Neuromuscular synaptic patterning requires the function of skeletal muscle dihydropyridine receptors

Abstract

Developing skeletal myofibers in vertebrates are intrinsically 'pre-patterned' for motor nerve innervation. However, the intrinsic factors that regulate muscle pre-patterning remain unknown. We found that a functional skeletal muscle dihydropyridine receptor (DHPR, the L-type Ca(2+) channel in muscle) was required for muscle pre-patterning during the development of the neuromuscular junction (NMJ). Targeted deletion of the β1 subunit of DHPR (Cacnb1) in mice led to muscle pre-patterning defects, aberrant innervation and precocious maturation of the NMJ. Reintroducing Cacnb1 into Cacnb1(-/-) muscles reversed the pre-patterning defects and restored normal development of the NMJ. The mechanism by which DHPRs govern muscle pre-patterning is independent of their role in excitation-contraction coupling, but requires Ca(2+) influx through the L-type Ca(2+) channel. Our findings indicate that the skeletal muscle DHPR retrogradely regulates the patterning and formation of the NMJ.

Figure 1. Loss of DHPR function leads to defects in muscle pre-patterning

Whole mounts of diaphragm muscles (E14.5) were double-labeled by α-bgt for AChR and by anti-neurofilament (NF150) and anti-synaptotagmin 2 (Syt2) for the nerve. Increased nerve branching and expansion of innervation territories were detected in Cacnb1^−/− muscle (f) compared with WT muscle (a). White dashed lines (a) delineate the myotendinous junction between the central tendon and the medial edge of the muscle fibers. The asterisk (*) (f) indicates a small branch of the intercostal nerve attached to the outer edge of the diaphragm muscle. b–e, g–h: high-magnification views of the dorsal regions of the diaphragm muscle. In the WT muscle, AChR clusters were aligned along the central region of the muscle, forming a pre-patterned end-plate band (arrow in b); the nerves extend fine branches and terminate within the central end-plate band (d, g). In contrast, in the Cacnb1^−/− muscle, AChR clusters were distributed broadly across the entire surface of the muscle, including the medial and lateral edges of the muscle (arrowheads in c); the nerves in the Cacnb1^−/− muscle also branched extensively and expanded their innervation territories across the entire muscle surface (e, h). At this stage, the majority of the AChR clusters were not directly apposed to the nerve terminal (g, h). i–j: AChR clusters were distributed along a central end-plate band in HB9^−/− muscles (i), but were distributed broadly in double null Cacnb1^−/− HB9^−/− muscles (j). Scale bar: a, f: 200 μm; b–e, g–h: 100 μm; i–j: 400 μm.

Figure 2. Loss of DHPR function leads to multiple synaptic sites and an expansion of innervation territories

a–b: Distribution of AChR clusters (labeled by α-bgt) in whole mounts of diaphragm muscles (E18.5). AChR clusters were aligned in a central endplate band in WT (arrow in a), but were scattered over a broad region in Cacnb1^−/− muscles (b). c-d: Dissociated myofibers stained with FITC-conjugated α-bgt and rhodamine-conjugated phalloidin. e: histogram distribution of the percentage (in log scale) of dissociated myofibers containing various numbers of endplates ranging from 1 to 7. Multiple endplates were frequently detected in dissociated Cacnb1^−/− myofibers (d); the majority of them (72%, 399 out of 554) contained 2 or more endplates per fiber (2 patches: 42.8%; 3 patches: 16.4%; 4 patches: 8.1%; 5 patches: 3.8%; 6 patches: 0.7% and 7 patches: 0.2%). In contrast, the majority of control myofibers (99.4%, 357 out of 359) contained single endplate; less than 1% of them (0.6%, 2 out of 359) contained two endplates per fiber. f–g: The innervation territories were confined within the central region in the WT (bordered by dashed lines f), but expanded to the entire muscle in Cacnb1^−/− (g). h–i: Distribution of AChE clusters (arrow) revealed by cholinesterase staining. AChE clusters were localized along the central region of the muscle in the WT (h), but were broadly distributed in the Cacnb1^−/− muscle (i). Inset in i shows a high power view of individual myofibers containing multiple AChE clusters (arrowheads) in Cacnb1^−/− muscle. Scale bars: a–b: 400 μm; c–d: 50 μm; f–g: 200 μm; h–i: 250 μm.

Figure 3. DHPR function is not required for synaptogenesis, but its absence leads to increased synaptic and muscle electrical activity

a–f: Confocal images of E18.5 diaphragm muscles doubly labeled by presynaptic markers (NF150 and Syt2) (a, d) and postsynaptic marker α-bgt (b, e). Every end-plate in both WT (b) and the Cacnb1^−/− muscle (e) was fully innervated by the nerves (arrows in c, f). Endplates in the control muscle appeared predominantly as ovoid-shaped plaques (b), whereas endplates in Cacnb1^−/− muscles were bigger, and some were perforated (arrowheads in e). g-h: Electron micrographs of the NMJ (E18.5, diaphragm muscle). In both WT (g) and Cacnb1^−/− (h), synaptic vesicles (SV) were abundantly present at the nerve terminal (NT), and the basal lamina (white arrow in g, h) was well-defined in the synaptic cleft. i–j: Sample traces of mEPPs from control (i) and Cacnb1^−/− (j) myofibers. The mEPP frequency was markedly increased in Cacnb1^−/− muscles. k–l: Spontaneous action potentials (arrowheads) in control (k) and Cacnb1^−/− (l) mice; the lower trace in k and l illustrates an expanded view of the portion of the upper trace indicated by the grey line. Arrow in k indicates the displacement of the trace resulting from muscle contraction in the control. Scale bars: a–f: 20 μm; g–h: 0.5 μm.

Figure 4. Muscle-specific expression of Cacnb1 rescues the patterning defects in Cacnb1^−/− muscle

a–b: Sample traces of mEPP recorded from P0 diaphragm muscles in control (a) and the Cacnb1^−/− mice that also carry a HSA::Cacnb1 transgene under the control of the muscle-specific HSA promoter, as shown in the schematic drawing [Cacnb1^−/−; HSA::Cacnb1, referred to as the rescued mice (b)]. c–h: Wholemount diaphragm muscles at E14.5 (c–e) and E18.5 (f–h) from the rescued mice (Cacnb1^−/−; HAS::Cacnb1) were double-labeled for AChRs (c, f) and the nerves (d, g). Similar to the WT muscles (compared to Fig. 1, Fig. 2), AChR clusters in the rescued muscles were aligned to a central endplate band (arrow in c, f) and nerve terminals were also confined to a central endplate band as shown in the merged images (e, h). i–l: AChE staining of wholemount diaphragm muscle (Dia, P0) and triangularis sterni muscle (TS, P90) from control (i, j) and the rescued mice (Cacnb1^−/−; HAS::Cacnb1) (k, l). The patterns of AChE staining (black arrow) were similar between control and the rescued mice. Scale bars: c–e, 100 μm, f–h, 200 μm; i–l, 1000 μm.

Figure 5. RyRs and DHPRs play different roles in muscle pre-patterning

a–f: Distribution of AChR clusters (a, d), nerves (b, e) and merged images (c, f) revealed by double-immunofluroscence staining of E14.5 diaphragm muscles from RyR1^−/−RyR3^−/− (d–f) and littermate control mice (RyR1^+/+RyR3^+/−, a–c). AChRs were clustered along central regions of the muscles in both RyR1^−/−RyR3^−/− and RyR1^+/+RyR3^+/− mice (arrows in a, d), in a pattern similar to that seen in the WT mice (Fig. 1b). Increased innervation was detected in RyR1^−/−RyR3^−/− (e) compared with the control (RyR1^+/+RyR3^+/−, b). Nevertheless, nerve terminals in both RyR1^−/−RyR3^−/− and RyR1^+/+RyR3^+/− mice were confined to the central region of the muscle as shown in the merged images (arrowheads in c, f). g–i: AChR distribution in E18.5 diaphragm muscles. Unlike Cacnb1^−/− muscle in which AChR clusters were broadly distributed (i), the majority of AChR clusters were aligned in a central end-plate band in E18.5 RyR1^−/−RyR3^−/− muscle (arrow in h), similar to the end-plate band seen in the E18.5 WT muscle (arrow in g). However, some AChR clusters were ectopically localized to the peripheral regions of the E18.5 RyR1^−/−RyR3^−/− muscle (* in h). Scale bar: a–f: 100 μm; g–i: 400 μm.

Figure 6. DHPRs pattern neuromuscular synapses by regulating the expression of AChR and MuSK

Wholemount in situ hybridization with DIG-labeled AChRα probes were carried out in E14.5 diaphragm muscles (a–b) or E18.5 intercostal muscles (c). AChRα transcripts were detected along the central regions in the WT (a), but were broadly distributed in the Cacnb1^−/− muscle (b). c: AChRα transcripts were localized to the central region of the WT muscle (left panel in c), but were detected in the entire Cacnb1^−/− muscle (middle panel in c). The normal distribution of AChRα transcripts was restored in the rescued mice (right panel in c). d: Wholemount in situ hybridization of intercostal muscles using DIG-labeled MuSK probes. MuSK transcripts were localized to the central region of the WT (left), but were broadly distributed in the Cacnb1^−/− muscle (right). e: Relative expression levels of AChR and MuSK assayed by quantitative real-time PCR in C2C12 myotube cultures treated with L-type Ca²⁺ channel antagonists (verapamil, isradipine) or agonist (Bay K 8644) compared to the untreated or vehicle (DMSO) treated controls. Verapamil (10 μM) significantly increased the relative expression levels of MuSK (2.21 ± 0.13, N = 5 cultures, P = 0.0002) and AChR (2.10 ± 0.32, N = 5 cultures, P = 0.009) compared with controls (MuSK, 1.03 ± 0.12; AChR, 1.04 ± 0.16, N = 5 cultures). Similarly, isradipine (1 μM) also significantly increased the relative expression levels of MuSK (1.84 ± 0.21, N = 3 cultures, P = 0.0003) and AChR (1.68 ± 0.31, N = 3 cultures, P = 0.0189) compared with controls (MuSK, 1.01 ± 0.10; AChR, 1.00 ± 0.02, N = 3 cultures). Bay K 8644 significantly decreased the expression of MuSK (0.60 ± 0.13, N = 3 cultures, P = 0.0012) and AChR (0.83 ± 0.07, N = 3 cultures, P = 0.0337) compared with controls (MuSK, 1.02 ± 0.02; AChR, 1.04 ± 0.10, N = 6 cultures). f: Wholemount diaphragm muscles were immunostained with anti-MuSK antibodies and α-bgt. MuSK protein was clustered along the central region of the WT (left column), but broadly distributed in the Cacnb1^−/− muscles (middle column). MuSK distribution was restored to normal in the rescued mice (right column). Data are presented as mean ± SEM. Scale bars: a–b: 500 μm; c: 500 μm; d: 400 μm; f: 50 μm.