X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

Abstract

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.

SUMMARY:

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

INTRODUCTION:

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 group^1–3 and others^4,5 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 consists 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 proportional to the number of attached cross-bridges which closely correlated to the force generated in mouse skeletal muscle³. The meridional reflections which report periodicities within the thick and thin filaments can be used to estimate filament extensibility^1,2,6,7. Diffraction features not on the meridian and the equator are called layer lines which arise from the quasi-helically ordered thick and thin filaments. The intensity of myosin layer lines is closely related to the degree of ordering of myosin heads under various conditions^3,8. 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.

PROTOCOL:

1. Pre-experiment Preparation.

1.1) Prepare 500mL Ringer’s solution (contains: 145 mM NaCl, 2.5 mM KCl, 1.0 mM MgSO₄, 1.0 mM CaCl₂, 10.0 mM HEPES, 11 mM glucose, pH 7.4) freshly for each day of the experiment. Fill 200ml Ringer’s solution in a spray bottle and store at 4 °C fridge. Fill the dissecting petri-dish with Ringer’s solution and perfuse with 100% oxygen.

1.2) Prepare mounting metal hooks. The longer hook should be about 5cm long, and the shorter hook about 1cm long. Arrange all the dissecting tools, scissors, suture tying forceps, micro-scissors handy for use.

1.3) Connect and turn on all the equipment. This includes a combined motor/force transducer, motor/force transducer controller a high-power bi-phasic current stimulator and a computer controlled data acquisition and control system.

1.3.1 Turn on the data acquisition system and calibrate it in advance of the experiment⁹.

1.3.2 Connect the hoses from the thermal block on the sample holder to a refrigerated circulating bath and set the temperature to maintain the desired temperature in the chamber to between 10°C and 40 °C. This needs to be determined empirically ahead of time by setting the circulating bath to a range of temperatures and measuring the temperature in the chamber with a thermocouple.

2. Muscle preparation

2.1) Euthanizing the mouse

2.1.1. Euthanize the mouse by carbon dioxide inhalation followed by cervical dislocation.

2.1.2. Spray the skin on the hind limb with cold Ringer’s solution to prevent hair from blowing into the preparation. Remove the skin quickly to expose the muscles.

2.1.3. Amputate the hind limb and transfer it to a dissecting dish that has been filled with oxygenized Ringer’s solution, and then place under a binocular dissecting microscope.

2.2) Preparing a soleus muscle

2.2.1. Pin the hind limb down with the gastrocnemius muscle facing upwards. Cut the distal tendon of the gastrocnemius/soleus muscle group and lift the muscles gently and slowly by cutting away the fascia on either side of the gastrocnemius muscle using fine scissors. Isolate the gastrocnemius/soleus muscle group from the limb after freeing the proximal tendon of the soleus muscle.

2.2.2. Pin the muscle group containing the gastrocnemius muscle and the distal tendon down in the petri dish. Lift the soleus muscle gently via the proximal tendon and separate it from the gastrocnemius muscle leaving as much of the soleus distal tendon intact as possible.

2.3) Preparing an extensor digitorium longus (EDL) muscle.

2.3.1. Pin the hind limb down in the petri dish with the tibialis anterior muscle facing upwards. Cut the fascia along the tibialis anterior (TA) muscle and pull it clear using forceps. Identify and cut the distal tendon of the TA muscle. Lift the TA muscle and cut it out carefully without pulling on the EDL muscle.

2.3.2. Cut open the lateral side of the knee and expose the two tendons. Cut the tendons and lift the EDL muscle (medial muscle) by gently pulling the tendon. Cut the distal tendon once it is exposed.

2.4) Mounting the muscle

2.4.1. Pin down the muscle via the tendons, and trim all the extra fat, fascia and tendon away as much as possible. Insert one tendon into a pre-tied knot and tie the suture tightly with suture tying forceps. Tie the second knot on around the metal hook. Repeat the same procedure with the long hook on the other end of the tendon. Make sure that none of the body of the muscle is tied by the sutures. This will damage the preparation.

2.4.2. Attach the short hook to the bottom of the experimental chamber and the long hook to the dual mode force transducer/motor. Bubble the solution in the experimental chamber with 100% oxygen.

2.5) Optimizing stimulation protocols and muscle length.

2.5.1. Stretch the muscle to generate a baseline tension between 15 to 20mN before finding the best stimulus parameters. The stimulation voltage is set to 40V. The stimulation current is systematically increased until there is no additional increase in twitch force. The highest current found is increased by about 50% to ensure supra-maximal activation.

2.5.2. Find the optimal length, L₀, defined as the muscle length that give maximum twitch force, by increasing the muscle length and activating the muscle with a single twitch until the active force (peak force minus baseline force) stops increasing.

2.5.3. Perform a short tetanic contraction (1s activation) to test the mounting and stretch the muscle back to optimal baseline force if necessary. Record the muscle length in mm with a digital caliper.

3. X-ray Diffraction.

Note: The following description is for X-ray diffraction experiments done using the small angle X-ray diffraction instrument on the BioCAT beamline 18ID at the Advanced Photon Source, Argonne National Laboratory but similar methods could be employed on other beamlines such as ID 02 at the ESRF (France) and BL45XU at SPring8 (Japan). Beamline 18ID is operated at a fixed X-ray beam energy of 12KeV (0.1033 nm wavelength) with an incident flux of ~10¹³ photons per second in the full beam.

3.1) Choose a specimen to detector distance (camera length). Use a 1.8 m camera length for experiments examining the 2.7nm actin and high order myosin reflections such as 2.8nm meridional reflections. Use a 4–6 m camera for other experiments, where one is primarily interested in fine detail on the meridian and layer lines

3.2) Optimizing the position of the sample in beam.

3.2.1. Align the video camera by using a piece of heat sensitive paper to record the beam position (“a burn”). You can then use a video cross-hair generator to record the beam position or simply make a mark on the video screen with a marker pen.

3.2.2. Use the BioCAT supplied graphical interface to the sample positioner to move the muscle to the middle of the muscle. Oscillate the sample chamber at ~ 10–20 mm/s by moving the sample stage in order to spread the X-ray dose over the muscle during the exposure. Observe the sample as it moves to avoid large regions of fascia (contains collagen which will pollute the diffraction patterns) and to ensure that it stays illuminated during the entire path of its travel.

3.3) Setting up the CCD detector for high resolution patterns from muscle in defined static states (resting, or during isometric contraction).

3.3.1. Set up the exposure time and exposure period in the graphical user interface to the control software. Take a dark background image before taking the exposure and repeat this procedure every two hours or after changing of exposure time to correct any drift in the detector readout electronics.

3.3.2. Attenuate the X-ray beam to desired value for the exposure. Then take an image. It is not possible to take sequences of images with this detector. The CCD detector also needs several seconds to read out an individual image.

3.4) Setting up the pixel array detector for a time resolved experiment

3.4.1. Set up the number of images, exposure time, exposure period in the graphical user interface. The pixel array detector needs at least 1ms to readout. The maximum frame frequency for photon counting detector is 500Hz. Use the photon counting detector output signal to control the X-ray shutter.

3.4.2. Attenuate the beam to the desired intensity. Arm the detector and wait for the trigger from the data acquisition system. Synchronize the mechanical and X-ray data by triggering them at the same time.

Note: The exact exposure time and exposure period should be determined on a case by case basis for the information desired and the observed lifetime of the sample in the beam. Attenuate the beam in order to use no more X-ray beam than is needed to provide analyzable data in the chosen exposure period.

4. Post-experiment muscle treatment.

4.1) Recover and weigh the muscle after each mechanical and X-ray experiment. Calculate the cross-sectional area of the muscle using the measured muscle length and the muscle mass¹⁰ assuming a muscle density of 1.06 g/ml¹¹.

4.2) Stretch the muscle to the experimental length and fix it in 10% formalin for 10 min. Measure the sarcomere length using a video sarcomere length measuring system (such as the model 900B system from Aurora Scientific Inc.) from a series of fixed fiber bundles selected from locations throughout the entire muscle cross section.

REPRESENTATIVE RESULTS:

Isometric tetanic contraction.

Any kind of classic muscle mechanical experiment, such as isometric or isotonic contractions, can be performed with simultaneous acquisition of X-ray patterns. Figure 1A shows the experimental setup for mechanical and X-ray experiments. An example diffraction pattern taken during an isometric tetanic contraction is shown in Fig. 1B. The muscle was held at resting for 0.5s before activated for 1s. The mechanical recording stops 1s after the stimulus. The X-ray patterns were collected continuously throughout the protocol at 1ms exposure time and 2ms exposure period.

Figure 1. Mechanical and X-ray experiment setup and protocol.

(A) The muscle is mounted on one end to a hook inside the experimental chamber and the other end to a dual mode motor/force transducer. It is held between two kapton film windows to allow the X-rays to pass through. The chamber is filled with Ringer solution perfused with 100% oxygen throughout the experiment. (B) The mechanical protocol for X-ray experiments on a muscle during tetanic contraction.

X-ray diffraction patterns.

The muscle X-ray diffraction pattern can give nanometer resolution structural information from structures inside the sarcomere. Muscle X-ray diffraction patterns are composed of four equivalent quadrants divided by the equator and the meridian. The equatorial pattern arises from the myofilament packing within the sarcomere perpendicular to the fiber axis, while the meridional patterns report structural information from the myofilaments along the muscle axis. The remaining reflections not on the equator or the meridian are called layer lines. Layer lines arise from the quasi-helically arrangement of molecular subunits within the myosin containing thick filaments and the actin containing thin filaments. The myosin-based layer lines are strong and sharp in patterns from resting muscle (Fig 2A), while actin-based layer lines are more prominent in patterns from contracting muscle (Fig 2B). Difference patterns obtained by subtracting the resting pattern from the contracting pattern (Fig 2C) can shed light on structural changes during force development in healthy and diseased muscle. By following these structural changes at the millisecond time scale of the molecular events during muscle contraction, the X-ray diffraction patterns can reveal substantial structural information (Fig 2D).

Figure 2. EDL X-ray diffraction patterns.

EDL muscle X-ray diffraction pattern from resting (A) and contracting (B) muscle. (C) The difference pattern between resting and contracting pattern. The blue region indicates high intensity in resting pattern, while the yellow region represents high intensity in contracting pattern. (D) X-ray diffraction pattern from a 1ms exposure with EDL muscle.

Data Analysis using MuscleX.

Here is an example of equatorial reflections analysis using the “equator” routine in the MuscleX package (Fig3). MuscleX is an open-source analysis software package developed at BioCAT¹². The equatorial intensity ratio (I_1,1/I_1,0) is an indicator of the proximity of myosin to actin in resting muscle(Fig 3A), while it is closely correlated to the number of attached cross-bridges in contracting (Fig 3B) murine skeletal muscle³. The intensity ratio, I_1,1/I_1,0, is about 0.47 in resting muscle and about 1.2 in contracting muscle. The distance between the two 1,0 reflection (2*S_1,0) is inversely related to the inter-filament spacing. Detailed documentations and manuals for MuscleX are available online¹².

Figure 3. Data analysis of equatorial patterns using MuscleX.

The background subtracted equatorial intensity ratio profile (while area) and first five orders (green lines) were fit to calculate the intensity of each peak.

DISCUSSION:

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 disease^1–3 especially with the increased availability of genetic modified myopathy mouse models. 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 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 1ms exposure (See Figure2D). For an analyzable first myosin layer line reflection, 10ms total exposure time is typically required. To collect higher order meridional reflections such as the M15 (2.8nm myosin meridional reflection) and the 2.7nm actin meridional reflection, typically at least 1s total exposure is required but more than 2s total exposure is recommended for high accuracy measurements.

The choice of the optimal X-ray detector for your experiment is important. For the most detailed X-ray patterns a customized CCD detector 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 new photon counting detector providing 75 μm real resolution at maximum 9000Hz and 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 patterns¹³, in the near term the only option is to measure it after the experiment as described here.