cAMP Imaging at Ryanodine Receptors Reveals β2-Adrenoceptor Driven Arrhythmias

Rationale: 3',5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which, upon β-adrenergic receptor (β-AR) stimulation, acts in microdomains to regulate cardiac excitation-contraction coupling by activating phosphorylation of calcium handling proteins. One crucial microdomain is in vicinity of the cardiac ryanodine receptor type 2 (RyR2) which is associated with arrhythmogenic diastolic calcium leak from the sarcoplasmic reticulum (SR) often occurring in heart failure. Objective: We sought to establish a real time live cell imaging approach capable of directly visualizing cAMP in the vicinity of mouse and human RyR2 and to analyze its pathological changes in failing cardiomyocytes under β-AR stimulation. Methods and Results: We generated a novel targeted fluorescent biosensor Epac1-JNC for RyR2-associated cAMP and expressed it in transgenic mouse hearts as well in human ventricular myocytes using adenoviral gene transfer. In healthy cardiomyocytes, β 1 -AR but not β 2 -AR stimulation strongly increased local RyR2-associated cAMP levels. However, already in cardiac hypertrophy induced by aortic banding, there was a marked subcellular redistribution of phosphodiesterases (PDEs) 2, 3 and 4, which included a dramatic loss of the local pool of PDE4. This was also accompanied by measurableβ2-AR/AMP signals in the vicinity of RyR2 in failing mouse and human myocytes, increased β2-AR-dependent RyR2 phosphorylation, SR calcium leak and arrhythmia susceptibility. Conclusions: Our new imaging approach could visualize cAMP levels in the direct vicinity of cardiac RyR2. Unexpectedly, in mouse and human failing myocytes, it could uncover functionally relevant local arrhythmogenic β2-AR/cAMP signals which might be an interesting antiarrhythmic target for heart failure.

Abstract 16575: Synthetic TBX18 mRNA Induces Durable Reprogramming of Cardiac Myocytes to Pacemaker Cells

Introduction: Adenoviral gene delivery of transcription factor, TBX18, has been proven to reprogram ordinary cardiomyocytes to pacemaker cells, and provide ventricular pacing in the rodent and porcine heart. Yet host innate immune response to recombinant viral therapies remains a hurdle to overcome for successful translation. Somatic cell reprogramming does not require persistent expression of the reprogramming factors after arriving at the final cell fate. We hypothesized that brief expression of synthetic TBX18 mRNA suffices to convert chamber cardiomyocytes to induced pacemaker cells, thus obviating issues inherent to adenoviral gene therapy. Methods: Synthetic mRNAs were in vitro transcribed as previously described. Neonatal rat ventricular myocytes (NRVMs) were used as an in vitro platform to test pacemaker reprogramming by TBX18 mRNA. GFP or fLuc encoded mRNA was used as controls in all experiments. Results: Three days after synthetic TBX18 mRNA transfection, Hcn4 transcript level increased >6-fold compared to control. After two weeks, genes enriched in the pacemaker tissue, e.g., Shox2, Hcn4, and Tbx3, continued to be expressed higher in TBX18-NRVMs compared to control by >4.2-fold. The level of endogenously expressed, rat Tbx18 increased by 50% in NRVMs transfected with synthetic human TBX18 mRNA, which point to long-term reprogramming. To identify and lineage-trace reprogrammed pacemaker cells in real time, we cultured ventricular cardiomyoytes from transgenic mouse line Hcn4 ( GFP/+ ) , in which de novo pacemaker cells expressing Hcn4 contain eGFP fluorescence. Twelve days after synthetic mRNA or adenoviral gene transfer of TBX18 or GFP control, a significantly higher number of Hcn4 ( GFP/+ ) cells were observed in both mRNA and adenoviral TBX18-treated wells compared to their respective controls. TBX18 mRNA-transfected NRVM monolayers exhibited >2-fold slower conduction velocities compared to fLuc-transfected myocytes. In line with slow conduction velocity in the induced pacemaker myocytes, expression of ventricular gap junction protein, Cx43, decreased significantly compared to control. Conclusions: Together, the data indicate durable nodal pacemaker cell reprogramming with transient expression of synthetic TBX18 mRNA.

The FMS-like tyrosine kinase-3 ligand/lung dendritic cell axis contributes to regulation of pulmonary fibrosis

RationaleDendritic cells (DC) accumulate in the lungs of patients with idiopathic lung fibrosis, but their pathogenetic relevance is poorly defined.ObjectivesTo assess the role of the FMS-like tyrosine kinase-3 ligand (Flt3L)-lung dendritic cell axis in lung fibrosis.Measurements and main resultsWe demonstrate in a model of adenoviral gene transfer of active TGF-β1 that established lung fibrosis was accompanied by elevated serum Flt3L levels and subsequent accumulation of CD11bpos DC in the lungs of mice. Patients with idiopathic pulmonary fibrosis also demonstrated increased levels of Flt3L protein in serum and lung tissue and accumulation of lung DC in explant subpleural lung tissue specimen. Mice lacking Flt3L showed significantly reduced lung DC along with worsened lung fibrosis and reduced lung function relative to wild-type (WT) mice, which could be inhibited by administration of recombinant Flt3L. Moreover, therapeutic Flt3L increased numbers of CD11bpos DC and improved lung fibrosis in WT mice exposed to AdTGF-β1. In this line, RNA-sequencing analysis of CD11bpos DC revealed significantly enriched differentially expressed genes within extracellular matrix degrading enzyme and matrix metalloprotease gene clusters. In contrast, the CD103pos DC subset did not appear to be involved in pulmonary fibrogenesis.ConclusionsWe show that Flt3L protein and numbers of lung DC are upregulated in mice and humans during pulmonary fibrogenesis, and increased mobilisation of lung CD11bpos DC limits the severity of lung fibrosis in mice. The current study helps to inform the development of DC-based immunotherapy as a novel intervention against lung fibrosis in humans.

Regulation of the metabolism of apolipoprotein M and sphingosine 1-phosphate by hepatic PPARγ activity

Apolipoprotein M (apoM) is a carrier and a modulator of sphingosine 1-phosphate (S1P), an important multifunctional bioactive lipid. Since peroxisome proliferator-activated receptor γ (PPARγ) is reportedly associated with the function and metabolism of S1P, we investigated the modulation of apoM/S1P homeostasis by PPARγ. First, we investigated the modulation of apoM and S1P homeostasis by the overexpression or knockdown of PPARγ in HepG2 cells and found that both the overexpression and the knockdown of PPARγ decreased apoM expression and S1P synthesis. When we activated or suppressed the PPARγ more mildly with pioglitazone or GW9662, we found that pioglitazone suppressed apoM expression and S1P synthesis, while GW9662 increased them. Next, we overexpressed PPARγ in mouse liver through adenoviral gene transfer and observed that both the plasma and hepatic apoM levels and the plasma S1P levels decreased, while the hepatic S1P levels increased, in the presence of enhanced sphingosine kinase activity. Treatment with pioglitazone decreased both the plasma and hepatic apoM and S1P levels only in diet-induced obese mice. Moreover, the overexpression of apoM increased, while the knockdown of apoM suppressed PPARγ activities in HepG2 cells. These results suggested that PPARγ regulates the S1P levels by modulating apoM in a bell-shaped manner, with the greatest levels of apoM/S1P observed when PPARγ was mildly expressed and that hepatic apoM/PPARγ axis might maintain the homeostasis of S1P metabolism.

Ultrasound targeted gene therapy in a mouse model of prostate cancer: Evaluation of immune response.

120 Background: Gene transfer to malignant sites using human adenoviruses (hAd) has been limited because of their immunogenicity. Murine cells often lack some of the receptors needed for hAd infection; therefore, are generally non-permissive for hAd infection and replication, which limits translational studies of adenoviral gene transfer techniques. We developed a gene transfer method, which uses a combination of lipid-encapsulated perfluorocarbon microbubbles (MBs) and ultrasound (US) to shield and deliver hAds to a specific tissue bypassing the requirement of the coxsackie and adenovirus receptor (CAR). Methods: Transduction efficiency and GFP protein expression of hAd.GFP was assessed by flow cytometry and fluorescence microscopy in murine TRAMP-C2 and human DU145 prostate cancer cells. Innate and acquired immunity response was determined by ELISA and CTL assay in C57BL/6 mice bearing TRAMP-C2 syngeneic tumor grafts following injections of MBs-Ad.GFP complexes in the presence or absence of ultrasound. Results: We observed that the murine prostate cancer cells TRAMP-C2 were transduced less efficiently by hAd.GFP than the human DU145 cells. We showed in vitro that the transduction rate was increased significantly in both TRAMP-C2 and DU145 prostate cancer cells when delivering the Ad particles by a combination of MBs and US. Moreover, we observed expression of the GFP transgene in both cell lines at 48 hours and 72 hours. Lack of activation of the innate and acquired immunity was observed in vivo by quantifying IL-6 and TNF-α cytokines, and by assaying neutralizing IgG antibodies and CTLs activity, following intratumoral or intravenous injections of MBs-Ad.GFP complexes in the presence or absence of ultrasound. Conclusions: This study demonstrates the feasibility of using the TRAMP-C2 murine model of prostate adenocarcinoma to translate our ultrasound-mediated MB-Ad delivery system from the bench to the clinic. Our data provides evidence that the TRAMP-C2 prostate cancer graft model is a suitable system to study in immune competent animals the capacity of lipid-encapsulated perfluorocarbon MBs and US, to shield and deliver hAds to a site-specific tissue bypassing the requirement of specific receptors.