Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction

Abstract

Huntington disease is a progressive and fatal genetic disorder with debilitating motor and cognitive defects. Chorea, rigidity, dystonia, and muscle weakness are characteristic motor defects of the disease that are commonly attributed to central neurodegeneration. However, no previous study has examined the membrane properties that control contraction in Huntington disease muscle. We show primary defects in ex vivo adult skeletal muscle from the R6/2 transgenic mouse model of Huntington disease. Action potentials in diseased fibers are more easily triggered and prolonged than in fibers from WT littermates. Furthermore, some action potentials in the diseased fibers self-trigger. These defects occur because of decreases in the resting chloride and potassium conductances. Consistent with this, the expression of the muscle chloride channel, ClC-1, in Huntington disease muscle was compromised by improper splicing and a corresponding reduction in total Clcn1 (gene for ClC-1) mRNA. Additionally, the total Kcnj2 (gene for the Kir2.1 potassium channel) mRNA was reduced in disease muscle. The resulting muscle hyperexcitability causes involuntary and prolonged contractions that may contribute to the chorea, rigidity, and dystonia that characterize Huntington disease.

Keywords: channelopathy; electrophysiology; myopathy; myotonia; trinucleotide repeat.

Fig. 1.

Action potentials from WT and HD fibers. (A) Representative WT and HD action potentials showing the rising and falling phases. (B) Repolarization phase of the action potentials from A plotted with the WT (black dotted line) and HD (red dotted line) curve fits. (C) Minimum 0.5-ms depolarizing stimulus current (mean ± SEM) needed to trigger an action potential (AP) in WT (n = 17) and HD (n = 12) fibers. (D) Representative spontaneous action potential that self-triggered after an apparent subthreshold stimulus. *Significant difference compared with WT fibers (P < 0.05).

Fig. 2.

Average chloride currents of WT (n = 14) and HD (n = 9) fibers. (A–D) Voltage clamp traces for WT (Left) and HD (Right) fibers. (A) Voltage protocol with a holding potential of −20 mV, a break during the 150-ms conditioning pulse to +60 mV (P1), a second 200-ms test pulse with steps from −140 to +120 mV in +20-mV increments (P2), and a third 50-ms test pulse to −100 mV (P3). For clarity, only half the traces are shown in A–D. (B–D) Current records normalized to fiber surface area (cm²). (B) Total currents composed of the chloride current (I_Cl) plus leak and capacitive currents. (C) Leak and capacitive currents measured by blocking chloride channels with 400 μM 9AC. For scaling purposes, the full capacitive transients in B and C are not shown. (D) Specific I_Cl records obtained by subtracting the leak and capacitive currents (C) from the total currents (B). (E) IV relationship of the peak I_Cl (mean ± SEM) from P2 for WT and HD fibers. G_ClC-1 was the slope of the IV relationship from −100 to −140 mV. (F) Relative open probability of WT and HD chloride channels (mean ± SEM) obtained by plotting the normalized peak currents from P3 as a function of steady-state voltage from P2. The WT and HD data were each fitted with a Boltzmann curve.

Fig. 3.

Average inward rectifying potassium currents of WT (n = 12) and HD (n = 11) fibers. (A and B) Voltage clamp traces for WT (Left) and HD (Right) fibers. (A) Voltage protocol with a holding potential of 0 mV and a series of 700-ms steps from −60 to +20 mV in +10-mV increments. (B) Specific inward rectifying potassium (I_Kir) currents obtained by recording the total currents and subtracting the leak and capacitive currents (analogous to method for obtaining I_Cl). Leak and capacitive currents records were made by blocking inward rectifying potassium channels with 5 mM Ba²⁺. (C) IV relationship of the peak I_Kir (mean ± SEM) from B for WT and HD fibers. G_Kir was the slope of the IV relationship from −40 to −60 mV.

Fig. 4.

Reduced expression of Clcn1 mRNA in HD muscle coincides with atypical splicing of Clcn1 pre-mRNA. (A) Gel showing aberrantly spliced Clcn1 mRNA that contains exon 7a (Exon 7a+, 420 bp) and normal adult Clcn1 mRNA that lacks exon 7a (Exon 7a−, 341 bp) in WT (Left) and HD interosseous (Right) muscle. (B) Aberrant Clcn1 mRNA with exon 7a was expressed at higher proportional levels in the HD (n = 2) compared with WT (n = 3) muscle. (C and D) The relative expression of total Clcn1 mRNA, normalized to β2-microglobulin using the ΔΔCT method, was significantly reduced in HD muscle as determined by real-time RT-PCR using two separate primer sets spanning exons 10–14 or exons 16–19. *Significant difference compared with WT muscle (P < 0.05). Average values (± SEM) of the resting or baseline membrane potential, the maximum rate-of-rise of the depolarization, the peak change in membrane potential, the initial repolarization time constant (τ₁), and the slower repolarization time constant (τ₂). *Significant difference compared with WT fibers (P < 0.05).