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

Physiologically intact mouse skeletal muscle can produce high quality X-ray diffraction patterns containing much structural information that can provide insight into physiological processes. X-ray diffraction is the only technique that allows the acquisition of high resolution structural information from living muscle tissue under real physiological conditions in real physiological time. Many muscle diseases are inherited.

With increased availability to genetically modify most models of myopathies, the X-ray diffraction can provide structural insights in disease mechanisms and indicate therapeutic strategies. Most extensor digitorum longus and soleus muscles are particularly convenient for this purpose. But many other muscles in small animals can be dissected intact and handled in a similar way.

Before beginning the procedure, turn on the combined motor force transducer, the motor force transducer controller with a high power biphasic current stimulator, and a computer control data acquisition control system. Next, spray the skin on the hind limb of the mouse with cold Ringer's solution and use fine dissection scissors to cut the skin around the thigh. Using number five forceps, quickly pull the skin down to expose the muscles and amputate the hind limb.

Place the limb in an elastomer-coated dissecting dish containing oxygenized Ringer's solution and place the dish under a binocular dissecting microscope. To harvest the soleus muscle, pin the hind limb with the gastrocnemius muscle facing upwards. Use fine scissors to cut the distal tendon of the gastrocnemius/soleus muscle group.

Cut away the fascia on either side of the gastrocnemius muscle to allow the muscles to be lifted gently and slowly away from the bone. Then free the proximal tendon of the soleus muscle. Pin down the muscle group containing the gastrocnemius muscle and the distal tendon in the dish.

Lift the soleus muscle gently via the proximal tendon to separate it from the gastrocnemius muscle, leaving as much of the soleus distal tendon intact as possible. To harvest the extensor digitorum longus or EDL muscle, pin the hind limb in the dish with the tibialis anterior muscle facing upwards and cut the fascia along the tibialis anterior muscle. Use forceps to pull the fascia clear and cut the distal tendon of the tibialis anterior muscle.

Lift the tibialis anterior muscle and cut it out carefully without pulling on the EDL muscle, and cut open the lateral side of the knee to expose the two tendons. Cut the proximal tendon, leaving us much of the tendon as possible still attached to the muscle and gently pull on the tendon to lift the EDL muscle. Then cut the distal tendon once it is exposed.

To mount the harvested muscle, pin down the muscle via the tendons and trim as much of the extra fat, fascia and tendon as possible. Insert one tendon into a pre-tied knot and use suture tying forceps to tie the suture tightly. Tie the second knot on around the metal hook and repeat the procedure on the other end of the tendon.

Then 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. To optimize the stimulation protocol and muscle length adjust the micro manipulators attached to the transducer motor to generate a baseline tension between 15 to 20 millinewtons to find the best stimulus parameters to stretch the muscle.

Set the stimulation voltage to 40 volts. The stimulation current will be systematically increased until there is no additional increase in twitch force. To find the optimal length, increase the muscle length and activate the muscle with a single twitch until the active force stops increasing.

Perform a one second tetanic contraction to test the mounting and stretch the muscle back to the optimal baseline force as necessary. Then record the muscle length in millimeters with a digital caliper. To determine the beam position, use a piece of X-ray sensitive paper that produces a dark spot in response to X-rays and a video crosshair generator to create a crosshair aligned with the burn mark on the paper.

Using the BioCAT-supplied graphical user interface to the sample positioner, center the muscle on the beam position and move the sample stage to oscillate the sample chamber at 10 to 20 millimeters per second to spread X-ray dose all over the muscle during the exposure. Observe the sample as it moves to avoid large regions of fascia that contain collagen and to ensure that it stays illuminated during the entire path of its travel. Arm the detector and wait for the trigger from the data acquisition system.

Then trigger the mechanical and X-ray data at the same time to synchronize them. The X-ray patterns will be collected continuously throughout the protocol with a 10-millisecond exposure time and a 50-millisecond exposure period. In this representative isometric tetanic contraction the EDL muscle was held at rest for 0.5 seconds before it was activated for one second, followed by a 1.5-second relaxation.

The muscle X-ray diffraction pattern can give nanometer resolution structural information from structures inside the sarcomere. Myosin-based layer lines containing thick filaments are strong and sharp in patterns from resting muscle, while actin-based layer lines containing thin filaments are more prominent in patterns from contracting muscle. Difference patterns obtained by subtracting the resting pattern from the contracting pattern can shed light on structural changes during force development in healthy and diseased muscle.

By following these structural changes at the millisecond timescale of the molecular events during muscle contraction and relaxation, the X-ray diffraction patterns can reveal substantial structural information. In this representative equatorial reflections analysis using the Equator routine in the open source MuscleX package, the equatorial intensity ratio indicates the proximity of myosin to actin in resting muscle and is closely correlated to the number of attached cross-bridges in contracting murine skeletal muscle. The distance between the two 1, 0 reflection is inversely related to the interfilament spacing.

A clean dissection is the key for a successful intact muscle X-ray experiment, so try to avoid any mechanical damage during muscle preparation. Any standard physiological protocol with whole muscles can be implemented in these experiments and can be used to study muscle activation, relaxation and cross-bridge behavior during rapid mechanical transience. Genetic manipulation of mice are becoming increasingly sophisticated.

New transgenic mouse models will allow more specific and insightful experiments to be designed to indicate new therapeutic directions for human myopathies.