Targeting the Expression of Long Noncoding RNAs in Murine Satellite Cells from Single Myofibers

[Abstract] LncRNAs have been recently implicated in the epigenetic control of muscle differentiation and their functional characterization has traditionally relied upon in vitro models of myogenic differentiation. However, the use of experimental paradigms to specifically target lncRNAs expression in muscle stem cells (MuSCs), also known as satellite cells, represents an important requisite to interrogate their function in more physiological contexts. Since isolation and culture of single myofibers preserves satellite cells within their physiological niche underneath the surrounding basal lamina, this procedure represents the optimal approach to follow satellite cell dynamics ex-vivo , such as activation from quiescence, expansion of committed progenitors, differentiation, and self-renewal. Here, we detail an optimized protocol to isolate viable single myofibers from the extensor digitorum longus (EDL) skeletal muscle of adult mice and to manipulate the expression of lncRNAs by antisense LNA GapmeRs-mediated knock-down (KD). Furthermore, we describe a method of EdU incorporation that, coupled to lncRNA KD and subsequent immunofluorescence analysis of proliferating, differentiating, and satellite cell-specific markers, permits the inference of lncRNAs function on muscle stem cells dynamics.

[Background] Long noncoding RNAs (LncRNAs) represent a class of transcripts longer than 200 nucleotides, which are mostly devoid of protein-coding capacity (Rinn and Chang 2012;Fatica and Bozzoni, 2014).They are found both in the cytoplasm and in the nucleus, and modulate gene expression by influencing genome organization via epigenetic, transcriptional, and post-transcriptional mechanisms (Quan et al., 2015;Cai et al., 2016).As such, they are emerging as critical modulators of several biological processes, including stem-cell mediated regeneration and differentiation.
LncRNAs have been recently implicated in the epigenetic control of different steps of muscle differentiation (Lu et al., 2013;Ballarino et al., 2015 and2018;Zhu et al., 2017;Desideri et al., 2020).However, examination of lncRNA function during myogenesis has traditionally relied on ablation studies performed on established myoblast cell lines (i.e., C2C12) or primary cells cultured in proliferating (highserum) and differentiating (low-serum) conditions.These experimental settings allow discrimination of the role of lncRNAs only in the proliferation and differentiation steps, preventing their functional characterization on other crucial phases of in vivo myogenesis.Indeed, formation of new skeletal have been usually referred to as satellite cells (Mauro, 1961).In resting adult muscles, satellite cells are present in a post-mitotic quiescent state.However, upon injury, satellite cells activate and proliferate to expand a pool of committed myogenic progenitors that, upon expression of muscle-regulatory factors, enter the muscle differentiation program to form new muscle fibers (Figure 1).A small fraction of the expanded pool avoids terminal differentiation to self-renew and replenish the pool of quiescent stem cells (Biferali and Mozzetta, 2019).
Therefore, to infer the function of lncRNAs along the different stages of satellite cell-mediated myogenesis, it is important to rely on an experimental model that allows for monitoring of satellite cell activation from the quiescent state to their expansion and commitment to differentiation, and even to self-renewal (Mozzetta, 2016).To this end, we optimized previous protocols to isolate intact and viable single myofibers (Pasut et al., 2013;Mozzetta, 2016;Pegoli et al., 2020) from the extensor digitorum longus (EDL) muscle of wild-type mice for the purposes of subsequent manipulation of lncRNAs expression and phenotypical characterization by EdU incorporation and immunostaining (Cipriano et al., 2021).This isolation procedure preserves satellite cells within their physiological niche underneath the surrounding basal lamina.Thus, once cultured ex-vivo in non-adherent conditions and in the presence of serum and growth factors, satellite cells associated with the myofibers start to proliferate, giving rise to a progeny visible as a round group of cells attached to the fiber (Figure 1).This cluster represents the progeny of a single satellite cell that, upon 72-96 h of culture, comprises either differentiating myoblasts or self-renewing cells.The protocol described here details how to interfere with lncRNAs expression by LNA GapmeRs-mediated knock-down and optimization of subsequent immunostaining assays for assessment of MuSCs-associated myofibers.

Procedure
Attention: Keep everything sterile if planning to culture the fibers.
A. Preparation 1. Fill an ice bucket with ice.
3. Pre-heat the "wash solution" and "growth medium for fibers" solution at 37°C. 4. Coat 2 ml tubes (one tube per sample) with FBS.Add 1 ml of FBS to the tube.Move it up and down until the sides of the tube are completely covered, and then discard the FBS.
Note: The tube needs to be coated to keep the myofibers in suspension; otherwise, they will stick to the tube's wall.We use FBS for coating as this will also be in the growth medium.
5. Add 2 ml of the "digestion solution" to each coated tube.Keep it on ice.
6. Pre-heat the water bath at 37°C.

B. EDL isolation
Attention: This step must be performed as fast as possible before rigor mortis.For this reason, we suggest performing EDL isolation from one mouse at a time.
Note: Always handle the muscle from tendon to tendon and avoid touching the bulk of the muscle to preserve myofiber integrity.
1. Sacrifice the mouse using a method approved for your research, such as CO2 or cervical dislocation.
2. Wet the skin and the fur of the mouse with 70% ethanol.

D. Single myofiber isolation
Attention: 1.This step needs to be performed as fast as possible to avoid fiber contraction.When the myofibers are kept for too long on the "digestion plate," they will start to shrink and contract, leading to myofiber damage.

2.
To avoid contraction, pre-heat all the solutions to 37°C.The myofibers are sensitive to temperature and should be kept constantly at 37°C.Tip: If it is possible, perform myofiber isolation from one mouse at a time.In any case, keep all the plates containing the fibers in the incubator while they are not being used.
1. Prepare two 60 mm plates per mouse with 4 ml of pre-heated "wash solution" for the cleaning step.Tip: It is possible to perform this step using the tip of a P1000 micropipette cut with scissors or with a sterile glass Pasteur pipette previously broken at the neck.
Note: Be sure to coat the tip with the media before using it.
5. When more myofibers are released, pick out single myofibers with the coated tip of a P200 micropipette (as done in Step D3) and place them in the 60 mm plate used before in the cleaning step.In this case, also avoid the broken pieces, taking only the single myofibers.
6.When all the myofibers are in the cleaning plate, pick single myofibers in the direction of the fiber and plate them in the final dish (e.g., 24 well plate ~10-15 fiber/well or 12 well plate ~20-25 fiber/well) for culture with the pre-heated "Growth medium for fiber." Return the plates to the incubator at 37°C.

Attention:
The myofibers are cultured in suspension, so the volume should not be too small to allow fibers to float in the medium (e.g., in a 12 well plate, it should not be less than 1 ml).
The fibers can be fixed and analyzed immediately or cultured for satellite cell amplification.

E. Culture and transfection
Attention: The myofibers are sensitive to changes in temperature.
1. Keep the myofiber in the incubator for at least 4 h before transfection.The first hour after plating is critical, so keep the myofibers in the incubator to stabilize them.2. To perform the transfection, mix 1 μl/ml of lipofectamine with 49 μl of Optimem in one 1.5 ml tube per sample.In another 1.5 ml tube, add 1 μl/ml of antisense oligos (GapmeRs) from a stock at a concentration of 50 μM per sample and mix 49 μl of Optimem.After 5 min, mix the content of the lipofectamine tube with the content of the GapmeRs tube and wait for 20 min.Pipet 100 μl of the mix directly to the culture media in each well, drop by drop.Incubate overnight (13-14 h).

Notes: a. The final volume should be 1 ml per well (for a 12 well plate).
b.We strongly recommended not performing more than one overnight (13-

H. Edu detection and immunofluorescence
Note: For immunofluorescence analysis, transfer approximately 50 myofibers in 2 ml tubes per condition.
1. Coat a 2 ml tube/each sample with FBS (as described in Step A3).
2. Under the microscope, remove as much supernatant as possible, and then pick the single myofibers (in the direction of the myofiber) with a coated P200 micropipette tip and transfer them to the bottom of the 2 ml coated tube.
Attention: When pipetting the myofibers, always coat the micropipette tip with FBS to prevent myofiber loss or disruption (they tend to attach to the sides of the uncoated tube or tip).
3. Let the fibers settle down to the bottom of the tube for 10 min and then remove the supernatant.
Leave the myofibers in no less than 150 μl of PBS.Tip: It is a good trick to compare the volume with another 2 ml tube containing 150 μl of water.
4. Add 500 μl of Triton 0.5% to each tube and incubate for 10 min at room temperature.

5.
Remove the supernatant and wash with 1 ml of PBS for 10 min.

3 .
Position the mouse under the hood.Pull up and cut the skin from the ankle to remove the skin and to expose the tibialis anterior (TA) and the extensor digitorum longus (EDL) (Video 1).www.bio-protocol.org/e4209Bio-protocol 11(21): e4209.DOI:10.21769/BioProtoc.4209Copyright Macino et al.This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).Please cite this article as: Macino, M. et al. (2021).Targeting the Expression of Long Noncoding RNAs in Murine Satellite Cells from Single Myofibers.Bio-protocol 11(21): e4209.DOI: 10.21769/BioProtoc.4209.Video 1. Procedure for EDL isolation.(This video was made at the Department of Biology and Biotechnologies of University Sapienza, according to guidelines approved by the Institutional Animal Care and Use Committee of the Department of Biology and Biotechnology of University Sapienza, the Italian Ministry of Health, and local authorities according to Italian law; Protocol N° 7FF2C.4-AuthorizationN° 746/2016-PR.)4. Carefully cut the tendon of the TA muscle from the ankle, grab the loose tendon with the tweezers, and gently pull up the muscle.Then cut the TA muscle under the knee to expose and have a good visualization of the EDL muscle (Video 1). 5. Cut the tendon of the EDL at the base, just over the ankle, and gently pull the muscle until it remains attached only by the tendon on the knee.By keeping the EDL from the ankle tendon, isolate the muscle cutting the tendon over the knee (Video 1).Place the muscle inside a coated tube with the "digestion solution" and leave the tube on ice.6. Repeat Steps B4 and B5 with the other leg.Add the second muscle to the coated tube with the "digestion solution" and place it on ice.C. Digestion 1. Incubate the tube from 45 min to 1 h in a pre-warmed bath at 37°C.Gently shake every 10 min.Note: Invert the tubes a couple of times; do not shake vigorously to avoid breaking the myofibers.Tip: It is possible to see the state of the digested muscle looking through the tube against a source of light.The muscle should be in a loose state.2. At the end of the incubation, give a strong shake to release the myofibers from the digested muscle.Tip: It is possible to see the released myofibers looking through the tube against light before and after the final shake.www.bio-protocol.org/e4209Bio-protocol 11(21): e4209.DOI:10.21769/BioProtoc.4209Copyright Macino et al.This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).Please cite this article as: Macino, M. et al. (2021).Targeting the Expression of Long Noncoding RNAs in Murine Satellite Cells from Single Myofibers.Bio-protocol 11(21): e4209.DOI: 10.21769/BioProtoc.4209.

2 .
Transfer the digestion solution containing the fibers to one of the 60 mm plates with the "wash solution."Note: Myofibers should appear long, smooth, unbroken, and slightly translucent (Figure 2, red arrow).

Figure 2 .
Figure 2. Representative image of a newly isolated single myofiber (red arrow) in the washing plate.Black arrows indicate broken pieces that should be avoided when collecting the single myofiber.

3 .
Coat the tip of a P200 micropipette by pipetting one time with the "wash solution."Under the dissecting microscope (Zeiss; Stemi DV4), with the coated tip, take one by one every single myofiber in the direction of the fiber to avoid damage, collect multiple fibers in the tip, and gently release them into the second clean 60 mm plate containing 4 ml of pre-heated "wash solution" (cleaning step).www.bio-protocol.org/e4209Bio-protocol 11(21): e4209.DOI:10.21769/BioProtoc.4209Copyright Macino et al.This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).Please cite this article as: Macino, M. et al. (2021).Targeting the Expression of Long Noncoding RNAs in Murine Satellite Cells from Single Myofibers.Bio-protocol 11(21): e4209.DOI: 10.21769/BioProtoc.4209.Tip: Slightly slope the plate (and consequently move the media) to better visualize the translucent myofibers.Attention: a. Avoid the broken or contracted pieces (Figure 2, black arrow) and take only the intact single myofibers.b.Critical step!Be as gentle and as fast as possible.All the cleaning procedures over the hood should not take more than 10 min.4.After picking all the visible intact myofibers, gently pipette the digested muscle to release the myofibers still attached to it.Under the microscope, it is possible to see the myofibers being released.

6 .
For EdU detection, myofibers are stained using Click-iT EdU Alexa Fluor 594 HCS Assay (Invitrogen): a. Dilute the 10× solutions (Click-iT EdU buffer additive Component E 10× and Click-iT reaction buffer Component C 10×) provided in the kit ten times to 1× by adding the proper volume of water (e.g., 50 μl of E 10× + 450 μl of water).b.Then prepare the Click-iT reaction cocktail mix, mixing 425 μl of Component C solution 1×, 20 μl of CuSO₄, 1.25 μl of Alexa Fluor azide, and 50 μl of Component E solution 1× to obtain ~500 μl of the mix.Note: Reagents should be added to the mix in this specified order.c.Then add 200 μl of cocktail mix per sample of fibers to be stained.Incubate 30 min in the dark at room temperature, and then wash with 200 μl of reaction rinse buffer provided in the kit.Notes:i.Perform the EdU detection assay before standard immunofluorescence.ii.If you perform EdU detection, all the steps of the immunofluorescence should be performed in the dark.7.Remove the supernatant and wash with 1 ml of PBS for 10 min.8.Remove the supernatant and add 1 ml of block solution (10% FBS in PBS).Leave for 1 hour at room temperature, preferably on a shaker in slow agitation.

9 .Recipes 1 .
Let the tube settle for 10 min in a vertical position and then remove the supernatant, leaving the myofibers in ~150 μl.Add the primary antibodies diluted in 10% FBS in PBS (Ki67 at 1:100 and Pax7 at 1:10).The final volume should not be less than 300 μl.Incubate the tube overnight (13-14 h) at 4°C.Tip: When preparing the mix with the antibodies, we recommend calculating the needed concentration of the antibody for the final volume but adding 150 μl less of FBS 10% in the mix www.bio-protocol.org/e4209Bio-protocol 11(21): e4209.DOI:10.21769/BioProtoc.4209Copyright Macino et al.This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).Please cite this article as: Macino, M. et al. (2021).Targeting the Expression of Long Noncoding RNAs in Murine Satellite Cells from Single Myofibers.Bio-protocol 11(21): e4209.DOI: 10.21769/BioProtoc.4209.manual trituration that could cause myofiber damage.Digestion solution (2 ml per mouse) Collagenase I 10 mg DMEM high-glucose + Pyruvate (Gibco) 2 ml Pen/Strep 100× 20 μl Attention: i.The quantity and the state of the Collagenase I is critical for the success of this procedure ii.Always keep the collagenase I and the solution on ice.When using mice with fluorescent reporter genes, we recommend keeping this solution in the dark.a. Measure the quantity of Collagenase I very carefully.b.Place it in a 50 ml tube.In a sterile environment, add the media and the antibiotic and vortex carefully until the Collagenase I is completely dissolved.c.Filter the solution through a 0.22 μm filter with a 50 ml syringe into a new 50 ml tube.Tip: It is possible to lose some of the volume in the filtering step, so it is better to start with a larger volume.

2 .
Wash solution (50 ml) DMEM high-glucose + Pyruvate (Gibco) 45 ml Horse serum 10% 5 ml Mix the ingredients Before use, pre-heat the solution at 37°C Note: Ensure that the solutions are sterile.Tip: If the fibers are to be transfected, we recommend not adding any antibiotics in this solution, as they will interfere with the transfection efficiency.3. Growth medium for fibers DMEM high-glucose + Pyruvate (Gibco), 39.5 ml FBS 20%, 10 ml FGF 2.5 ng/ml Chick embryo extract (CEE) 1%, 500 μl Mix the ingredients and filter them through a 0.22 μm filter with a 50 ml syringe into a new 50 ml tube.Before use, pre-heat the solution at 37°C.Note: Ensure that the solutions are sterile.Tip: If the fibers are to be transfected, we recommend not adding any antibiotics in this solution, as they will interfere with the transfection efficiency.