Isoform-specific expression of the Coxsackie and adenovirus receptor (CAR) in neuromuscular junction and cardiac intercalated discs

Abstract

Background: The Coxsackie and adenovirus receptor (CAR) has a restricted expression pattern in the adult. In skeletal muscle, although CAR is expressed in immature fibers, its transcript levels are barely detectable in mature muscle. This is in contrast to the robust expression observed in the heart. However, both heart and skeletal muscle are susceptible to infection with the Coxsackie B virus which utilizes primarily CAR for cellular internalization. The specific point of viral entry in skeletal and heart muscle remains unknown.

Results: Using antibodies directed against the extracellular and the cytoplasmic domains of CAR, we show CAR in normal human and mouse skeletal muscle to be a novel component of the neuromuscular junction. In cardiac muscle, CAR immunoreactivity is observed at the level of intercalated discs. We demonstrate a single isoform of CAR to be expressed exclusively at the human neuromuscular junction whereas both predominant CAR isoforms are expressed at the intercalated discs of non-diseased human heart.

Conclusion: The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus.

Figure 1

Depiction of the CAR sequences recognized by the anti-C-terminal antibodies. The chicken antibody ChCT was raised against a fusion protein containing C-terminal sequence common to both CAR isoforms (from aa 259 – 339). The rabbit antibodies (RP291 and RP194) were raised against peptides that were specific to each isoform. The diagram does not show the immunogen of the rabbit antibody 2240 which is the extracellular N-terminal domain of murine CAR. Note that each antibody has a distinct recognition sequence. TM – transmembrane sequence.

Figure 2

Immunolocalization of CAR to the neuromuscular junction in human skeletal muscle. Frozen sections of human muscle were incubated with Alexa Fluor-conjugated α-bungarotoxin and antibodies to CAR as described in Materials and Methods. Panels A and D show α-bungarotoxin staining of acetylcholine receptors at neuromuscular junctions (green). Panel B shows immunofluorescent staining of the section in panel A with a polyclonal antibody (ab 2240) to the extracellular domain of CAR (red). Panel E shows immunofluorescent staining of the section in panel D with a mixture of the isoform-specific C-terminal antibodies RP194 and RP 291 (red). Panel C is a merge of panels A and B. Panel F is a merge of panels D and E. These merges [C, F] show that CAR colocalizes with α-bungarotoxin at neuromuscular junctions (in yellow). Of the two C-terminal antibodies, only RP291 (G) demonstrates the typical neuromuscular junction staining while signal is absent when sections are incubated under similar conditions with RP194 (H) although RP194 does label blood vessels (I). Sections incubated with secondary antibody alone did not reveal any signals (J). Bar = 25 μm (A, D); 50 μm (G, I).

Figure 3

Immunolocalization of CAR to the neuromuscular junction in mouse skeletal muscle. Frozen sections of mouse muscle were incubated with Alexa Fluor-conjugated α-bungarotoxin and antibodies to CAR as described in Materials and Methods. Panels A and D show α-bungarotoxin staining of acetylcholine receptors at neuromuscular junctions (green). Panel B shows immunofluorescent staining of the section in panel A with a polyclonal antibody (ab 2240) to the extracellular domain of CAR (red). Panel E shows immunofluorescent staining of the section in panel D with a chicken polyclonal antibody (ChCT) directed against a C-terminal portion of CAR conserved in both CAR isoforms (red). Panel C is a merge of panels A and B. Panel F is a merge of panels D and E. These merges [C, F] show that CAR colocalizes with α-bungarotoxin at murine neuromuscular junctions (in yellow). Sections incubated with the secondary antibodies alone did not give any signal – anti-rabbit IgG [G], anti-chicken IgY [H]. Bar = 35 μm

Figure 4

Immunolocalization of CAR to intercalated discs in human and murine cardiac muscle. Human [A, B] and murine [C, D] cardiac muscle sections were reacted with the polyclonal anti-C-terminal antibodies RP194 [A, C] and RP291 [B, D]. Note the intense staining of both isoforms at the human intercalated discs (arrow) [A, B] and murine intercalated discs [C, D]. Neither the human cardiac tissue [E] nor the murine cardiac muscle [F] gave any signal when incubated with the anti-rabbit IgG. Bar = 10 μm (A, E); 30 μm (C, F).

Figure 5

Murine cardiac muscle homogenates express both isoforms of CAR. A. Immunoblot analysis of cardiac muscle homogenates (10 μg) reveals that a single polypeptide of 46 kDa is detected by the anti-C-terminal antibodies recognizing the two different CAR isoforms (RP194 and RP291). B. Murine cardiac homogenates were immunoprecipitated with either RP194 (lanes 1,2) or RP291 (lanes 3,4) followed by immunoblotting with the chicken ChCT antibody that recognizes the C-terminal portion of CAR that is common to both isoforms. The ChCT antibody detected as a single band the increasing amounts of CAR that was immunoprecipitated with increasing amounts of the rabbit polyclonal antibodies (2 μl, lanes 1,3; 4 μl, lanes 2,4).