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Nav1.4 deregulation in dystrophic skeletal muscle leads to Na+ overload and enhanced cell death.

Hirn C, Shapovalov G, Petermann O, Roulet E, Ruegg UT - J. Gen. Physiol. (2008)

Bottom Line: Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice.Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4.Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmacology, Geneva-Lausanne School of Pharmaceutical Sciences, University of Geneva, CH 1211 Geneva 4, Switzerland.

ABSTRACT
Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca(2+) concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na(+) concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Na(v)1.4, leading to an increased Na(+) concentration under the sarcolemma. Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4. Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.

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Steady-state activation and inactivation. (A) Representative steady-state activation and inactivation curves. Individual curves were fit with the Boltzmann function, and midpoints were averaged per patch and then overall averages were calculated, yielding no significant differences between activation thresholds (−33.8 ± 1.5 mV for control and −29.6 ± 2 mV for mdx5cvpatches). However, a shift of ∼10 mV toward more positive potentials for inactivation thresholds was observed in mdx5cv (−89.4 mV ± 2 mV for control and −79.5 mV ± 2.5 mV for the mdx5cv fibers, P = 0.01). The stimulation protocol used to produce currents for the inactivation curves is shown on the inset. (B) Window currents calculated from the determined average activation/inactivation thresholds and slopes at midpoint. Vertical line at −50 mV represents measured resting membrane potential, emphasizing an ∼2× increase in opening probability of Nav1.4 channels at rest in mdx fibers.
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fig4: Steady-state activation and inactivation. (A) Representative steady-state activation and inactivation curves. Individual curves were fit with the Boltzmann function, and midpoints were averaged per patch and then overall averages were calculated, yielding no significant differences between activation thresholds (−33.8 ± 1.5 mV for control and −29.6 ± 2 mV for mdx5cvpatches). However, a shift of ∼10 mV toward more positive potentials for inactivation thresholds was observed in mdx5cv (−89.4 mV ± 2 mV for control and −79.5 mV ± 2.5 mV for the mdx5cv fibers, P = 0.01). The stimulation protocol used to produce currents for the inactivation curves is shown on the inset. (B) Window currents calculated from the determined average activation/inactivation thresholds and slopes at midpoint. Vertical line at −50 mV represents measured resting membrane potential, emphasizing an ∼2× increase in opening probability of Nav1.4 channels at rest in mdx fibers.

Mentions: Stimulation protocols used to study activation and inactivation properties of VGSCs are shown on the insets of Fig. 3 B and Fig. 4 A. In short, patches were held at −100 mV between stimulation applications. The inactivation-recovery prepulses to −120 mV were applied for 30 ms and then potential was stepped up to voltages ranging from −70 to +40 mV or, during the determination of Na+ reversal potential, to +100 mV in steps of 10 mV for 20 ms. To study inactivation properties, the patches were held for 500 ms at voltages ranging from −120 to −10 mV in steps of 10 mV and currents were sampled at 10 mV for 50 ms. For the purpose of leak subtraction, the stimulation protocols were extended by a 20-ms step from the holding potential of −100 mV to −80 mV. Additionally, after performing recordings as described, currents were acquired using the online leak subtraction as implemented in Clampex program (pClamp-9 suite, Axon Instruments). Both methods allowed equivalently good removal of leakage currents during the study of activation properties of Nav1.4 channels. Only online leak subtraction was used to study the inactivation properties of these channels.


Nav1.4 deregulation in dystrophic skeletal muscle leads to Na+ overload and enhanced cell death.

Hirn C, Shapovalov G, Petermann O, Roulet E, Ruegg UT - J. Gen. Physiol. (2008)

Steady-state activation and inactivation. (A) Representative steady-state activation and inactivation curves. Individual curves were fit with the Boltzmann function, and midpoints were averaged per patch and then overall averages were calculated, yielding no significant differences between activation thresholds (−33.8 ± 1.5 mV for control and −29.6 ± 2 mV for mdx5cvpatches). However, a shift of ∼10 mV toward more positive potentials for inactivation thresholds was observed in mdx5cv (−89.4 mV ± 2 mV for control and −79.5 mV ± 2.5 mV for the mdx5cv fibers, P = 0.01). The stimulation protocol used to produce currents for the inactivation curves is shown on the inset. (B) Window currents calculated from the determined average activation/inactivation thresholds and slopes at midpoint. Vertical line at −50 mV represents measured resting membrane potential, emphasizing an ∼2× increase in opening probability of Nav1.4 channels at rest in mdx fibers.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483333&req=5

fig4: Steady-state activation and inactivation. (A) Representative steady-state activation and inactivation curves. Individual curves were fit with the Boltzmann function, and midpoints were averaged per patch and then overall averages were calculated, yielding no significant differences between activation thresholds (−33.8 ± 1.5 mV for control and −29.6 ± 2 mV for mdx5cvpatches). However, a shift of ∼10 mV toward more positive potentials for inactivation thresholds was observed in mdx5cv (−89.4 mV ± 2 mV for control and −79.5 mV ± 2.5 mV for the mdx5cv fibers, P = 0.01). The stimulation protocol used to produce currents for the inactivation curves is shown on the inset. (B) Window currents calculated from the determined average activation/inactivation thresholds and slopes at midpoint. Vertical line at −50 mV represents measured resting membrane potential, emphasizing an ∼2× increase in opening probability of Nav1.4 channels at rest in mdx fibers.
Mentions: Stimulation protocols used to study activation and inactivation properties of VGSCs are shown on the insets of Fig. 3 B and Fig. 4 A. In short, patches were held at −100 mV between stimulation applications. The inactivation-recovery prepulses to −120 mV were applied for 30 ms and then potential was stepped up to voltages ranging from −70 to +40 mV or, during the determination of Na+ reversal potential, to +100 mV in steps of 10 mV for 20 ms. To study inactivation properties, the patches were held for 500 ms at voltages ranging from −120 to −10 mV in steps of 10 mV and currents were sampled at 10 mV for 50 ms. For the purpose of leak subtraction, the stimulation protocols were extended by a 20-ms step from the holding potential of −100 mV to −80 mV. Additionally, after performing recordings as described, currents were acquired using the online leak subtraction as implemented in Clampex program (pClamp-9 suite, Axon Instruments). Both methods allowed equivalently good removal of leakage currents during the study of activation properties of Nav1.4 channels. Only online leak subtraction was used to study the inactivation properties of these channels.

Bottom Line: Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice.Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4.Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmacology, Geneva-Lausanne School of Pharmaceutical Sciences, University of Geneva, CH 1211 Geneva 4, Switzerland.

ABSTRACT
Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca(2+) concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na(+) concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Na(v)1.4, leading to an increased Na(+) concentration under the sarcolemma. Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4. Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.

Show MeSH
Related in: MedlinePlus