? Skeletal muscles are highly plastic tissues, continually responding to a variety of imposed demands. This plasticity arises from the ability of individual muscle fibers to change size (hypertrophy and atrophy) or fiber type. Muscle fiber type is largely determined by the type of myosin heavy chain (MHC) present within an individual fiber, since the MHC isoform in a muscle fiber directly affects muscle shortening velocity, power output, and contractile efficiency. Within the past 10 years, a significant number of studies have revealed that many individual fibers possess two or more MHC isoforms (hybrid fibers). While some of these fibers may be in transition from one 'pure' phenotype to another, many represent stable phenotypes. In fact, recent studies suggest that hybrid fibers are often more prevalent than pure phenotypes. Surprisingly few studies have focused on the basic biology of these hybrid fibers. We currently know little about the distribution of hybrid fibers in different muscles, the cellular basis of the multiple isoform expression, or about the responses of the hybrid fibers to exercise.
The specific aims are: #1: Quantify the levels of single fiber MHC polymorphism in mouse muscles at the level of protein and mRNA. SDS-PAGE techniques are used to determine MHC isoforms in single fibers at the protein level, and real-time quantitative RT PCR is used to measure the levels of mRNA coding for different MHC isoforms. #2: Determine whether the polymorphism within single fibers is due to multiple isoform expression within single nuclei, or if multiple nuclei each express a single isoform. In situ hybridization with RNA probes is used to identify the isoform-specific expression patterns among the multiple nuclei within individual muscle fibers. #3: Determine the effects of running exercise on MHC gene expression and single fiber polymorphism. Mice are run on a multi-lane, variable speed treadmill acutely, or for 6 weeks of training. Following the exercise protocol, muscles are harvested and the effects on MHC gene expression in hybrid fibers is assessed. Results from the current proposal will help fill a major gap in our understanding of the molecular complexity of skeletal muscles. The long-term goal is to elucidate the cellular and molecular mechanisms responsible for determining skeletal ? muscle phenotype and plasticity. An understanding of muscle plasticity is central to our understanding of adaptation to exercise and to certain diseases such as cancer cachexia and age-related sarcopenia. ? ? ?
