We present detailed protocols for performing small-angle X-ray diffraction experiments using intact mouse skeletal muscles. With the wide availability of transgenic mouse models for human diseases, this experimental platform can form a useful test bed for elucidating the structural basis of genetic muscle diseases
Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal muscle. Mouse skeletal muscle has been shown to produce high quality X-ray diffraction patterns on third generation synchrotron beamlines providing an opportunity to link changes at the level of the genotype to functional phenotypes in health and disease by determining the structural consequences of genetic changes. We present detailed protocols for preparation of specimens, collecting the X-ray patterns and extracting relevant structural parameters from the X-ray patterns that may help guide experimenters wishing to perform such experiments for themselves.
Synchrotron small-angle X-ray diffraction is the method of choice for studying the nm-scale structure of actively contracting muscle preparations under physiological conditions. Importantly, structural information from living or skinned muscle preparations can be obtained in synchrony with physiological data, such as muscle force and length changes. There has been increasing interest in applying this technique to study the structural basis of inherited muscle diseases that have their basis in point mutations in sarcomeric proteins. The muscle biophysics community has been very active in generating transgenic mouse models for these human disease conditions that could provide ideal test beds for structural studies. Recent publications from our group1,2,3 and others4,5 have indicated that the X-ray patterns from the mouse extensor digitorum longus (EDL) and soleus muscles can provide all the diffraction information available from more traditional model organisms such as frog and rabbit psoas skeletal muscle. An advantage of the mouse skeletal muscle preparation is the ease of dissection and performing basic membrane-intact, whole muscle physiological experiments. The dimensions of the dissected muscle have sufficient mass to yield highly detailed muscle patterns in very short X-ray exposure times (~millisecond per frame) on third generation X-ray beamlines.
Muscle X-ray diffraction patterns consist of the equatorial reflections, the meridional reflections as well as the layer line reflections. The equatorial intensity ratio (ratio of the intensity of the 1,1 and 1,0 equatorial reflections, I11/I10), is closely correlated to the number of attached cross-bridges, which is proportional to the force generated in mouse skeletal muscle2. The meridional reflections that report periodicities within the thick and thin filaments can be used to estimate filament extensibility1,3,6,7. Diffraction features not on the meridian and the equator are called layer lines, which arise from the approximately helically ordered myosin heads on the surface of thick filament backbone as well as the approximately helically ordered thin filaments. The intensity of myosin layer lines is closely related to the degree of ordering of myosin heads under various conditions2,8. All of this information can be used study the behaviors of sarcomeric proteins in situ in health and disease.
Synchrotron X-ray diffraction of muscle has been historically done by teams of highly specialized experts but advances in technology and the availability of new data reduction tools indicate that this need not always be the case. The BioCAT Beamline 18ID at the Advanced Photon Source, Argonne National Laboratory has dedicated staff and support facilities for performing muscle X-ray diffraction experiments that can help newcomers to the field get started in using these techniques. Many users choose to formally collaborate with BioCAT staff, but an increasing number of users find they can do the experiments and analysis themselves reducing the burden on beamline staff. The primary goal of this paper is to provide training that provides potential experimenters with the information they need to plan and execute experiments on the mouse skeletal muscle system either at the BioCAT beamline or at other high flux beamlines around the world where these experiments would be possible.
Recent publications from our group showed that X-ray patterns from the mouse skeletal muscle can be used to shed light on sarcomeric structural information from muscle in health and disease1,2,3 especially with the increased availability of genetic modified mouse models for various myopathies. High resolution mechanical studies on single fibers or small bundles combined with X-ray diffraction is best done by experts. If, however, more modest mechanical information will suffice for your purposes, the whole muscle preparation allows collection of detailed X-ray patterns from a simple preparation.
A clean dissection is key to a successful combined mechanical and X-ray experiment. It is very important not to pull on the target muscle as well as other muscles associated with the soleus or EDL muscles during dissection since this could tear parts of the muscle and lead to reduced force. It can also lead to damaged internal structure that will degrade the X-ray patterns. Since everything will scatter in the X-ray beam, it is important to cleaning away any extra fat, the collagen in fascia as well as any hairs or loose bits of tissue while doing the following protocol. To reduce additional compliance in the muscle preparation, it is also important to securely tie the tendons to the hooks, as close as possible to the muscle body without damaging it.
Different X-ray exposure times can provide different kinds of information from the same muscle. Using the full beam on 18ID, an analyzable equatorial pattern can be obtained in a 1 ms exposure (See Figure 2D). For an analyzable first myosin layer line reflection, a 10 ms total exposure time is typically required. To collect higher order meridional reflections such as the M15 (2.8 nm myosin meridional reflection) and the 2.7 nm actin meridional reflection, typically at least 1 s total exposure is required but more than 2 s total exposure is recommended for high accuracy measurements.
The choice of the optimal X-ray detector for the experiment is important. For the most detailed X-ray patterns a customized CCD detector, such as the one at BioCAT with ca. 40 µm pixels and ~65 µm point spread functions in the phosphor, can provide patterns with high dynamic range and good spatial resolution but can only take one frame at a time. For time resolved experiments, the photon counting pixel array detector at BioCAT can collect X-ray patterns at 500 Hz. The 172 µm pixel size with this detector, however, does not provide sufficient spatial resolution for detailed studies of the inner part of the meridian but is adequate for most other purposes. BioCAT acquired a high-resolution photon counting detector providing 75 µm real resolution at maximum frame rate of 9,000 Hz. Similar detectors of this type are expected to supplant current detectors for muscle studies over the next few years.
With the very high fluxes of X-rays at third generation synchrotrons, radiation damage is a serious concern. It is always a good choice to attenuate the beam to deliver no more beam than is needed to observe the desired diffraction features. The same total X-ray exposure can be achieved by prolonging the exposure time from an attenuated beam. An advantage of photon counting pixel array detectors is that individual frames can be summed together with no noise penalty. Even then, radiation damage is possible. Signs of radiation damage includes drop of maximum force of contraction, smearing of layer line reflections, even change of muscle color.
One of the limitations of the intact mouse skeletal muscle preparation is the difficulty in obtaining sarcomere length from the intact muscle during the experiments. The muscles are too thick for video microscopy and laser diffraction. While with future developments it may be possible to estimate sarcomere length directly from the diffraction patterns14, in the near term the only option is to measure it after the experiment as described here.
The authors have nothing to disclose.
This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This project was supported by grant P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090-01 from NIGMS. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institute of General Medical Sciences or the National Institutes of Health.
#5 forceps | WPI | 500342 | |
4/0 surgical suture | Braintree Sci | SUT-S 108 | |
aquarium air stone | uxcell | a regular air stone from a pet store would be fine | |
CaCl2 | Sigma-Aldrich | C5670 | |
CCD detector | Rayonix Inc | MAR 165 CCD | |
data accquisition system | Aurora Scientific Inc | 610A | |
elastomer compound | Dow Corning | Sylgard 184 | |
Glucose | Sigma-Aldrich | G8270 | |
HEPES | Sigma-Aldrich | H3375 | |
High resolution photon counting detector | Dectris Inc | EIGER X 500K | |
high-power bi-phasic current stimulator | Aurora Scientific Inc | 701 | |
Iris Scissors | WPI | 501263-G | |
KCl | Sigma-Aldrich | P9541 | |
MgSO4 | Sigma-Aldrich | M7506 | |
micro scissor | WPI | 503365 | |
motor/force transducer | Aurora Scientific Inc | 300C-LR | |
NaCl | Sigma-Aldrich | S9888 | |
petri-dish | Sigma-Aldrich | CLS430167 | |
photon counting detector | Dectris Inc | Pilatus 3 1M | |
Stainless Steel wire | McMaster-carr | 8908K21 | |
Suture Tying Forceps | WPI | 504498 | |
Video sarcomere length measuring system | Aurora Scientific Inc | 900B |