THRIFTY; A Novel Method For Rapid Fiber Type Identification Of Isolated Skeletal Muscle Fibers

Abstract

Human skeletal muscle is composed of a mixture of fiber types with distinct contractile and metabolic properties, and adaptions to exercise, aging and disease have been reported to occur in a fiber type specific manner. Fiber type specific analysis of skeletal muscle is therefore crucial to fully understand its heterogenous response to external stimuli. However, such analyses are rarely performed due to the labor-intensive process of fiber typing. Purpose: To develop a valid and fast method that enables high-throughput fiber typing of individually isolated muscle fibers. Methods: Employing THRIFTY (High THRoughput Immunofluorescence Fibre Typing), a ≤ 0.5 mm end was cut off individual fibers. The fiber ends were fixed onto a microscope slide with a pre-printed coordinated grid system. Slides were then submerged in acetone to permeabilize the fiber ends. Subsequently, the slides were stained with primary antibodies targeting MHC I & II (BA-F8 & SC-71, respectively), washed three times in PBS, and stained with fluorescent secondary antibodies. After three additional washes, a cover glass was mounted on the slide using anti-fade reagent. Fiber ends were then typed using a fluorescent microscope under 4X magnification. The THRIFTY protocol was then compared to a previously validated protocol for fiber typing using dot blot 1. A time requirement comparison between the two protocols was also carried out by a technician novice to both methods. Results: Both THRIFTY and dot blot produced pools of type I & type II muscle fibers free from contamination validated with SDS-PAGE and silver stain. Out of 400 fibers stained with both THRIFTY and dot blot, 9 fibers were considered contaminated in one of the methods, while being identified as free from contamination in the other. No fibers were identified as BA-F8 positive in one method, and SC-71 positive in the other, or vice versa. The agreement between the two methods was considered strong, κ = 0,955 (95% CI, 0,928 to 0,982), p < 0.001. Time requirements for THRIFTY was less than half that of the dot-blot protocol. Conclusion: THRIFTY is a novel and valid method for fiber typing individual muscle fibers yielding fiber pools free from contamination while drastically reducing the labor requirement, thus enabling a high throughput analyses for large scale investigations. 1) PMID: 31019216