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Rationale: Pulmonary arterial hypertension (PAH) is characterized by structural remodeling of pulmonary arteries and arterioles. Underlying biological processes are likely reflected in a perturbation of circulating proteins. Objectives: To quantify and analyze the plasma proteome of patients with PAH using inherited genetic variation to inform on underlying molecular drivers. Methods: An aptamer-based assay was used to measure plasma proteins in 357 patients with idiopathic or heritable PAH, 103 healthy volunteers, and 23 relatives of patients with PAH. In discovery and replication subgroups, the plasma proteomes of PAH and healthy individuals were compared, and the relationship to transplantation-free survival in PAH was determined. To examine causal relationships to PAH, protein quantitative trait loci (pQTL) that influenced protein levels in the patient population were used as instruments for Mendelian randomization (MR) analysis. Measurements and Main Results: From 4,152 annotated plasma proteins, levels of 208 differed between patients with PAH and healthy subjects, and 49 predicted long-term survival. MR based on cis-pQTL located in proximity to the encoding gene for proteins that were prognostic and distinguished PAH from health estimated an adverse effect for higher levels of netrin-4 (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.16-2.08) and a protective effect for higher levels of thrombospondin-2 (OR, 0.83; 95% CI, 0.74-0.94) on PAH. Both proteins tracked the development of PAH in previously healthy relatives and changes in thrombospondin-2 associated with pulmonary arterial pressure at disease onset. Conclusions: Integrated analysis of the plasma proteome and genome implicates two secreted matrix-binding proteins, netrin-4 and thrombospondin-2, in the pathobiology of PAH.

Pulmonary arterial hypertension (PAH), group 1 pulmonary hypertension (PH), is a fatal disease that is characterized by vasoconstriction, increased pressure in the pulmonary arteries, and right heart failure. PAH can be described by abnormal vascular remodeling, hyperproliferation in the vasculature, endothelial cell dysfunction, and vascular tone dysregulation. The disease pathomechanisms, however, are as yet not fully understood at the molecular level. Purinergic receptors P2Y within the G-protein-coupled receptor family play a major role in fluid shear stress transduction, proliferation, migration, and vascular tone regulation in systemic circulation, but less is known about their contribution in PAH. Hence, studies that focus on purinergic signaling are of great importance for the identification of new therapeutic targets in PAH. Interestingly, the role of P2Y2 receptors has not yet been sufficiently studied in PAH, whereas the relevance of other P2Ys as drug targets for PAH was shown using specific agonists or antagonists. In this review, we will shed light on P2Y receptors and focus more on the P2Y2 receptor as a potential novel player in PAH and as a new therapeutic target for disease management.

Pulmonary hypertension (PH) is characterized by a progressive elevation of mean arterial pressure followed by right ventricular failure and death. Previous studies have indicated that numerous inhibitors of receptor tyrosine kinase signaling could be either beneficial or detrimental for the treatment of PH. Here we investigated the therapeutic potential of the multi-kinase inhibitor regorafenib (BAY 73-4506) for the treatment of PH. A peptide-based kinase activity assay was performed using the PamStation®12 platform. The 5-bromo-2′-deoxyuridine proliferation and transwell migration assays were utilized in pulmonary arterial smooth muscle cells (PASMCs). Regorafenib was administered to monocrotaline- and hypoxia-induced PH in rats and mice, respectively. Functional parameters were analyzed by hemodynamic and echocardiographic measurements. The kinase activity assay revealed upregulation of twenty-nine kinases in PASMCs from patients with idiopathic PAH (IPAH), of which fifteen were established as potential targets of regorafenib. Regorafenib showed strong anti-proliferative and anti-migratory effects in IPAH-PASMCs compared to the control PASMCs. Both experimental models indicated improved cardiac function and reduced pulmonary vascular remodeling upon regorafenib treatment. In lungs from monocrotaline (MCT) rats, regorafenib reduced the phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2. Overall, our data indicated that regorafenib plays a beneficial role in experimental PH.

PKGs are serine/threonine kinases. PKG1 has two isoforms—PKG1α and β. Inositol trisphosphate receptor (IP3R)-associated cGMP-kinase substrate 1 (IRAG1) is a substrate for PKG1β. IRAG1 is also known to further interact with IP3RI, which mediates intracellular Ca2+ release. However, the role of IRAG1 in PH is not known. Herein, WT and IRAG1 KO mice were kept under normoxic or hypoxic (10% O2) conditions for five weeks. Animals were evaluated for echocardiographic variables and went through right heart catheterization. Animals were further sacrificed to prepare lungs and right ventricular (RV) for immunostaining, western blotting, and pulmonary artery smooth muscle cell (PASMC) isolation. IRAG1 is expressed in PASMCs and downregulated under hypoxic conditions. Genetic deletion of IRAG1 leads to RV hypertrophy, increase in RV systolic pressure, and RV dysfunction in mice. Absence of IRAG1 in lung and RV have direct impacts on PKG1β expression. Attenuated PKG1β expression in IRAG1 KO mice further dysregulates other downstream candidates of PKG1β in RV. IRAG1 KO mice develop PH spontaneously. Our results indicate that PKG1β signaling via IRAG1 is essential for the homeostasis of PASMCs and RV. Disturbing this signaling complex by deleting IRAG1 can lead to RV dysfunction and development of PH in mice.

BackgroundPulmonary arterial hypertension (PAH) is a progressive disease characterised by pro-proliferative and anti-apoptotic phenotype in vascular cells, leading to pulmonary vascular remodelling and right heart failure. Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1), a highly conserved enzyme, which binds to and catalyses the isomerisation of specific phosphorylated Ser/Thr-Pro motifs, acts as a molecular switch in multiple coordinated cellular processes. We hypothesised that Pin1 plays a substantial role in PAH, and its inhibition with a natural organic compound, Juglone, would reverse experimental pulmonary hypertension.ResultsWe demonstrated that the expression of Pin1 was markedly elevated in experimental pulmonary hypertension (i.e. hypoxia-induced mouse and Sugen/hypoxia-induced rat models) and pulmonary arterial smooth muscle cells of patients with clinical PAH. In vitro Pin1 inhibition by either Juglone treatment or short interfering RNA knockdown resulted in an induction of apoptosis and decrease in proliferation of human pulmonary vascular cells. Stimulation with growth factors induced Pin1 expression, while its inhibition reduced the activity of numerous PAH-related transcription factors, such as hypoxia-inducible factor (HIF)-α and signal transducer and activator of transcription (STAT). Juglone administration lowered pulmonary vascular resistance, enhanced right ventribular function, improved pulmonary vascular and cardiac remodelling in the Sugen/hypoxia rat model of PAH and the chronic hypoxia-induced pulmonary hypertension model in mice.ConclusionOur study demonstrates that targeting of Pin1 with small molecule inhibitor, Juglone, might be an attractive future therapeutic strategy for PAH and right heart disease secondary to PAH.

Progression of pulmonary arterial hypertension (PAH) is associated with pathological remodeling of the pulmonary vasculature and the right ventricle (RV). Oxidative stress drives the remodeling process through activation of MAPKs (mitogen-activated protein kinases), which stimulate apoptosis, inflammation, and fibrosis.We investigated whether pharmacological inhibition of the redox-sensitive apical MAPK, ASK1 (apoptosis signal-regulating kinase 1), can halt the progression of pulmonary vascular and RV remodeling.A selective, orally available ASK1 inhibitor, GS-444217, was administered to two preclinical rat models of PAH (monocrotaline and Sugen/hypoxia), a murine model of RV pressure overload induced by pulmonary artery banding, and cellular models.Oral administration of GS-444217 dose dependently reduced pulmonary arterial pressure and reduced RV hypertrophy in PAH models. The therapeutic efficacy of GS-444217 was associated with reduced ASK1 phosphorylation, reduced muscularization of the pulmonary arteries, and reduced fibrotic gene expression in the RV. Importantly, efficacy was observed when GS-444217 was administered to animals with established disease and also directly reduced cardiac fibrosis and improved cardiac function in a model of isolated RV pressure overload. In cellular models, GS-444217 reduced phosphorylation of p38 and JNK (c-Jun N-terminal kinase) induced by adenoviral overexpression of ASK1 in rat cardiomyocytes and reduced activation/migration of primary mouse cardiac fibroblasts and human pulmonary adventitial fibroblasts derived from patients with PAH.ASK1 inhibition reduced pathological remodeling of the pulmonary vasculature and the right ventricle and halted progression of pulmonary hypertension in rodent models. These preclinical data inform the first description of a causal role of ASK1 in PAH disease pathogenesis.

Although p38 mitogen-activated protein kinase (MAPK) is known to have a role in ischemic heart disease and many other diseases, its contribution to the pathobiology of right ventricular (RV) hypertrophy and failure is unclear. Therefore, we sought to investigate the role of p38 MAPK in the pathophysiology of pressure overload-induced RV hypertrophy and failure. The effects of the p38 MAPK inhibitor PH797804 were investigated in mice with RV hypertrophy/failure caused by exposure to hypoxia or pulmonary artery banding. In addition, the effects of p38 MAPK inhibition or depletion (by small interfering RNA) were studied in isolated mouse RV fibroblasts. Echocardiography, invasive hemodynamic measurements, immunohistochemistry, collagen assays, immunofluorescence staining, and Western blotting were performed. Expression of phosphorylated p38 MAPK was markedly increased in mouse and human hypertrophied/failed RVs. In mice, PH797804 improved RV function and inhibited cardiac fibrosis compared with placebo. In isolated RV fibroblasts, p38 MAPK inhibition reduced transforming growth factor (TGF)-b-induced collagen production as well as stress fiber formation. Moreover, p38 MAPK inhibition/depletion suppressed TGF-b-induced SMAD2/3 phosphorylation and myocardin-related transcription factor A (MRTF-A) nuclear translocation, and prevented TGF-b-induced cardiac fibroblast transdifferentiation. Moreover, p38 MAPK inhibition in mice exposed to pulmonary artery banding led to diminished nuclear levels of MRTF-A and phosphorylated SMAD3 in RV fibroblasts. Together, our data indicate that p38 MAPK inhibition significantly improves RV function and inhibits RV fibrosis. Inhibition of p38 MAPK in RV cardiac fibroblasts, resulting in coordinated attenuation of MRTF-A cytoplasmic-nuclear translocation and SMAD3 deactivation, indicates that p38 MAPK signaling contributes to distinct disease-causing mechanisms.

Background and objectives: Pulmonary hypertension (PH) is characterized by the vasoconstriction and abnormally proliferative vascular cells. The available allopathic treatment options for PH are still not able to cure the disease. Alternative medicine is becoming popular and drawing the attention of the general public and scientific communities. The entomogenous fungus Yarsagumba (Cordyceps sinensis) and its biologically active ingredient cordycepin may represent the therapeutic option for this incurable disease, owing to their anti-inflammatory, vasodilatory and anti-oxidative effects. Methods: In this study, we investigated whether Yarsagumba extract and cordycepin possess anti-proliferative and vasorelaxant properties in the context of PH, using 5-bromo-2&rsquo, deoxyuridine assay and isolated mice lungs, respectively. Results: Our results revealed that Yarsagumba extract and its bioactive compound cordycepin significantly attenuated the proliferation of human pulmonary artery smooth muscle cells derived from donor and PH subjects. In isolated murine lungs, only Yarsagumba extract, but not cordycepin, resulted in vasodilatation, indicating the probable existence of other bioactive metabolites present in Yarsagumba that may be responsible for this outcome. Conclusion: Future comprehensive in vivo and in vitro research is crucially needed to discover the profound mechanistic insights with regard to this promising therapeutic potency of Yarsagumba extract and to provide further evidence as to whether it can be used as a strategy for the treatment of PH.

The ability of the right ventricle to compensate pressure overload determines survival in pulmonary arterial hypertension (PAH). Nitric oxide (NO) reduces the right ventricular afterload through pulmonary vasodilation, but excessive NO amounts cause oxidative stress. Oxidative stress drives remodeling of pulmonary arteries and the right ventricle. In the present study, we hypothesized that nitric oxide synthase 2 (NOS2) induction leads to excessive NO amounts that contribute to oxidative stress and impair right ventricular adaptation to PAH. We used a surgical pulmonary artery banding (PAB) mouse model in which right ventricular dysfunction and remodeling occur independently of changes in the pulmonary vasculature. Three weeks after PAB, NOS2 expression was increased twofold in the hypertrophied right ventricle on transcript and protein levels together with increased NO production. Histomorphology localized NOS2 in interstitial and perivascular cardiac fibroblasts after PAB, which was confirmed by cell isolation experiments. In the hypertrophied right ventricle, NOS2 induction was accompanied by an increased formation of reactive oxidants blocked by ex vivo NOS inhibition. We show that reactive oxidant formation in the hypertrophied right ventricle is in part NOS2 dependent (in NOS2-deficient mice [NOS2

Apoptosis, or programmed cell death, is an essential physiological process for proper embryogenesis as well as for homeostasis during aging. In addition, apoptosis is one of the major mechanisms causing cell loss in pathophysiological conditions such as heart failure. Thus, inhibition of apoptosis is an important approach for preventive and therapeutic strategies. Here we show that the histone 3 lysine 4- and lysine 36-specific methyltransferase Smyd2 acts as an endogenous antagonistic player of p53-dependent cardiomyocyte apoptosis. Smyd2 protein levels were significantly decreased in cardiomyocytes upon cobalt chloride-induced apoptosis or myocardial infarction, while p53 expression was enhanced. siRNA-mediated knockdown of Smyd2 in cultured cardiomyocytes further enhanced cobalt chloride-induced cardiomyocyte apoptosis. In contrast, Smyd2 overexpression resulted in marked methylation of p53 and prevented its accumulation as well as apoptotic cell death in an Hsp90-independent manner. Moreover, overexpression, of Smyd2, but not Smyd2Y240F lacking a methyl transferase activity, significantly rescued CoCl2-induced apoptosis in H9c2 cardioblasts. Finally, Smyd2 cardiomyocyte-specific deletion in vivo promoted apoptotic cell death upon myocardial infarction, which correlated with enhanced expression of p53 and pro-apoptotic Bax. Collectively, our data indicate Smyd2 as a cardioprotective protein by methylating p53.

Fibroblast growth factors (FGFs) signal through FGF receptors (FGFRs) mediating a broad range of cellular functions during embryonic development, as well as disease and regeneration during adulthood. Thus, it is important to understand the underlying molecular mechanisms that modulate this system. Here, we show that FGFR-1 can interact with the TNF receptor superfamily member fibroblast growth factor-inducible molecule 14 (Fn14) resulting in cardiomyocyte cell cycle reentry. FGF1-induced cell cycle reentry in neonatal cardiomyocytes could be blocked by Fn14 inhibition, while TWEAK-induced cell cycle activation was inhibited by blocking FGFR-1 signaling. In addition, costimulation experiments revealed a synergistic effect of FGF1 and TWEAK in regard to cardiomyocyte cell cycle induction via PI3K/Akt signaling. Overexpression of Fn14 with either FGFR-1 long [FGFR-1(L)] or FGFR-1 short [FGFR-1(S)] isoforms resulted after FGF1/TWEAK stimulation in cell cycle reentry of >40% adult cardiomyocytes. Finally, coimmunoprecipitation and proximity ligation assays indicated that endogenous FGFR-1 and Fn14 interact with each other in cardiomyocytes. This interaction was strongly enhanced in the presence of their corresponding ligands, FGF1 and TWEAK. Taken together, our data suggest that FGFR-1/Fn14 interaction may represent a novel endogenous mechanism to modulate the action of these receptors and their ligands and to control cardiomyocyte cell cycle reentry.

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by remodeling of the pulmonary vasculature and a rise in right ventricular (RV) afterload. The increased RV afterload leads to right ventricular failure (RVF) which is the reason for the high morbidity and mortality in PAH patients. The objective was to evaluate the therapeutic efficacy and antiremodeling potential of the phosphodiesterase type 5 (PDE5) inhibitor sildenafil and the soluble guanylate cyclase stimulator riociguat in a model of pressure overload RV hypertrophy induced by pulmonary artery banding (PAB). Mice subjected to PAB, one week after surgery, were treated with either sildenafil (100 mg/kg/d, n=5), riociguat (30 mg/kg/d, n=5), or vehicle (n=5) for 14 days. RV function and remodeling were assessed by right heart catheterization, magnetic resonance imaging (MRI), and histomorphometry. Both sildenafil and riociguat prevented the deterioration of RV function, as determined by a decrease in RV dilation and restoration of the RV ejection fraction (EF). Although both compounds did not decrease right heart mass and cellular hypertrophy, riociguat prevented RV fibrosis induced by PAB. Both compounds diminished TGF-beta1 induced collagen synthesis of RV cardiac fibroblasts in vitro. Treatment with either riociguat or sildenafil prevented the progression of pressure overload-induced RVF, representing a novel therapeutic approach.

Significance Adhesion G protein-coupled receptors (GPCRs) are expressed in many developing organs, immune cells, and cancer cells, suggesting that they might play an important role in physiological and pathological functions. Compared with their potential importance, their function and signaling mechanisms are poorly understood. Disruption of the G protein-coupled receptor 126 ( Gpr126 ) gene in mice leads to lack of myelination in the peripheral nervous system (PNS) and heart abnormalities. Similarly, the zebrafish mutant line gpr126 st49 exhibits PNS abnormalities but, in contrast, no heart phenotype. Here we provide an explanation for these discrepancies. The presented data suggest that in the heart, the N-terminal fragment of Gpr126 can act independently as a ligand or coreceptor. Taken together, our data provide evidence of tissue- and domain-specific adhesion GPCR function.

Background: Pulmonary arterial hypertension (PAH) is devastating disease with no cure. Pulmonary arterial vascular smooth muscle cells (PASMCs) proliferation and migration, and overproduction collagen, are important pathological processes in pulmonary vascular remodeling. Pirfenidone, the first anti-fibrotic drug approved for the treatment of Idiopathic pulmonary fibrosis (IPF). We hypothesize that pirfenidone may inhibit collagen synthesis and cell proliferation of PASMCs induced by growth factors in pulmonary hypertension. Method: Effects of pirfenidone on human PASMCs proliferation, migration and collagen secretion were studied. Proliferation of PASMCs was monitored by newly synthesized DNA with BrdU. PASMCs migration assay was performed with Ibidi chamber method. Collagen secretion in the medium was measured by Sircol assay. Real time PCR was applied for quantifying the pro-inflammatory cytokines interleukin rnRNA expression. Results: Pirfenidone inhibited the PDGF induced proliferation of PASMCs in dose depended manner. Pirfenidone significantly decreased PDGF-induced PASMCs migration compare to vehicle treatment (p

The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.

Aims Although the fundamental role of the E2F transcription factor family in cell proliferation is well established, the specific function of E2F4 is unclear. On the basis of findings from cell culture experiments, E2F4 is generally considered as an inhibitor of cell proliferation. Accumulating evidence suggests, however, that E2F4 acts as an activator of cell proliferation in certain contexts. Here, we have investigated the role of E2F4 during heart development and in proliferating cardiomyocytes. Methods and results Nuclear E2F4 expression in cardiomyocytes declined during mouse heart development, which correlates with the loss of proliferative capacity of cardiomyocytes. Re-induction of proliferation in postnatal cardiomyocytes increased nuclear E2F4 expression. E2F4 accumulated in the nucleus at the end of the S phase, remained nuclear during mitosis, and disappeared at the end of cytokinesis. siRNA-mediated inhibition of E2F4 in proliferating postnatal cardiomyocytes resulted in a significant reduction in mitosis, but not in DNA synthesis. Co-staining of E2F4 and Crest revealed that E2F4 co-localizes with kinetochores. Moreover, chromatin immunoprecipitation showed that E2F4 binds to centromeric α-satellite DNA during mitosis. Conclusion Our data indicate that E2F4 is required for cardiomyocyte proliferation and suggest a function for E2F4 in mitosis.

Aims Proliferation of mammalian cardiomyocytes stops during the first weeks after birth, preventing the heart from regenerating after injury. Recently, several studies have indicated that induction of cardiomyocyte proliferation can be utilized to regenerate the mammalian heart. Thus, it is important to identify novel factors that can induce proliferation of cardiomyocytes. Here, we determine the effect of TNF-related weak inducer of apoptosis (TWEAK) on cardiomyocytes, a cytokine known to regulate proliferation in several other cell types. Methods and results Stimulation of neonatal rat cardiomyocytes with TWEAK resulted in increased DNA synthesis, increased expression of the proliferative markers Cyclin D2 and Ki67, and downregulation of the cell cycle inhibitor p27KIP1. Importantly, TWEAK stimulation resulted also in mitosis (H3P), cytokinesis (Aurora B), and increased cardiomyocyte numbers. Loss of function experiments revealed that re-induction of proliferation was dependent on tumour necrosis factor receptor superfamily member 12A (FN14) signalling. Downstream signalling was mediated through activation of extracellular signal-regulated kinases and phosphatidylinositol 3-kinase as well as inhibition of glycogen synthase kinase-3beta. In contrast to neonatal cardiomyocytes, TWEAK had no effect on adult rat cardiomyocytes due to developmental downregulation of its receptor FN14. However, adenoviral expression of FN14 enabled efficient induction of cell cycle re-entry in adult cardiomyocytes after TWEAK stimulation. Conclusion Our data establish TWEAK as a positive regulator of cardiomyocyte proliferation.

Alternative splicing emerges as one of the most important mechanisms to generate transcript diversity. It is regulated by the formation of protein complexes on pre-mRNA. We demonstrate that protein phosphatase 1 (PP1) binds to the splicing factor transformer2-beta1 (tra2-beta1) via a phylogenetically conserved RVDF sequence located on the RNA recognition motif (RRM) of tra2-beta1. PP1 binds directly to tra2-beta1 and dephosphorylates it, which regulates the interaction between tra2-beta1 and other proteins. Eight other proteins, including SF2/ASF and SRp30c, contain an evolutionary conserved PP1 docking motif in the beta-4 strand of their RRMs indicating that binding to PP1 is a new function of some RRMs. Reducing PP1 activity promotes usage of numerous alternative exons, demonstrating a role of PP1 activity in splice site selection. PP1 inhibition promotes inclusion of the survival of motoneuron 2 exon 7 in a mouse model expressing the human gene. This suggests that reducing PP1 activity could be a new therapeutic principle to treat spinal muscular atrophy and other diseases caused by missplicing events. Our data indicate that the binding of PP1 to evolutionary conserved motifs in several RRMs is the link between known signal transduction pathways regulating PP1 activity and pre-mRNA processing.

Objective. The serotonin (5-HT) pathway was shown to play a role in pulmonary hypertension (PH), but its functions in right ventricular failure (RVF) remain poorly understood. The aim of the current study was to investigate the effects of Terguride (5-HT2A and 2B receptor antagonist) or SB204741 (5-HT2B receptor antagonist) on right heart function and structure upon pulmonary artery banding (PAB) in mice.Methods. Seven days after PAB, mice were treated for 14 days with Terguride (0.2 mg/kg bid) or SB204741 (5 mg/kg day). Right heart function and remodeling were assessed by right heart catheterization, magnetic resonance imaging (MRI), and histomorphometric methods. Total secreted collagen content was determined in mouse cardiac fibroblasts isolated from RV tissues.Results. Chronic treatment with Terguride or SB204741 reduced right ventricular fibrosis and showed improved heart function in mice after PAB. Moreover, 5-HT2B receptor antagonists diminished TGF-beta1 induced collagen synthesis of RV cardiac fibroblastsin vitro.Conclusion. 5-HT2B receptor antagonists reduce collagen deposition, thereby inhibiting right ventricular fibrosis. Chronic treatment prevented the development and progression of pressure overload-induced RVF in mice. Thus, 5-HT2B receptor antagonists represent a valuable novel therapeutic approach for RVF.

Introduction: The p38 mitogen-activated protein kinases (MAPK) considered as a key signaling pathway that responds to varied stresses, including those that contribute to heart failure. However, the role of p38 MAPK in pulmonary vascular and right ventricular remodeling is still need to be elucidated. Methods: The effects of p38 MAPK inhibitor PHA-00797804 (Pfizer, USA) investigated in mice subjected either to hypoxia-induced PH or to pulmonary artery banding (PAB). After 21 days, mice exposed to hypoxia were randomized and treated by vehicle or 5 mg/kg bw PHA-00797804 for 14 days. PAB mice were treated by vehicle or PHA-00797804 in a dose 5 mg/kg bw from day 7 to 21. The echocardiography, invasive hemodynamic measurement and hitomorphometry were performed. The myocardin-related transcription factor (MRTF) translocation and stress fibers formation were determined by immunofluorescence staining on isolated mouse cardiac fibroblasts. Results: The 38αMAPK inhibition significantly reversed pulmonary vascular remodeling and improved RV functions in hypoxia-induced PAH. However, to determine whether the effect in hypoxic mice was due to a direct impact of pharmacological interventions on RV or an indirect impact due to reduced pulmonary vascular resistance, we have investigated the effects of p38 MAPK inhibition in an established model of RVH and failure by PAB. The PHA-00797804 significantly increased cardiac output (p Conclusion: Inhibition of p38 MAPK improved RV remodeling and function in experimental models of RV hypertrophy and failure.

Tumor necrosis factor (TNF) has been firmly established as a pathogenic factor in heart failure, a significant socio-economic burden. In this review, we will explore the role of other members of the TNF/TNF receptor superfamily (TNFSF/TNFRSF) in cardiovascular diseases (CVDs) focusing on TWEAK and its receptor Fn14, new players in myocardial remodeling and heart failure. The TWEAK/Fn14 pathway controls a variety of cellular activities such as proliferation, differentiation, and apoptosis and has diverse biological functions in pathological mechanisms like inflammation and fibrosis that are associated with CVDs. Furthermore, it has recently been shown that the TWEAK/Fn14 axis is a positive regulator of cardiac hypertrophy and that deletion of Fn14 receptor protects from right heart fibrosis and dysfunction. We discuss the potential use of the TWEAK/Fn14 axis as biomarker for CVDs as well as therapeutic target for future treatment of human heart failure based on supporting data from animal models and in vitro studies. Collectively, existing data strongly suggest the TWEAK/Fn14 axis as a potential new therapeutic target for achieving cardiac protection in patients with CVDs.

Cardiac pressure overload-induced hypertrophy and pathological remodelling frequently leads to right ventricular dysfunction, which is the most frequent cause of death in patients with pulmonary arterial hypertension. Nowadays, accumulating reports support the concept that proinflammatory cytokines and growth factors play crucial roles in the failing heart. We recently identified Fn14 as an endogenous key regulator in cardiac fibrosis in the PAB (Pulmonary Artery Banding) pressure-overload model. Right ventricular overload after PAB is also characterized by hypertrophy. The aim of this study was to determine whether right ventricular (RV) cardiac hypertrophy induced by PAB is mediated by the TWEAK/Fn14 axis. After baseline MRI, Fn14 −/− mice and wild-type (WT) littermates were randomly assigned to two groups: (1) SHAM-operated ( n ⩾ 4, per genotype) and (2) PAB ( n ⩾ 11, per genotype). The results of MRI and histological analysis demonstrated that Fn14 −/− mice exhibit less PAB-induced cardiac hypertrophy compared to WT littermates. Moreover, Fn14 overexpression in cultured adult rat cardiomyocytes enhanced cardiomyocyte size. Collectively, our studies demonstrate that Fn14 ablation attenuates RV hypertrophy after PAB and that activation of TWEAK/Fn14 signaling promotes cardiomyocyte growth in vitro. These results nominate Fn14 as a potential novel target for the treatment of heart hypertrophy.

Regulated alternative splice site selection emerges as one of the most important mechanisms to control the expression of genetic information in humans. It is therefore not surprising that a growing number of diseases are either associated with or caused by changes in alternative splicing. These diseases can be caused by mutation in regulatory sequences of the pre-mRNA or by changes in the concentration of trans-acting factors. The pathological expression of mRNA isoforms can be treated by transferring nucleic acids derivatives into cells that interfere with sequence elements on the pre-mRNA, which results in the desired splice site selection. Recently, a growing number of low molecular weight drugs have been discovered that influence splice site selection in vivo. These findings prove the principle that diseases caused by missplicing events could eventually be cured.

Regulated alternative splice site selection emerges as one of the most important mechanisms to control the expression of genetic information in humans. It is therefore not surprising that a growing number of diseases are either associated with or caused by changes in alternative splicing. These diseases can be caused by mutation in regulatory sequences of the pre-mRNA or by changes in the concentration of trans-acting factors. The pathological expression of mRNA isoforms can be treated by transferring nucleic acids derivatives into cells that interfere with sequence elements on the pre-mRNA, which results in the desired splice site selection. Recently, a growing number of low molecular weight drugs have been discovered that influence splice site selection in vivo. These findings prove the principle that diseases caused by missplicing events could eventually be cured.

The Sam68-like mammalian protein SLM-1 is a member of the STAR protein family and is related to SAM68 and SLM-2. Here, we demonstrate that rSLM-1 interacts with itself, scaffold-attachment factor B, YT521-B, SAM68, rSLM-2, SRp30c, and hnRNP G. rSLM-1 regulates splice site selection in vivo via a purine-rich enhancer. In contrast to the widely expressed SAM68 and rSLM-2 proteins, rSLM-1 is found primarily in brain and, to a much smaller degree, in testis. In the brain, rSLM-1 and rSLM-2 are predominantly expressed in different neurons. In the hippocampal formation, rSLM-1 is present only in the dentate gyrus, whereas rSLM-2 is found in the pyramidal cells of the CA1, CA3, and CA4 regions. rSLM-1, but not rSLM-2, is phosphorylated by p59 fyn . p59 fyn -mediated phosphorylation abolishes the ability of rSLM-1 to regulate splice site selection, but has no effect on rSLM-2 activity. This suggests that rSLM-1-positive cells could respond with a change of their splicing pattern to p59 fyn activation, whereas rSLM-2-positive cells would not be affected. Together, our data indicate that rSLM-1 is a tissue-specific splicing factor whose activity is regulated by tyrosine phosphorylation signals emanating from p59 fyn .

Pulmonary arterial hypertension (PAH) is a fatal disease for which no cure is yet available. The leading cause of death in PAH is right ventricular (RV) failure. Previously, the TNF receptor superfamily member fibroblast growth factor-inducible molecule 14 (Fn14) has been associated with different fibrotic diseases. However, so far there is no study demonstrating a causal role for endogenous Fn14 signaling in RV or LV heart disease. The purpose of this study was to determine whether global ablation of Fn14 prevents RV fibrosis and remodeling improving heart function. Here, we provide evidence for a causative role of Fn14 in pulmonary artery banding (PAB)-induced RV fibrosis and dysfunction in mice. Fn14 expression was increased in the RV after PAB. Mice lacking Fn14 (Fn14−/−) displayed substantially reduced RV fibrosis and dysfunction following PAB compared to wild-type littermates. Cell culture experiments demonstrated that activation of Fn14 induces collagen expression via RhoA-dependent nuclear translocation of myocardin-related transcription factor-A (MRTF-A)/MAL. Furthermore, activation of Fn14 in vitro caused fibroblast proliferation and myofibroblast differentiation, which corresponds to suppression of PAB-induced RV fibrosis in Fn14−/− mice. Moreover, our findings suggest that Fn14 expression is regulated by endothelin-1 (ET-1) in cardiac fibroblasts. We conclude that Fn14 is an endogenous key regulator in cardiac fibrosis and suggest this receptor as potential new target for therapeutic interventions in heart failure. Electronic supplementary material The online version of this article (doi:10.1007/s00395-012-0325-x) contains supplementary material, which is available to authorized users.