The inherited mutation (R14del) in the calcium regulatory protein phospholamban (PLN) is linked to malignant ventricular arrhythmia with poor prognosis starting at adolescence. However, the underlying early mechanisms that may serve as prognostic factors remain elusive. This study generated humanized mice in which the endogenous gene was replaced with either human wild type or R14del-PLN and addressed the early molecular and cellular pathogenic mechanisms. R14del-PLN mice exhibited stress-induced impairment of atrioventricular conduction, and prolongation of both ventricular activation and repolarization times in association with ventricular tachyarrhythmia, originating from the right ventricle (RV). Most of these distinct electrocardiographic features were remarkably similar to those in R14del-PLN patients. Studies in isolated cardiomyocytes revealed RV-specific calcium defects, including prolonged action potential duration, depressed calcium kinetics and contractile parameters, and elevated diastolic Ca-levels. Ca-sparks were also higher although SR Ca-load was reduced. Accordingly, stress conditions induced after contractions, and inclusion of the CaMKII inhibitor KN93 reversed this proarrhythmic parameter. Compensatory responses included altered expression of key genes associated with Ca-cycling. These data suggest that R14del-PLN cardiomyopathy originates with RV-specific impairment of Ca-cycling and point to the urgent need to improve risk stratification in asymptomatic carriers to prevent fatal arrhythmias and delay cardiomyopathy onset.
Immunonutrition as a therapeutic approach is rapidly gaining interest in the fight against infection. Targeting l-arginine metabolism is intriguing, considering this amino acid is the substrate for antimicrobial NO production by macrophages. The importance of l-arginine during infection is supported by the finding that inhibiting its synthesis from its precursor l-citrulline blunts host defense. During the first few weeks following pulmonary mycobacterial infection, we found a drastic increase in l-citrulline in the lung, even though serum concentrations were unaltered. This correlated with increased gene expression of the l-citrulline–generating (i.e., iNOS) and l-citrulline–using (i.e., Ass1) enzymes in key myeloid populations. Eliminating l-arginine synthesis from l-citrulline in myeloid cells via conditional deletion of either Ass1 or Asl resulted in increased Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis H37Rv burden in the lungs compared with controls. Our data illustrate the necessity of l-citrulline metabolism for myeloid defense against mycobacterial infection and highlight the potential for host-directed therapy against mycobacterial disease targeting this nutrient and/or its metabolic pathway.
The goal of this study was to define the functional role of adipocyte-specific expression of the RNA binding protein Human antigen R (HuR). Mice with an adipocyte-specific deletion of HuR (Adipo-HuR −/-) were generated by crossing HuR floxed (HuR fl/fl) mice with mice expressing adiponectin-driven cre-recombinase (Adipoq-cre). Our results show that Adipo-HuR −/mice display a lean phenotype compared to wild-type littermate controls. HuR deletion results in a diet-independent reduction in percent body fat composition along with an increase in energy expenditure. Functionally, Adipo-HuR −/mice show a significant impairment in acute adaptive thermogenesis (six hours at 4°C), but uncoupling protein 1 (UCP1) protein expression in brown adipose tissue (BAT) is unchanged compared to control. Pharmacological inhibition of HuR also results in a marked decline in core body temperature following acute cold challenge independent of UCP1 protein expression. Among the 588 HuR-dependent genes in BAT identified by RNA-seq analysis, gene ontology analysis shows a significant enrichment in mediators of calcium transport and signalling, almost all of which are decreased in Adipo-HuR −/mice compared to control. In conclusion, adipocyte expression of HuR plays a central role in metabolic homoeostasis and mediates UCP1-independent thermogenesis in BAT, potentially through post-transcriptional control of intracellular calcium transport.
Adipose tissue homeostasis plays a central role in cardiovascular physiology, and the presence of thermogenically active brown adipose tissue (BAT) has recently been associated with cardiometabolic health. We have previously shown that adipose tissue-specific deletion of HuR (Adipo-HuR-/-) reduces BAT-mediated adaptive thermogenesis, and the goal of this work was to identify the cardiovascular impacts of Adipo-HuR-/-. We found that Adipo-HuR-/- mice exhibit a hypercontractile phenotype that is accompanied by increased left ventricle wall thickness and hypertrophic gene expression. Furthermore, hearts from Adipo-HuR-/- display increased fibrosis via picrosirius red staining and periostin expression. To identify underlying mechanisms, we applied both RNA-seq and weighted gene co-expression network analysis (WGCNA) across both cardiac and adipose tissue to define HuR-dependent changes in gene expression as well as significant relationships between adipose tissue gene expression and cardiac fibrosis. RNA-seq results demonstrated a significant increase in pro-inflammatory gene expression in both cardiac and subcutaneous white adipose tissue (scWAT) from Adipo-HuR-/- mice that is accompanied by an increase in serum levels of both TNF-ᵯC; and IL-6. In addition to inflammation-related genes, WGCNA identified a significant enrichment in extracellular vesicle-mediated transport and exosome-associated genes in scWAT whose expression most significantly associated with degree of cardiac fibrosis observed in Adipo-HuR-/- mice, implicating these processes as a likely adipose-to-cardiac paracrine mechanism. These results are significant in that they demonstrate the spontaneous onset of cardiovascular pathology in an adipose tissue-specific gene deletion model and contribute to our understanding of how disruptions in adipose tissue homeostasis may mediate cardiovascular disease.
Tranilast is clinically indicated for the treatment of allergic disorders and is also a nonselective blocker of the transient receptor potential vanilloid 2 (TRPV2) channel. Previous studies have found that it has protective effects in various animal models of cardiac disease. Our laboratory has found that genetic deletion of TRPV2 results in a blunted hypertrophic response to increased afterload; thus, this study tested the hypothesis that tranilast through cardiomyocyte TRPV2 blockade can inhibit the hypertrophic response to pressure overload in vivo through transverse aortic constriction and ex vivo through isolated myocyte studies. The in vivo studies demonstrated that tranilast blunted the fibrotic response to increased afterload and, to a lesser extent, the hypertrophic response. After 4 weeks, this blunting was associated with improved cardiac function, although at 8 weeks, the cardiac function deteriorated similarly to the control group. Finally, the in vitro studies demonstrated that tranilast was not inhibiting these responses at the cardiomyocyte level. In conclusion, we demonstrated that tranilast blunting of the fibrotic and hypertrophic response occurs independently of cardiac TRPV2 channels and may be cardioprotective in the short term but not after prolonged administration.
Summary Background: Full-length tissue factor (flTF) and alternatively spliced TF (asTF) contribute to growth and spread of pancreatic ductal adenocarcinoma. It is unknown, however, if flTF and/or asTF contribute to pathobiology of pancreatic neuroendocrine tumors (pNET). Objective: To assess TF expression in pNET and the effects of mTOR complex 1/2 (mTORC1/2) inhibition on pNET growth. Methods: Human pNET specimens were immunostained for TF. Human pNET cell lines QGP1 and BON were evaluated for TF expression and responsiveness to mTOR inhibition. shRNA were used to knock down TF in BON. TF cofactor activity was assessed using a two-step FXa generation assay. TF promoter activity was assessed using transient transfection of human TF promoter-driven reporter constructs into cells. Mice bearing orthotopic BON tumors were treated with the mTORC1/2 ATP-site competitive inhibitor sapanisertib / MLN0128 (3 mg/kg, oral gavage) for 34 days. Results: Immunostaining of pNET tissue revealed flTF and asTF expression. BON and QGP1 expressed both TF isoforms, with BON exhibiting higher levels. shRNA directed against TF suppressed BON proliferation in vitro. Treatment of BON with sapanisertib inhibited mTOR signaling and suppressed TF levels. BON tumors grown in mice treated with sapanisertib had significantly less TF protein and co-factor activity, and were smaller compared to tumors grown in control mice. Conclusions: TF isoforms are expressed in pNET. Sapanisertib suppresses TF mRNA and protein expression as well as TF co-factor activity in vitro and in vivo. Thus, further studies are warranted to evaluate the clinical utility of TF-suppressing mTORC1/2 inhibitor sapanisertib in pNET management.
Myocardial ischemia/reperfusion (I/R) injury and the resulting cardiac remodeling is a common cause of heart failure. The RNA binding protein Human Antigen R (HuR) has been previously shown to reduce cardiac remodeling following both I/R and cardiac pressure overload, but the full extent of the HuR-dependent mechanisms within cells of the myocardium have yet to be elucidated. In this study, we applied a novel small molecule inhibitor of HuR to define the functional role of HuR in the acute response to I/R injury and gain a better understanding of the HuR-dependent mechanisms during post-ischemic myocardial remodeling. Our results show an early (two hours post-I/R) increase in HuR activity that is necessary for early inflammatory gene expression by cardiomyocytes in response to I/R. Surprisingly, despite the reductions in early inflammatory gene expression at two hours post-I/R, HuR inhibition has no effect on initial infarct size at 24-hours post-I/R. However, in agreement with previously published work, we do see a reduction in pathological remodeling and preserved cardiac function at two weeks post-I/R upon HuR inhibition. RNA-sequencing analysis of neonatal rat ventcricular myocytes (NRVMs) at two hours post-LPS treatment to model damage associated molecular pattern (DAMP)-mediated activation of toll like receptors (TLRs) demonstrates a broad HuR-dependent regulation of pro-inflammatory chemokine and cytokine gene expression in cardiomyocytes. Utilizing a conditioned media transfer experiment from LPS-treated NRVMs to bone marrow derived macrophages (BMDMs), we show that conditioned media from NRVMs pre-treated with HuR inhibitor loses the ability to induce inflammatory gene expression in BMDMs compared to NRVMs treated with LPS alone. Functionally, HuR inhibition in NRVMs also reduces their ability to induce endocrine migration of peripheral blood monocytes in vitro and reduces post-ischemic macrophage infiltration to the heart in vivo. In summary, these results suggest a HuR-dependent expression of pro-inflammatory gene expression by cardiomyocytes that leads to subsequent monocyte recruitment and macrophage activation in the post-ischemic myocardium.
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