Assessing the contractile properties of myofibrils isolated from striated muscle can be used to determine whether sarcomere dysfunction is a primary cause of muscle weakness due to mutations in sarcomeric proteins. This technique can be used to acquire data with a very high signal-to-noise ratio with nanonewton resolution. This protocol is also suitable to measure contractility in permeabilized cardiomyocytes.
Keep in mind that the force transducer is very fragile. So when removing the glue from the mounting needle or when gluing a myofibril, you need to have a steady hand, a lot of practice, and a lot of patience. Before mounting the tissue, place a homogenizer rod in the tube containing the muscle tissue.
Keeping the tube on ice, spin the rotor for 15 seconds on speed five. At the end of the homogenization, transfer 50 microliters of the resulting myofibril suspension and 250 microliters of relaxing solution onto a poly-HEMA coated slide in a tissue bath. Cover the bath with a lid to protect the mixture from dust and wait 5 to 10 minutes to allow the myofibrils to sink to the bottom of the drop.
During the sinking of the myofibrils, heat Shellac an ethanol glue at 65 degrees Celsius for 30 to 60 seconds before adding about six microliters of glue onto an uncoated glass slide. Repeatedly dip the tip of each mounting needle into the glue until a layer of glue is visible. Then use micro-manipulators to move the probe and piezo vertically to make room for the tissue bath on the microscope stage and remove the glass slide containing the glue.
After mounting the myofibrils, to measure the sarcomere length, move the piezo and/or force probe to set the initial sarcomere length of the myofibril to 2.5 micrometers. Using the vessel function of the system controller software, measure the myofibril length and width. Use the microscope stage to position the myofibril in the center of the video image and stretch a rectangle from one side of the myofibril to the other, taking care to include the dark edge of the glue droplets.
To start recording the data, click start. After five seconds, click pause. The length will have been recorded.
To measure the width, rotate the camera 90 degrees to view the contrast of the edge of the myofibril itself. Adjust the rectangle and click start to start recording the data. After five seconds, click pause.
The width will have been recorded. To position the theta glass, use the eyepiece and the manipulator to carefully move the theta glass toward the myofibril. Align the top channel of the theta glass with the myofibril and perform a fast-step to check the position.
Turn on the background relaxing flow to check the theta glass alignment and use a Luer valve lever to turn on the inflow of the flow chamber. Then to start draining the flow chamber and to prevent overflowing of the flow chamber, set the outflow pump valve to bath valve two, the micro step mode to micro, the plunger target to 48, 000, and the plunger speed to 38 to 40. To measure the rate of tension redevelopment, calculate the piezo movement necessary to slacken the myofibril by 15%and enter this value into the signal generator.
Click resume to continue recording the data and open valves one and six to start the flows of the relaxing solution and the different calcium concentrations through the theta glass respectively. Select reset range on the interferometer to reset the range of the interferometer so that the baseline force is zero volts. When the force trace is stable, perform the theta glass fast-step with a 100 micrometer step size.
When the force plateau is reached, perform the shortening re-stretch with the piezo. An activation relaxation trace will be recorded. Click pause.
To perform a step-wise stretch, click start and reset the range of the interferometer so that the baseline force is zero volts. Perform a step-wise stretch with the signal generator. When the stretch is finished, use the piezo to shorten the myofibril to the slack length.
Then press pause and stop and save the data. Here, force traces of an active force experiment with a myofibril isolated from healthy human quadriceps muscle are shown. The myofibril was activated five times with solutions with varying calcium concentrations, with an average maximum force of all of the myofibrils of approximately 123 millinewtons per square millimeter.
The construction of a force calcium concentration curve from the plateau forces reached during each activation in each of the five calcium curves allows calculation of the calcium concentration at 50%of the maximum force production. As illustrated in this representative analysis, the myofibrils can be treated with multiple compounds in a single experiment to answer additional questions about the myofibril type. The active force and sarcomere length can also be measured to allow calculation of the rates of redevelopment, activation and relaxation.
The steep rise shown in the dotted red box is caused by both viscosity and elasticity. The plateau resembles only the elastic component. As viscosity resists strain linearly, the force dropped after the strain was removed.
When positioning the theta glass, make sure you align it properly with the myofibril. Otherwise, the activating solution might come in between the optical fiber and the cantilever of the force probe and this causes artifacts to occur in your data. Also, the myofibril may not activate properly.
This method of perfusion is also suitable to determine the muscle fiber type of the myofibril. For example, you can first perfuse the myofibril with a normal calcium solution followed by perfusion of that same solution, but then add a muscle fiber-type specific force-enhancing compound.
