Sodium channel mRNAs at the neuromuscular junction: distinct patterns of accumulation and effects of muscle activity

Abstract

Voltage-gated sodium channels (VGSCs) are highly concentrated at the neuromuscular junction (NMJ) in mammalian skeletal muscle. Here we test the hypothesis that local upregulation of mRNA contributes to this accumulation. We designed radiolabeled antisense RNA probes, specific for the "adult" Na(V)1.4 and "fetal" Na(V)1.5 isoforms of VGSC in mammalian skeletal muscle, and used them in in situ hybridization studies of rat soleus muscles. Na(V)1.4 mRNA is present throughout normal adult muscles but is highly concentrated at the NMJ, in which the amount per myonucleus is more than eightfold greater than away from the NMJ. Na(V)1.5 mRNA is undetectable in innervated muscles but is dramatically upregulated by denervation. In muscles denervated for 1 week, both Na(V)1.4 and Na(V)1.5 mRNAs are present throughout the muscle, and both are concentrated at the NMJ. No Na(V)1.5 mRNA was detectable in denervated muscles stimulated electrically for 1 week in vivo. Neither denervation nor stimulation had any significant effect on the level or distribution of Na(V)1.4 mRNA. We conclude that factors, probably derived from the nerve, lead to the increased concentration of VGSC mRNAs at the NMJ. In addition, the expression of Na(V)1.5 mRNA is downregulated by muscle activity, both at the NMJ and away from it.

Fig. 1.

Generation of isoform-specific probes for VGSCs.A, Primary structure of α-subunit of VGSC (adapted from Catterall, 1992). The location of sequences used to make probes for Na_V1.4 and Na_V1.5 isoforms are indicated bybroken lines in cytoplasmic domains I and II.B, Agarose gel electrophoresis (1%) of RT-PCR products from skeletal (Sk) and cardiac (C) muscles using isoform-specific oligonucleotide primers. The Na_V1.4-specific primer set (Materials and Methods) amplified the correct 349 bp product from skeletal but not from cardiac cDNA (left panel). The Na_V1.5-specific fragment (410 bp) is amplified from both skeletal cardiac muscle cDNAs (right panel). No products are detected in the absence of reverse transcriptase using either primer set (−RT lanes). The first lane in each panel contains a molecular size marker (Life Technologies). C, RPA using isoform-specific antisense RNA probes. The Na_V1.4-specific (top panel) and Na_V1.5-specific (bottom panel) probes were labeled with [α-³²P]UTP and hybridized with 10 μg of total RNA from brain, liver, heart, and innervated (Sol+N) and denervated (Sol-N) soleus muscles (see Materials and Methods). The Na_V1.4 probe protected a fragment of expected size from both innervated and denervated soleus but none from brain, liver, or cardiac RNA. Hybridization with the Na_V1.5-specific probe resulted in protection of two fragments from cardiac and denervated soleus RNA, one smaller than the expected size. No protected fragments are detected from liver, brain, or innervated soleus muscle RNA (middle panel). Digested (Dig) and undigested (Undig) probe controls were run in adjacent lanes in each gel. Thebottom panel shows a denaturing agarose gel electrophoresis (1%) of an aliquot total cytosolic RNA used for RPA, demonstrating intact 28 S and 18 S ribosomal RNA bands and 5 S RNA at the bottom of the ethidium bromide-stained gel.

Fig. 2.

Na_V1.4, but not Na_V1.5, mRNA is concentrated at the NMJ in normal rat soleus muscle. Na_V1.4 mRNA visualized after in situ hybridization and autoradiography as silver grains (A) is densely accumulated at the NMJ (arrow), identified by the reddish–browncholinestrase reaction product. Accumulation of Na_V1.4 mRNA is also visible in XJ regions (arrowheads) as occasional smaller clusters. No such accumulation is seen after hybridization with the Na_V1.5-specific antisense (C) or the sense transcripts of either Na_V1.4 or Na_V1.5 probes (B and D, respectively). Scale bar, 50 μm.

Fig. 3.

Na_V1.5 mRNA is increased after denervation of adult muscle. Accumulation of silver grains is visible at the NMJ and in XJ regions after ISH with Na_V1.5 antisense transcript in 7 d denervated soleus (C) but not in control muscle (A). Na_V1.4 mRNA is concentrated at the majority of NMJs and XJ regions in both control (B) and denervated (D) rat soleus. Scale bar, 50 μm. Time course of Na_V1.5 mRNA accumulation after denervation of adult rat muscle (E). Mean intensity of labeling within a circle of 12 μm radius centered on J clusters starts to increase by the second day, reaching a peak at the third day and is sustained for up to 4 weeks after denervation of adult soleus muscles (●). The same temporal pattern is seen for fiber cross sections (▴) and clusters (▪) in XJ regions (40–50 J and 30–40 XJ clusters and 45 XJ fibers were analyzed for each time point).

Fig. 4.

Electrical stimulation reverses Na_V1.5 mRNA accumulation in denervated adult muscle. Na_V1.5 mRNA clustering at the NMJ and XJ regions in 7 d denervated soleus (A) is abolished by chronic electrical stimulation in vivo (C) for 1 week as outlined in Materials and Methods. Na_V1.4 mRNA is accumulated at the NMJ and XJ regions in both denervated (B) and stimulated (D) soleus muscle. Scale bar, 50 μm.

Fig. 5.

Na_V1.5 mRNA accumulation in adult muscle is controlled by activity. Intensity of Na_V1.5 labeling per nucleus is nearly fourfold greater at J than XJ nuclei (top panel) in denervated muscle, which is reduced to control levels after electrical stimulation (p < 0.001). Labeling intensity with Na_V1.4 probe at J and XJ nuclei is not affected by either denervation or stimulation of adult soleus muscle (bottom panel). Thirty to 40 J clusters and XJ fibers were analyzed for each category.