Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level.

FKBP12.6, a binding protein to the immunosuppressant FK506, which also binds the ryanodine receptor (RyR2) in the heart, has been proposed to regulate RyR2 function and to have antiarrhythmic properties. However, the level of FKBP12.6 expression in normal hearts remains elusive and some controversies still persist regarding its effects, both in basal conditions and during ß-adrenergic stimulation. We quantified FKBP12.6 in the left ventricles (LV) of WT (wild-type) mice and in two novel transgenic models expressing distinct levels of FKBP12.6, using a custom-made specific anti-FKBP12.6 antibody and a recombinant protein. FKBP12.6 level in WT LV was very low (0.16 ± 0.02 nmol/g of LV), indicating that <15% RyR2 monomers are bound to the protein. Mice with 14.1 ± 0.2 nmol of FKBP12.6 per g of LV (TG1) had mild cardiac hypertrophy and normal function and were protected against epinephrine/caffeine-evoked arrhythmias. The ventricular myocytes showed higher [Ca2+]i transient amplitudes than WT myocytes and normal SR-Ca2+ load, while fewer myocytes showed Ca2+ sparks. TG1 cardiomyocytes responded to 50 nM Isoproterenol increasing these [Ca2+]i parameters and producing RyR2-Ser2808 phosphorylation. Mice with more than twice the TG1 FKBP12.6 value (TG2) showed marked cardiac hypertrophy with calcineurin activation and more arrhythmias than WT mice during ß-adrenergic stimulation, challenging the protective potential of high FKBP12.6. RyR2R420Q CPVT mice overexpressing FKBP12.6 showed fewer proarrhythmic events and decreased incidence and duration of stress-induced bidirectional ventricular tachycardia. Our study, therefore, quantifies for the first time endogenous FKBP12.6 in the mouse heart, questioning its physiological relevance, at least at rest due its low level. By contrast, our work demonstrates that with caution FKBP12.6 remains an interesting target for the development of new antiarrhythmic therapies.

Identification of a tetrahydroquinoline analog as a pharmacological inhibitor of the cAMP-binding protein Epac.

The cAMP-binding protein Epac is a therapeutic target for the treatment of various diseases such as cardiac hypertrophy and tumor invasion. This points out the importance to develop Epac inhibitors to better understand the involvement of these cAMP sensors in physiology and pathophysiology. Here, we have developed a functional fluorescence-based high-throughput assay with a Z' value around 0.7 for screening Epac-specific antagonists. We identified an Epac1 inhibitor compound named CE3F4 that blocked Epac1 guanine nucleotide exchange activity toward its effector Rap1 both in cell-free systems and in intact cells. CE3F4 is a tetrahydroquinoline analog that fails to influence protein kinase A holoenzyme activity. CE3F4 inhibited neither the interaction of Rap1 with Epac1 nor directly the GDP exchange on Rap1. The kinetics of inhibition by CE3F4 indicated that this compound did not compete for binding of agonists to Epac1 and suggested an uncompetitive inhibition mechanism with respect to Epac1 agonists. A structure-activity study showed that the formyl group on position 1 and the bromine atom on position 5 of the tetrahydroquinoline skeleton were important for CE3F4 to exert its inhibitory activity. Finally, CE3F4 inhibited Rap1 activation in living cultured cells, following Epac activation by either 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac-selective agonist, or isoprenaline, a non-selective ß-adrenergic receptor agonist. Our study shows that CE3F4 and related compounds may serve as a basis for the development of new therapeutic drugs.

The (R)-enantiomer of CE3F4 is a preferential inhibitor of human exchange protein directly activated by cyclic AMP isoform 1 (Epac1).

Isoform 1 and isoform 2 of exchange protein directly activated by cAMP (Epac1 and Epac2) contribute to cAMP signaling in numerous cellular processes. Their guanine-nucleotide exchange factor (GEF) activity toward the small GTP-binding protein Rap1 is stimulated by the agonist cAMP. CE3F4, a tetrahydroquinoline analog, prevents Epac1 activation in vitro and in living cultured cells by inhibiting the GEF activity of Epac1. However, the activity of the (R)- and (S)-enantiomers of CE3F4, as well as the ability of CE3F4 and its analogs to inhibit Epac2 GEF activity, have not yet been investigated. In this study, we report that (R)-CE3F4 is a more potent cAMP antagonist than racemic CE3F4 and (S)-CE3F4, inhibiting the GEF activity of Epac1 with 10-times more efficiency than (S)-CE3F4. Epac2, in contrast to Epac1, is activated more efficiently by cAMP than by 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (007), an Epac-selective cAMP analog. (R)-CE3F4 displays Epac isoform preference, with 10-fold selectivity for Epac1 over Epac2. Deletion of the N-terminal cyclic nucleotide-binding domain of Epac2 does not affect the characteristics of activation of Epac2 by cAMP and by 007, nor its inhibition by CE3F4. Finally, the evaluation of a series of CE3F4 structural analogs as GEF inhibitors allowed identifying structural features that are important for high Epac1 inhibitory activity of CE3F4. We conclude that the (R)-enantiomer of CE3F4 is a preferential inhibitor of Epac1 with high potency in the low micromolar range, and we suggest that this compound may be a useful pharmacological tool for investigating the functional role of Epac1 in cAMP signaling.

Membranes prime the RapGEF EPAC1 to transduce cAMP signaling.

EPAC1, a cAMP-activated GEF for Rap GTPases, is a major transducer of cAMP signaling and a therapeutic target in cardiac diseases. The recent discovery that cAMP is compartmentalized in membrane-proximal nanodomains challenged the current model of EPAC1 activation in the cytosol. Here, we discover that anionic membranes are a major component of EPAC1 activation. We find that anionic membranes activate EPAC1 independently of cAMP, increase its affinity for cAMP by two orders of magnitude, and synergize with cAMP to yield maximal GEF activity. In the cell cytosol, where cAMP concentration is low, EPAC1 must thus be primed by membranes to bind cAMP. Examination of the cell-active chemical CE3F4 in this framework further reveals that it targets only fully activated EPAC1. Together, our findings reformulate previous concepts of cAMP signaling through EPAC proteins, with important implications for drug discovery.

OPA1 (dys)functions.

Mitochondrial morphology varies according to cell type and cellular context from an interconnected filamentous network to isolated dots. This morphological plasticity depends on mitochondrial dynamics, a balance between antagonistic forces of fission and fusion. DRP1 and FIS1 control mitochondrial outer membrane fission and Mitofusins its fusion. This review focuses on OPA1, one of the few known actors of inner membrane dynamics, whose mutations provoke an optic neuropathy. Since its first identification in 2000 the characterization of the functions of OPA1 has made rapid progress thus providing numerous clues to unravel the pathogenetic mechanisms of ADOA-1.

The BH3-only Bnip3 binds to the dynamin Opa1 to promote mitochondrial fragmentation and apoptosis by distinct mechanisms.

Opa1 modulates mitochondrial fusion, cristae structure and apoptosis. The relationships between these functions and autosomal dominant optic atrophy, caused by mutations in Opa1, are poorly defined. We show that Bnip3 interacts with Opa1, leading to mitochondrial fragmentation and apoptosis. Fission is due to inhibition of Opa1-mediated fusion and is counteracted by Opa1 in an Mfn1-dependent manner. Bnip3-Opa1 interaction is necessary to trigger Opa1 complex disruption in a Bax- and/or Bak-dependent manner, ultimately leading to apoptosis. Our results uncover a direct link between Opa1 on the inner mitochondrial membrane and the apoptotic machinery on the outer membrane that modulates fusion and cristae structure by separate mechanisms. These findings might help to unravel optic atrophy aetiology as retinal ganglion cells are particularly prone to hypoxia, an inductor of Bnip3 expression.

Identification of Small Molecules Inhibiting Cardiomyocyte Necrosis and Apoptosis by Autophagy Induction and Metabolism Reprogramming.

Improvement of anticancer treatments is associated with increased survival of cancer patients at risk of cardiac disease. Therefore, there is an urgent need for new therapeutic molecules capable of preventing acute and long-term cardiotoxicity. Here, using commercial and home-made chemolibraries, we performed a robust phenotypic high-throughput screening in rat cardiomyoblast cell line H9c2, searching for small molecules capable of inhibiting cell death. A screen of 1600 compounds identified six molecules effective in preventing necrosis and apoptosis induced by H2O2 and camptothecin in H9c2 cells and in rat neonatal ventricular myocytes. In cells treated with these molecules, we systematically evaluated the expression of BCL-2 family members, autophagy progression, mitochondrial network structure, regulation of mitochondrial fusion/fission, reactive oxygen species, and ATP production. We found that these compounds affect autophagy induction to prevent cardiac cell death and can be promising cardioprotective drugs during chemotherapy.

A novel class of ethacrynic acid derivatives as promising drug-like potent generation of anticancer agents with established mechanism of action.

The well-known diuretic Ethacrynic acid (EA, Edecrin), showing low anti-proliferative activities, was chemically modified at different positions. The new EA derivatives have been tested in vitro in anti-proliferative assays on both tumor KB (epidermal carcinoma) and leukemia HL60 (promyelocytic) cells suitable targets for anticancer activity. Reduction of the α-ß double bond of EA completely abolished anti-cancer activities, whereas introduction of either 2-(4-substituted phenyl)ethanamine (series A) or 4-(4-substituted phenyl)piperazine (series B) moieties generated compounds showing moderate to strong anti-proliferative activities against human cancer cell lines. Several substitutions on the phenyl of these two moieties are tolerated. The mechanism of action of the EA derivatives prepared in this study is more complex than the inhibition of glutathione S-transferase π ascribed as unique effect to EA and might help to overcome tumor resistances.

Hyaluronic acid-conjugated lipoplexes for targeted delivery of siRNA in a murine metastatic lung cancer model.

We have investigated the impact of hyaluronic acid (HA)-coating on the targeting capacity of siRNA lipoplexes to CD44-overexpressing tumor cells. Cellular uptake and localization of HA-lipoplexes were evaluated by flow cytometry and fluorescence microscopy and both methods showed that these lipoplexes were rapidly internalized and localized primarily within the cytoplasm. Inhibition of luciferase expression on the A549-luciferase lung cancer cell line was achieved in vitro using an anti-Luc siRNA. 81% of luciferase gene expression inhibition was obtained in vitro with HA-lipoplexes at +/- ratio 2. In vivo, in a murine A549 metastatic lung cancer model, the treatment with HA-lipoplexes carrying anti-luciferase siRNA led to a statistically significant decrease of luciferase expression as opposed to progressive increase with non-modified lipoplexes or NaCl 0.9%. The reduction of the expression of luciferase mRNA tumor of mice treated with HA-lipoplexes supported the inhibition effect due to siRNA. These results highlight the potential of HA-lipoplexes in CD44-targeting siRNA delivery.

Phosphatidylinositol 5-phosphate regulates invasion through binding and activation of Tiam1.

PtdIns5P is a lipid messenger acting as a stress-response mediator in the nucleus, and known to maintain cell activation through traffic alterations upon bacterial infection. Here, we show that PtdIns5P regulates actin dynamics and invasion via recruitment and activation of the exchange factor Tiam1 and Rac1. Restricted Rac1 activation results from the binding of Tiam1 DH-PH domains to PtdIns5P. Using an assay that mimics Rac1 membrane anchoring by using Rac1-His and liposomes containing Ni(2+)-NTA modified lipids, we demonstrate that intrinsic Tiam1 DH-PH activity increases when Rac1 is anchored in a PtdIns5P-enriched environment. This pathway appears to be general since it is valid in different pathophysiological models: receptor tyrosine kinase activation, bacterial phosphatase IpgD expression and the invasive NPM-ALK(+) lymphomas. The discovery that PtdIns5P could be a keystone of GTPases and cytoskeleton spatiotemporal regulation opens important research avenues towards unravelling new strategies counteracting cell invasion.

Tetrabromobisphenol-A disrupts thyroid hormone receptor alpha function in vitro: use of fluorescence polarization to assay corepressor and coactivator peptide binding.

Thyroid hormone receptors (TRs) recruit corepressor or coactivator factors to the promoters of target genes to regulate their transcription. Corepressors such as nuclear hormone receptor corepressor (NCoR) are recruited by unliganded TRs, whereas coactivators such as steroid receptor coactivator-2 (SRC2) are recruited when triiodothyronine (T3) is bound to TRs. These coregulator proteins interact with the ligand binding domain (LBD) of TRs via short, conserved peptide sequences that can be used to probe the conformational changes induced in TR LBD by TR ligands. Recombinant LBD of the human TRα1 isoform (hTRα1 LBD) was produced as a fusion with glutathione S-transferase, and used to develop assays based on fluorescence polarization to quantify the binding of either NCoR- or SRC2-derived fluorescent peptides to the hTRα1 LBD. The optimum concentrations of recombinant hTRα1 LBD, and of peptide probes were adjusted in order to produce the greatest possible T3-dependent signal variations in fluorescence polarization. Under these conditions, T3 induced a dose-dependent decrease in NCoR peptide binding, and a reciprocal dose-dependent increase in SRC2 peptide binding, in both cases at similar 50%-effective doses. The TR agonists triiodothyroacetic acid and thyroxine were also effective in preventing NCoR peptide binding and increasing SRC2 peptide binding, whereas reverse-triiodothyronine was less efficient and the biologically inactive thyronine had no effect on either process. These experiments validate cell-free assays based on the use of binding of corepressor or coactivator peptide probes, as measured by fluorescence polarization, for investigating the conformational changes of TRα1 LBD induced by potentially TR-interfering compounds. Both these methods were used to elucidate the mechanism of the disrupting effects of tetrabromobisphenol-A (TBBPA) on the hTRα1 LBD conformation related to the transcriptional activity of the receptor. TBBPA is a flame retardant that is released into the environment, and is a suspected disrupter of thyroid homeostasis. The present results indicate that TBBPA did indeed interfere with the ability of the hTRα1 LBD to bind both NCoR and SRC2. TBBPA behaved similarly to T3 in promoting the release of NCoR from LBD, whereas it failed to promote LBD interactions with SRC2. However, it did reduce the T3-induced interactions between LBD and the coactivator peptide. This study therefore suggests that TBBPA in the micromolar range can affect the regulation of transcription by both the apo- and the holo-TRα1, with potential disruption of the expression of genes that are either up- or down-regulated by T3.

A cardiac-specific robotized cellular assay identified families of human ligands as inducers of PGC-1α expression and mitochondrial biogenesis.

BACKGROUND: Mitochondrial function is dramatically altered in heart failure (HF). This is associated with a decrease in the expression of the transcriptional coactivator PGC-1α, which plays a key role in the coordination of energy metabolism. Identification of compounds able to activate PGC-1α transcription could be of future therapeutic significance. METHODOLOGY/PRINCIPAL FINDINGS: We thus developed a robotized cellular assay to screen molecules in order to identify new activators of PGC-1α in a cardiac-like cell line. This screening assay was based on both the assessment of activity and gene expression of a secreted luciferase under the control of the human PGC-1α promoter, stably expressed in H9c2 cells. We screened part of a library of human endogenous ligands and steroid hormones, B vitamins and fatty acids were identified as activators of PGC-1α expression. The most responsive compounds of these families were then tested for PGC-1α gene expression in adult rat cardiomyocytes. These data highly confirmed the primary screening, and the increase in PGC-1α mRNA correlated with an increase in several downstream markers of mitochondrial biogenesis. Moreover, respiration rates of H9c2 cells treated with these compounds were increased evidencing their effectiveness on mitochondrial biogenesis. CONCLUSIONS/SIGNIFICANCE: Using our cellular reporter assay we could identify three original families, able to activate mitochondrial biogenesis both in cell line and adult cardiomyocytes. This first screening can be extended to chemical libraries in order to increase our knowledge on PGC-1α regulation in the heart and to identify potential therapeutic compounds able to improve mitochondrial function in HF.