Assessing Repeatability In Muscle Fiber Typing: Variability In Myosin Heavy Chain-based Phenotyping And Sample Size Optimization

Identification of myosin heavy chain proteins (MHC) of single muscle fibers is a widely used method and considered the “gold standard” in determining skeletal muscle fiber types (FT) in muscle. Human muscle fibers can express a single MHC (resulting in a pure Type I, IIa, or IIx) or multiple MHCs (leading to hybrid I/IIa and IIa/IIx). Distribution of the types of fibers can change as a result of physical activity, exercise training, or disease state. PURPOSE: To determine the repeatability of the determination of muscle fiber phenotype using pools containing either 25, 100, or 150 muscle fibers selected randomly from a sample of ~200 muscle fibers per individual. METHODS: Following IRB approval, muscle biopsies were collected from the vastus lateralis muscle from six healthy individuals (M/F = 3/3, age = 29 ± 3 yrs, BMI range 22 - 41). Approximately 200 muscle fibers were isolated from the muscle sample of each individual with fine tweezers under a stereo microscope and analyzed via SDS PAGE and silver stain for MHC isoform content. Random samples of groups of 25, 100, and 150 fibers were taken in five replicates for each group to calculate the coefficient of variation (CV) associated with the sampling of the 25, 100, or 150 fiber replicates. T-tests were performed to test for differences in CV values between groups containing 25, 100, and 150 fiber replicates. RESULTS: Type I fibers had mean CV (mCV) at 29% ± 7%, 18% ± 6%, and 11% ± 2% at 25, 100, and 150 fibers, respectively, and significantly lower at 100 (p = 0.005), and 150 (p = 0.008) fibers when compared to 25. Type IIa mCV was 21% ± 3%, 13% ± 2%, and 10% ± 2% at 25, 100, and 150 fibers respectively and significantly lower at 100 (p = 0.05), and 150 (p = 0.02) fibers compared to 25. Notably, mCV for IIa/IIx hybrids were also significantly lower at 100 (mCV = 23% ± 6%, p = 0.04) and 150 (mCV = 19% ± 5%, p = 0.01) compared to 25 fibers (mCV = 38% ± 9%). IIx fibers, while only significant at 150 fibers (mCV = 44% ± 8%, p = 0.04) compared to 25 (mCV = 117% ± 39%), did not result in an acceptable CV due to rare existence of these fibers identified within the ~200 fibers isolated. Understanding the repeatability of MHC-determined muscle fiber type distribution is key for studies designed to evaluate differences in muscle fiber distribution between subject groups or within groups as a result of an intervention. This project was funded by NIDDK RO1DK123441