BACKGROUND AND PURPOSECardiac toxicity is a major concern in drug development and it is imperative that clinical candidates are thoroughly tested for adverse effects earlier in the drug discovery process. In this report, we investigate the utility of an impedance-based microelectronic detection system in conjunction with mouse embryonic stem cell-derived cardiomyocytes for assessment of compound risk in the drug discovery process.
EXPERIMENTAL APPROACHBeating of cardiomyocytes was measured by a recently developed microelectronic-based system using impedance readouts. We used mouse stem cell-derived cardiomyocytes to obtain dose-response profiles for over 60 compounds, including ion channel modulators, chronotropic/ionotropic agents, hERG trafficking inhibitors and drugs known to induce Torsades de Pointes arrhythmias.
KEY RESULTSThis system sensitively and quantitatively detected effects of modulators of cardiac function, including some compounds missed by electrophysiology. Pro-arrhythmic compounds produced characteristic profiles reflecting arrhythmia, which can be used for identification of other pro-arrhythmic compounds. The time series data can be used to identify compounds that induce arrhythmia by complex mechanisms such as inhibition of hERG channels trafficking. Furthermore, the time resolution allows for assessment of compounds that simultaneously affect both beating and viability of cardiomyocytes.
CONCLUSIONS AND IMPLICATIONSMicroelectronic monitoring of stem cell-derived cardiomyocyte beating provides a high throughput, quantitative and predictive assay system that can be used for assessment of cardiac liability earlier in the drug discovery process. The convergence of stem cell technology with microelectronic monitoring should facilitate cardiac safety assessment.
AbbreviationsBRI, beating rhythm irregularity; hERG, human ether a go go; MEA, multi elelctrode array; mESCC, mouse embryonic stem cell
An O‐glycosylated protein of approximately 18 kDa responsible for mating type specific agglutination has been isolated from Saccharomyces cerevisiae a cells, purified to homogeneity and via peptide sequences the gene was cloned by PCR. An open reading frame codes for a protein of 69 amino acids. A minimum of five serine and five threonine residues of the mature protein are glycosylated. alpha‐Agglutinin is a highly N‐glycosylated protein of approximately 250 kDa. Both purified agglutinins form a specific 1:1 complex in vitro. Pretreatment of alpha‐agglutinin, but not of alpha‐agglutinin, with diethylpyrocarbonate (DEPC) prevents formation of the complex; treatment of alpha‐agglutinin in the presence of alpha‐agglutinin protects the former from DEPC inactivation. By carboxy terminal shortening of the alpha‐agglutinin gene and by replacing three of its eight histidyl residues by arginine, the active region of alpha‐agglutinin for interaction with alpha‐agglutinin has been defined. Neither the N‐ nor the O‐linked saccharides of the two agglutinins seem to be essential for their interaction.
A cell surface glycoprotein induced by the mating pheromone alpha factor in Saccharomyces cerevisiae a cells has been purified to homogeneity. At 4 x 10(‐9) M it strongly inhibits mating‐type‐specific agglutination between a and alpha cells. The protein is solely O‐glycosylated. It consists of 29% carbohydrate and its apparent molecular mass is 22 kd on SDS gels. After HF treatment it behaves like a protein of 13 kd; therefore its true molecular mass probably is close to 18 kd. Mild periodate treatment destroys the biological activity of the purified protein. The protein contains one cysteine, no arginine, and 27% of the amino acids are serine and threonine residues, two thirds of which are glycosylated. With a polyclonal antibody the glycoprotein can already be detected at the cell surface 15 min after pheromone addition. The inducible antigen is not expressed in a specific phase of the cell cycle; it first appears exclusively on the growing bud. Mother cells express the antigen on their surface only after the daughter cells have separated; it is then localized at the tip of the pear‐shaped ‘shmoo’. Using the secretory ts‐mutant sec 18 is shown that a mannosylated precursor of a agglutinin accumulates at the endoplasmic reticulum.
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