Depyrogenation of digestive enzymes reduces lipopolysaccharide tolerance in isolated cardiac myocytes

The isolated myocyte is useful for examining the direct cardiac effects of substances such as lipopolysaccharide (LPS) and cytokines. However, the digestive enzymes used for standard cell isolation procedures are contaminated by several hundred ng/ml LPS. We depyrogenated the digestive enzymes with a series of Triton X-114 and Polymyxin B washes to remove 93.7-99.9% of the LPS. This lowered LPS contamination levels from 100-300 ng/ml to 0.15-0.70 ng/ml, while maintaining good quality cell isolations from the left ventricle of New Zealand white rabbits. We evaluated whether brief exposure to LPS contaminant levels, as occur during standard cell isolations, induces LPS tolerance. Cardiac myocytes (isolated with depyrogenated enzymes) were pre-exposed to 100 ng/ml LPS for 1 h, washed, then exposed to a challenge dose with 100 ng/ml LPS. The LPS challenge dose induced a time-dependent decrease in cell shortening over 6 h in myocytes without pre-exposure, but not in myocytes pre-exposed to an earlier dose of LPS. We examined whether LPS tolerance develops in myocytes isolated with untreated enzymes, compared with depyrogenated enzymes. In myocytes isolated with untreated enzymes, there was a significant decrease in cell shortening after 6 h exposure to 1000-10 000 ng/ml LPS. In myocytes isolated with depyrogenated enzymes, it required only 5-50 ng/ml LPS to induce a comparable cardiac depression. We conclude that brief exposure to LPS contaminant. levels, which occur with standard cell isolation procedures, induces a hyporesponsiveness or tolerance to subsequent doses of LPS in isolated cardiac myocytes.