Increased Titin Compliance Reduced Length-Dependent Contraction and Slowed Cross-Bridge Kinetics in Skinned Myocardial Strips from Rbm20ΔRRM Mice

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.

Effects of activation on the elastic properties of intact soleus muscles with a deletion in titin

“…Horowits and Podolsky (1987) and Horowits and colleagues (1989) also showed that without force transmission by titin, force generation by the cross-bridges is highly inefficient because energy from crossbridge interactions is lost to thick filament displacement at the expense of longitudinal tension. Numerous other studies also show that changes in titin isoforms and consequent changes in titin-based stiffness are associated with changes in force production (Ottenheijm et al, 2012;Pulcastro et al, 2016). We suggest that the force deficit in mdm muscles is due to impaired transmission of force from the cross-bridges to the Z-lines.…”

Titin‐based mechanosensing modulates muscle hypertrophy

“…However, these studies were performed in intact, contracting muscle. Consequently, the results might be confounded by the presence of calcium cycling: calcium binds to the PEVK spring‐domain of titin, changing its stiffness,15, 16, 17 and titin‐based stiffness modulates contractility20, 41, 42, 43, 44 which in turn might affect muscle growth. The importance of the current findings is that they show that the effects of titin‐based mechanosensing occur independent of contractile activity, and can be ascribed to titin's elastic properties.…”

New Insights in RBM20 Cardiomyopathy

“…Interestingly, a slowdown of cross-bridge detachment normally leads to an increased Ca 2+ sensitivity, while the opposite is the case when RBM20 is lost [65,67]. This may be explained by the altered titin, as titin plays a large part in modulation of the sarcomeric complex and interacts both with the thin and thick filament [65,114]. Interestingly, heightened levels of collagen are observed in the RBM20-deficient heart, which may be a compensatory effect working against the reduced titin stiffness [63].…”

“…Functionally, compliance of the heart increases, and passive tension of the sarcomere is reduced [23,63]. Additionally, the Frank-Starling mechanism is impaired due to a diminished length-dependent activation (a reduction of the force generated by an increase in muscle length), which was reduced in both heterozygous and homozygous Rbm20-deficient mice [63,65]. This results in a reduced fractional shortening (FS), and an even further reduced FS when the afterload is increased (for example through application of noradrenergic stress) [63].…”

“…This results in a reduced fractional shortening (FS), and an even further reduced FS when the afterload is increased (for example through application of noradrenergic stress) [63]. Furthermore, the length-dependent Ca 2+ sensitivity of the sarcomere is also affected by loss of RBM20 [23,65]. A study in human RBM20-deficient cardiomyocytes found an increase in Ca 2+ sensitivity, which could be counteracted by application of protein kinase A (PKA), while control cardiomyocytes showed no change after PKA application [23].…”