A Direct Method to Measure the Restoring Force and Slack Sarcomere Length of Intact Cardiomyocytes

Abstract

Molecular mechanisms underlying diastolic suction are poorly understood. Several proteins have been implicated to play a role, including extra-cellular proteins, titin, and cytoskeletal proteins. An in vitro measurement of diastolic suction at the cell level is restoring force (or stiffness), a force which is difficult to measure as it requires a cell to be passive below the slack sarcomere length (SL). Previous restoring force studies were made utilizing calcium independent shortening of cardiomyocytes to below their slack length and then upon relaxation measuring the force that developed as the cell is stretched back to its slack length. However, these studies used chemically permeablized cells and, thus, eliminated the membrane and soluble intracellular proteins as possible contributors to restoring force. In the current study we developed a novel method to determine restoring force and the slack sarcomere length of mouse cardiomyocytes that were intact. Intact cardiac myocytes that were below their slack length due to a low level of active force development were attached to flexible carbon fibers. We then added butanedione monoxime (BDM) to inhibit actomyosin interactions and abolish active tension. This led to an increase in the sarcomere length and a negative force. From this new baseline force (-1.34±0.34 mN/mm2 (mean±SE)), we stretched the cells across the physiological range from ∼1.8-2.2 μm. We determined the sarcomere length at which force is zero (slack SL) to be 1.93±0.019 μm. Plotting the stress-SL relationship we then determined the restoring stiffness from the slope of this plot as 16.1 mN/mm2/μm. Thus we successfully measured the restoring force - SL relation of intact cardiac myocytes.