Pre-Activation of Cardiomyocytes Determines Contractile Force and Speed of Contraction; Role of Titin and Calcium

Abstract

Cardiomyocytes have, through splicing, an exquisite control over the length of titin, enabling it to regulate passive stiffness. As titin sets the preload of the cardiomyocyte, we hypothesized that together with diastolic Ca2+ it pre-activates the cardiomyocyte during diastole and that this is a major determinant of force production in the subsequent systolic phase. Through this mechanism titin would play an important role in active force development and length-dependent activation. Mutations in the splicing factor RNA binding motif protein (RBM20) results in the expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work-loops that mimic the cardiac cycle in wildtype (WT) and heterozygous (HET) RBM20 deficient rats, with long compliant titin. In addition we studied membrane-permeabilized cardiomyocytes with different amounts of diastolic calcium. At low pacing frequencies, myocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT), but this difference disappeared when diastolic calcium increased at high pacing frequencies. HET myocytes operated at higher SL to achieve the same level of work (2.10 µm vs. 1.94 µm at 6 Hz). To delineate the effects of diastolic Ca2+ and titin pre-activation on force generation, measurements were performed in detergent-permeabilized cardiomyocytes isolated from rat hearts. In these cells cardiac twitches were simulated by transiently (1 s) exposing the cell to a Ca2+-concentration of 2 µM. Increasing pre-activation by pre-stretching the myocyte increased the kinetics of force development and total force development. Increasing diastolic Ca2+ did increase force development, but only within a small concentration range. These findings are consistent with our hypothesis that pre-activation can increase force development. Highly compliant titin allows cells to function at higher diastolic Ca2+, but this is expected to result in limited contractile reserve.