Hyperactive intracellular calcium signaling associated with localized mitochondrial defects in skeletal muscle of an animal model of amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by degeneration of motor neurons and atrophy of skeletal muscle. Mutations in the superoxide dismutase (SOD1) gene are linked to 20% cases of inherited ALS. Mitochondrial dysfunction has been implicated in the pathogenic process, but how it contributes to muscle degeneration of ALS is not known. Here we identify a specific deficit in the cellular physiology of skeletal muscle derived from an ALS mouse model (G93A) with transgenic overexpression of the human SOD1(G93A) mutant. The G93A skeletal muscle fibers display localized loss of mitochondrial inner membrane potential in fiber segments near the neuromuscular junction. These defects occur in young G93A mice prior to disease onset. Fiber segments with depolarized mitochondria show greater osmotic stress-induced Ca(2+) release activity, which can include propagating Ca(2+) waves. These Ca(2+) waves are confined to regions of depolarized mitochondria and stop propagating shortly upon entering the regions of normal, polarized mitochondria. Uncoupling of mitochondrial membrane potential with FCCP or inhibition of mitochondrial Ca(2+) uptake by Ru360 lead to cell-wide propagation of such Ca(2+) release events. Our data reveal that mitochondria regulate Ca(2+) signaling in skeletal muscle, and loss of this capacity may contribute to the progression of muscle atrophy in ALS.

Figures

FIGURE 1.

Structural and functional defects of mitochondria in G93A muscle. A, electron microscopy images of FDB muscles from 3-month-old G93A and WT mice. In the WT, mitochondria (arrows) aligned within the sarcomeric I band and had uniform sizes (panel 2). In contrast, G93A muscle (panel 1) had enlarged mitochondria with vacuoles (panels 3–5), invading the sarcomeric A band. B, live muscle fibers probed simultaneously with MitoTracker Deep Red (panels 1 and 3) and TMRE (panels 2 and 4). A segment with depolarized mitochondria is identified in the G93A fiber (panel 2, bracket). The corresponding segment in the MitoTracker Deep Red image shows fuzzy staining with loss of contrast, due to morphological alterations of mitochondria (panel 1). Both mitochondrial probes stain the WT fiber equally and homogeneously (panels 3 and 4). C, overlays of TMRE (green) and α-BTX images (red) of dually stained G93A FDB fibers. Areas of mitochondrial defects had variable size, but they always faced the NMJ.

FIGURE 2.

Hyperactive Ca2+ release in G93A muscle fibers. A, the response of a WT fiber to osmotic shock. TMRE stains the entire fiber homogeneously (panel 1). Osmotic stress-induced Ca2+ release events (after panel 2) are mainly confined to the peripheral region of the fiber. B, the responses of G93A fibers. Lack of TMRE staining identifies fiber segments with depolarized mitochondria (panel 1). The shock-induced Ca2+ release activity was greater and not restricted to the periphery of the fiber in the segments with depolarized mitochondria (panels 3 and 4). C, evolution of fluo-4 fluorescence in regions with normal or defective mitochondria in G93A muscle fibers. Mean (±S.E. (error bars)) over experiments of area-averaged fluorescence, normalized to values before the osmotic shock, is shown. The difference between normal and lesioned areas was highly significant (n = 6, p < 0.0001).

FIGURE 3.

Stress-induced Ca2+ waves stop in the region with normal mitochondria. Images of ΔΨ (TMRE) and cytosolic [Ca2+]i (fluo-4) simultaneously obtained in G93A fibers are shown. A, transversely scanned xy images (with the rapidly changing abscissa x perpendicular to the fiber axis), showing Ca2+ waves for the 20 min following the osmotic shock. Ca2+ waves occur only in the region with depolarized mitochondria. B, longitudinally scanned images, with abscissa x along the fiber axis. Ca2+ waves invade the normal region for not more than ∼30 μm.

FIGURE 4.

Blocking mitochondrial Ca2+ uptake by FCCP and Ru360. A, Ca2+ release events triggered by osmotic shock (panels 2 and 3). The application of FCCP (1 μm, added 5 min after the shock) caused an increase in Ca2+ events (panels 4–7). At 280 s, Ca2+ release engulfed the entire fiber (panel 8). TMRE staining in the normal areas also decreased (panel 9). B, increased Ca2+ release activity in a G93A fiber after the application of Ru360 (panels 4–9). The mitochondrial potential was not changed by Ru360 after 45 min (panel 10).

FIGURE 5.

Proposed pathogenic sequence in muscle of the ALS mouse model. A, in the early presymptomatic stages, the situation is similar to the WT. mito, mitochondria. B, accumulation of mutant SOD1 inside mitochondria results in functional deficits, including a reduced ΔΨ and Ca2+ removal by mitochondria. The alterations lead to progressive withdrawal of the nerve terminal. These changes can be self-reinforcing consequences, including loss of mutual trophic signals between muscle and nerve cells, uncontrolled release of Ca2+ from the SR, local cytosolic and mitochondrial increase in [Ca2+] (in both cells), and more Ca2+ stress on the normal mitochondria. The process may be characterized as a dual interplay between abnormal mitochondria and uncontrolled SR and between muscle fibers and altered nerve terminals, which eventually leads to destruction of both cells. nAChR, nicotinic acetylcholine receptor.