Name: Video: Isolation of Single-Fiber from Freeze-Dried Human Skeletal Muscle
Uploaded: 2023-09-22T14:15:00
Description: 302 Views. Isolation of Single-Fiber from Freeze-Dried Human Skeletal Muscle

isolation of single-fiber from freeze-dried human skeletal muscle

MyDoBID- A Novel Technique for Identifying Muscle Fiber Types Efficiently

Skeletal muscle is a heterogeneous tissue, with distinct cellular metabolic and contractile properties that are dependent on whether the cell (fiber) is slow twitch (type I) or fast twitch (type II). The fiber type can be identified by examining the myosin heavy chain (MHC) isoforms, which differ from each other in several ways, including contraction time, speed of shortening, and fatigue resistance1. Major MHC isoforms include type I, type IIa, type IIb, and type IIx and their metabolic profiles are either oxidative (type I and IIa) or glycolytic (IIx, IIb)1. The proportion of these fiber types varies in muscle type and between species. Type IIb is widely found in rodent muscle. Human muscles do not contain any type IIb fibers and consist predominantly of MHC isoforms type I and IIa fibers, with a small proportion of IIx fibers2. Protein expression profiles vary between different fiber types and can be altered with aging3, exercise4,5, and disease6.
Measuring cellular responses in different skeletal muscle fiber types is often overlooked or not possible due to the examination of muscle homogenates (a mix of all fiber types). Single-fiber western blot allows for the investigation of multiple proteins in individual muscle fibers7. This methodology has previously been utilized to produce novel and informative single-fiber characteristics that were not possible to obtain using homogenate preparations. However, there are some limitations of the original single-fiber western blot methodology, including the time-consuming nature, the inability to generate sample replicates, and the use of expensive, sensitive enhanced chemiluminescence (ECL) reagents. If fresh tissue is being used, this method is further limited due to the time constraints of needing to isolate individual fibers within a limited timeframe (i.e., 1-2 h). Fortunately, this restraint is mitigated by isolating single-fiber segments from freeze-dried tissue8. However, fiber collection from freeze-dried samples is limited by the size and quality of the biopsied tissue.
Fiber type identification using the dot blotting method9 has been significantly elaborated upon and expanded in this comprehensive protocol. Previously, it has been demonstrated that as little as ~2-10 mg of wet weight muscle tissue is adequate for freeze-drying and single-fiber MHC isoform protein analysis9. Christensen et al.9 used 30% of a ~1 mm fiber segment to detect the MHC isoform present by dot blotting, which was confirmed by western blotting. This work showed that by substituting western blotting with dot blotting, the overall costs were reduced by ~40-fold (for 50 fiber segments). Fibers were then &#34;pooled&#34; into type I and type II samples, which allowed for experimental replication9. Nevertheless, a limitation was that only two fiber type-specific samples were obtained: type I (MHCI positive) and type II (MHCII positive fibers), with type II samples containing a mixture of MHCIIa and MHCIIx6,10. Notably, the current protocol demonstrates how pure type IIx fibers can be identified and provides a highly detailed workflow (summarized in Figure 1), including troubleshooting strategies for common protocol issues.

Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique 

Fiber Type Identification of Human Skeletal Muscle

Watch this Scientific Journal Video about Isolation of Single-Fiber from Freeze-Dried Human Skeletal Muscle at JoVE.com

Isolation of Single-Fiber from Freeze-Dried Human Skeletal Muscle  

isolation-of-single-fiber-from-freeze-dried-human-skeletal-muscle

Research

JoVE Journal

Biochemistry

302 Views. Isolation of Single-Fiber from Freeze-Dried Human Skeletal Muscle

Video: Isolation of Single-Fiber from Freeze-Dried Human Skeletal Muscle  

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.