Abstract
Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20ΔRRM) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20ΔRRM animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20ΔRRM on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca2+-activated, skinned papillary muscle strips from Rbm20ΔRRM and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca2+-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20ΔRRM vs. WT mice. As sarcomere length increased from 1.9 to 2.2 μm, length-dependent activation of contraction was eliminated in the Rbm20ΔRRM myocardium, even though myosin MgADP release rate decreased ~20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.
Highlights
Titin is the largest protein that has been identified, spanning from the Z-disk to the M-band of the cardiac sarcomere (LeWinter et al, 2007)
Ca2+-sensitivity of the tensionpCa relationship increased with sarcomere length in the wild-type mice (WT) strips, but this sarcomere length-dependent increase in Ca2+-sensitivity of tension was lost in Rbm20∆RNA Recognition Motif (RRM) strips (Table 1)
At 2.2 μm sarcomere length, nH was smaller for WT strips than Rbm20∆RRM strips; there were no differences in nH between genotypes at 1.9 μm sarcomere length
Summary
Titin is the largest protein that has been identified, spanning from the Z-disk to the M-band of the cardiac sarcomere (LeWinter et al, 2007). RNA Binding Motif Protein 20 (RBM20) suppresses differential titin splicing such that wild-type mice (WT) predominantly express the stiffer N2B titin isoform and homozygous Rbm20∆RRM mice express a very large, more compliant N2BA titin isoform (Guo et al, 2013; Li et al, 2013; Methawasin et al, 2014). We have recently shown that cross-bridge cycling kinetics slowed at longer sarcomere length due to slowing of MgATP binding and MgADP release (Tanner et al, 2015). This led to the hypothesis that increased sarcomeric compliance in Rbm20∆RRM hearts could affect cross-bridge cycling kinetics differently at short vs long sarcomere lengths, which may provide an explanation for compromised myocardial function in Rbm20∆RRM vs WT myocardium
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