Pre-Activation of Cardiomyocytes Determines Speed of Contraction: Role of Titin

Abstract

The giant myofilament protein titin has an extendable region that functions as a molecular spring. Cardiomyocytes have exquisite control over the length of titin, through splicing, enabling it to regulate passive stiffness. We hypothesized that titin as it sets the preload on the cardiomyocyte when stretched, together with diastolic Ca2+ pre-activates the cardiomyocyte during diastole and that this pre-activation is a major determinant for force production in the subsequent systolic phase. Via this route titin is thought to play an important role in active force development. Mutations in the splicing factor RNA binding motif protein (RBM20) results in the expression of large, highly compliant titin isoforms. In the present study we aimed to investigate the effect of long, highly compliant titin on the contractile properties of single cardiomyocytes. We measured single cardiomyocyte work-loops that mimic the cardiac cycle, in wildtype (WT) and heterozygous (HET) RBM20 deficient rats. In addition we studied detergent-permeabilized human patient samples that had known variations in titin based passive stiffness. 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 (>6 Hz). HET myocytes operated at higher SL to achieve the same level of work (2.1µm vs. 1.94µm at 6 Hz). In detergent-permeabilized cardiomyocytes isolated from human and rat heart we simulated cardiac twitches by transiently (0.5 s) exposing the cell to a physiological calcium concentration of pCa 5.7. Increasing pre-activation by bathing the cells in pCa 6.7 or pre-stretching the myocyte increased the kinetics of force development and thus the total force development within a transient activation. This is consistent with our hypothesis that pre-activation can increase force development in a time limited contraction such as a cardiac twitch. Pre-activation was pre-load dependent as the sarcomere length to which the myocytes had to be stretched for equivalent levels of pre-activation varied with the compliance of titin.