THRIFTY: a novel high‐throughput method for rapid fibre type identification of isolated skeletal muscle fibres

Abstract

Fibre type‐specific analyses are required for broader understanding of muscle physiology, but such analyses are difficult to conduct due to the extreme time requirements of dissecting and fibre typing individual fibres. Investigations are often confined to a small number of fibres from few participants with low representativeness of the entire fibre population and the participant population. To increase the feasibility of conducting large‐scale fibre type‐specific studies, a valid and rapid method for high‐throughput fibre typing of individually dissected fibres was developed and named THRIFTY (for high‐THRoughput Immunofluorescence Fibre TYping). Employing THRIFTY, 400 fibre segments were fixed onto microscope slides with a pre‐printed coordinated grid system, probed with antibodies against myosin heavy chain (MyHC)‐I and MyHC‐II and classified using a fluorescence microscope. The validity and speed of THRIFTY was compared to a previously validated protocol (dot blot) on a fibre‐to‐fibre basis. Fibre pool purity was evaluated using ‘gold standard’ SDS‐PAGE and silver staining. A modified THRIFTY‐protocol using fluorescence western blot equipment was also validated. THRIFTY displayed excellent agreement with the dot blot protocol, κ = 0.955 (95% CI: 0.928, 0.982), P < 0.001. Both the original and modified THRIFTY protocols generated type I and type II fibre pools of absolute purity. Using THRIFTY, 400 fibres were typed just under 11 h, which was approximately 3 times faster than dot blot. THRIFTY is a novel and valid method with high versatility for very rapid fibre typing of individual fibres. THRIFTY can therefore facilitate the generation of large fibre pools for more extensive mechanistic studies into skeletal muscle physiology. Key points Skeletal muscle is composed of different fibre types, each with distinct physiological properties. To fully understand how skeletal muscle adapts to external cues such as exercise, nutrition and ageing, fibre type‐specific investigations are required. Such investigations are very difficult to conduct due to the extreme time requirements related to classifying individually isolated muscle fibres. To bypass this issue, we have developed a rapid and reliable method named THRIFTY which is cheap as well as versatile and which can easily be implemented in most laboratories. THRIFTY increases the feasibility of conducting larger fibre type‐specific studies and enables time‐sensitive assays where measurements need to be carried out in close connection with tissue sampling. By using THRIFTY, new insights into fibre type‐specific muscle physiology can be gained which may have broad implications in health and disease.

Pro-Brain-Derived Neurotrophic Factor (BDNF), but Not Mature BDNF, Is Expressed in Human Skeletal Muscle: Implications for Exercise-Induced Neuroplasticity

“…For the fiber type-specific analyses, type I and type II fibers were isolated from the resting muscle samples from project 3. Following fiber type determination with the THRIFTY method, 37 all type I and type II fibers from 1 sample were pooled separately (∼100 fibers per sample), weighed, and then prepared for immunoblotting and enzyme-linked immunosorbent assay (ELISA) as described below. In the case of the 4 muscle samples used for IHC, samples were frozen in isopentane chilled in liquid nitrogen for subsequent cryosectioning.…”

Need for speed: Human fast-twitch mitochondria favor power over efficiency

“…The petri dish was placed on a bed of ice under a stereomicroscope and 80 individual muscle fibers were dissected out of the sample and placed in individual droplets of BIOPS allocated in a gridded plastic container. The dissected muscle fibers were fiber typed according to our recently developed THRIFTY protocol [ 17 , 18 ]. Briefly, an end of each muscle fiber (≈0.5 mm) was cut off and placed on a microscope slide pre-printed with a coordinate grid system.…”

“…Freeze-dried samples were manually dissected under a stereomicroscope. Approximately 50–100 fibers were dissected from each biopsy sample and subsequently typed according to the THRIFTY protocol [ 17 , 18 ]. Ten fibers of each type from every volunteer were homogenized individually in 20 μl of ice-cooled homogenization buffer with 1× Laemmli sample buffer.…”

“…To investigate this, we performed fiber type-specific single muscle fiber analysis on intrinsic molecular markers regulating mitochondrial structure-, and ultrastructure as well as oxidative phosphorylation (OXPHOS) enzyme content. Furthermore, using the novel THRIFTY technique developed in our lab [ 17 , 18 ], we were able to rapidly identify fiber types in individually dissected muscle fibers and for the first time, assess mitochondrial respiration in fiber-type specific pools from human skeletal muscle. This approach coupled with single fiber analysis of molecular markers allowed us to provide novel insights into muscle fiber type-specific mitochondrial function in human skeletal muscle.…”

The dawn of the functional genomics era in muscle physiology

“…To facilitate these types of experiments, a recent wet‐lab method has been published that allows for relatively high‐throughput and scalable fibre typing of isolated single myofibres. Using a fluorescence‐based method with antibodies against myosin heavy chain isoforms, the THRIFTY method enables robust, rapid and high‐throughput profiling of fibre types of isolated single myofibres (Horwath et al., 2022). Further experimental developments have now made it possible to visualise the composition of myofibre types in vivo and could enable the ex vivo sorting of fast myofibre types, an approach that could be highly complementary to fibre‐type genomic analyses.…”