, white blood cells, hemoglobin and hematocrit concentrations which were associated with increase in cardiac enzymes markers like creatine kinase (CK), CK-MB and lactate dehydrogenase. Treatment with DZR reversed the hematological statistics and the biochemical markers of CFZ-induced cardiotoxicity. Furthermore, DZR also attenuated the effects of CFZ-induced toxic effect on redox markers such as malondialdehyde and reduced glutathione. Above findings were further confirmed by beta-myosin heavy chain (β-MHC) and alpha-MHC (α-MHC) gene expression. Histopathological reports suggested that DZR ameliorates CFZ-induced changes in cardiac cellular architecture in rats. These results confirm that DZR protects heart from CFZ-induced cardiotoxicity.

Histone deacetylase inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats.

CG200745 is a novel inhibitor of histone deacetylases (HDACs), initially developed for treatment of various hematological and solid cancers. Because it is water-soluble, it can be administered orally. We hypothesized that the HDAC inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in deoxycorticosterone acetate (DOCA)-induced hypertensive rats. For establishment of hypertension, 40 mg/kg of DOCA was subcutaneously injected four times weekly into Sprague-Dawley rats. All the rats used in this study including those in the sham group had been unilaterally nephrectomized and allowed free access to drinking water containing 1% NaCl. Systolic blood pressure was measured by the tail-cuff method. Blood chemistry including sodium, potassium, glucose, triglyceride, and cholesterol levels was analyzed. Sections of the heart were visualized after trichrome and hematoxylin and eosin stain. The expression of hypertrophic genes such as atrial natriuretic peptide A (Nppa) and atrial natriuretic peptide B (Nppb) in addition to fibrotic genes such as Collagen-1, Collagen-3, connective tissue growth factor (Ctgf), and Fibronectin were measured by quantitative real-time PCR (qRT-PCR). Injection of DOCA increased systolic blood pressure, heart weight, and cardiac fibrosis, which was attenuated by CG200745. Neither DOCA nor CG200745 affected body weight, vascular contraction and relaxation responses, and blood chemistry. Injection of DOCA increased expression of both hypertrophic and fibrotic genes, which was abrogated by CG200745. These results indicate that CG200745 attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats.

Rutin Attenuates Carfilzomib-Induced Cardiotoxicity Through Inhibition of NF-κB, Hypertrophic Gene Expression and Oxidative Stress.

Carfilzomib is a proteasome inhibitor, commonly used in multiple myeloma, but its clinical use may be limited due to cardiotoxicity. This study was aimed to evaluate the influence of rutin in carfilzomib-induced cardiotoxicity in rats. Wistar albino male rats weighing 200-250g (approximately 10weeks old) were taken for this study. Animals were divided into four groups of six animals each. Group 1 served as normal control (NC), received normal saline; group 2 animals received carfilzomib (dissolved in 1% DMSO) alone; group 3 animals received rutin (20mg/kg)+carfilzomib; and group 4 animals received rutin (40mg/kg)+carfilzomib. Hematological changes, biochemical changes, oxidative stress, hypertrophic gene expression, apoptotic gene expression, NFκB and IκB-α protein expression and histopathological evaluation were done to confirm the finding of carfilzomib-induced cardiotoxicity. Treatment with rutin decreased the carfilzomib-induced changes in cardiac enzymes such as lactate dehydrogenase, creatine kinase (CK) and CK-MB. For the assessment of cardiotoxicity, we further evaluated cardiac hypertrophic gene and apoptotic gene expression such as α-MHC, β-MHC and BNP and NF-κB and p53 gene expression, respectively, using RT-PCR. Western blot analysis showed that rutin treatment prevented the activation of NF-κB by increasing the expression of IκB-α. Rutin also attenuated the effects of carfilzomib on oxidant-antioxidant including malondialdehyde and reduced glutathione. Histopathological study clearly confirmed that rutin attenuated carfilzomib-induced cardiotoxicity in rats.

RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure.

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload-induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease.

Cardiac mTOR complex 2 preserves ventricular function in pressure-overload hypertrophy.

Mammalian target of rapamycin (mTOR), a central regulator of growth and metabolism, has tissue-specific functions depending on whether it is part of mTOR complex 1 (mTORC1) or mTORC2. We have previously shown that mTORC1 is required for adaptive cardiac hypertrophy and maintenance of function under basal and pressure-overload conditions. In the present study, we aimed to identify functions of mTORC2 in the heart. Using tamoxifen-inducible cardiomyocyte-specific gene deletion, we generated mice deficient for cardiac rapamycin-insensitive companion of mTOR (rictor), an essential and specific component of mTORC2. Under basal conditions, rictor deficiency did not affect cardiac growth and function in young mice and also had no effects in adult mice. However, transverse aortic constriction caused dysfunction in the rictor-deficient hearts, whereas function was maintained in controls after 1 week of pressure overload. Adaptive increases in cardiac weight and cardiomyocyte cross-sectional area, fibrosis, and hypertrophic and metabolic gene expression were not different between the rictor-deficient and control mice. In control mice, maintained function was associated with increased protein levels of rictor, protein kinase C (PKC)βII, and PKCδ, whereas rictor ablation abolished these increases. Rictor deletion also significantly decreased PKCε at baseline and after pressure overload. Our data suggest that reduced PKCε and the inability to increase PKCβII and PKCδ abundance are, in accordance with their known function, responsible for decreased contractile performance of the rictor-deficient hearts. Our study demonstrates that mTORC2 is implicated in maintaining contractile function of the pressure-overloaded male mouse heart.

Abstract 15544: GRK2 RGS Domain-Specific Interaction With Gαq Inhibits Hypertrophy and Cardiac Dysfunction During Pressure Overload

G protein-coupled receptor (GPCR) kinases (GRK) play a critical role in cardiac function through regulation of GPCR activity. Canonically, GRK2 phosphorylates active GPCRs leading to desensitization and down-regulation of the receptors to regulate signaling in the heart. Mounting evidence, however, supports non-canonical regulation of cardiac function by GRK2 through a dynamic “interactome” in which GRK2 can uncouple GPCRs via novel protein-protein interactions. Several GRK2 interacting partners are important for adaptive and maladaptive myocyte growth including Gq, the signaling trigger for maladaptive cardiac hypertrophy, leading to HF. Importantly, GRK2 contains a putative amino-terminal Regulator of G protein Signaling (RGS) domain, that directly interacts with Gq and appears to inhibit signaling without altering Gq enzymatic activity. In the present study we generated transgenic (Tg) mice with cardiac-specific expression of the RGS domain of GRK2 (βARKrgs) and subjected them to a trans-aortic constriction (TAC) model of pressure overload hypertrophy. The βARKrgs Tg mice exhibited a marked anti-hypertrophic effect, with reduced left ventricular wall thickness and hypertrophic gene expression relative to their littermate controls. Use of genetically engineered lines demonstrated that this was through an interaction of the βARKrgs peptide with Gq-mediated signaling that inhibited hypertrophy as well as the progression to heart failure (HF). These data support our hypothesis that the RGS domain of GRK2 may serve as a non-canonical inhibitor of Gq-mediated hypertrophic signaling in the heart and highlight how this research may pave the way for novel GRK2-based therapeutic approaches to prevent hypertrophy and HF.

BAPX-1/NKX-3.2 acts as a chondrocyte hypertrophy molecular switch in osteoarthritis.

Osteoarthritis (OA) development involves a shift of the articular chondrocyte phenotype toward hypertrophic differentiation via still poorly characterized mechanisms. The purpose of this study was to test our hypothesis that the function of BAPX-1/NKX-3.2 is impaired in OA chondrocytes and leads directly to loss of hypertrophic protection of the articular chondrocyte, which is central in the changing chondrocyte phenotype that drives OA. Human articular chondrocytes (HACs; from healthy and OA donors) and SW-1353 chondrocytic cells were exposed to bone morphogenetic protein 7 (BMP-7), interleukin-1β (IL-1β), tumor necrosis factor, or OA synovial fluid (SF; 20% [volume/volume]). Loss-of-function and gain-of-function experiments for BAPX-1/NKX-3.2 were performed. Mouse experimental models of OA were used, and (immuno)histochemistry of tissue sections was performed. Gene and protein expression of BAPX-1/NKX-3.2 and chondrogenic, hypertrophic, and OA-related mediators were determined by real-time quantitative polymerase chain reaction analysis and immunoblotting. In addition, alkaline phosphatase (AP) activity and prostaglandin E2 levels were measured. BAPX-1/NKX-3.2 expression correlated negatively with expression of chondrocyte hypertrophic markers (RUNX-2, COL10A1, AP), cartilage-degrading enzymes (matrix metalloproteinase 13, ADAMTS-5), and mediators of inflammation (cyclooxygenase 2, IL-6) in healthy and OA chondrocytes, as well as in OA induced chondrocytes. BAPX-1/NKX-3.2 positivity was diminished in articular chondrocytes in the knee joints of mice with experimental OA. Knockdown of BAPX-1/NKX-3.2 in HACs did not influence the expression of SOX9, COL2A1, or aggrecan, but led to an acute hypertrophic shift in the HAC phenotype. Overexpression of BAPX-1/NKX-3.2 decreased hypertrophic gene expression in HACs. Furthermore, the hypertrophic OA chondrocyte phenotype could be counteracted by overexpression of BAPX-1/NKX-3.2 and by BMP-7 in a BAPX-1/NKX-3.2 dependent manner. Our findings indicate that BAPX-1/NKX-3.2 is a molecular switch that is involved in controlling the hypertrophic phenotype of the postdevelopmental (OA) articular chondrocyte.

Orphan nuclear receptor Nur77 affects cardiomyocyte calcium homeostasis and adverse cardiac remodelling.

Distinct stressors may induce heart failure. As compensation, β-adrenergic stimulation enhances myocardial contractility by elevating cardiomyocyte intracellular Ca2+ ([Ca2+]i). However, chronic β-adrenergic stimulation promotes adverse cardiac remodelling. Cardiac expression of nuclear receptor Nur77 is enhanced by β-adrenergic stimulation, but its role in cardiac remodelling is still unclear. We show high and rapid Nur77 upregulation in cardiomyocytes stimulated with β-adrenergic agonist isoproterenol. Nur77 knockdown in culture resulted in hypertrophic cardiomyocytes. Ventricular cardiomyocytes from Nur77-deficient (Nur77-KO) mice exhibited elevated diastolic and systolic [Ca2+]i and prolonged action potentials compared to wild type (WT). In vivo, these differences resulted in larger cardiomyocytes, increased expression of hypertrophic genes, and more cardiac fibrosis in Nur77-KO mice upon chronic isoproterenol stimulation. In line with the observed elevated [Ca2+]i, Ca2+-activated phosphatase calcineurin was more active in Nur77-KO mice compared to WT. In contrast, after cardiac pressure overload by aortic constriction, Nur77-KO mice exhibited attenuated remodelling compared to WT. Concluding, Nur77-deficiency results in significantly altered cardiac Ca2+ homeostasis and distinct remodelling outcome depending on the type of insult. Detailed knowledge on the role of Nur77 in maintaining cardiomyocyte Ca2+ homeostasis and the dual role Nur77 plays in cardiac remodelling will aid in developing personalized therapies against heart failure.

Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca(2+). The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCeδB are resistant to oxidative stress induced by hydrogen peroxide (H2O2) relative to CPCe, whereas knockdown of CaMKIIδB resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and α-smooth muscle actin in CPCeδB compared with CPCe, suggesting increased differentiation. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages.

Adiponectin is required for cardiac MEF2 activation during pressure overload induced hypertrophy

Cardiomyocyte (CM) hypertrophy and increased heart mass in response to pressure overload are associated with hyper-activation of the myocyte enhancer factor-2 (MEF2) family of transcriptional regulators, and concomitant initiation of the fetal gene program. Adiponectin, an adipokine that is reduced in individuals with obesity and diabetes, has been characterized both as a negative regulator or permissive factor in cardiac hypertrophy. We therefore sought to analyze temporal regulation of MEF2 activity in response to pressure overload (PO) and changes in adiponectin status. To address this we crossed a well characterized transgenic MEF2 “sensor” mouse (MEF2-lacZ) with adiponectin null mice (Ad-KO) to create compound MEF2 lacZ/Ad-KO mice. Initially, we established that transverse aortic banding induced PO in wild-type (WT) mice increased heart mass and CM hypertrophy from 1 to 4weeks following surgery, indicated by increased CM diameter and heart weight/tibia length ratio. This was associated with cardiac dysfunction determined by echocardiography. Hypertrophic changes and dysfunction were observed in Ad-KO mice 4weeks following surgery. MEF2 lacZ activity and endogenous ANF mRNA levels, used as indicators of hypertrophic gene activation, were both robustly increased in WT mice after MTAB but attenuated in the Ad-KO background. Furthermore, activation of the pro-hypertrophic molecule p38 was increased following MTAB surgery in WT mice, but not in Ad-KO animals, and treatment of primary isolated CM with recombinant adiponectin induced p38 phosphorylation in a time dependent manner. Adiponectin also increased MEF2 activation in primary cardiomyocytes, an effect attenuated by p38 MAPK inhibition. In conclusion, our data indicate that robust hypertrophic MEF2 activation in the heart in vivo requires a background of adiponectin signaling and that adiponectin signaling in primary isolated CM directly enhances MEF2 activity through activation of p38 MAPK. We conclude that adiponectin is required for full induction of cardiomyocyte MEF2 activation, thus contributing to the myocardial hypertrophic gene expression program in response to PO.

Abstract 216: Genetic Deletion of Cardiomyocyte Mineralocorticoid Receptors Prevents Cardiac Dysfunction and Premature Death in Mice Lacking Glucocorticoid Receptors in the Heart

Heart failure is one of the leading causes of death in the Western world, and stress is increasingly associated with adverse cardiac outcomes. Glucocorticoids are primary stress hormones, but their direct role in heart physiology and pathology is poorly understood. The actions of glucocorticoids are mediated classically by the glucocorticoid receptor (GR); however, in cardiomyocytes glucocorticoid occupancy and activation of the mineralocorticoid receptor (MR) may also contribute to the observed glucocorticoid response. To elucidate the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte-specific deletion of GR (cardioGRKO). The cardioGRKO mice spontaneously develop cardiac hypertrophy by 2 months of age and left ventricular systolic dysfunction and dilatation between 3-6 months of age, and they die prematurely from heart failure. The mean survival age of the cardioGRKO mice was 9.3 months, and 97% (38 of 39) of the mice died prior to reaching 12 months of age. Loss of GR in the heart was not accompanied by an increase in fibrosis. To investigate whether cardiomyocyte MR signaling contributes to the pathology in the cardioGRKO mice, we generated for the first time mice lacking both GR and MR in the heart (cardioGRMRKO). Despite showing increased expression of classic hypertrophic marker genes, the cardioGRMRKO mice were protected from the development of cardiac hypertrophy, and no major alterations were observed in the function and chamber size of the left ventricle. Moreover, the cardioGRMRKO mice were protected from premature death as 94% (15 of 16) of the mice survived past the 13 month study endpoint. Microarray analysis revealed marked differences in gene expression profiles between the cardioGRKO and cardioGRMRKO hearts. These findings reveal that cardiomyocyte MR signaling, when unopposed by GR signaling, plays a major role in the progression of cardiac disease. Moreover, they suggest that combining GR agonists with MR antagonists may represent an improved therapeutic approach for treating heart failure.