A Cell-Based Phenotypic Assay to Identify Cardioprotective Agents

• Published in: Circulation research 2012-03, Vol.110 (7), p.948-957
• Main Authors: Guo, Stephanie, Olm-Shipman, Adam, Walters, Andrew, Urciuoli, William R, Devito, Stefanie, Nadtochiy, Sergiy M, Wojtovich, Andrew P, Brookes, Paul S
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Cardiology. Vascular system; Cardiotonic Agents - pharmacology; Cardiotonic Agents - therapeutic use; Cell Death; Cell Line; Cells, Cultured; Coronary heart disease; Disease Models, Animal; Heart; High-Throughput Screening Assays - instrumentation; High-Throughput Screening Assays - methods; Hydrogen-Ion Concentration; Medical sciences; Myocardial Reperfusion Injury - metabolism; Myocardial Reperfusion Injury - pathology; Myocardial Reperfusion Injury - prevention & control; Myocytes, Cardiac - drug effects; Myocytes, Cardiac - metabolism; Myocytes, Cardiac - pathology; Neurology; Phenotype; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Vascular diseases and vascular malformations of the nervous system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/CIRCRESAHA.111.263715

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 instrumentʼs 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, r=0.66) and infarct size (positive, r=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.