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

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

Our research centers around skeletal muscle, which is comprised of cells with distinctive metabolic and contractile properties, characterized as slow or fast twitch fibers. We delve into the factors contributing to these differences and explore their implications in the context of aging, exercise, and various diseases. Measuring cellular responses in different skeletal muscle fibers is often overlooked, or not possible due to the examination of muscle homogenates.The MyDoBID technique allows us to repeatedly analyze fiber type protein differences that were previously impossible to measure. This protocol accurately and efficiently identifies type one and type two muscle fibers from freeze dried human skeletal muscle samples. This newly revised method provides a straightforward approach for researchers to investigate protein differences in skeletal muscle at the cellular level.Notably, it facilitates the identification of type two X fibers, and these have historically been challenging to distinguish. The technique allows researchers to shed light on all fiber types, including the elusive type two X fiber. With the MyDoBID technique, numerous questions can now be answered.For example, one can discover the properties of type two X muscle fibers, and determine if there's evidence indicating preferential atrophy of type two X muscle fibers during aging or disease. One can also observe how a specific exercise intervention impacts each fiber type.

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  

To begin, position the freeze dried muscle sample under the stereo microscope and view it at low magnification. With one pair of fine tissue dissecting forceps, hold the muscle in place and separate the small bundles of fibers with the other forceps. Isolate one bundle of fibers and gently tease them apart until single fiber segments are extracted from the bundle.Then shift the fiber to a clear area of the Petri dish. Investigate each single fiber segment under 50 times magnification. Attempt to further separate the fiber at one end.If the fiber begins to break, it is a single fiber. Carefully collect the fiber with a pair of forceps and place it into the previously prepared aliquot of denaturing buffer. Tap the tube firmly on the bench three times.Afterward, vortex the fiber samples and briefly centrifuge them for five seconds at 2, 500G to ensure the samples settles at the bottom of the tube. Let the samples rest at room temperature for one hour before storing them at minus 80 degrees Celsius for future use.