Impaired organization and function of myofilaments in single muscle fibers from a mouse model of Pompe disease

Abstract

Pompe disease, a deficiency of lysosomal acid alpha-glucosidase, is a disorder of glycogen metabolism that can affect infants, children, or adults. In all forms of the disease, there is progressive muscle pathology leading to premature death. The pathology is characterized by accumulation of glycogen in lysosomes, autophagic buildup, and muscle atrophy. The purpose of the present investigation was to determine if myofibrillar dysfunction in Pompe disease contributes to muscle weakness beyond that attributed to atrophy. The study was performed on isolated myofibers dissected from severely affected fast glycolytic muscle in the alpha-glucosidase knockout mouse model. Psoas muscle fibers were first permeabilized, so that the contractile proteins could be directly relaxed or activated by control of the composition of the bathing solution. When normalized by cross-sectional area, single fibers from knockout mice produced 6.3 N/cm2 of maximum Ca2+-activated tension compared with 12.0 N/cm2 produced by wild-type fibers. The total protein concentration was slightly higher in the knockout mice, but concentrations of the contractile proteins myosin and actin remained unchanged. Structurally, X-ray diffraction showed that the actin and myosin filaments, normally arranged in hexagonal arrays, were disordered in the knockout muscle, and a lower fraction of myosin cross bridges was near the actin filaments in the relaxed muscle. The results are consistent with a disruption of actin and myosin interactions in the knockout muscles, demonstrating that impaired myofibrillar function contributes to weakness in the diseased muscle fibers.

Fig. 1.

Pathological features of myofibers derived from fast (psoas) muscle of mice with Pompe disease. Top: differential interference contrast microscopy images of unstained single muscle fibers from a 6-mo-old wild-type (WT) and an acid α-glucosidase (GAA) knockout (KO) mouse. Centrally located autophagic accumulation is clearly visible throughout the length of the fiber from the GAA-KO mouse. Scale bar, 20 μm. Bottom: immunostaining of single fibers for mono- and polyubiquitinated conjugates (FK2, red; BIOMOL International, Philadelphia, PA) and for lysosomal-associated membrane protein 1 (green; BD Pharmingen, San Diego, CA). Procedure for isolation, fixation, and immunostaining of single fibers is described elsewhere (21). Centrally located area devoid of muscle striation corresponds to the region of autophagic accumulation in the GAA-KO fiber. Ubiquitin-positive structures are clearly seen in the GAA-KO fiber, and they are confined to the autophagic area. Lysosomal-associated membrane protein 1 staining is present throughout the fiber, in the autophagic area and on the enlarged lysosomes in the GAA-KO fiber. Lysosomes appear as small dots in the WT fiber, and there is no accumulation of the ubiquitin-positive structures. Scale bar, 20 μm.

Fig. 2.

Records of isometric tension generated by a single permeabilized psoas muscle fiber from a WT (A) and a GAA-KO (B) mouse. In A, muscle fiber was initially in the relaxed state at time 0. Activating solution was delivered to the muscle fiber at 5 s for 0.5 s; a second round of activating solution was delivered after a 5-s interval. After ∼30 s of steady tension, relaxing solution was delivered for 0.5 s, and another round of relaxing solution was delivered after a 5-s interval. In B, 3 relaxing-activating cycles demonstrate stability of the fiber. Similar cycles were observed for the WT mouse. Note different time scales in A and B. Mice were 6 mo of age, fiber diameters were 66 μm (A) and 59 μm (B), sarcomere length was 2.4 μm, and temperature was 20°C.

Fig. 3.

Concentrations of total protein, myosin heavy chain, and actin in WT and GAA-KO muscle fibers. Each parameter was normalized to the mean corresponding value in WT fibers.

Fig. 4.

X-ray intensity profiles of equatorial reflections obtained from psoas muscle from WT and GAA-KO mice. Reflections from the GAA-KO mouse are significantly broader, indicating disorder of the filament lattice in the muscle cells. Intensity ratio (I₁₁/I₁₀) of the reflections is greatly reduced, suggesting decreased interaction between myosin and actin. Inset: cross-sectional view of the hexagonal lattice formed by myosin and actin filaments that give rise to the equatorial reflections.