Eric Fabbrizio

Utrophin localization in normal and dystrophin-deficient heart.

The localization of dystrophin at the sarcolemma of cardiac skeletal fibers and cardiac Purkinje fibers has been described. Dystrophin deficiency produces clinical manifestations of disease in skeletal muscles and hearts of patients with Duchenne and Becker muscular dystrophy. Utrophin (or dystrophin-related protein), a dystrophin homologous protein, was found to be expressed in fetal muscles and reexpressed in dystrophin-deficient skeletal muscle fibers. We therefore examined utrophin expression in normal and in dystrophindeficient hearts. Methods and ResultsThe expression and subcellular distribution of utrophin was examined in cardiac muscle by immunoblot and immunofluorescence analysis in normal bovine heart compared with dystrophin. Utrophin expression was also examined in normal and dystrophin-deficient hearts of MDX mice. Three monoclonal antibodies reacting with dystrophin and utrophin solely or reacting with both proteins along with two polyclonal antibodies reacting with either utrophin or dystrophin and utrophin were tested. In normal bovine heart, utrophin was not expressed at the periphery of fibers but was strongly expressed in intercalated disks and in the cytoplasm of cardiac Purkinje fibers. In cardiocytes, utrophin was colocalized along transverse T tubules with dystrophin. Dystrophin was present at the periphery of cardiocytes and cardiac Purkinje fibers as well as in transverse T tubules but was absent or faintly expressed in intercalated disks. The results with monoclonal and polyclonal antibodies were identical. Western blot analysis revealed that the detected molecules corresponded only to a 400-kD protein band and not to possible shorter transcripts of utrophin or dystrophin (apo-utrophin or apo-dystrophin). In dystrophin-deficient hearts of MDX mice, utrophin alone was abundant but not organized in the same networklike distribution. ConclusionsThis first localization of utrophin in normal heart (in Purkinje fibers, transverse tubules, and intercalated disks) showed a distinct subcellular localization of this protein with dystrophin, suggesting an important function of this protein in intercellular communication. In dystrophin-deficient hearts of MDX mice, utrophin alone is overexpressed as in skeletal muscle sarcolemma, an area normally occupied by dystrophin but not organized in the same networklike distribution.

Monoclonal antibodies targeted against the C-terminal domain of dystrophin or utrophin

The structure‐function relationships of dystrophin, a protein which is absent or defective in patients with Duchenne or Becker muscular dystrophies, and utrophin can only be compared if specific antibodies are produced. We expressed C‐terminal parts of dystrophin and utrophin in expression vectors. Mice were immunized with recombinant proteins and 26 monoclonal antibodies were produced and analyzed. Their respective epitopes were determined using other overlapping recombinant products. We observed antibody specificity towards 400 kDa dystrophin and/or utrophin protein bands, either by Western blot analysis or immunodetection in human skeletal (quadriceps) and smooth (uterus) muscles. These antibodies have been used to compare the relative abundance of both dystrophin and utrophin relative to the structures analyzed.

N-terminal domain of dystrophin

Contro‐versial experiments have been published on calmodulin binding of dystrophin. In this study, we used recombinant proteins and the techniques of affinity chromatography and ELISA to show that the N‐terminal part of dystrophin binds calmodulin specifically in a calcium‐dependent manner. The calcium‐dependent interaction of calmodulin and dystrophin does not directly regulate binding of actin to dystrophin, but may regulate dystrophin interactions with other associated proteins.

The dystrophin superfamily: variability and complexity

Duchenne muscular dystrophy (DMD) is a progressive muscular disease caused by the absence of a molecule called dystrophin (Hoffman et al., 1987; Koenig et al., 1987, 1988). It affects one male birth in 3500 and death generally occurs by respiratory or heart failure in the third decade of life. Dystrophin was one of the first genes identified by positional cloning and has been characterized in detail (Zatz et aL, 1981; Davies et aL, 1983; Kunkel et aL, 1985). It spans over 2.3 Mb along the X-chromosome, locus p2Z (Kingston et al., 1984). An allelic myopathy called Becker muscular dystrophy (BMD) is a milder form of the disease in which dystrophin is present but in an altered form. Sixty percent of Duchenne and Becker muscular dystrophies result from gene deletions (Koenig et al., 1989), while I0 and 30% correspond to duplications and mutations respectively (DenDunnen et al., 1987). Not long after the dystrophin gene was identified other related proteins were characterized. Presently, five distinct promoters have been found to generate various transcripts of the dystrophin gene (Nudel et al., 1989; Bar et al., 1990; Klamut et al., 1990; Gorecki et al., 1992; Byers et al., 1993; Tinsley et al., 1993), as presented in Fig. 1. In addition, a dystrophin related-protein (DRP or utrophin) was identified encoded by a gene localized on chromosome 6 in humans (Love et al., 1989; Tinsley et al., 1992) and 10 in mice (Buckle et al., 1990). We review the modular structure of dystrophin, its subcellular localization and interactions with other cell components. Structural homologies between the members of the dystrophin family are discussed in relation to their potential function.

Dystrophin and dystrophin-related protein expression in Torpedo marmorata electric organ

The presence of different dystrophin-related protein forms was investigated in electric organ as compared to cardiac, white or red skeletal muscles from Torpedo marmorata. Two strategies were followed. First, we used specific C-terminal dystrophin and dystrophin-related protein monoclonal antibodies which we characterized in the present study. 400 kDa protein bands were detected in the tissues mentioned above with both specific types of antibodies. Second, we produced monoclonal antibodies raised against a dystrophin-enriched preparation from T. marmorata electric organ. Western blot and immunofluorescence analyses showed the tissue specificity of T. marmorata antibodies and allowed us to classify them as types I, II and III. Vessel walls and neuromuscular junctions were labeled with T. marmorata type II and III antibodies in human muscles (skeletal and smooth). Both approaches demonstrated that the T. marmorata electric organ contained different proteins related with dystrophin: a dystrophin form, a dystrophin-related protein form and a dystrophin-related protein isoform, homologous to the dystrophin-related protein present in muscle vessel walls and at the neuromuscular junctions of human tissues. The presence of dystrophin and dystrophin-related protein is finally discussed relative to their functions and organ specificities.

Properties of chicken cardiac dystrophin

Summary— We investigated the presence of dystrophin by immunoblot and immunofluorescence analyses, negative staining, rotatory shadowing and immunogold electron microscopy in chicken cardiac muscle. Saponin was found to be better than Triton X‐100 for providing a new ‘dystrophin‐enriched’ solution for use in biochemical studies of the molecule. By Western blot analysis, only a 400‐kDa band was revealed with polyclonal antibodies directed against a central region (residues 1178–1723) of the dystrophin molecule and no cross‐reactions with other proteins or degraded products were observed. Specific cleavage of the dystrophin molecule showed that the central rod‐shaped domain corresponded to a resistant ‘core’. This structure might rigidify the protein. By immonofluorescence, dystrophin was localized at the periphery of cardiac ventricular cells. The molecule was examined by electron microscopy and found to have variable lengths (140–160 nm for the monomeric from and about 260 ± 10 nm or more for oligomeric forms). These oligomeric structures are considered to be associated molecules which are only partially overlapped lengthwise. The precise distribution of dystrophin within the cardiac muscle was determined by visualisation of gold particles in immuno‐electron microscopy. Gold particles were found on the sarcolemma with no evidence of any association with cytoplasmic structures. The present data provide further details on the cardiac dystrophin molecule and suggest that its capacity of self‐association may elasticize the dystrophin dimer.