Skeletal Muscle - Science topic

Hi Aleksandar Dishkelov I am using high glucose DMEM.

Hi Aman Ullah,

Thank you so much for your reply.

Sonny.

Dropping muscles directly into liquid nitrogen causes severe freezing artifacts -- ice crystals will form, which causes crushing/tearing of the muscle fibers. To avoid these artifacts, muscles can be frozen in isopentane cooled in liquid nitrogen: place a small container of isopentane in a larger container of liquid nitrogen; wait until the isopentane starts to freeze; you are ready to put in your muscle.

Good luck!

To answer your question, the values of kc, ke, and d in the force-velocity relationship may not be the same for all types of muscles. The force-velocity relationship is influenced by several factors, including the muscle fiber type, the size and length of the muscle, and the contractile history of the muscle. Therefore, the parameters kc, ke, and d may vary depending on the specific muscle being modeled.

In order to determine the values of kc, ke, and d for different muscles, experimental data is typically needed. These parameters can be determined by fitting the force-velocity relationship to experimental data obtained from measurements of muscle force and shortening velocity under different conditions, such as varying levels of activation or different types of contractions. Alternatively, these parameters can be estimated using a combination of experimental and modeling techniques, such as inverse modeling or parameter optimization.

It is also important to note that the force-velocity relationship is often simplified in muscle models, and may not fully capture the complex interactions that occur within the muscle during contraction. Therefore, while the parameters kc, ke, and d can provide useful insights into the behavior of skeletal muscles, their values should be interpreted with caution and should be validated against experimental data whenever possible.

Traditionally, Non-ionic detergent is used for isolating nuclei from tissues. It is the detergent that causes nuclear clamping. You can try detergent-free method or a detergent -containing kit but is able to generating single nuclei.

It seems that the fixation and also the cutting procedures had some problems. Moreover, my feeling is that you may had some problems in freezing the tissue, as it seems that some cells were de-freezed.

Georgia State University Alumni Association's 40 Under 40 Class of 2023 (gsu.edu)

It will depend on the species studied. For exemple, in chicken cells slow MyHC are expressed and sarcomeric myosin (MF20 antibody) will be also used.

Dear Rabeah Al-Temaimi, thank you for your response!

Images shown in the mentioned article are somewhat similar to ours, it's quite possible that our mitochondria may also lose their membrane potential. Early or late apoptotic events are also possible, as I find in muscles with severely disorganized myofibrils.

Cristae swelling is rather unlikely (yet still possible) - on image taken on higher magnification (attached below) I cannot see swollen cristae, those hyperintensities seem to be independent of visible cristae location. Specimens for current study were fixed in 2.5% glutaraldehyde right after biopsy - however, I am not present during tissue collection, it's possible that between biopsy and muscle fixation more time passed.

Alpers-Huttenlocher syndrome can be rather excluded - it has early onset and we have adult patients only - although, from what I see, muscle mitochondria looks similar indeed.

Thank you again for your response, it was very helpful! Now I have some foothold for further research.

Jeffrey J Brault Thank you for your response and recommendations. We'll optimize the time of treatment following these and others in literature through a time point study.

Interesting, I'll read more on how serum starvation increases pAMPK and whether the effects are additive. This wasn't my question but will definitely look into this too.

Attached is a review article we wrote several years back about the linkage of muscle loading to bone properties and how that changes with aging.

Check if your AB is ok by SDS PAGE (Coomassie staining) and/or Western Blot (probe the blot with anti-species HRP).

If it is ok, do you have a positive control for PFK?

If in doubt, include another positive control for your detection system, i.e. an AB or serum from rabbit in this case.

I would not blame brands in general, esp. not CST. Have you called their tech service for assistance?

I also want to calculate muscle fibers CSA in H&E staining using imageJ. Thresholding cannot be used but manually it can be done and it will be time taking process. I want to know about how many and how the fibers are randomly selected in a sample field, how many sample fields will be needed and what should be the magnification?

Thank you Yasuhiro Nishida I will check for it

Did you load the cells using the acetoxymethylester form of Fura-2? Your comments suggest you did. Use fresh DMSO to solubilize the Fura-2 AM. Which excitation filter set are you using? Which emission filter? Also ensure your cells are in glass and not plastic chambers. Plastic absorbs UV wavelengths more so than glass.

If you can't get Fura-2 to work, switch, at least temporarily, to using Fluo-3 or Fluo-4. They are easier to work with.

Proteinase K should not ever digest DNA or RNA, unless it is contaminated with nucleases. Make sure you use a DNase- and RNase-free source of the enzyme.

If you want to study calcium dynamics in skeletal muscle fibers, many calcium sensitive dyes works but the selection depends on many factors including: Imaging equipment available (Framerate, UV and/or Visible excitation); necessity of real calcium concentration (ratiometric vs single wavelength dyes); which type of calcium event (quiescent, stimulated or small events like sparks) and even the location (cytosol, sarcoplasmic reticulum or mitochondria).

Without knowing your specific need and equipment, is difficult to recommend something. However, For general use:

I had successfully used in isolated skeletal muscle fibers and whole EDL muscle ratiometric UV dyes (Indo-1, Fura-2) and singlewave visible dyes (Fluo-5F and Rhod-2) for stimulated calcium events (single twitch or 40 Hz tetanic).

Fluo-4 is good for smaller calcium events, but almost too bright/sensitive for stimulated events.

Fluo-5N can be used for study calcium in sarcoplasmic reticulum.

Hope that helps!

The holes seem to have formed due to ice crystals rupturing the tissue. If the experimental animal wasn't perfused with saline followed by PFA, I would recommend a post-fixation of the tissue.

The most common procedure to avoid artifacts is to freeze muscle in isopentane cooled in liquid nitrogen. In other words, the isopentane is added to a small container, and this container is put in a larger container containing liquid nitrogen. When the isopentane is cooled to its freezing temperature -- it starts to becomes a white solid -- then it is ready.

I assume you are talking about fixation for light microscopy and not both light and electron microscopy. If the former, why fix at all? Why not cut frozen sections from unfixed muscle in a cryostat? If a staining procedure requires fixation (many don't), you can do it during the staining procedure and the fixation procedure can be done custom to the staining procedure.

The above may be helpful. Trizol is for RNA purification, if it does not work, you could try another anothermethod. good luck.

Hello Sabrina Salvatore

Here are some posibillities:

Journal of Exercise Physiology - but there are charges for submission and publication

- they do not state what is the length required for a review

Journal of Clinical Exercise Physiology

- for review/commentary type articles, you need to write to them (at the journal) first to discuss your topic before they will consider your idea for an article

As per the protocol, protein estimation is needed. I don't run western blots but run a coomassie protein assay to determine protein levels of the samples against standards. In our lab, we have never used western quantification as it is not needed.

Ta Hu I use the Aurora system as well to study both dorsi and plantar flexion. We do the study in vivo using the foot plate so we can assess it weekly. If you want gastrocnemius specific force then you will have to do a non survival surgery where you cut off the tendon and attach solely the gastrocnemius to the lever arm. That will provide more information such as optimal length. When I use the foot plate i don't measure solely gastrocnemius, since it is plantar flexion,, both gastroc and soleus are involved.

My guess would be that the kit might not have a specific protocol for preparation of muscle samples... and the inhibitors in the kit might not be specific enough for the other ATPases in skeletal muscle. This kit was used quite recently to study activity in skeletal muscle (Journal of Physiology), so it might be worth looking into:

I have done this a few thousand times. Your procedure should work. I might do a couple things differently. 100 ml of isopentane is more than I would use. It will take a while for the isopentane to cool down. However, if you are willing to wait, you can use 100 ml. The most important step, I believe, to getting artifact-free sections is to cool the muscle specimen/OCT as quickly as possible. I will plunge the specimen/OCT to the bottom of the cooled isopentane as fast as possible. I do not let the specimen/OCT stay in the isopentane more than 10-12 seconds. If it stays longer, I often see cracking of the OCT.

Javier Russell-Guzmán Thank you for your response. Yes I am also doing western blotting for the GLUT 4 as I am mainly looking to determine its quantity in my samples after treatment. The part about the GLUT 4 translocation is also really insightful and helpful, I will read more about it. How about the ELISA though? Is it not recommended to use for determining the GLUT 4 quantity in skeletal cells?

Dear Cristiane Matsuura

I suggest you go through these articles first:

1. https://www.microscopemaster.com/histochemistry.html

2. https://www.sciencedirect.com/topics/immunology-and-microbiology/silver-impregnation

3. https://pubmed.ncbi.nlm.nih.gov/29701111/

4.

5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048572/

6.

7. https://www.nature.com/articles/s41467-018-07676-y

8. https://iopscience.iop.org/article/10.1088/2053-1591/aa6922

Kind Regards

Sten Knutsen's latency relaxation of the thin filaments

Thanks, Gordon for your reply.

Actually, I want to make sure that my calculation is correct.

Briefly, the final concentrations were normalized based on muscle weight and then multiplied by 2, because I separated extracted lipids in two vials equally. This is how I calculated lipid concentration.

Dan Robbins Thank you for your reply. Yes, I am aware of that paper that you mentioned. But I am really looking for more of a "real-time" method of glycogen synthesis.

These journal articles should address what you have requested:

Laumonier T, Jacques Menetrey J. Muscle injuries and strategies for improving their repair. J Exp Orthop. 2016;3:15. doi: 10.1186/s40634-016-0051-7.

Karalaki M, et al. Muscle regeneration: Cellular and molecular events. In Vivo. 2009 (Sept);23(5):779-796.

L. Baoge, E. Van Den Steen, S. Rimbaut, et al. Treatment of skeletal muscle injury: A review, Int Scholarly Res Notices. 2012; doi: 10.5402/2012/689012.

Spinal muscular atrophy is inherited in an autosomal recessive pattern

There 4 known types

Spinal muscular atrophy type 0 is evident before birth and is the rarest and most severe form of the condition. Affected infants move less in the womb, and as a result they are often born with joint deformities (contractures). They have extremely weak muscle tone (hypotonia) at birth. Their respiratory muscles are very weak and they often do not survive past infancy due to respiratory failure. Some infants with spinal muscular atrophy type 0 also have heart defects that are present from birth (congenital).

Spinal muscular atrophy type I (also called Werdnig-Hoffmann disease) is the most common form of the condition.

Spinal muscular atrophy type II (also called Dubowitz disease) is characterized by muscle weakness that develops in children between ages 6 and 12 months. Children with this type can sit without support, although they may need help getting to a seated position.

Spinal muscular atrophy type III (also called Kugelberg-Welander disease) typically causes muscle weakness after early childhood. Individuals with this condition can stand and walk unaided, but over time, walking and climbing stairs may become increasingly difficult. Many affected individuals require wheelchair assistance later in life. People with spinal muscular atrophy type III typically have a normal life expectancy.

Spinal muscular atrophy type IV is rare and often begins in early adulthood. Affected individuals usually experience mild to moderate muscle weakness, tremors, and mild breathing problems. People with spinal muscular atrophy type IV have a normal life expectancy.

In autosomal recessive inheritance, a person who has SMA has inherited two altered (mutated) copies of the SMN1 gene from his or her parents. The parents of an individual with an autosomal recessive inherited disorder such as SMA are carriers of one copy of the altered gene

RPS6 protein has phosphorylation sites, and its phosphorylated at S235 & S236.

1. The showing up of 15kDa in western blot might be due to loss of phosphorylation sites due to denaturing and reducing condition of SDS PAGE in western blot. Probably your antibody is detecting the non-phosphorylated form only.

2. When probed for another protein, no band might be due to reason that your antibody is recognizing non-phosphorylated form and there is only phosphorylated protein RPS6 in muscle.

Hi Adele,

You may try to remove myoglobin from your skeletal muscle extracts, which tends to interfere with many enzymatic reactions. My 2 cents...

Gedas

Thank you Gordon L Warren.

Muscle cell cultures are already in an atrophy state, i.e., there is minimal (if any) contractile loading. Perhaps it would be better to go in the other direction, i.e., increase contractile loading, so as to promote "hypertrophy". Electrically stimulate the cell cultures. This is harder than it sounds. Too much stimulation can kill cells.

Hello Steven,

Key in this kind of drying it a path out of the tisssue for the water. Muscle or other animal tissues can be a complex mix of substances, and it is therefore challenging to model the drying process well. Typically, dry until you reach a constant mass is the method, however, how you weigh the sample in the dryer is a challenge. This leads to long, perhaps over long, methods for drying tissues.

Challenge here is to keep the water all frozen. The tissues is complex, but also the places that the water exists are complex too, full of peptides, sugars proteins and salts, all of which change the freezing behaviour of water. The low pressure, 200mTorr = -40°C freezing / sublimation point, is a funtion of making sure that the whole sample remains frozen so that there is minimal tissue damage and creation of artefact in your sample. Remember, the vacuum pressure in the system will control the sublimation temperature of the water.

The thing on your side is that your samples are low mass. I would, if you can, ensure they are thin, and prefreeze them. However, the challenge is that most tissues processors use a shelf based freeze dryer which enables the tissue to be frozen on the shelf, this gives far superior control to a benchtop basic lab dryer. It also allows the addition of heat, which when in steady contition speeds up drying to a certain extent. To speed things up in a Benchtop, the best we can suggest is put the dryer in a sunny window such that IR from the sun will help out.

If you can, I suggest a couple of experiments with samples that you don't need for your research, e.g. use some meat from the store, and see how that goes. If you prep this like your samples, load up 3 or 4 flasks and then you can remove them sequentially say at 6 or 8 hour intervals to evaluate the process of drying. With such small samples, counterintuitive though this may sound, it may help the process to pre-freeze them a samll block of buffer to overcome the antifreeze effects of plasma.

Kind regards

Rob

Lara Ivanković why don't you try to analyze the samples with an additional method for verification of the results? Inhibition of 1,2,3-trihydroxybenzene is often used ( . Some comparisons with standard kits have also been published ( eg. https://faseb.onlinelibrary.wiley.com/doi/abs/10.1096/fasebj.21.6.a814)

Kits are often ungrateful as, in reality, each analytical method should be optimized for a specific purpose and quality parameters should be satisfied for given conditions (eg. not all kits work with all buffers, with the same sample preparation procedures, ...)

If you decide to double-check with the pyrogallol method I'd be happy to help.

Best,

Jan

I am not sure about this specific commercial kit, but for the original colorimetric DNPH method, the precipitation is normal and desirable after we add trichloroacetic acid to both test and blank samples, right after the derivatization with DNPH.

I recommend monoclonal antibodies from Abcam, Roche, Biocompare, and Sigma. They work excellent.

Thanks for your advice

sales@immunotex.ru

Hi Jawad, I've always felt it was ideal to at least stick with the same analysis method for both the submaximal and maximal contractions. So if the submaximal stimulation is a series of spikes at a certain frequency, and peak-to-peak (P-P) is the most appropriate method to get the amplitude, then you'd want the same for the maximal signal. However, P-P does not work well with MVCs, so a maximal M-wave from electrical nerve stimulation would be optimal. If that is not an option, and normalization can only occur with an MVC, then I'd use RMS as your analysis method for both the muscle stimulation and MVC (and not P-P).

Hello, did you successfully culture skeletal muscle organoids ?

Hi Peter,

That is what I always asked myself. And here is where I found a plausible answer

Regards

I did RNA isolation from skeletal muscles with double TRIzol method or combination of Trizol/ RNEasy column and got very good quality RNA. I believe that you might be taking too much tissue and too little RNAzol. Try to increase TRIzol volume (at least to 1ml) and decrease amount of tissue. I collected tissue (appr 20-30mg, or 5mmx5mmx5mm piece) at room temperature- (it's protected from RNAses in Trizol- no need to keep it at LN2 or 4C before final processing), placed it immediately into half of final volume of Trizol (0.5ml), dissected it with scissors into small pieces directly in Trizol, then homogenized directly in Trizol later in a day after collection of all the samples. RNA is protected in Trizol at least 24h at RT. After homogenization added another half of TRIzol (final volume of Trizol is minimum 9 volumes of tissue size but never less than 1mL)- divided TRIzol volumes in half just to avoid spill during homogenization. Then followed regular Trizol protocol. Your protocol looks OK except need of addition of EQUAL to aqueos phase volume of isopropanol. I also usually do 2 washes with 70% ethanol to get rid of salts before air drying of RNA pellet. After resuspension in water and DNAse I treatment I do either Trizol, either RNeasy column RNA purification again. If you don't plan DNAse I treatment (exon spanning primers and verified -RT controls), single round of RNA purification is enough. Once I had larger than normal piece of tissue and this caused exactly the same problem as you described.

hi paper,

you may coupling your macrophage staining with anti myosin heavy chain or perilipin immunostaining to mark fibers or adipocytes, respectively. Alternatively, you can perform a hematoxylin and eosin stain. In this case, adipocytes appear as white vacuolar unstained cells while fibers will be stained in red

Did you consider the approach followed in this article ?

Cardiotoxin works but is not my preferred way for inducing a traumatic skeletal injury. About 20 years ago, we compared it against using a freeze injury. The freeze injury was more consistent in the amount of injury induced. The problem with using cardiotoxin seemed to be related to the volume of cardiotoxin injected into the muscle. In our case, we were using the mouse tibialis anterior muscle. When injection volume exceeded a few microliters, there would be leakage of cardiotoxin from the muscle. FYI, I am attaching an article that describes our freeze injury technique. We do use it for studying muscle regeneration.

If you haven't already, the first thing you should do is confirm successful knock out of your gene/s. If a specific antibody is available, confirm the absence of protein by western blot or, even better, confirm its absence in skeletal muscle using IHC. Can you detect evidence of nonsense-mediated decay by in situ hybridisation?

If the knock out is successful, then you can explore potential reasons for the lack of phenotype, where one might be expected. One possibility is compensation by paralogous genes with similar function. Can you detect an increase in mRNA transcripts which might be compensating? Are they expressed in muscle?

Another possibility is that you are missing a phenotype. How are you looking for evidence of poor sarcomere assembly? You should, for example, stain for a range of myofibrillar proteins (e.g. MyHC, actin, alpha-actinin, titin.... etc)...

Cheers,

Mike.

Jana Tumova Thanks Jana!

The toothpaste may affect gut microbiota balance of the digestive tract thus affecting natural PH levels. The triclosan is proving s extremely aggressive. https://stm.sciencemag.org/content/10/443/eaan4116

What is the size of the structures, etc. you wish to assess? Are these small intracellular structures (e.g., calcium release channels) or ones that cross multiple cells (e.g., components of the extracellular matrix)? How heterogeneous are these structures, etc.? If they are very homogeneous from one cell to the next, then you can get by with a "small" specimen. On the other hand, if the structures are heterogeneous, your specimen will need to be "large" in order to provide a reliable assessment.

Thanks@Valeria Tananska

Hi Andrew,

It's many years since you posted this question, but I'm having similar difficulties at present and wonder if/how you resolved this?

Best,

David

https://www.gene-quantification.de/norm.html

Dear Prof. Andersson, Apologies for the delay in responding. Somehow I did notice any notification for your reply from research gate. Previously, our lab has developed a method to measure recovery kinetic of creatine following plantor flexion exercise. That method could only image 2D slice and hence, could distinguish inter-muscular difference between say lateral gastroc, medial gastroc etc from a single inplane image. I extended the method to enable mapping along the muscular length i.e. my method is 3D. So, I can measure difference between various muscle groups as well as if there is an abnormality within same muscle. I am writing "introduction" of my paper and would like to demonstrate the significance of my work in various creatine kinease disorders, which are non systemic and hence, the ability to scan multiple slice may bring unique information.

Dear Yulia Panina,

Thank you for your reply and I am sorry to write you back late. Could you please elaborate a bit in what perspectives piggybac was better than lenti? I cam to know through One of my colleagues that someone used sleeping beauty method which is similar to piggybac and was not happy with it. Thanks in advance.

Best practice (also enforced by many journals including JBC) is to first identify a protein that remains unchanged under your experimental conditions in your hands. If you choose a HKP from literature, you need to still prove stability of the protein under control and experiential conditions. Them after identifying the combined linear range for your target and HKP perform normalization expt.

https://www.licor.com/documents/fwf274p9k35hwym6p2keq397p4edho5o

Hi Akbar

Your results are highly dependent on the degrees of freedom definition. For instance, if you don't personnlize the axis of rotation of the knee, you may have unaccurate values. More particularly for moment which are computed with forces lever arms. How do you define this axis of rotation? Is it constant ?

And, if you wish to better understand your results you should have a look to the decomposition of the joint moment, more particularly for hips (abduction-adduction, flexion-extension and internal-external rotation moments). Or you could compute joint energies and powers.

Even if walking movement is mainly included into the saggital plane, some interesting information may be measured in the other ones.

And, depending on your objective you may also define the upper limbs. More particularly if at last, you would like to estimate muscle force production.

Dear you can do that by relating to lymphocytes ratio.

You might try using a sample clean-up kit like the Zymo Research DNA Clean & Concentrator Kit (https://bit.ly/2HfWwpX), or similar kits from other vendors.We use these often in our Lab.

Mouse skin is considerably thinner than a 3-mm diameter cylinder of muscle. Mouse skin thickness is in the 0.5-0.8 mm range.

Sure, I can send you the protocol.

About the sensitivity, I can not 100% guarantee that one reviewer won't ask for ATPase staining... However, I think it will be more likely that someone will ask for a myosin separation gel. At least, this is what happened to us, some years ago.

Moreover, I stayed many years in Stefano Schiaffino's lab, and I can tell you that we NEVER performed a single ATPase staining!

Hai,

You take 300 mg of tissue, cut and slice well. After that, you go for homogenate because, skeletal muscles are very hard. Then follow the QiagenRNEasy kit. You should wash properly in each step. Read at 260/280 nm. The ratio is 1.72 to 2.1.

You will get low ration means, run the Gel. Check the two bands.

After go for DNAse treatment.

Here is a quick methods paper describing lipid extraction from muscle tissue (human).

Michael Plank Benzonase would be a solution, but depends on the next steps - it is a contaminating protein added to do the sample, so if this sample will be used e.g. for MS, then I would be very careful with adding any new protein.

No it's the problem, my sample is really precious that's why i can use my classical protocol (Trizol for RNA, and special kit for DNA)

Hi Bodvael,

In my experience, I would suggest you to use AAV9 and AAV-DJ, which are good at in vitro cell infection. AAV-DJ is better in most of cell lines，but AAV9 may work better in primary cells.

Genemedi is expert in AAV production and genetherapy, you could find more information on this website: www.genemedi.net/i/aav-packaging

In general the tropism of AAV is determined by its capsid structure. The capsid of AAV9 preferentially binds to terminal N-linked galactose, a certain sugar structure which is present on the surface of a variety of cells (e.g. as part of glycoproteins). Additional protein(s) which serve as AAV9 receptor(s) need to be present on the cell membrane as well. One candidate is the transmembrane protein KIAA0319L (also termed AAVR). After binding to AAVR the AAVs are taken up by the cell via clathrin-mediated endocytosis togehter with AAVR. The amount of N-linked galactose in the cell membrane together with AAVR and/or other protein(s) that serve as AAV9 receptor(s) determines the amount of AAV9 being taken up by the cell. Skeletal muscle cells seem to present a substantial amount of these molecules on their surface. Keep in mind though, that AAV9 is not 100% specific for skeletal muscle cells and is also able to transduce a large number of different other cell types that also display the relevant AAV9 receptors.

You might find our article „redistribution of glucose from skeletal muscle to adipose tissue during catch-up fat“ published in Diabetes useful. The article can be downloaded from my profile and you can find there absolute values for glucose uptake in different tissues during a hyperinsulinemic-euglycemic clamp

Hi Annelise, this is a very nice paper.