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A NOVEL APPROACH TO DISTINGUISH HUMAN MYOSIN HEAVY CHAIN ISOFORM DURING SINGLE MUSCLE FIBER DISSECTION

Abstract

G.E. Privett, A. Ricci, S.J. Mongold, M.R. Fruto, D.M. Callahan

University of Oregon, Eugene, OR

Due to the important impact of myosin heavy chain (MHC) isoform on contractile and metabolic characteristics, it is advantageous to distinguish MHC isoform during cellular-level experimentation of skeletal muscle. While animal models offer conveniently homogenous samples by anatomical muscle group, human skeletal muscle is far more heterogeneous with respect to MHC distribution within single samples. PURPOSE: The aim of our study was to develop an effective approach for distinguishing “slow-contracting” MHCI muscle fibers from “faster-contracting” MHCIIA/MHCIIX fibers during mechanical dissection. METHODS: Single fibers were dissected from bundles of ~100 fibers on multiple occasions. Fiber type predictions were based on elasticity of fibers during extraction from the bundle and fiber length compared to the bundle length following extraction. Fibers were then allocated to one of two groups: 1) stiff (maintained length similar to bundle length during extraction) or 2) compliant (stretched beyond bundle length). Stiff fibers were categorized as MHC I, while compliant fibers were considered to be “Not MHC I” (MHC types IIa and IIx) based on known associations between passive stiffness and the predominant MHC isoform in skeletal muscle fibers. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the MHC isoform of categorized fibers. Receiver Operating Characteristics and a Confusion Matrix were used to determine estimate accuracy. RESULTS: In total, 65 fibers were dissected, assigned a group, and assessed for MHC isoform via SDS-PAGE. Our stiffness-based estimates of fiber types yielded Sensitivity of 0.55, Specificity of 0.86, and Accuracy of 0.75. CONCLUSION: Our findings demonstrate the feasibility of early efforts to distinguish human skeletal muscle fibers by MHC isoform during mechanical dissection, thus reducing the temporal and sample resource burden of conducting skeletal muscle analyses that benefit from a homogenous MHC population.

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