Thin filament length in the cardiac sarcomere varies with sarcomere length but is independent of titin and nebulin

Abstract

Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05 μm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titin’s spring region is a factor controlling TFL by studying the Rbm20ΔRRM mouse which expresses titins that are ~500 kDa (heterozygous mice) and ~1000 kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20ΔRRM mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1 μm per 0.35 μm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0 μm where TFL was ~1.05 μm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin.

The complexity of titin splicing pattern in human adult skeletal muscles

“…For example, it is well known that the alternatively spliced is7 region, encoded by exon 363, binds the calcium-dependent protease calpain 3 (CAPN3) [ 31 ]. On the other hand, the role of the titin, and also nebulin, filament length (as a result of splicing events) on the sarcomere length and its passive elastic properties is still under debate [ 32 – 34 ].…”

Overview of the Muscle Cytoskeleton

“…Nebulin is highly abundant in skeletal muscle, with very low amounts detected in the heart (42,307). Cardiac-specific nebulin knockout mice do not have altered thin filament length indicating that nebulin has a distinct, yet undefined, role in the heart (326). …”

Nebulin: big protein with big responsibilities

“…In cardiac muscle, nebulin expression is very low and it is controversial whether nebulin contributes to thin filament length regulation in the heart. Nebulin deficiency in vivo did not result in abnormal thin filament length (Kolb et al 2016).…”

“…Thin filaments are composed of actin polymers which are embedded in the Z-disk with the barbed end while the pointed end is located in the centre Littlefield and Fowler 1998). Additionally, thin filament length is able to change in response to sarcomere lengths to allow for an optimal thin/thick filament overlap during muscle contraction (Kolb et al 2016).…”