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* Department of Neurology, Caritas St. Elizabeth’s Medical Center, Tufts University School of Medicine,
Department of Neurobiology and Behaviour, University of California, Irvine, California, USA;
Whitaker Cardiovascular Institute, Boston University School of Medicine, and
Department of Anaesthesia, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
1Correspondence: Department of Neurology, Caritas St. Elizabeth’s Medical Center of Boston, Tufts University School of Medicine, 736 Cambridge St., CBR419, Boston. MA 02135, USA. E-mail: henry.querfurth{at}tufts.edu
1. SPECIFIC AIMS
The intracellular deposition of the ßbeta;-amyloid (Aßbeta;) peptide in skeletal muscle fibers is a recognized pathological feature of human inclusion body myositis (IBM). An abnormal up-regulation of ßbeta;APP gene expression is one possible contributing factor. We found that overexpression of Aßbeta; is detrimental to the ionic homeostasis and viability of myotubes in culture. Since deposits of misfolded proteins are selectively toxic to subpopulations of cells in the brain, presumably based on metabolic vulnerabilities, we reasoned that ßbeta;APP gene expression confined to muscle fiber type can reproduce some aspects of the disease phenotype in an age-dependent manner. We generated a transgenic mouse in which wild-type (WT) ßbeta;APP751 production is restricted to fast-twitch fibers through control by a myosin light chain (MLC) 1/3 promoter/enhancer. Hemizygous transgenic mice (MLC-ßbeta;APP) were examined for deposits of ßbeta;APP, Aßbeta;42, and inclusions in type II skeletal muscle fibers, as well as electrophysiological abnormalities and calcium deregulation in a corresponding subpopulation of myofibers. Correlation to ultrastructural changes was made. The functional significance of these findings to clinical weakness with advancing age in IBM is discussed.
2. PRINCIPAL FINDINGS
2.1. IBM-like histopathology in MLC-ßbeta;APP mice
Transgene integration was analyzed in total DNA extracted from distal tail samples by polymerase chain reaction (PCR) and genomic Southern blot hybridizations. Most results are reported from a single hemizygous line. Steady-state levels of transgene-derived human ßbeta;APP were determined by Western blot on total protein extracted from skeletal muscle of transgenic mice compared with aged-matched control littermates. Serially sectioned tibialis anterior dissected from transgenic mouse no. 10 showed intracellular deposits of immunoreactive ßbeta;APP (Fig. 1
A). The same deposits were immunoreactive for antibodies vs. Aßbeta; (Fig 1C
). The proteinaceous aggregates were shown to correspond with large intramyofiber vacuoles or tubular aggregates easily identified on Gomori trichrome-developed sections (Fig. 1D
). We confirmed ßbeta;APP immunoreactivity in serially sectioned triceps from transgenic mouse no. 27, where ßbeta;APP deposits (Fig. 1E
) were exclusively localized to fast twitch fibers (Fig. 1F
). Subsarcolemmal inclusions were confirmed by Gomori trichrome staining in a cross section of tibialis anterior of transgenic animal 27 (Fig. 1G
). The paler cytosolic stain suggests relative mitochondrial paucity, such as characterized type II fibers that are expected to bear the transgene. We next probed for immunoreactive Aßbeta; deposits (Fig. 1H
) in serially sectioned hamstring from animal no. 15 and confirmed that Aßbeta; immunoreactivity was localized to fast-twitch muscle fibers. Thus, Aßbeta;-bearing myofibers were ATPase 4.6 negative (Fig. 1I
) and were fast myosin heavy chain positive (Fig. 1J
). We probed for Aßbeta;1–42 immunoreactivity using either of two end-specific antibodies, followed by thioflavin S staining, in cross sections of hamstring from transgenic animals 27 (Fig. 1K
) and 28 (Fig. 1M
). Several Aßbeta;1–42-positive fibers were also thioflavin S positive (Fig 1L, N
), suggesting the presence of beta-sheet structure. ßbeta;APP and Aßbeta; immunoreactivity were not widely distributed throughout the tissue but tended to affect clusters of fast-twitch muscle fibers within discrete fascicles. H and E staining of hamstring sections from transgenic mouse no. 27 revealed centralized nuclei (Fig. 1O
) compared with the normal peripheral location of nuclei in nontransgenic control littermates (Fig. 1P
).
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2.2. MLC-ßbeta;APP mice exhibit electromyographic abnormalities and muscle weakness
Transgenic mice did not display any obvious behavioral or movement disorders, and their body weight and life span were not diminished compared with nontransgenic littermates (data not shown). Transgenic mice with increased levels of ßbeta;APP in skeletal muscle were significantly weaker (84.9±1.3 g-force) as early as 6 months of age (Fig. 2
A) in an isometric forelimb strength test compared with nontransgenic control littermates (124.9±3.5 g-force). Transgene harboring animals remained weak and developed palpable muscle atrophy and waddling gait changes by 2.5 years of age (77.1±4.2 g-force) compared with nontransgenic control littermates (133.9±4.8 g-force). Hind limb isometric grip strength (Fig. 2B
) was also decreased by 6 months (77.4±6 g-force) and up to 2.5 years of age (98.5±4.5 g-force) in transgenic compared with nontransgenic control littermates (140.3±4.8 and 159.9±7.9 g-force, respectively). Muscle atrophy and markers of injury and inflammation were documented (see full text). EMG recordings (Fig. 2)
revealed a greater proportion of smaller MUAP in the transgene-affected mice. These were typically small in amplitude and duration (Fig. 2C
, middle) and in some instances, polyphasic (Fig. 2C
, bottom). Both the mean amplitude (309.4±23) and mean area (179.6±14) of MUAP (Fig. 2D, E
, respectively) were significantly larger in control mice than their transgenic littermates (220.6±18 and 135.8±11, respectively). The frequency distribution of MUAP amplitude in transgenic mice was shifted left (65% <200 µV) relative to control (Fig. 2D
). Similarly, the frequency distribution of MUAP area (Fig. 2E
) was shifted left (
75% <100 µV.ms) in transgenic compared with nontransgenic littermates.
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2.3. ßbeta;APP overexpression leads to Ca2+ dyshomeostasis and relative membrane depolarization
A decrease in membrane potential (Vm) in muscle fibers from transgenic mice (–71.8±0.7) was observed compared with nontransgenic (–83.9±0.4) littermates (Fig. 2F
). Moreover, this change was associated with a 2-fold increase in resting intracellular calcium [Ca2+]i (356.8±21.5) compared with nontransgenic (176.0±10.0) control littermates (Fig. 2F
). We next classified the transgenic fibers in Fig 2G
into subpopulations [Tg (1) and Tg (2)], based on a Vm cutoff of –80 mV, corresponding to the upper limit of the control values. This split is clearly shown to differentiate the Vm of the two groups. The same subpopulation of Tg (1) fibers from transgenic mice exhibited a mean resting [Ca2+]i (Fig. 2H
) not dissimilar to nontransgenic control littermates. Thus, a smaller population of fibers remain unaffected in MLC-ßbeta;APP mice, whereas the majority of fibers in subpopulation Tg (2), all having relatively depolarized Vm, displayed an increase in mean myoplasmic [Ca2+]i (Fig. 2H
). Comparison recordings were carried out on previously reported muscle creatine kinase (MCK)-ßbeta;APP mice, which express a non-IBM pathogenic Swedish double mutation of ßbeta;APP without specificity for muscle fiber type involvement. These mice exhibited a comparable increase in resting [Ca2+]i in all fibers tested (Supplemental Fig. 1 of full text). Thus, the distribution of resting calcium values into two fiber populations [Tg (1) and (2)] observed in our MLC-ßbeta;APP mice is consistent with the expected chimeric gene expression; type II transgene-bearing fibers having an abnormally raised [Ca2+]i.
Transgenic mice were examined for ultrastructural changes (see full text). The findings include mitochondrial cytopathy, tubular aggregates, vacuolar inclusions bearing amorphous and filamentous material, membrane whorls, and multivesicular body-like structures.
3. CONCLUSIONS AND SIGNIFICANCE
The MLC-ßbeta;APP transgenic mouse exhibited many of the clinical and myopathological features characteristic of the human IBM condition. Directed overexpression of human WT ßbeta;APP751 into type II skeletal muscle leads to Aßbeta; accumulation into vacuolar inclusions affecting only fast-twitch skeletal muscle. Intramyofiber accumulation of ßbeta;-amyloid is associated with membrane depolarization and an increase in the level of resting calcium in skeletal muscle (Fig. 2)
. This is the first demonstration that WT holoßbeta;APP expression in skeletal muscle tissue is not only sufficient to produce these disease markers in vivo, but also when restricted to fast twitch myofibers. The gradual accumulation of Aßbeta;42 over time into aggregates also resembles sIBM, a disorder affecting aged adults. Our results are consistent with culture-based models of ßbeta;APP and Aßbeta; mismetabolism in muscle. Furthermore, the observed increase in resting calcium and relative membrane depolarization in muscle fibers of MLC-ßbeta;APP transgenic mice provides a mechanism relating ßbeta;APP mismetabolism to abnormal excitation-contraction coupling and clinical weakness (Fig. 3
). Myocellular degeneration, muscle injury, and atrophy also contribute.
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WT ßbeta;APP expression was selectively targeted to postnatal fast-twitch skeletal muscle by choosing the developmentally sensitive MLC 1/3 promoter/enhancer. In previous models, the mutant ßbeta;APP and mutant C99 fragment transgene products were controlled by a general ßbeta;-actin/cytomegalovirus (CMV) promoter and were contrived to accelerate Aßbeta; genesis. Accordingly, they are removed to a degree from the central WT ßbeta;APP-IBM hypothesis. Several features of IBM, however, were not represented in this mouse model, including a lack of endomysial inflammatory infiltrates, small angulated myofibers and dense aggregates of phosphorylated neurofilaments. However, changes in tau expression and conformation were observed in our model (see full text). Further studies are planned to identify the molecular cause of calcium derangement and correct the phenotypic changes by manipulations of calcium handling, amyloid load, and survival signal pathways.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5763fje
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