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. P o 2 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 2 =0.66) and infarct size (positive, r 2 =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.