We sought to develop an approach to examine fiber type specific protein content from human muscle biopsy samples. Of consideration was an approach that would accurately identify fiber types across the continuum, have the ability to assess a large number of proteins from a given fiber type, and be reasonable from both a time and cost perspective when applying to human based exercise studies. The primary findings were: 1) fiber type specific protein content can be measured via Western blotting in conjunction with SDS-PAGE fiber typing across the continuum of human skeletal muscle fiber types (MHC I, I/IIa, IIa, IIa/IIx, IIx), 2) further support for the method outlined here was the expected findings of anaerobic (GAPDH), aerobic (citrate synthase), and regulatory (p38) protein content in slow (MHC I) and fast (MHC IIa) muscle fiber types, and 3) for the first time, hybrid (I/IIa, IIa/IIx) and the MHC IIx fibers were shown to have metabolic protein content that generally existed in a hierarchal fashion along the continuum of human muscle fiber types.
The method we present here was shown to be consistent and adaptable for applications that require low amounts of protein. Support for this assay approach with human muscle biopsy samples comes from the consistent protein load and transfer studies (1 vs. 2 μg) and low coefficient of variation among the multiple loading paradigms employed in this investigation. For fiber typing we chose to use SDS-PAGE due to our familiarity with the technique and to decrease the likelihood of fiber misclassification compared to other approaches for identifying fiber types in humans [7
]. The SDS-PAGE approach to fiber typing enables a large volume of muscle fibers (200-400) to be examined relatively rapidly (<24 h) with identification of the five main fiber types in human skeletal muscle (MHC I, I/IIa, IIa, IIa/IIx, IIx) without the need for fiber type specific antibodies.
The fiber-pooling approach in the current study generated enough total protein to allow the use of a commercially available protein assay kit. This is highly favorable for Western blot analysis as it enables a known amount of total protein to be loaded and serve as a control, and thereby eliminates the need for a control/housekeeping protein, which may be challenging to define for different fiber types, age groups, and interventions. The protein assay analysis also enabled us to perform a loading spectrum step for each protein of interest to establish the optimal amount of protein to be loaded for the Western blot analysis. The load spectrum is important since proteins have a wide range of molecular weight and abundance in human skeletal muscle. This was highlighted in the current investigation, as each protein of interest required a different optimal loading amount for the Western blot analysis (GAPDH = 1 μg, CS = 3 μg, p38 = 5 μg). The load spectrum step optimizes loading for Western blot analysis, which increases Western blotting accuracy [35
] and saves samples.
Another consideration for our approach was to develop a method that would yield enough total protein to allow for assessment of multiple proteins for a given fiber type. The fiber-pooling model accomplished this goal. This is important when examining numerous proteins in a particular pathway or any type of analysis that may require several proteins to be analyzed. Compared to an approach of analyzing one muscle fiber at a time, fiber pooling helps reduce the volume of Western blot work, which saves time and resources. It should be noted that obtaining 20 (or more) fibers of a given MHC type is not always possible (i.e. targeting less abundant isoforms in limited tissue samples). However, this is not a major limitation as the MHC I/IIa and MHC IIx pools analyzed here only contained 5 and 11 fibers, respectively, but still generated enough total protein to assess some proteins of interest. The fiber-pooling model outlined here is also advantageous for researchers conducting human based experiments. In these types of experiments where multiple treatment groups and pre-to-post interventions are common, examination of protein content across the continuum of fiber types can be easily managed when using the fiber-pooling model.
Several laboratories have used alternative techniques (e.g. fluorescent staining or immunohistochemistry) to assess metabolic [36
] and size regulating [39
] proteins in human MHC I and IIa fibers. The methodology presented here was the first to employ SDS-PAGE fiber typing and the first to assess protein content in hybrid (MHC I/IIa and MHC IIa/IIx) and pure MHC IIx fibers via Western blots. The inclusion of the hybrid muscle fibers is a valuable aspect of our model as they are prevalent in most human populations and are particularly responsive to chronic perturbation (e.g., aging, unloading, exercise) [11
]. Conversely, the MHC IIx fibers in human muscle are generally rare (<2%) in active healthy individuals [7
], but can become more prominent in inactive and diseased states [15
]. When assessing various aspects of skeletal muscle health and plasticity, the continuum of fiber types should be considered to better understand muscle adaptation.
Application of this technique indicates both metabolic (GAPDH and CS) and signaling proteins (p38) exist in a hierarchal manner across all fiber types. GAPDH is a major facilitator of glycolysis and thus not surprisingly, was more abundant in the fast-twitch fibers. Previous literature supports this contention as GAPDH mRNA abundance [46
], activity [47
], and protein content exist preferentially in fast-twitch fibers in active [48
], inactive [49
], diseased [51
], and aging [46
] humans. Inversely, CS content (a common marker of mitochondrial abundance and oxidative capacity) was greatest in slow-twitch fibers. These data complement the well accepted notion that glycolytic enzymes are more abundant in fast-twitch fibers and oxidative enzymes are more abundant in slow-twitch fibers [1
]. However, for the first time this concept is extended to include hybrid fiber types. We also found a signaling protein (p38) to be more abundant in fast-twitch fibers. Future research will be required to determine if this is consistent in the human vastus lateralis or if factors such as the subjects’ long history of aerobic endurance exercise influenced the findings.
In summary, we present a new methodology for the assessment of human skeletal muscle fiber type specific protein content that is compatible with SDS-PAGE fiber typing, a standard protein assay kit, and Western blotting. The initial results indicate a hierarchal relationship in specific protein content and contractile function across the entire continuum of human skeletal muscle fiber types. This technique will allow future research to determine changes in protein content in response to various perturbations (e.g., aging, disease, exercise) in MHC I and MHC IIa as well as the understudied hybrid and MHC IIx fibers, providing a more complete profile of skeletal muscle health in humans.