Role of MRFs and calcineurin in modulating muscle fiber type

Porcine meat quality can be affected by variation in muscle fiber type composition. Therefore, understanding the molecular control of muscle fiber plasticity is a critical step toward improving the production of high quality pork. The objectives of these studies were to test in vivo the recent hypothesis suggesting that the myogenic regulator factor myogenin and the serine/threonine phosphatase calcineurin (CaN) control muscle fiber phenotype. The full-length porcine CaN delta isoform catalytic subunit gene (GenBank accession number AF193515) was cloned and CaN expression was characterized in several adult muscles. Surprisingly, we detected by northern blot a novel 5.2 kb mRNA transcript and have shown that CaN mRNA isoform expression is greatest in fast muscles. Using immunohistochemistry, we have shown that CaN protein is mainly localization in cytoplasm of fast fibers. The effects of myogenin and calcineurin overexpression on muscle fiber phenotype was studied using direct DNA injection technology. First plasmid DNA encoding for epitope-tagged porcine myogenin and mouse MyoD proteins was injected in fast skeletal muscles of 10-day-old piglet (n = 6). At day 3, 10 and 17, piglets were euthanized and injected muscles were collected. Data showed that overexpression of myogenin does not induce slow/β myosin heavy chain (MHC) mRNA in transfected fibers. However, strongly staining succinate dehydrogenase was detected specifically in myogenin positive fibers. Therefore, myogenin may participate in controlling metabolism shifts in muscle metabolism. Plasmid DNA encoding myc epitope-tagged constitutively active form of CaN was also injected into pig muscle. Control plasmid DNA containing sequences encoding for the green fluorescent protein (GFP) were also injected into fast piglet muscle. Injected muscle samples were collected at 3, 10 and 17 days post-injection. Positive myc-transfected fibers were observed only a day 3 post-injection, whereas GFP positive fibers were observed until 17 days post-injection. Conclusive evidence regarding the role of calcineurin in controlling skeletal muscle fiber type were not possible. However, data suggest that overexpression of calcineurin may trigger apoptosis