Progressive impairment of CaV1.1 function in the skeletal muscle of mice expressing a mutant type 1 Cu/Zn superoxide dismutase (G93A) linked to amyotrophic lateral sclerosis

Abstract

Background: Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3-5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca(2+) channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation.

Methods: We recorded L-type currents, charge movements, and myoplasmic Ca(2+) transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1(G93A)).

Results: In FDB fibers obtained from "symptomatic" global SOD1(G93A) mice, we observed a substantial reduction of SR Ca(2+) release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1(G93A) fibers when compared to control fibers. Ca(2+) transients were not significantly reduced in similar experiments performed with FDB fibers obtained from "early-symptomatic" SOD1(G93A) mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca(2+) release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1(G93A) expression was restricted to skeletal muscle.

Conclusions: We report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1(G93A) mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1(G93A) and skeletal muscle autonomous MLC/SOD1(G93A) mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status.

Keywords: ALS; Amyotrophic lateral sclerosis; CaV1.1; Excitation-contraction coupling; L-type; Neuromuscular disease; SOD1; Skeletal muscle.

Fig. 1

EC coupling is impaired in symptomatic global SOD1^G93A FDB fibers. Representative voltage-induced myoplasmic Ca²⁺ transients elicited by 25 ms depolarizations from −80 to −60, −40, −20, 0, and 20 mV from FDB fibers harvested from a wild-type C57BL/6J mouse (a) and a symptomatic SOD1^G93A mouse (b). The peak ΔF/F-V relationships for wild-type and symptomatic SOD ^G93A fibers are presented in panel (c). Ca²⁺ transients were evoked at 0.1 Hz by test potentials ranging from −70 mV through +60 mV in 10 mV increments. The smooth curves are plotted according to Eq. 1 with the respective fit parameters shown in Table 1. The numbers of analyzed fibers are indicated in parentheses. Throughout, error bars represent ± SEM

Fig. 2

SR Ca²⁺ store content is similar in wild-type and symptomatic SOD1^G93A FDB fibers. Representative records of 4-CmC (1 mM)-induced SR Ca²⁺ release in dissociated FDB fibers harvested from a wild-type mouse (a) and from a symptomatic SOD1^G93A mouse (b). Insets show images of Fluo 3-loaded fibers before 4-CmC application (left) and at the peak of fluorescence (right). c Data summary

Fig. 3

L-type currents and charge movements are reduced in symptomatic SOD1^G93A FDB fibers. Representative whole-cell recordings of L-type currents elicited by 500 ms step depolarizations from −80 to −20, 0, 20, and 40 mV are shown for a wild-type FDB fiber (a) and a symptomatic SOD1^G93A fiber (b). c Peak I-V relationships corresponding to the current families shown in panels (a) and (b). Currents were evoked at 0.1 Hz by test potentials ranging from −40 mV through +80 mV in 10 mV increments. Representative recordings of intramembrane charge movements elicited by 25 ms depolarizations from −80 to −40, −20, 0, and 20 mV are shown for a wild-type (d) and a symptomatic SOD1^G93A fiber (e). f Q-V relationships corresponding to the charge movements shown in panels (d) and (e). Charge movements were evoked at 0.1 Hz by test potentials ranging from −70 mV through +50 mV in 10 mV increments. The smooth curves in panels (c) and (f) are plotted according to Eqs. 2 and 3, respectively, with fit parameters displayed in Table 2

Fig. 4

Western blot analysis of Ca_V1.1 protein levels in symptomatic SOD1^G93A skeletal muscle. a western blots of lysates obtained from four different tibialis anterior (b) gastrocnemius muscles of age-matched wild-type and symptomatic SOD1^G93A mice are shown (N = 4 for both groups). In each case, the blots were probed with a monoclonal antibody directed to Ca_V1.1 (top panels), then stripped and reprobed sequentially for α-actin (middle panels) and then histone H3 (bottom panels). A protein ladder standard resides in lane 1 of each blot; molecular weights are indicated in kDa. c A western blot of lysates obtained from FDB muscles of age-matched wild-type and symptomatic SOD1^G93A mice are shown (N = 2 for both groups). The blot was first probed with a monoclonal antibody directed to Ca_V1.1 (top panel) and then stripped and reprobed sequentially with antibodies directed to α-actin (middle panel) and then histone H3 (bottom panel). For each blot, changes in expression were determined by comparing the intensity of each Ca_V1.1 band in the top panel to the corresponding actin band the middle panel. For each panel, similar results were observed in at least three experiments

Fig. 5

EC coupling is not significantly impaired in early-symptomatic SOD1^G93A FDB fibers. Representative Ca²⁺ transients elicited by 25 ms depolarizations from −80 to −60, −40, −20, 0, and 20 mV from fibers harvested from a wild-type mouse (a) and from an early-symptomatic SOD1^G93A mouse (b). ΔF/F-V relationships are shown in (c)

Fig. 6

L-type currents and charge movements are reduced in early-symptomatic SOD1^G93A FDB fibers. Representative L-type currents elicited by 500 ms depolarizations from −80 to −20, 0, 20, and 40 mV are shown for a wild-type FDB fiber (a) and an early-symptomatic SOD1^G93A fiber (b). c Peak I-V relationships corresponding to the current families are shown in panels (a) and (b). Representative charge movement recordings elicited by 25 ms depolarizations from −80 to −40, −20, 0, 20 mV are shown for a wild-type (d) and a pre-symptomatic SOD1^G93A fiber (e). f Q-V relationships. The smooth curves in panels (c) and (f) are plotted according to Eq. 2 and Eq. 3, respectively, with fit parameters displayed in Table 2

Fig. 7

Summary of results obtained with global SOD1^G93A fibers. EC coupling (ΔF/F), L-type Ca²⁺ current (peak I_L)_, and maximal charge movement (Q _max) for early-symptomatic (105 ± 1 d; N = 6; white bars) and symptomatic global SOD1^G93A (149 ± 1 d; N = 9; black bars) fibers are represented as a percentage of corresponding values for the respective age-matched wild-type fiber groups

Fig. 8

EC coupling is impaired in MLC/SOD1^G93A FDB fibers. Representative Ca²⁺ transients elicited by 25 ms depolarizations from −80 to −60, −40, −20, 0, and 20 mV in fibers obtained from a wild-type FVB/NJ mouse (a) and a MLC/SOD1^G93A mouse (b). ΔF/F-V relationships are shown in (c)

Fig. 9

L-type currents and charge movements are reduced in MLC/SOD1^G93A FDB fibers. Representative L-type currents elicited by 500 ms depolarizations from −80 to −20, 0, 20, and 40 mV are shown for a wild-type FDB fiber (a) and an MLC/SOD1^G93A fiber (b). c Peak I-V relationships corresponding to the current families shown in panels (a) and (b). Representative charge movement recordings elicited by 25 ms depolarizations from −80 to −40, −20, 0, and 20 mV are shown for a wild-type (d) and an MLC/SOD1^G93A fiber (e). f Q-V relationships corresponding to the charge movements shown in panels (d) and (e). The smooth curves in panels (c) and (f) are plotted according to Eqs. 2 and 3, respectively, with fit parameters displayed in Table 2