Fig. 3

Histopathology of the patient muscle biopsy and MYPN analysis. A Minimal changes of the muscle architecture with hematoxylin-eosin (HE) and Gomori modified trichrome staining (TRG). A clear predominance of slow type muscle fibers is evident with anti-myosin heavy chain 7 antibody (MYH7) labeling. Scale bar, 100 μm. B Western blot analysis of muscle lysates from two healthy controls (ctr 1 and ctr 2) and from the patient loaded at the same protein concentration of controls (60 μg) and at higher concentration (100 μg). In control lysates, MYPN antibody identifies a clear band at the predicted molecular weight of the full-length MYPN isoform (red arrow), and a lower additional band which could correspond to the 114 kD MYPN isoform 2 (blue arrow). Additional bands (black arrows) corresponding to uncharacterized isoforms or nonspecific signal are also present. In patient, the full-length MYPN is absent, as well as the band around 100 kD, while the bands at 25 and 30 kD are similar to the controls. GAPDH was used as loading control. C Immunofluorescence analysis of MYPN. Note the diffuse reduction of protein, a negative fiber (asterisk), and protein accumulation in rare subsarcolemmal areas (insert) in patient muscle fibers if compared with the even distribution of control muscle. Blue staining, DAPI. Scale bar, 100 μm. D Double labeling of MYPN (green) and α-actinin (α-ACTN, red) in control (ctr, upper panels) and patient (lower panels) muscle sections. In patient muscle, the residual MYPN does not colocalize at Z line with α-actinin and appears diffuse among myofibrils. Scale bar, 2 μm. E Transmission electron microscope analysis of control (a, b) and patient (c–f). Note the interruption of the square arrangement (arrows in c) and the focal loss of actin filaments at the Z line (arrows in d) in patient muscle fibers when compared with the ordered organization in control sections (a, b). Aspects of myofilament disorganization (asterisk in e) and tiny nemaline-like structures (arrowheads in f) in subsarcolemmal areas of rare patient fibers. Scale bar, 200 nm