ERG potassium channels and T-type calcium channels contribute to the pacemaker and atrioventricular conduction in zebrafish larvae.

AIM: Bradyarrhythmias result from inhibition of automaticity, prolonged repolarization, or slow conduction in the heart. The ERG channels mediate the repolarizing current IKr in the cardiac action potential, whereas T-type calcium channels (TTCC) are involved in the sinoatrial pacemaker and atrioventricular conduction in mammals. Zebrafish have become a valuable research model for human cardiac electrophysiology and disease. Here, we investigate the contribution of ERG channels and TTCCs to the pacemaker and atrioventricular conduction in zebrafish larvae and determine the mechanisms causing atrioventricular block. METHODS: Zebrafish larvae expressing ratiometric fluorescent Ca2+ biosensors in the heart were used to measure Ca2+ levels and rhythm in beating hearts in vivo, concurrently with contraction and hemodynamics. The atrioventricular delay (the time between the start of atrial and ventricular Ca2+ transients) was used to measure impulse conduction velocity and distinguished between slow conduction and prolonged refractoriness as the cause of the conduction block. RESULTS: ERG blockers caused bradycardia and atrioventricular block by prolonging the refractory period in the atrioventricular canal and in working ventricular myocytes. In contrast, inhibition of TTCCs caused bradycardia and second-degree block (Mobitz type I) by slowing atrioventricular conduction. TTCC block did not affect ventricular contractility, despite being highly expressed in cardiomyocytes. Concomitant measurement of Ca2+ levels and ventricular size showed mechano-mechanical coupling: increased preload resulted in a stronger heart contraction in vivo. CONCLUSION: ERG channels and TTCCs influence the heart rate and atrioventricular conduction in zebrafish larvae. The zebrafish lines expressing Ca2+ biosensors in the heart allow us to investigate physiological feedback mechanisms and complex arrhythmias.

Early calcium and cardiac contraction defects in a model of phospholamban R9C mutation in zebrafish.

The phospholamban mutation Arg 9 to Cys (R9C) has been found to cause a dilated cardiomyopathy in humans and in transgenic mice, with ventricular dilation and premature death. Emerging evidence suggests that phospholamban R9C is a loss-of-function mutation with dominant negative effect on SERCA2a activity. We imaged calcium and cardiac contraction simultaneously in 3 and 9 days-post-fertilization (dpf) zebrafish larvae expressing plnbR9C in the heart to unveil the early pathological pathway that triggers the disease. We generated transgenic zebrafish lines expressing phospholamban wild-type (Tg(myl7:plnbwt)) and phospholamban R9C (Tg(myl7:plnbR9C)) in the heart of zebrafish. To measure calcium and cardiac contraction in 3 and 9 dpf larvae, Tg(myl7:plnbwt) and Tg(myl7:plnbR9C) fish were outcrossed with a transgenic line expressing the ratiometric fluorescent calcium biosensor mCyRFP1-GCaMP6f. We found that PlnbR9C raised calcium transient amplitude, induced positive inotropy and lusitropy, and blunted the ß-adrenergic response to isoproterenol in 3 dpf larvae. These effects can be attributed to enhanced SERCA2a activity induced by the PlnbR9C mutation. In contrast, Tg(myl7:plnbR9C) larvae at 9 dpf exhibited ventricular dilation, systolic dysfunction and negative lusitropy, hallmarks of a dilated cardiomyopathy in humans. Importantly, N-acetyl-L-cysteine rescued this deleterious phenotype, suggesting that reactive oxygen species contribute to the pathological pathway. These results also imply that dysregulation of calcium homeostasis during embryo development contributes to the disease progression at later stages. Our in vivo model in zebrafish allows characterization of pathophysiological mechanisms leading to heart disease, and can be used for screening of potential therapeutical agents.

The p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.

Inhibition of the mitotic kinase PLK1 overcomes therapeutic resistance to BET inhibitors in triple negative breast cancer.

The inhibition of bromo- and extraterminal domains (BET) has shown an anti-proliferative effect in triple negative breast cancer (TNBC). In this article we explore mechanisms of resistance to BET inhibitors (BETi) in TNBC, with the aim of identifying novel ways to overcome such resistance. Two cellular models of acquired resistance to the BET inhibitor JQ1 were generated using a pulsed treatment strategy. MTT, colony formation, and cytometry assays revealed that BETi-resistant cells were particularly sensitive to PLK1 inhibition. Targeting of the latter reduced cell proliferation, especially in resistant cultures. Quantitative PCR analysis of a panel of mitotic kinases uncovered an increased expression of AURKA, TTK, and PLK1, confirmed by Western blot. Only pharmacological inhibition of PLK1 showed anti-proliferative activity on resistant cells, provoking G2/M arrest, increasing expression levels of cyclin B, pH3 and phosphorylation of Bcl-2 proteins, changes that were accompanied by induction of caspase-dependent apoptosis. JQ1-resistant cells orthotopically xenografted into the mammary fat pad of mice led to tumours that retained JQ1-resistance. Administration of the PLK1 inhibitor volasertib resulted in tumour regression. These findings open avenues to explore the future use of PLK1 inhibitors in the clinical setting of BETi-resistant patients.

Failure of prion protein oxidative folding guides the formation of toxic transmembrane forms.

The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrP(C)) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.

Imaging local estrogen production in single living cells with recombinant fluorescent indicators.

Estrogens are steroid hormones with many systemic effects in addition to development and maintenance of the female reproductive system, and ligands of estrogen receptors are of clinical importance because of their use as oral contraceptive, hormone replacement and antitumoral therapy. In addition, tumoral tissues have been found to express aromatase and other steroidogenic enzymes synthesizing estradiol. To aid in the understanding of these processes, we have developed assays to image the local production of estrogens in isolated living mammalian cells. We constructed biosensors based on estrogen receptor α ligand binding domain and fluorescent proteins by following two approaches. First, the ligand binding domain and a short fragment of steroid receptor coactivator-1 were appended to a circularly permuted yellow fluorescent protein to construct an excitation ratio estrogen indicator. In the second strategy, we constructed emission ratio sensors based on fluorescence resonance energy transfer, containing the ligand binding domain flanked by donor and acceptor fluorescent proteins. Estrogens altered the fluorescence signal of cells transfected with the indicators in a dose-dependent manner. We imaged local estrogen production in adrenocortical H295 cells expressing aromatase and transfected with the fluorescent sensors. In addition, paracrine detection was observed in HeLa cells harboring the indicators and co-cultured with H295 cells. This imaging approach may allow detection of physiological levels of these hormones in suitable animal models.

Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange.

Membrane protein function is determined by the relative organization of the protein domains with respect to the membrane. We have experimentally verified the topology of a protein with diverse orientations arising from a single primary sequence (the cellular prion protein, PrP(C)), a novel somatostatin truncated receptor, and the Golgi-associated protein GPBP(91). Tagging with fluorescent proteins (FP) allows location of their expression at the plasma membrane or at endomembranes, but does not inform about their orientation. Exploiting the pH dependency of some FPs, we developed a pH exchange assay in which extracellularly exposed FPs are quenched by application of low pH buffer. We constructed standards to demonstrate and calibrate the assay, and the method was adapted for acidic organelle membrane proteins. This method can serve as a proof of concept, experimentally confirming and/or discriminating in living cells among theoretical topology predictions, providing the proportion of inside/outside orientation for proteins with multiple forms.

Glitazones differentially regulate primary astrocyte and glioma cell survival. Involvement of reactive oxygen species and peroxisome proliferator-activated receptor-gamma.

The glitazones or thiazolidinediones are ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma). The glitazones are used in the treatment of diabetes, regulate adipogenesis, inflammation, cell proliferation, and induce apoptosis in several cancer cell types. High grade astrocytomas are rapidly growing tumors derived from astrocytes, for which new treatments are needed. We determined the effects of two glitazones, ciglitazone and the therapeutic rosiglitazone, on the survival of serum-deprived primary rat astrocytes and glioma cell lines C6 and U251, which were assessed by the methylthiazolyl tetrazolium assay and lactate dehydrogenase release. Rosiglitazone (5-20 microM) decreased survival of glioma cells without affecting primary astrocytes, whereas ciglitazone at 20 microM was toxic for both cell types. Ciglitazone at 10 microM was cytoprotective for primary astrocytes but toxic to glioma cells. Cell death induced by ciglitazone, but not rosiglitazone, presented apoptotic features (Hoechst staining and externalization of phosphatidylserine). Two mechanisms to explain cytotoxicity were investigated: activation of PPARgamma and production of reactive oxygen species (ROS). PPARgamma does not seem to be the main mechanism involved, because the order of efficacy for cytotoxicity, ciglitazone > rosiglitazone, was inverse of their reported affinities for activating PPARgamma. In addition, GW9662, an inhibitor of PPARgamma, only slightly attenuated cytotoxicity. However, the rapid increase in ROS production and the marked reduction of cell death with the antioxidants ebselen and N-acetylcysteine, indicate that ROS have a key role in glitazone cytotoxicity. Ciglitazone caused a dose-dependent and rapid loss (in minutes) of mitochondrial membrane potential in glioma cells. Therefore, mitochondria are a likely source of ROS and early targets of glitazone cytotoxicity. Our results highlight the potential of rosiglitazone and related compounds for the treatment of astrogliomas.