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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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Nav1.4 channel clusters in sarcolemma of control and mdx fibers. (A) Confocal images of Nav1.4 localization. Isolated fibers were immunolabeled with mouse antibodies to Nav1.4. A fluorescent signal was observed primarily at the sarcolemma. The luminescence distribution plot (inset) shows nonuniform density corresponding to apparent clusters of Na+ channels. (B) Distribution of Nav1.4 expression levels in cell-attached patches (model parameter A in fits with the Hodgkin-Huxley model) displays a similar non-Gaussian shape corresponding to that observed in confocal microscopy. The continuous curve represents the distribution density of amplitudes calculated using a Gaussian kernel density estimate with the bandwidth of 40 pA from the data points plotted below the histogram as thin vertical lines.
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fig5: Nav1.4 channel clusters in sarcolemma of control and mdx fibers. (A) Confocal images of Nav1.4 localization. Isolated fibers were immunolabeled with mouse antibodies to Nav1.4. A fluorescent signal was observed primarily at the sarcolemma. The luminescence distribution plot (inset) shows nonuniform density corresponding to apparent clusters of Na+ channels. (B) Distribution of Nav1.4 expression levels in cell-attached patches (model parameter A in fits with the Hodgkin-Huxley model) displays a similar non-Gaussian shape corresponding to that observed in confocal microscopy. The continuous curve represents the distribution density of amplitudes calculated using a Gaussian kernel density estimate with the bandwidth of 40 pA from the data points plotted below the histogram as thin vertical lines.

Mentions: Individual responses at every sampling potential were extracted and fit with the Hodgkin-Huxley model with the help of custom made software, which can be downloaded at http://www.unige.ch/sciences/pharm/fagie/software/elphys_utils-20080407.tar.bz2. Fit convergence was verified by recalculating the peak currents from the obtained fit parameters and comparing them with the apparent peak currents, as well as by fitting only tail regions of VGSC responses using Clampfit 9 software (pClamp-9 suite) and comparing the resulting inactivation rates (τi). Averages were computed by first calculating per patch means of the corresponding individual values and then establishing the overall averages. Typically, three recordings were performed for every patch. Patches where a significant rundown of signal or nonspecific activity was detected were discarded. Reversal potential for Na+ was determined by fitting tails of individual IV curves in the region where the tails were linear (typically 10–30 or, for patches with large currents, 40 mV) and averaging the calculated intersections with I = 0 first per patch and then for all patches. These data analyses were supplemented by traces with the sampling voltage steps extended to 100 mV, in order to perform control measurements of zero-current crossings. Details of these data analyses can be found in online supplemental material. Statistical modeling and composition of histograms (Fig. 5 B) were performed with the help of the R project for statistical computing (http://www.r-project.org/). Plots of sample Nav1.4 traces, steady-state activation and inactivation curves, and fit parameters were prepared using Microcal Origin software (Origin Laboratory).


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)

Nav1.4 channel clusters in sarcolemma of control and mdx fibers. (A) Confocal images of Nav1.4 localization. Isolated fibers were immunolabeled with mouse antibodies to Nav1.4. A fluorescent signal was observed primarily at the sarcolemma. The luminescence distribution plot (inset) shows nonuniform density corresponding to apparent clusters of Na+ channels. (B) Distribution of Nav1.4 expression levels in cell-attached patches (model parameter A in fits with the Hodgkin-Huxley model) displays a similar non-Gaussian shape corresponding to that observed in confocal microscopy. The continuous curve represents the distribution density of amplitudes calculated using a Gaussian kernel density estimate with the bandwidth of 40 pA from the data points plotted below the histogram as thin vertical lines.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Nav1.4 channel clusters in sarcolemma of control and mdx fibers. (A) Confocal images of Nav1.4 localization. Isolated fibers were immunolabeled with mouse antibodies to Nav1.4. A fluorescent signal was observed primarily at the sarcolemma. The luminescence distribution plot (inset) shows nonuniform density corresponding to apparent clusters of Na+ channels. (B) Distribution of Nav1.4 expression levels in cell-attached patches (model parameter A in fits with the Hodgkin-Huxley model) displays a similar non-Gaussian shape corresponding to that observed in confocal microscopy. The continuous curve represents the distribution density of amplitudes calculated using a Gaussian kernel density estimate with the bandwidth of 40 pA from the data points plotted below the histogram as thin vertical lines.
Mentions: Individual responses at every sampling potential were extracted and fit with the Hodgkin-Huxley model with the help of custom made software, which can be downloaded at http://www.unige.ch/sciences/pharm/fagie/software/elphys_utils-20080407.tar.bz2. Fit convergence was verified by recalculating the peak currents from the obtained fit parameters and comparing them with the apparent peak currents, as well as by fitting only tail regions of VGSC responses using Clampfit 9 software (pClamp-9 suite) and comparing the resulting inactivation rates (τi). Averages were computed by first calculating per patch means of the corresponding individual values and then establishing the overall averages. Typically, three recordings were performed for every patch. Patches where a significant rundown of signal or nonspecific activity was detected were discarded. Reversal potential for Na+ was determined by fitting tails of individual IV curves in the region where the tails were linear (typically 10–30 or, for patches with large currents, 40 mV) and averaging the calculated intersections with I = 0 first per patch and then for all patches. These data analyses were supplemented by traces with the sampling voltage steps extended to 100 mV, in order to perform control measurements of zero-current crossings. Details of these data analyses can be found in online supplemental material. Statistical modeling and composition of histograms (Fig. 5 B) were performed with the help of the R project for statistical computing (http://www.r-project.org/). Plots of sample Nav1.4 traces, steady-state activation and inactivation curves, and fit parameters were prepared using Microcal Origin software (Origin Laboratory).

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