This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.
Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.
Individual myofibers are large, highly organized syncytial cells. There are a few cell-culture models for muscle; however, these models have as their major limitation that they do not fully differentiate into mature myofibers. For example, the C2C12 and L6 cell lines are derived from mice and rats, respectively 1-4. Under conditions of serum starvation, the mononuclear "myoblast-like" cells cease proliferation, undergo cell cycle withdrawal, and enter into the myogenic program forming multinucleated cells with sarcomeres, referred to as myotubes. With prolonged culture conditions, myotubes may exhibit contractile properties and "twitch" in culture. Human cell lines have also now been established 5. In addition to these immortalized cell lines, mononuclear myoblasts can be isolated from muscle and under the similar conditions of serum starvation will form myotubes. These cell lines and primary myoblast cultures are highly useful because they can be transfected with plasmids or transduced with viruses and used to study basic cell biological processes. However, these cells, even when induced to form myotubes, lack many of the salient features of mature muscle organization. Specifically, myotubes are much smaller than individual mature myofibers and lack the normal shape of myofibers. Critically, myotubes lack transverse (T-) tubules, the membranous network required for efficient Ca2+ release throughout the myoplasm.
An alternative method to primary myoblast or myogenic cell lines entails using mature myofibers. Transgenesis can be employed to establish expression of tagged proteins, but this method is costly and time consuming. In vivo electroporation of mouse muscle has emerged as a preferred method for its speed and reliability 6-10.
Methods for in vivo electroporation and the efficient isolation of myofibers have been optimized for the mouse flexor digitorum brevis (FDB) muscle 6. The methods can be completed readily and are minimally invasive to induce in vivo expression from plasmids. This approach is now combined with high resolution imaging methods including imaging after laser disruption of the sarcolemma 6,7. The combination of fluorescent dyes and expression of fluorescently labeled proteins can be used to monitor cell biological processes in mature myofibers.
The methods in this study were performed in ethical accordance with the Northwestern University Feinberg School of Medicine Institutional Animal Care and Use Committee (IACUC) approved guidelines. All efforts were made to minimize suffering.
1. Experimental Procedure for in vivo Electroporation of the Flexor Digitorum Brevis (FDB) Muscle Bundle in Mouse
2. Experimental Procedure for Isolation of the Flexor Digitorum Brevis (FDB) Fibers
3. Experimental Procedure for Visualization of Laser-induced Membrane Damage using Confocal Microscopy
Electroporation is a minimally invasive technique that efficiently introduces purified plasmid DNA into the flexor digitorum brevis (FDB) muscle bundle (Figure 1A). Seven days post transfection fluorescently tagged protein is visualized in isolated myofibers (Figure 1B). Isolated fibers are then plated and prepared for imaging on a confocal microscope. Fibers can be subjected to laser induced injury. FM dye can be used to identify the site of membrane damage (Figure 1C)
During the digestion process, a small number of myofibers may be injured (white arrow) (Figure 2A). Damaged myofibers can be identified by membrane blebbing present at the sarcolemma (black arrow). The damaged fiber is not suitable for further experimentation and should not be used. Representative confocal images showing the expression of a fluorescent fusion protein within the isolated myofibers Figure 2B. Utilization of the CMV promoter drives optimal protein expression in myofibers. DIC imaging shows an optimal fiber. FM 4-64 is present at the membrane at low levels prior to damage (Figure 2C).
Representative image of a myofiber loaded with FM 4-64 imaged on a Leica SP 5 confocal microscope. The sarcolemma is centered in the field of view (white boxes) and ROI crosshair (white x) is aligned on the crisp, fluorescent edge of the sarcolemma in an area devoid of myonuclei (Figure 3, middle). Nuclei are avoided as they can influence FM dye analysis post imaging. Upon membrane damage, FM dye minimally enters normal myofibers (Figure 3, right). Muscle fibers defective in membrane repair will display increased levels of FM dye uptake upon laser-induced damage.
Figure 1. Schematic of in vivo Electroporation and Laser Damage Protocol. (A) Hyaluronidase is injected into the footpad of an anesthetized mouse. Plasmid DNA is then injected and voltage is applied. (B) Seven days post injection, the flexor digitorum brevis (FDB) bundle (white arrow middle panel) is isolated from the footpad leaving behind exposed tendons (black arrow). (C) Fibers are plated and laser-induced damage is performed on live myofibers. Left panel,scale 50 µm. Right panel, scale 5 µm. Please click here to view a larger version of this figure.
Figure 2. Isolated Myofibers Expressing Fluorescently Tagged Proteins. Seven days post electroporation, protein expression is detected throughout the myofiber. (A) The isolation procedure results in a small number of unusable myofibers with distinct membrane blebbing (black arrow) and membrane disruptions (white arrow). Scale 5 µm. (B) Representative image of a healthy myofiber. Evenly spaced sarcomeres are visualized in DIC imaging (left). A myofiber expressing annexin A6 tagged with GFP (center panel). There is minimal FM dye signal prior to damage (right panel). Scale 5 µm. Please click here to view a larger version of this figure.
Figure 3. Alignment of Laser Crosshairs on the FM-positive Sarcolemma. The region of interest on the sarcolemma is centered within the field of view. The sarcolemma is magnified (white dotted box) and ROI crosshairs aligned on the sharp FM-positive edge of the sarcolemma in an area devoid of nuclei (center panel). Upon damage, FM dye enters at the site of injury (right panel, white arrow). Scale 5 µm. Please click here to view a larger version of this figure.
The use of electroporation to study fluorescently tagged proteins in vivo is ideally suited for the study of muscle given the absence of suitable cell line models that faithfully replicate the entire cell structure of muscle. This protocol describes the utilization of electroporation and high-resolution confocal microscopy to provide detailed imaging of protein localization and translocation after laser wounding. This method can be adapted to study other cellular processes including cytoskeleton reorganization, trafficking, and fusion.
Using this protocol, up to 90% of myofibers within the FDB muscle bundle express plasmid, and this can be seen readily by fluorescence microscopy both before and after myofiber dissociation. There is typically a gradient of expression with higher expression observed nearer to the injection site. Because of the gradient of expression, the effect of varying protein expression can be monitored from a single experiment. As with all transfection methods, this approach is limited by issues related to overexpression. Notably, with high level expression, protein aggregation can occur. In our experience, the mCherry fluorescent tag demonstrates more aggregation than the enhanced green fluorescent protein (eGFP) tag reducing resolution and sharpness of protein domains (for example see Figure 4 in 7). Additionally, mTurquise2 and Venus fluorescent tags both demonstrate sufficient fluorescence expression. However, both proteins are less bright than eGFP.
This method can be used to image living myofibers under a variety of conditions including testing pharmaceutical agents and cell wounding. We used laser injury to disrupt the sarcolemma, but other wounding processes could be adapted to this method. To this end, formation of membrane blebs created by osmotic shock as well as membrane injury induced by rolling glass beads can also be assessed utilizing electroporated myofibers 11,12. For laser wounding, the type and power of the laser, as well as the objective will influence the length of time needed to create a membrane lesion 13,14. Furthermore, external buffers, specifically the concentration of calcium and FM dye within the buffer will influence the repair process 13,15. This methodology has been tested on both Nikon and Leica confocal imaging systems and both are capable of performing the technique. FM 4-64 is well suited for experimentation with green fluorescent protein 7. Others have used FM 1-43, another lipophilic dye that binds negatively charged phospholipids with an excitation peak at 470 nm and emission peak at 610 nm, which limits the application of this dye 13,15,16. Photobleaching and phototoxicity are both possible outcomes due to over-imaging. Determining the proper balance between exposure time, image frequency and number of channels imaged are parameters easily manipulated to reduce these effects.
The authors have nothing to disclose.
This work was supported by National Institutes of Health grants NS047726, NS072027, and AR052646.
DMEM+A2:D32D42A2:D31AA2:D29 | Life Technologies | 11995-073 | Dissection Media Dilution: 50ml +100g BSA |
Collagenase, Type II | Life Technologies | 17101-015 | Fiber Digestion Dilution: 160mg / 4ml DMEM (stock 40mg/ml aliquot) |
Falcon 12-well dishes | Fisher Scientific | 877229 | Fiber Digestion |
Ringers Solution | Fiber Digestion and Imaging Dilution: 146 mM NaCl 5 mM KCl 2 mM CaCl2 1 mM MCl2 10mM HEPES pH7.4 in H2O |
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1ml Pipettes | Axygen | T-1000-C-R | Digestion |
Razor Blades | Personna | 94-120-71 | Digestion |
Precision Glide 30G needle | BD Biosciences | 305128 | Dissection |
1x PBS (-/-) | Life Technologies | 14190-250 | Dissection |
Sylgard 184 | Fisher Scientific | 50-366-794 | Dissection |
Forceps | FST by Dumont | #5/45 | Dissection |
Forceps | Roboz | RS-4913 | Dissection |
Scissors | World Precision Instruments | 500260 | Dissection |
BSA | Sigma | A7906 | Dissection media Dilution: 100mg / 50ml DMEM |
Falcon 60mm dishes | Fisher Scientific | 08772B | Dissection- used to hold sylgard |
Clay | Electroporation | ||
Stimulator | Grass | s88x | Electroporation |
Clamps | Radio Shack | 270-356 | Electroporation |
Triadine | Aplicare | 82-220 | Electroporation |
Precision Glide 27G needle | BD Biosciences | 305136 | Electroporation |
Simulator | Grass | SIU-V | Electroporation |
Gauze | Covidien | 2146 | Electroporation |
Hyaluronidase | Sigma | H4272 | Injection Dilution: Add 160ul PBS (1X) to 40µl 5x stock |
1cc U-100 Insulin Syringe (28G1/2) | BD Biosciences | 329420 | Injection |
Eppendorf Tubes | Fisher Scientific | 02-682-550 | Injection |
35mm glass bottom Microwell dish | MaTek | P35G-1.5-14-C | Microscopy |
FM 4-64 | Life Technologies | T-13320 | Microscopy Dilution: Final 2.5 ug/ml |
FM 1-43 | Life Technologies | T-35356 | Microscopy Dilution: Final 2.5 ug/ml |
Endotoxinfree Plasmid Maxi Kit | Qiagen | 12362 | Plasmid Purification |