real-time reverse transcriptase-polymerase chain reaction; electrophoresis; myosin heavy chain gene regulation; muscle plasticity IN THE RAT DIAPHRAGM MUSCLE (Dia m ), significant changes in myosin heavy chain (MHC) isoform expression occur during the first 4 wk of postnatal development (22-24, 38, 39, 41). Most fibers express protein for the MHC Neo isoform at birth (P-0), but the relative expression of this isoform gradually decreases until it disappears by postnatal day 28 (P-28). In contrast, MHC 2X and MHC 2B protein isoforms are not expressed in the rat Dia m until at least the second postnatal week. Postnatal development of the rat Dia m is also characterized by changes in muscle contractile properties and by significant growth of muscle fibers, most notably those expressing MHC 2X and MHC 2B (15,22,36,38,42,43,45,47). Thus the relative MHC isoform composition changes during postnatal development of the Dia m , and ultimately it determines Dia m function. At the present time, very little is known about the underlying mechanisms regulating MHC phenotype expression.Previous studies of postnatal development, both in limb muscles and the Dia m , have focused exclusively on changes in the relative protein expression of the different MHC isoforms in the whole muscle (9, 11, 22-24, 30, 31, 33, 38, 39, 45). However, during postnatal development of the rat Dia m , MHC content (measured by MHC mass per half-sarcomere volume and fiber cross-sectional area) increases markedly (15). This increase varies across fiber types (classified based on MHC isoform expression). At single Dia m fibers, the developmental increase in MHC content is greater in fibers expressing MHC 2X and/or MHC 2B isoforms than in those expressing MHC Slow or MHC 2A . Thus, during postnatal development, not only do Dia m fibers increase in size but also they increase their MHC protein content. This substantial increase in total MHC protein expression across Dia m fiber types is not evident if only the relative expression of MHC isoform is examined.The period from birth to adulthood provides a unique and significant opportunity to examine the mechanisms underlying rapid muscle growth and phenotype transitions. In rat limb muscles, postnatal muscle phenotype is regulated by MHC gene expression either as a result from mechanical stretch induced by skeletal growth and/or increased muscle activity (18). At present, there is no direct information regarding the transcriptional regulation of MHC isoform expression during postnatal development of the rat Dia m .An increase in MHC protein of the rat Dia m during postnatal development could be due to transcriptional, translational, and/or posttranslational mechanisms, which may vary depending on fiber type (10). Assuming transcriptional rate remains constant, an increase in mRNA expression would result in increased protein expression. For example, patterns of MHC isoform-specific mRNA and protein expression are very similar during postnatal development in pigs (25). We hypothesized that postnatal transitions ...