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Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.
Myofibers are the functional contractile units of skeletal muscle. While myofibers are established during embryogenesis by fusion of myoblasts into myotubes, processes involved in their growth and repair continue throughout life. These processes are supported by myogenic progenitors known as satellite cells that are located between the basal lamina and the plasmalemma of the myofiber (1,2);; for a schematic and electron microscope image see Fig. 1. In a growing muscle at least some of the satellite cells are proliferating, and contribute myonuclei to the enlarging myofibers, whereas in intact adult muscles most satellite cells are quiescent. In response to a variety of conditions, ranging from increased muscle utilization to muscle injury, satellite cells can enter the cell cycle, producing progeny that fuse into existing myofibers, or form new myofibers (3,4);. Satellite cells are considered stem cells because in addition to giving rise to progeny needed for myofiber repair, they can self-renew (5,6);. It is not known, however, if all satellite cells are identical with regard to their amplification and renewal potential (6,7);. Insights into the cascade of cellular and molecular events controlling satellite cell myogenesis are essential for understanding the mechanisms controlling muscle maintenance as well as for developing strategies to enhance muscle repair after trauma or in myopathic diseases (8–11);.
Satellite cells were initially identified using electron microscopy by their location under the myofiber basal lamina (1,12,13); (Fig. 1). More recently it has become possible to monitor satellite cells by light microscopy based on the expression of a range of markers that can be detected by immunostaining (14);. In particular, the specific expression of the paired box transcription factor Pax7 by satellite cells and the availability of an excellent antibody for immunodetection of this protein provide a uniform means to identify satellite cells in their native position in a range of species, such as mouse, rat, and chicken (15–18);. In humans, however, Pax7 expression may not necessarily identify all satellite cells (14);. Additionally, genetically manipulated reporter mice permit direct detection of satellite cells based on specific expression of a fluorescent tag or of beta-galactosidase (7,16,19,20);. We demonstrated that transgenic expression of GFP under the control of nestin regulatory elements (NES-GFP) allows detection of satellite cells in freshly isolated myofibers. NES-GFP mice also facilitate the isolation of satellite cells using fluorescent-activated cell sorting (FACS) and subsequent studies of purified populations (7,16);.
Satellite cell progeny can be distinguished from their quiescent progenitors based on distinctive gene expression patterns (2,4,7);. In particular, the myogenic regulatory factors MyoD and myogenin have been used extensively to monitor progeny of satellite cells (21–24);. Proliferating progeny (myoblasts) continue to express Pax7, but distinctly from their quiescent progenitors, also express MyoD. A decline in Pax7 along with the induction of the muscle-specific transcription factor myogenin mark myoblasts that have entered into the differentiation phase and subsequently fuse into myotubes. Re-emergence of cells that express Pax7, but not MyoD (reserve cells), define a self-renewing population of satellite cells (2,5–7,22–24);.
Two main cell culture approaches have been employed in the study of satellite cells: (i) primary myogenic cultures prepared from mononucleated cells dissociated from whole muscle; and (ii) cultures of isolated myofibers (also referred to below as “fibers”) where the satellite cells remain in their in situ position underneath the myofiber basal lamina. Protocols for obtaining primary myogenic cultures involve releasing satellite cells from their niche. Steps of mincing, enzymatic digestion and repetitive triturations of the muscle are required for breaking down both the connective tissue network and the myofibers in order to release the satellite cells from the muscle bulk. These steps are followed by procedures for removing tissue debris and reducing the contribution of non-myogenic cells typically present in primary isolates of myogenic cells (6,16,22,25–29);. In contrast, protocols for isolating individual muscle fibers result in the release of intact myofibers that retain satellite cells in their native position underneath the basal lamina (16,21–23,26);. These protocols allow the study of satellite cells and their progeny in their in situ position on the myofiber, and after they migrate from the parent myofiber.
This chapter describes two protocols used in our laboratory for isolation and culture of single myofibers from mouse skeletal muscles (22,30);. One protocol, first introduced by Bekoff and Betz (31); and further developed by Bischoff (32,33);, has been adopted by us for studies of satellite cells in isolated myofibers from both rats (21,26,34); and mice (22,35,36);. In this case, single myofibers are isolated from the flexor digitorum brevis (FDB) muscle of the hind feet. Because these FDB myofibers are short and do not get tangled, typically multiple myofibers are processed and cultured together. A second approach, introduced by Rosenblatt and colleagues (37,38);, allows isolation of longer myofibers from a variety of muscles, including extensor digitorum longus (EDL), tibialis anterior (TA) and soleus (5,20,37,38);, and has been used extensively by our laboratory as well (2,7,16,22,39);. These longer myofibers can get tangled, and therefore, when working with muscles such as the EDL, the released myofibers are typically processed and cultured individually. The EDL single myofiber isolation procedure described here has also been adapted in our laboratory for diaphragm, masseter and extraocular muscles. Both the short and long myofibers are cultured in dishes that have been pre-coated with commercially available matrices that facilitate rapid and firm adherence of the myofibers to the dish surface, as detailed below. It is worth noting, however, that in addition to the matrix-attached myofiber cultures described herein, other laboratories have introduced approaches where isolated myofibers are cultured in suspension (23,40);.
The current chapter contains background, procedural and reagent updates for FDB and EDL myofiber isolation. We also include figures depicting step-by-step “real live” images of respective muscle dissection and harvesting, not illustrated in our previous 2005 report on myofiber isolation and culture (30);. In addition, new to this chapter is a description of myofiber isolation from diaphragm, masseter and extraocular muscles. Table 1 compares the two approaches of myofiber isolation from FDB and EDL muscles and the specific use of each procedure, while representative micrographs of FDB and EDL myofiber cultures are shown in Figs. 2 and and3,3, respectively. Figs. 4 and and55 are presented later in the method section to assist the investigator in the dissection of the FDB and the EDL muscles. Protocols for immunocytochemical analysis of satellite cells in cultures of FDB and EDL myofibers and of freshly isolated myofibers are also included in the chapter.
Altogether, to achieve the isolation of intact myofibers, it is of utmost importance to delicately manipulate the muscle of interest. Following the procedures and protocol notes detailed in this chapter, investigators can successfully isolate, culture and analyze myofibers from well studied EDL and FDB fibers, and also use these protocols as a framework for the study of other muscles.
The following facilities are required for the cultures described in this chapter:
C57BL/6 mice, 2– 5 months old, maintained according to institutional animal care regulations. Aged mice (up to 33 months old) and other mouse strains have also been used in our studies following the same myofiber isolation procedures (e.g., (7,22);; see Note 3);. When harvesting muscles for fiber preparation, we prefer cervical dislocation for euthanizing mice as this method is more rapid and minimizes muscle stiffening that occurs after death. Muscle stiffening can make the isolation of single fibers more difficult and decrease overall fiber yield.
The information in this introductory section is provided to assist in the identification of the FDB muscles. The FDB is a superficial, multipennate, broad and thin muscle of the foot and paw (33,42);; it arises from the tendon of the plantaris as three slender muscles converging into long tendons. At the base of the first phalanx it divides into two, passes around the tendon of the flexor hallucis longus obliquely across the dorsum of the foot, and ends as the tendons insert into the second phalanx of the 2nd through the 5th digits. As the FDB contracts, digits 2–5 are flexed. For additional details about the anatomy of the FDB muscle see Note 15.
For uniformity, we typically use only the hindlimb muscles in our studies. Fig. 4 depicts “real-live” images of steps in FDB muscle isolation that emphasize the location of the specific tendons that are handled during the process. It is of utmost importance to delicately manipulate the muscle of interest only at the tendons during its excision and further processing.
Isotonic PureCol collagen can be prepared during the settling of myofibers. The isotonic mixture should be kept on ice. Stock PureCol is an acidic solution, and when made isotonic, it gels rapidly if not maintained at 4°C (see Note 1);.
The information in this introductory section is provided to assist in the identification of the EDL muscles. The EDL muscle is situated at the ventral-lateral aspect of the hindlimb, running from the knee to the ankle, extending to the 2nd – 5th digits (42);. The EDL actually consists of four combined muscle bellies and their tendons; the bellies arise from the lateral condyle of the tibia and the front edge of the fibula (2 tendons at the origin of the muscle). The tendons lie close to each other and appear as one glistening white tendon that continues down to the surface of the ankle. At the ankle joint it separates to 4 tendons, each attached to one of the 2nd-5th digits. As the EDL contracts, the 4 digits are extended. For additional details about the anatomy of the EDL muscle see Note 15.
As detailed in Subheading 3.1. we typically use only the hindlimb muscles in our studies. Fig. 5 depicts “real-live” images of the steps in EDL muscle isolation with emphasis on the location of the specific tendons that are handled during the process. It is of utmost importance to delicately manipulate the muscle of interest only at the tendons during its excision and further processing.
The EDL single myofiber isolation procedure described here has also been adapted in our laboratory for the isolation of myofibers from the diaphragm, masseter and extraocular muscles (see Note 18);.
Steps 1–6 are done on ice, unless otherwise noted.
This is done to minimize adherence of myofibers to plasticware and glassware used during the isolation process.
Use a stereo dissecting microscope throughout the procedure, which involves rinses of the digested muscle bulk and a 3-step sequence of muscle bulk trituration to release myofibers. All Pasteur pipettes used in this process should be fire-polished. It is recommended to spend no more than 5–7 min at a time per each trituration step. When processing multiple EDLs it is a good strategy to alternate between muscle bulks so that only one EDL is outside of the incubator at a time in order to minimize muscle cooling. Additionally, the recommended number of rinses of the digested muscle and of individual myofibers as detailed in this section should not be overlooked. The myofiber rinses are essential for minimizing the contribution of non-myogenic cells that are released from the muscle bulk during the enzymatic digestion. Unless myofibers are well rinsed, such non-myogenic cells will be co-isolated with the myofibers and eventually produce many progeny in the rich culture conditions.
This section details current protocols used in our laboratory to fix myofiber cultures for immunofluorescent studies of satellite cells and their progeny. FDB myofiber cultures are typically fixed with ice-cold methanol (the preferred fixative when working with dishes coated with PureCol collagen), whereas the EDL myofiber cultures are typically fixed with paraformaldehyde that is pre-warmed to room temperature. Ideal fixatives for FDB or EDL myofiber cultures are not necessarily the optimal fixatives for specific antigen detection. Thus, when analyzing single myofibers via immunofluorescence, fixatives should be optimized for both preserving the myofibers and the antigens being analyzed. Fixation protocols described in this section are also appropriate for detecting proliferating satellite cells in single myofibers by autoradiography following labeling with 3H-thymidine (32,34); or when analyzing proliferation using bromodeoxyuridine (2,16,41,43);. All steps are done in a sterile manner. Handling antibodies strictly in the tissue culture hood minimizes possible bacterial contamination and helps maintain antibody stocks for years.
EDL myofiber cultures are fixed by slightly different approaches when fixing long term cultures (detailed in this section) or when fixing freshly isolated (Time 0; T0 fibers; detailed in the following section). Importantly, when fixing T0 cultures and early time points, use a stereo dissecting microscope throughout the procedure to ensure that the fibers are not lost or become damaged. All additional wash steps should be performed using a 9” fire-polished Pasteur pipette. At later time points, when fibers and emanating cells are adhering strongly to the matrix, one may not necessarily require the aid of a microscope when fixing or rinsing the cultures.
Plate EDL fibers as previously described in Subheading 3.2.7. but instead of plating the fiber in a well containing 500-µl medium, transfer the fiber with residual DMEM (~150 µl) into the center of a Matrigel-coated well that has not received growth medium. The fiber should be sitting in a droplet of DMEM, on top of the Matrigel to ensure that it does not dry out. After the desired number of fibers has been dispensed (1 per well), place the plate back in the incubator for 3 h to allow the fibers to adhere to the Matrigel. Minimize the amount of time that the fibers remain outside of the incubator and do not subject the plate to sudden motion as this can cause the fibers to contract or lose contact with the plate substrate.
The authors are grateful to the granting agencies that funded this study. Our current research is supported by grants to Z.Y.R. from the National Institutes of Health (AG021566; AG035377; AR057794) and the Muscular Dystrophy Association (135908). The development the FDB myofiber isolation protocol described in this chapter could not be possible without the valuable contribution of our former lab member, Anthony Rivera, and previous funding from the Muscular Dystrophy Association, the Cooperative State Research, Education and Extension Service / US Department of Agriculture (National Research Initiative), the National Institutes of Health, and the Nathan Shock Center of Excellence in the Basic Biology of Aging, University of Washington.