Small molecule screening platform for assessment of cardiovascular toxicity on adult zebrafish heart

<p>Abstract</p> <p>Background</p> <p>Cardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance <it>ex vivo</it>, and a setup to perform quick small molecule throughput screening, including an in-house implemented analysis script.</p> <p>Results</p> <p>Adult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for <it>ex vivo </it>culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens.</p> <p>Conclusion</p> <p>The new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.</p

Human tetraspanin superfamily members used for expression screening in <i>S</i>. <i>cerevisiae</i>.

<p>^a tetraspanin superfamily member lacking the CCG motif [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref044" target="_blank">44</a>].</p><p>Human tetraspanin superfamily members used for expression screening in <i>S</i>. <i>cerevisiae</i>.</p

Size exclusion chromatogram of purified CO-029.

<p>Size exclusion of the purified CO-029 protein after proteolytic cleavage and removal of the GFP-tag. The elution was monitored with the absorbance at 280 nm.</p

Sequence alignment of selected tetraspanins.

<p>Predicted localization of transmembrane helices with TOPCONS server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref078" target="_blank">78</a>] are indicated with a gray background. Residues conserved across the family–indicated in yellow and predicted glycosylation sites [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref079" target="_blank">79</a>] are indicated with a red background. Sequence alignments were generated with the ClustalW server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref080" target="_blank">80</a>] by feeding 219 tetraspanin sequences from diverge spices to improve alignment statistics.</p

Experimental studies reporting tetraspanin production or isolation.

<p>^a A tetraspanin superfamily member lacking the CCG motif [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref044" target="_blank">44</a>].</p><p>MDCK, Madin-Darby Canine Kidney Epithelial Cells; HEK293, Human Embryonic Kidney 293 cells</p

Solubilization efficiency of TSPAN-GFP fusion proteins.

<p>The solubilization degree of five tetraspanin-containing yeast membranes with the help of detergents and SMA polymers. The solubilization ratio was determined by comparing the fluorescence counts of solubilized material after ultracentrifugation to the mix before separation of solubilized and non-solubilized material.</p

Tetraspanin topology scheme.

<p>Transmembrane helices are numbered 1–4, conserved helices in the large extracellular domain indicated with letters A,B,E according to nomenclature by Seigneuret et al. <b>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref032" target="_blank">32</a>]</b>. Conserved residues are shown in circles, where x stands for any amino acid. Possible post-translational modifications are indicated as palmitoylation sites shown as waves close to the intracellular side of the protein and available N-linked glycosylation sites shown as forks on the extracellular domains.</p

Detergents and hydrolyzed forms of SMA polymers used in screening for solubilization efficiency of membranes containing TSPAN-GFP fusion proteins.

<p>Listed are also the final concentration used for solubilization.</p