Perturbations in intracellular Ca²⁺ handling in skeletal muscle in the G93A*SOD1 mouse model of amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by skeletal muscle atrophy and weakness, ultimately leading to respiratory failure. The purpose of this study was to assess changes in skeletal muscle excitation-contraction (E-C) coupling and intracellular Ca(2+) handling during disease progression in the G93A*SOD1 ALS transgenic (ALS Tg) mouse model. To assess E-C coupling, single muscle fibers were electrically stimulated (10-150 Hz), and intracellular free Ca(2+) concentration was assessed using fura-2. There were no differences in peak fura-2 ratio at any stimulation frequency at 70 days (early presymptomatic). However, at 90 days (late presymptomatic) and 120-140 days (symptomatic), fura-2 ratio was increased at 10 Hz in ALS Tg compared with wild-type (WT) fibers (0.670 ± 0.02 vs. 0.585 ± 0.02 for 120-140 days; P < 0.05). There was also a significant increase in resting fura-2 ratio at 90 days (0.351 ± 0.008 vs. 0.390 ± 0.009 in WT vs. ALS Tg; P < 0.05) and 120-140 days (0.374 ± 0.001 vs. 0.415 ± 0.003 in WT vs. ALS Tg; P < 0.05). These increases in intracellular Ca(2+) in ALS Tg muscle were associated with reductions in the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase proteins SERCA1 (to 54% and 19% of WT) and SERCA2 (to 56% and 11% of WT) and parvalbumin (to 80 and 62% of WT) in gastrocnemius muscle at 90 and 120-140 days, respectively. There was no change in dihydropyridine receptor/l-type Ca(2+) channel at any age. Overall, these data demonstrate minimal changes in electrically evoked Ca(2+) transients but elevations in intracellular Ca(2+) attributable to decreased Ca(2+)-clearance proteins. These data suggest that elevations in cellular Ca(2+) could contribute to muscle weakness during disease progression in ALS mice.

Intact single muscle fibres from SOD1G93A amyotrophic lateral sclerosis mice display preserved specific force, fatigue resistance and training‐like adaptations

“…), and increased resting [Ca 2+ ] i due to reduced SERCA protein expression (Chin et al . ). However, our data from mechanically dissected muscle fibres of SOD1 G93A mice showed no signs of impaired membrane excitability both in the stimulation voltage required to elicit a tetanus and in the downstream process of SR Ca 2+ release, as shown by the similar tetanic [Ca 2+ ] i in fibre from SOD1 G93A vs .…”

“…) and elevated resting [Ca 2+ ] i in enzymatically dissociated single muscle fibres from the predominantly fast‐twitch hindlimb flexor digitorum brevis (FDB) muscle of SOD1 G93A mice (Chin et al . ). Although reduced force generation in muscles of mice expressing the SOD1 G93A mutation has been observed (Dobrowolny et al .…”

“…In SOD1 G93A mice, reduced whole muscle force has been attributed to selective degeneration and loss of fast-fatigable type II muscle fibres (Hegedus et al 2008). However, other evidence points to intrinsic defects within muscle fibres that could contribute to impaired contractile function (Dobrowolny et al 2008;Hegedus et al 2008;Wong & Martin, 2010;Chin et al 2014;Beqollari et al 2016). The global SOD1 G93A hereditary ALS mutation is present in all tissues including skeletal muscle.…”

Calcium dysregulation, functional calpainopathy, and endoplasmic reticulum stress in sporadic inclusion body myositis

“…Samples were prepared as previously described [10]. Briefly, rectus femoris muscle biopsy samples were transferred from liquid nitrogen directly to a tube containing chilled RIPA buffer (0.15 M NaCl, 0.01 M Tris–HCl pH 8, 0.005 M EDTA, 0.5% Sodium Deoxycholate, 0.1% SDS, 1% Triton-X100) and 1 cOmplete™ protease inhibitor cocktail (Sigma-Aldrich) on ice.…”

“…Antigen-antibody complexes were visualized after incubation with Western Clarity ECL (Bio-Rad) and analyzed using either the Bio-Rad Image Lab™ software or ImageStudioLite™. Equal protein loading was confirmed with total protein staining, as previously discussed [10], as well as with probes for β-actin or vinculin.…”