Masatake Shimokawa

Developmental regulation of myotonic dystrophy protein kinase in human muscle cells in vitro.

From our previous studies, myotonic dystrophy protein kinase: gene product of myotonic dystrophy is localized at the terminal cisternae of sarcoplasmic reticulum of human adult muscle. Now we have studied the developmental expression of myotonic dystrophy protein kinase in aneurally cultured human muscles and contracting cross-striated muscles innervated with fetal rat spinal cord using a semi-quantitative reverse transcription-polymerase chain reaction method for myotonic dystrophy protein kinase messenger RNA expression, Western blot analysis, immunohistochemical examinations by laser scanning confocal microscopy and immunoelectron microscopy. About 65,000 mol. wt myotonic dystrophy protein kinase was detected in aneurally cultured muscles. Myotonic dystrophy protein kinase messenger RNA was expressed in both aneurally and innervated cultured muscles, but in early innervated cultured muscles the message was transiently lower than in aneurally cultured muscles and innervated cultured muscles in long-term co-culture. In aneurally cultured muscles, immature aneurally cultured muscles show a diffuse and irregular distribution of myotonic dystrophy protein kinase in the deeper cytoplasm near the nuclei. Ultrastructurally the immuno-products against myotonic dystrophy protein kinase were observed as dense deposits in parts of the membranes near the mitochondria. In innervated cultured muscles, immunofluorescent microscopy showed myotonic dystrophy protein kinase to be localized regularly in the I bands and A-I junctions. Ultrastructurally myotonic dystrophy protein kinase was localized in branched duct-like membranes in the early stage of innervated cultured muscles and then in small sacs at the I bands and A-I junctions of the sarcolemma in the mature stage. Our present studies strongly suggest that innervation plays an important role in the localization of myotonic dystrophy protein kinase in human skeletal muscle during development. We conclude that the expression of myotonic dystrophy protein kinase during development is under neuronal influence.

Immunolocalization of myotonic dystrophy protein kinase in corbular and junctional sarcoplasmic reticulum of human cardiac muscle

The subcellular localization of myotonic dystrophy protein kinase has been examined in human cardiac muscles with confocal laser-scanning microscopy and electron microscopy. A polyclonal antibody was produced against the synthesized peptide from a human kinase cDNA clone. We checked the antibody specificity for cardiac myotonic dystrophy protein kinase using an immunoblotting technique. Immunoblotting of extract from human cardiac muscles showed mainly 70 kDa and 55 kDa molecular weight bands. Confocal images of the protein kinase immunostaining showed striated banding patterns similar to those of skeletal muscles. In addition, the kinase was strongly detected around the intercalated disc. Immunoelectron microscopy showed that the kinase was mainly expressed in both corbular and junctional sarcoplasmic reticulum, but not in network sarcoplasmic reticulum. These results suggest that myotonic dystrophy protein kinase may be involved in the modulation of Ca2+ homeostasis in cardiac myofibres.

Immunocytochemical localization of a full-length myotonin protein kinase in rat L6 myoblasts ☆

Expansion mutation of CTG-repeat motifs within myotonin protein kinase (MtPK) gene is responsible for pathological changes in myotonic dystrophy (DM). To explore its pathological role in skeletal muscle, a full-length human MtPK cDNA was transfected into rat L6 myogenic cell line. Recombinantly expressed human MtPK protein in L6 cell line has a predicted molecular mass of 70 kDa. We have raised a polyclonal antibody against a synthetic peptide in the deduced sequence of the C-terminal portion of MtPK. MtPK in L6 cell is localized to perinuclear region, that resembles with sarcoplasmic reticulum. The MtPK-transfected myoblast cells established in this study will allow us to elucidate the molecular pathomechanism of muscle manifestations in DM.