Measuring Work Loops in Intact Isolated Cardiac Myocytes by Controlling Pre- and Afterload using a New Generation Force Transducer

Abstract

A recent exciting development in cardiac research is the ability to do mechanical experiments on intact isolated cardiac myocytes. Here we mimic cardiac PV loops by imposing a ‘pre-load’ and ‘after-load’ on the myocyte. For these experiments we developed a new generation force transducer as currently available force transducers were not sufficiently sensitive or had insufficiently stable base line levels to achieve force control. A cantilever with a spring constant of 7 Nm is interrogated using an interferometer via an optical fiber. The resolution of the interferometer/probe system is 2nm, resonance frequency > 2 kHz and force resolution < 10 nN. As the probe is small enough to be fully submerged in water, variations in solution level have no effect on the force measurement, resulting in a stable baseline (drift < 50 nN over a 10 minute period at 21C). Software was written to take in the signal from the force transducer, process it and return a signal to a linear motor that could stretch or shorten the myocyte in order to control force levels. Using this software we were able to achieve a two-sided force clamp (setting ‘pre-load’ and ‘after-load’ ) to measure work loops and re-create the Frank-Starling relation at the single cell level. Experiments show that in isolated cardiac mouse myocytes residual active force at the end of diastole limits the effective work the mycoyte can produce. Small concentrations of BDM, thought to inhibit strong crossbridge formation by stabilizing weak cross bridges, allow the myocyte to relax at end-diastole, shifting the pre-load-sarcomere length relation upwards. The resulting increase in length dependent activation outweighs the effects of crossbridge inhibition, leading to strongly increased mechanical work per contractile cycle.