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A cell-based phenotypic assay to identify cardioprotective agents.

ABSTRACT: RATIONALE:Tissue ischemia/reperfusion (IR) injury underlies several leading causes of death such as heart-attack and stroke. The lack of clinical therapies for IR injury may be partly due to the difficulty of adapting IR injury models to high-throughput screening (HTS). OBJECTIVE:To develop a model of IR injury that is both physiologically relevant and amenable to HTS. METHODS AND RESULTS:A microplate-based respirometry apparatus was used. Controlling gas flow in the plate head space, coupled with the instrument's mechanical systems, yielded a 24-well model of IR injury in which H9c2 cardiomyocytes were transiently trapped in a small volume, rendering them ischemic. After initial validation with known protective molecules, the model was used to screen a 2000-molecule library, with post-IR cell death as an end point. Po2 and pH monitoring in each well also afforded metabolic data. Ten protective, detrimental, and inert molecules from the screen were subsequently tested in a Langendorff-perfused heart model of IR injury, revealing strong correlations between the screening end point and both recovery of cardiac function (negative, r2=0.66) and infarct size (positive, r2=0.62). Relationships between the effects of added molecules on cellular bioenergetics and protection against IR injury were also studied. CONCLUSIONS:This novel cell-based assay can predict either protective or detrimental effects on IR injury in the intact heart. Its application may help identify therapeutic or harmful molecules.

SUBMITTER: Guo S

PROVIDER: S-EPMC3329894 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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A cell-based phenotypic assay to identify cardioprotective agents.

Guo Stephanie S Olm-Shipman Adam A Walters Andrew A Urciuoli William R WR Devito Stefanie S Nadtochiy Sergiy M SM Wojtovich Andrew P AP Brookes Paul S PS

Circulation research 20120306 7

<h4>Rationale</h4>Tissue ischemia/reperfusion (IR) injury underlies several leading causes of death such as heart-attack and stroke. The lack of clinical therapies for IR injury may be partly due to the difficulty of adapting IR injury models to high-throughput screening (HTS).<h4>Objective</h4>To develop a model of IR injury that is both physiologically relevant and amenable to HTS.<h4>Methods and results</h4>A microplate-based respirometry apparatus was used. Controlling gas flow in the plate ...[more]

PMID: 22394516

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Project description:BackgroundInterpatient variability in immune and chemotherapeutic cytotoxic responses is likely due to complex genetic differences and is difficult to ascertain in humans. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at examining interstrain differences in viability on normal, noncancerous immune cells following chemotherapeutic cytotoxic insult. Drug effects were investigated by comparing selective chemotherapeutic agents, such as BEZ-235 and selumetinib, against conventional cytotoxic agents targeting multiple pathways, including doxorubicin and idarubicin.MethodsSplenocytes were isolated from 36 isogenic strains of mice using standard procedures. Of note, the splenocytes were not stimulated to avoid attributing responses to pathways involved with cellular stimulation rather than toxicity. Cells were incubated with compounds on a nine-point logarithmic dosing scale ranging from 15 nM to 100 ?M (37°C, 5% CO2). At 4 hours posttreatment, cells were labeled with antibodies and physiological indicator dyes and fixed with 4% paraformaldehyde. Cellular phenotypes (eg, viability) were collected and analyzed using flow cytometry. Dose-response curves with response normalized to the zero dose as a function of log concentration were generated using GraphPad Prism 6.ResultsPhenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. The flow cytometry assays produced over 16,000 data points that were used to generate dose-response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. The calculated heritability for the viability of immune cells was higher with anthracyclines than the novel agents, making them better suited for downstream genetic analysis.ConclusionUsing this approach, we identify cell lines of variable sensitivity to chemotherapeutic agents and aim to identify robust, replicable endpoints of cellular response to drugs that provide the starting point for identifying candidate genes and cellular toxicity pathways for future validation in human studies.

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A cell-based phenotypic assay to identify cardioprotective agents.

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A cell-based phenotypic assay to identify cardioprotective agents.

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