Expressing a Z-disk nebulin fragment in nebulin-deficient mouse muscle: effects on muscle structure and function

Abstract

Background: Nebulin is a critical thin filament-binding protein that spans from the Z-disk of the skeletal muscle sarcomere to near the pointed end of the thin filament. Its massive size and actin-binding property allows it to provide the thin filaments with structural and regulatory support. When this protein is lost, nemaline myopathy occurs. Nemaline myopathy causes severe muscle weakness as well as structural defects on a sarcomeric level. There is no known cure for this disease. Methods: We studied whether sarcomeric structure and function can be improved by introducing nebulin's Z-disk region into a nebulin-deficient mouse model (Neb cKO) through adeno-associated viral (AAV) vector therapy. Following this treatment, the structural and functional characteristics of both vehicle-treated and AAV-treated Neb cKO and control muscles were studied.Results: Intramuscular injection of this AAV construct resulted in a successful expression of the Z-disk fragment within the target muscles. This expression was significantly higher in Neb cKO mice than control mice. Analysis of protein expression revealed that the nebulin fragment was localized exclusively to the Z-disks and that Neb cKO expressed the nebulin fragment at levels comparable to the level of full-length nebulin in control mice. Additionally, the Z-disk fragment displaced full-length nebulin in control mice, resulting in nemaline rod body formation and a worsening of muscle function. Neb cKO mice experienced a slight functional benefit from the AAV treatment, with a small increase in force and fatigue resistance. Disease progression was also slowed as indicated by improved muscle structure and myosin isoform expression. Conclusions: This study reveals that nebulin fragments are well-received by nebulin-deficient mouse muscles and that limited functional benefits are achievable.

Nebulin and Lmod2 are critical for specifying thin-filament length in skeletal muscle

“…S3, B and C). It is known that the Z-disk thickness varies in different muscle types (17), in the mouse the Z-disk thickness is ~50 nm in EDL and ~100 nm in soleus muscle (18,19). Thus, nebulin exon inclusion in the Z-disk positively correlates with Z-disk thickness.…”

“…In addition, measurements assume that nebulin's C terminus is at the middle of the Z-disk, while in reality, nebulin is likely to penetrate the Z-disk (30,31). This will underestimate the measured length of nebulin by half of the Z-disk thickness, i.e., 50 nm in soleus and 25 nm in EDL (18,19). Hence, the corrected measured nebulin length is 1128 nm for soleus (1070 + 8 + 50) and 1078 nm for EDL (1035 + 8 + 25).…”

Role of Actin-Binding Proteins in Skeletal Myogenesis

“…Inhibit actomyosin ATPase, regulate and stabilize actin filament motility [61] Drebrin (Dbn) Binds to the F-actin side and promotes F-actin formation [62] Microtubule associated monooxygenase (MICAL) Induces redox reactions on F-actin, makes certain disaggregation, and prevents polymerization [63,64] Nexilin (Nelin, NEXN) F-actin cross-linking activity through binding along the sides of F-actin [65] XIN-repeating protein (XIN) Prevents depolymerization by binding to F-actin [66] ABPs related to muscle contraction α-actinin, myosin IIs (NM IIs), Nebulin, Tropomyosins (Tpms) Stabilize and regulate F-actin [67][68][69][70][71][72]…”

“…3.8. Muscle Contractile-Related ABPs in Myogenic Differentiation 3.8.1. α-Actinin α-Actinin, along with myosin IIs (NM IIs), nebulin, and tropomyosin, is a specific protein expressed in mature skeletal muscle tissue and is instrumental in muscle contraction when coupled with myosin motors [67][68][69][70]237]. These proteins, pivotal in the architecture and function of mature skeletal muscles, are also implicated in Nemaline myopathies (NM) and are under investigation for potential clinical therapies [237].…”

Mature Myotubes Generated From Human-Induced Pluripotent Stem Cells Without Forced Gene Expression

“…Many congenital and late-onset myopathies are characterized by the malformation of intracellular structures, such as the triad structure, which is composed of t-tubules and the sarcoplasmic reticulum (SR) ( Buj-Bello et al, 2008 ; Al-Qusairi et al, 2009 ; Cowling et al, 2014 ), sarcomeric structures ( Li et al, 2020 ), and nuclei morphology ( Davidson and Lammerding, 2014 ). To investigate the pathophysiology of these disorders in vitro , it is necessary to generate mature myotubes with intramuscular structures that form in the late-stage development.…”