Muscle cell and motor protein function in patients with a IIa myosin missense mutation (Glu-706 to Lys)

Abstract

The pathogenic events leading to the progressive muscle weakness in patients with a E706K mutation in the head of the myosin heavy chain (MyHC) IIa were analyzed at the muscle cell and motor protein levels. Contractile properties were measured in single muscle fiber segments using the skinned fiber preparation and a single muscle fiber in vitro motility assay. A dramatic impairment in the function of the IIa MyHC isoform was observed at the motor protein level. At the single muscle fiber level, on the other hand, a general decrease was observed in the number of preparations where the specific criteria for acceptance were fulfilled irrespective of MyHC isoform expression. Our results provide evidence that the pathogenesis of the MyHC IIa E706K myopathy involves defective function of the mutated myosin as well as alterations in the structural integrity of all muscle cells irrespective of MyHC isoform expression.

Figure 1

Serial cross sections of muscle biopsy from patient 1 showing increased variability of fiber size and slight increase of interstitial connective tissue. Major structural changes are confined to type 2A muscle fibers (two of them are indicated as 2A) but structural alterations with internalized myonuclei are also present in some type 1 fibers (arrows). a) Haematoxylin-eosin b) ATPase pH 4.6 c) NADH-tetrazolium reductase

Figure 2

Electrophotretic separation of MyHC isoforms from 10 μm cross-sections from a healthy control subject (C) and the four patients with the IIa MyHC mutation (1-4). The two fast (type IIa and IIX) and the β/slow (type I) MyHC isoforms are indicated on the silver stained 6% SDS-PAGE. Only the section corresponding to the MyHC region of the gel is included.

Figure 3

Specific tension in muscle cells expressing different MyHC isoforms from control subjects (open symbols) and from patients carrying the type IIa myosin missense mutation (filled symbols). All fibers from the control subjects fulfilled the criteria for acceptance (see Materials & Methods), but only 4 of the 96 fibers from the patients were acceptable and all these fibers expressed the type I MyHC.

Figure 4

Actin filament motility speed propelled by different myosin heavy chain isoforms extracted from single muscle fiber segments from control subjects (open symbols) and from patients carrying the type IIa myosin missense mutation (filled symbols).