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

MyDoBID - טכניקה חדשנית לזיהוי יעיל של סוגי סיבי שריר

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.

מחבר זרקור: פענוח המסתורין של סוגי סיבי שריר השלד באמצעות טכניקת MyDoBID 

זיהוי סוג סיבים של שרירי השלד האנושיים

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  

בידוד סיב בודד משרירי השלד האנושיים המיובשים בהקפאה

כדי להתחיל, מקמו את דגימת השריר המיובש בהקפאה מתחת למיקרוסקופ הסטריאו וצפו בה בהגדלה נמוכה. בעזרת זוג אחד של רקמות עדינות המנתחות מלקחיים, החזיקו את השריר במקומו והפרידו את צרורות הסיבים הקטנים עם המלקחיים האחרים. בודדו צרור אחד של סיבים והפרידו אותם בעדינות עד למיצוי מקטעי סיבים בודדים מהצרור.לאחר מכן העבירו את הסיב לאזור ברור של צלחת הפטרי. בדקו כל מקטע סיב בודד בהגדלה של פי 50. נסו להפריד עוד יותר את הסיב בקצה אחד.אם הסיב מתחיל להישבר, זהו סיב יחיד. בזהירות לאסוף את הסיב עם זוג מלקחיים ומניחים אותו לתוך aliquot מוכן מראש של מאגר denaturing. הקש על הצינור בחוזקה על הספסל שלוש פעמים.לאחר מכן, מערבלים את דגימות הסיבים וצנטריפוגות אותן לזמן קצר למשך חמש שניות ב -2, 500G כדי להבטיח שהדגימות מתיישבות בתחתית הצינור. תנו לדגימות לנוח בטמפרטורת החדר למשך שעה לפני אחסונן בטמפרטורה של מינוס 80 מעלות צלזיוס לשימוש עתידי.

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Research

JoVE Journal

Biochemistry

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

289 Views.  La Trobe University. כדי להתחיל, מקמו את דגימת השריר המיובש בהקפאה מתחת למיקרוסקופ הסטריאו וצפו בה בהגדלה נמוכה. בעזרת זוג אחד של רקמות עדינות המנתחות מלקחיים, החזיקו את השריר במקומו והפרידו את צרורות הסיבים הקטנים עם המלקחיים האחרים. בודדו צרור אחד של סיבים והפרידו אותם בעדינות עד למיצוי מקטע...

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

Fiber Type Identification of 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.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

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.

MHCI and MHCII Analysis of Single Fibers Isolated from Freeze-Dried Muscle Samples Using Dot Blot

To start, prepare the four sheets of filter paper, each measuring 12.5 by 7.5 centimeters. Stack two sheets together and place the stack in transfer buffer to soak through. Place the PVDF membrane in 95%ethanol and agitate the membrane on a rocker for one minute.Recollect the ethanol. Immerse the membrane in transfer buffer. And agitate for two minutes.Next, lay the soaking filter paper stack on a flat, movable surface, such as a large lid and flatten it using a roller. Using a gel releaser or tweezers, carefully position the PVDF membrane on the stack. Afterward, place a single sheet of dry filter paper on this membrane and glide the roller over the paper to absorb any excess buffer that's on the membrane's surface.Lift off the top filter paper without rubbing against the membrane. Take isolated fiber samples from the freezer and thaw them at room temperature before centrifuging and thoroughly re-suspending the sample. Place a one microliter droplet of each sample in the center of the designated membrane area.Avoid touching the membrane with the pipette tip. Allow the sample droplets to absorb entirely into the membrane for 15 minutes. Then using a gel releaser or tweezers, carefully lift the membrane from the damp filter paper stack.Place it onto a dry sheet of filter paper and leave it for at least five minutes to dehydrate. Reactivate the membrane after the sample spots have turned completely white. Then proceed with immunolabeling the target protein.Learn how to use the signal panel intensity to classify the intensity of the MHC isoforms and Actin signals from the dot blot images. Strong, medium and little target protein detection is shown by saturated, moderate and faint signals respectively. For fiber type identification, initially compare the myosin heavy chain IIA and myosin heavy chain one results.Document the fibers that display only a single myosin heavy chain isoform with either saturated or moderate signal intensity. Record the fibers detected with Actin and no detection of myosin heavy chain one or IIA as a potential type 2X. If a faint myosin heavy chain isoform signal is present with a moderate desaturated Actin signal, record it as unidentified.Discard samples with faint or undetected target proteins. Fibers displaying saturated or moderate signals of both MHCIIA and MHC1 should not be used for fiber type-specific preparation. After MYO1D ID, prepare type one and two samples by combining fibers with saturated or moderate signals for the respective MHC isoform.Use 10 microliters of each fiber type-specific sample and separate them on a precast gel by SDS page, following the manufacturer's guidelines. Use a gel imager to detect the target MHC isoform, following the manufacturer's protocol. Compare the signal intensity of each MHC isoform in all fiber type-specific samples.Fiber type ID is confirmed by the correct MHC isoform at moderate or saturated signal intensity. Immunolabeling of the dot blot allowed the identification of type two fibers, type one fibers and potential type 2X fibers. The fiber type-specific samples were validated, using western blotting and myosin heavy chain specific antibodies.