Maintenance of sarcomeric integrity in adult muscle cells crucially depends on Z-disc anchored titin

Abstract

The giant protein titin is thought to be required for sarcomeric integrity in mature myocytes, but direct evidence for this hypothesis is limited. Here, we describe a mouse model in which Z-disc-anchored TTN is depleted in adult skeletal muscles. Inactivation of TTN causes sarcomere disassembly and Z-disc deformations, force impairment, myocyte de-stiffening, upregulation of TTN-binding mechanosensitive proteins and activation of protein quality-control pathways, concomitant with preferential loss of thick-filament proteins. Interestingly, expression of the myosin-bound Cronos-isoform of TTN, generated from an alternative promoter not affected by the targeting strategy, does not prevent deterioration of sarcomere formation and maintenance. Finally, we demonstrate that loss of Z-disc-anchored TTN recapitulates muscle remodeling in critical illness ‘myosinopathy’ patients, characterized by TTN-depletion and loss of thick filaments. We conclude that full-length TTN is required to integrate Z-disc and A-band proteins into the mature sarcomere, a function that is lost when TTN expression is pathologically lowered.

Graded titin cleavage progressively reduces tension and uncovers the source of A-band stability in contracting muscle

“…Compared to a single N2A titin band (~3.7 MDa) in control samples (including treated and untreated Wt), after TEV protease treatment, two smaller bands were clearly visible in Het and Hom TC muscles at the expected sizes of ~2.3 MDa for A-M-band titin and ~1.4 MDa for Z-I-band titin, indicating specific titin cleavage. The ~2.3 MDa titin cleavage product was slightly smaller than a doublet running at ~2.4 MDa, which contains proteolytic titin fragment T2 and the alternative titin isoform, Cronos (Swist et al, 2020) (Fig. 1E).…”

“…To determine the contribution of elastic titin to radial compressive forces in myocytes, isolated, relaxed psoas fibers were probed by nanoindentation using an atomic force microscope (Nanowizard 3 AFM system, JPK, Germany), as described recently (Swist et al, 2020). Permeabilized fibers were prepared as mentioned above, extensively washed in relaxing solution, and homogenized at a relatively low speed (T10, IKA).…”

“…During experiments, performed at room temperature, the pre-calibrated AFM cantilever (spring constant kC = 0.03-0.04 N m -1 , Novascan Technologies, USA) affixed with a mounted spherical polystyrene bead (diameter, 10 μm) periodically indented the fiber at 2 μm s -1 up to a pre-set indentation force of 3 nN. From these measurements, the indentation distance and elastic modulus (Young’s modulus; E) were calculated as explained previously (Li et al, 2016; Swist et al, 2020). Indentation and Young’s modulus data were pooled by genotype (Wt / Hom) and treatment type (with or without TEV protease incubation) for statistical analysis.…”

“…To study titin’s role in passive and active force production, the most straightforward experimental strategy is to compare the mechanical properties of muscle before and after the functional removal of titin. Classic techniques include genetically impairing titin synthesis (Radke et al, 2019; Swist et al, 2020), chemical degradation of titin via trypsin digestion (Higuchi, 1992), or protein destruction via target ionizing radiation (Horowits et al, 1986). However, almost all animal models with a mutational change in titin present a phenotype of muscle dystrophy and wasting (e.g.…”

“…However, almost all animal models with a mutational change in titin present a phenotype of muscle dystrophy and wasting (e.g. Radke et al, 2019; Swist et al, 2020), and all previous methods to degrade titin also degrade other proteins (Horowits, 1999). Therefore, it is difficult to identify data trends that are caused directly by the changes in titin or indirectly by the consequences of the ensuing “disease” state.…”