To validate these findings independently and address the spatial distribution of M-band constituents, we examined frozensections by immunofluorescence microscopy. The α-all-Myom1 antibody detects known isoforms of Myom1, the EH-myomesin antibody detects only the EH splice variant of Myom1, and the Myom2 antibody detects known isoforms of Myom2.
Figure 7A(left) shows that TA was evenly labeled with both the all-Myom1 (top row) and Myom2 (middle row) but not significantly labeled with the EH-myomesin (bottom row) antibodies. In EOMs (middle column higher magnification, right column lower magnification), all fibers were labeled with all-Myom1 antibodies, with slightly greater signal in the OL. Consistent with our mRNA data, the major structural constituents of M-bands are present in EOMs at the protein level. To further define the complex layer-specific expression pattern, we performed double labeling using the EH-myomesin and the Myom2 antibodies. As shown in
Figure 7B , this combination labeled both the OL and GL, with EH-myomesin being predominantly expressed in OL (
Fig. 7B , left column), whereas Myom2 labeling restricted to the GL (
Fig. 7B , middle column). The reciprocal expression pattern of these antibodies (
Fig. 7B , middle column) also extended to the GL, where apart from the reciprocity observed in the OL, the GL fibers were noted to express either EH-myomesin or Myom2, but not both together. A subset of GL fibers was not labeled with either antibodies. Thus, EH-myomesin expression was detectable in EOMs and not in the TA. In contrast, the Myom2 antibodies labeled only ∼30% of fibers in the GL, whereas the OL was mainly negative for Myom2. Conversely, the EH-myomesin antibodies evenly labeled OL-fibers but only a few fibers with strong labeling for EH-myomesin were detected in the GL.
To extend and confirm these results, we performed confocal microscopy of whole mount preparations of EOMs in longitudinal optical planes. As shown in
Figures 7C and 7D , confocal microscopy revealed a similar pattern of labeling and unequivocally demonstrates M-bands that are seen to occur in a regular pattern of repeating transverse lines. The all-Myom1 antibodies labeled M-bands in both OL and GL fibers, EH-myomesin predominantly labeled OL fibers, whereas Myom2 labeling was restricted to some GL fibers. Using this sensitive method, we detected complex patterns of myomesin expression: We demonstrated that some fibers coexpressed both EH-myomesin and Myom2, even with one of the constituents being expressed only at minor levels. Fiber D II strongly expressed Myom2 with traces of EH-myomesin.