In vivo characterization of skeletal muscle function in nebulin-deficient mice

Abstract

Introduction: The conditional nebulin knockout mouse is a new model mimicking nemaline myopathy, a rare disease characterized by muscle weakness and rods within muscle fibers. We investigated the impact of nebulin (NEB) deficiency on muscle function in vivo.

Methods: Conditional nebulin knockout mice and control littermates were studied at 10 to 12 months. Muscle function (force and fatigue) and anatomy (muscles volume and fat content) were measured in vivo. Myosin heavy chain (MHC) composition and nebulin (NEB) protein expression were assessed by protein electrophoresis.

Results: Conditional nebulin knockout mice displayed a lower NEB level (-90%) leading to a 40% and 45% reduction in specific maximal force production and muscles volume, respectively. Nebulin deficiency was also associated with higher resistance to fatigue and increased MHC I content.

Discussion: Adult nebulin-deficient mice displayed severe muscle atrophy and weakness in vivo related to a low NEB content but an improved fatigue resistance due to a slower contractile phenotype.

Keywords: in vivo; fatigue; muscle wasting; muscle weakness; nemaline myopathy; neuromuscular disorder.

FIGURE 1

Nebulin protein expression in gastrocnemius and soleus muscles from 12-month-old WT (n = 6) and Neb-cKO (n = 4–6) mice. A, Example of agarose gels showing nebulin and MHC (left) and nebulin expression in gastrocnemius muscle (right). B, Example of agarose gels showing nebulin and MHC (left) and nebulin expression in soleus muscle (right). Values are mean ± SEM. ***P <0 .001, significantly different from WT mice. MHC, myosin heavy chain; Neb-cKO, conditional nebulin knockout; WT, wildtype

FIGURE 2

Hindlimb muscles volume and mass from 12-month-old WT (n = 17) and Neb-cKO mice (n = 17). A, Typical representative axial MR images of hindlimb muscles. B, Hindlimb muscles volume. C, Muscle mass. Values are mean ± SEM. ***P < 0.001, significantly different from WT mice. EDL, extensor digitorum longus; Neb-cKO, conditional nebulin knockout; WT, wildtype

FIGURE 3

MHC composition of gastrocnemius and soleus muscles from 12-month-old WT (n = 6) and Neb-cKO mice (n = 6). A, Example of typical electrophoretic pattern of whole gastrocnemius muscle lysate (left) and MHC type composition (right). B, Example of typical electrophoretic pattern of whole soleus muscle lysate (left) and MHC type composition (right). Data are presented as percentage of total MHC. Values are mean ± SEM. ***P <0 .001, significantly different from WT mice. cKO, conditional knockout; MHC, myosin heavy chain; Neb-cKO, conditional nebulin knockout; Sol, soleus; TA, tibialis anterior; WT, wildtype

FIGURE 4

Submaximal and maximal forces from 12-month-old WT (n = 18) and Neb-cKO mice (n = 17). A, Representative force traces of the plantar flexor muscles recorded during a 150-Hz tetanic stimulation. B, Plantar flexor muscles specific maximal force. C,D Plantar flexor muscles force recorded during a 20-HZ stimulation (C) and a 40-Hz stimulation (D). Results in C and D are expressed relative to the maximal tetanic force (measured at 150 Hz). Values are mean ± SEM. ***P < 0.001, significantly different from WT mice. Neb-cKO, conditional nebulin knockout; WT, wildtype

FIGURE 5

Force measurements during the fatigue protocol consisting of 80 contractions performed at 40 Hz (1.5 seconds on and 6 seconds off) in 12-month-old WT (n = 18) and Neb-cKO mice (n = 17). A, Representative force traces of the plantar flexor muscles recorded during the fatigue protocol in WT mice (top) and Neb-cKO mice (bottom). B, Plantar flexor muscles specific force production during the fatigue protocol. C, Mean specific force of the plantar flexor muscles during the whole fatigue protocol. D, Fatigue index calculated from the ratio between the last five and the first five contractions of the fatigue protocol. Values are mean ± SEM. ***P <0 .001, significantly different from WT mice. Neb-cKO, conditional nebulin knockout; WT, wildtype