Utrophin localization in normal and dystrophin-deficient heart.

Abstract

BACKGROUND 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 dystrophin-deficient hearts. METHODS AND RESULTS The 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. CONCLUSIONS This 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.