High-throughput assessment of calcium sensitivity in skinned cardiac myocytes

Isolated permeabilized cardiac myocytes have been used in the study of myof ilament calcium sensitivity through measurement of the isometric force-pCa curve. Determining this force-pCa relationship in skinned myocytes is relat ively expensive and carries a high degree of variability. We therefore atte mpted to establish an alternative high-throughput method to measure calcium sensitivity in cardiac myocytes. With the use of commercially available so ftware that allows for precise measurement of sarcomere spacing, we measure d sarcomere length changes in unloaded skinned cardiac myocytes over a rang e of calcium concentrations. With the use of this technique, we were able t o accurately detect acute increases or decreases in myofilament calcium sen sitivity after exposure to 10 mM caffeine or 5 mM 2,3-butanedione monoxime, respectively. This technique allows for the simple and rapid determination of myofilament calcium sensitivity in cardiac myocytes in a reproducible a nd inexpensive manner and could be used for high-throughput screening of ph armacological agents and/or transgenic mouse models for changes in myofilam ent calcium sensitivity.