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

Frank Li,Henk Granzier,Paola Tonino,Justin Kolb,John E Smith,Julie Crudele,Jeffrey S Chamberlain,Zaynab Hourani

doi:10.1186/s13395-019-0219-9

Frank Li, Henk Granzier + Show 6 more

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https://doi.org/10.1186/s13395-019-0219-9

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Abstract

BackgroundNebulin 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.MethodsWe 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.ResultsIntramuscular 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.ConclusionsThis study reveals that nebulin fragments are well-received by nebulin-deficient mouse muscles and that limited functional benefits are achievable.

Highlights

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
Expression of a nebulin Z-disk fragment in control (CTRL) and nebulin-deficient (Neb Conditional knockout (cKO)) mice To test the effect of expressing the Z-disk region of nebulin on the structure and function of the skeletal muscle sarcomere, an adeno-associated viral (AAV) vector expressing the Z-disk region plus nebulin’s final superrepeat was created (Fig. 1a)
This AAV construct, estimated to be 139.9 kDa, was injected into the anterior compartment of the lower hindlimb of the mouse as previously described [31]. This allows for the AAV to enter muscle fibers and the construct to be expressed in both the tibialis cranialis (TC) and the extensor digitorum longus (EDL) muscles [59]

Summary

Introduction

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. Nebulin is one of the largest proteins in the human body [25] It is a massive linear protein of ~ 700 kDa that extends from the Z-disks of the skeletal muscle sarcomeres out toward the pointed ends of the thin filaments [26]. Its core structure is comprised of 206 homologous, repeat modules which each contain an SDxxYK actin-binding sequence [27,28,29] These modules allow nebulin to associate closely with the actin thin filaments and contribute to its primary role as a thin filament length regulator. While the glutamic acid-rich region remains unstudied, the serine-rich region and the SH3 domains are thought to contribute to the regulation of other sarcomeric proteins as well as the development of the Z-disk [31]

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