The emergence of human stem cell-derived cardiomyocyte (hSCCM)-based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling "fingerprint" in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ? control. This rank order of drug-induced Ca(2+) signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework.
The ability of neutrophils to rapidly change shape underlies their physiological functions of phagocytosis and spreading. A major problem in establishing the mechanism is that conventional microinjection of substances and indicators interferes with this dynamic cell behaviour. Here we show that electroinjection, a "no-touch" point-and-shoot means of introducing material into the cell, is sufficiently gentle to allow neutrophils to be injected whilst undergoing chemokinesis and spreading without disturbing cell shape change behaviour. Using this approach, a fluorogenic calpain-1 selective peptide substrate was introduced into the cytosol of individual neutrophils undergoing shape changes. These data showed that (i) physiologically elevated cytosolic Ca(2+) concentrations were sufficient to trigger calpain-1 activation, blockade of Ca(2+) influx preventing calpain activation and (ii) calpain-1 activity was elevated in spreading neutrophil. These findings provide the first direct demonstration of a physiological role for Ca(2+) elevation in calpain-1 activation and rapid cell spreading. Electroinjection of cells undergoing dynamic shape changes thus opens new avenues of investigation for defining the molecular mechanism underlying dynamic cell shape changes.
Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.
Glycogen synthase kinase-3 (GSK3) is a highly conserved protein kinase that is involved in several important cell signaling pathways and is associated with a range of medical conditions. Previous studies indicated a major role of the Dictyostelium homologue of GSK3 (gskA) in cell fate determination during morphogenesis of the fruiting body; however, transcriptomic and proteomic studies have suggested that GSK3 regulates gene expression much earlier during Dictyostelium development. To investigate a potential earlier role of GskA, we examined the effects of loss of gskA on cell aggregation. We find that cells lacking gskA exhibit poor chemotaxis toward cAMP and folate. Mutants fail to activate two important regulatory signaling pathways, mediated by phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and target of rapamycin complex 2 (TORC2), which in combination are required for chemotaxis and cAMP signaling. These results indicate that GskA is required during early stages of Dictyostelium development, in which it is necessary for both chemotaxis and cell signaling.
The epithelial lining of the gastrointestinal tract is the major interface between the external world (e.g., the gut lumen) and the body, and as such the proper maintenance and regulation of epithelial barrier function is a key determinant of digestive health and host well-being. Many enteropathies are associated with increased gut permeability, including inflammatory bowel disease (IBD). Maintaining the barrier function of the epithelium, independent of whether paracellular or transcellular permeation pathways are considered, is an energy-dependent process. Here we present an overview of the impact that metabolic stress (e.g., reductions in epithelial ATP synthesis) can have on permeability characteristics of epithelial monolayers and show that metabolic stress in the presence of a commensal flora results in a significant loss of epithelial integrity, and that this increase in epithelial permeability can be enhanced by the presence of tumor necrosis factor-alpha (TNFalpha). We speculate that the combination of these factors in vivo would result in significant perturbations in epithelial barrier function that could be of pathophysiological significance and contribute to the initiation of IBD or the induction of disease relapses.
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