This protocol describes a method to measure mechanical control of relaxation or the strain dependence of muscle relaxation in cardiac trabecula. The main advantage of this technique is that it focuses on cardiac relaxation. Given the lack of treatment options for diastolic diseases, this technique might be used to identify novel indexes and drug targets relating to cardiac relaxation.
This method will be shown with a rodent heart, but it can be used on any intact muscle. Demonstrating this procedure will be myself and Anita Abbo, an undergraduate research assistant in my lab. To begin, remove the gross dissected small animal model's heart from the cannula and place it in a silicone elastomer coated weigh dish to prepare for trabecular isolation.
Then place and illuminate the heart under a stereo microscope. After locating the right ventricular outflow tract, pin the left atrium and the ventricular apex to the silicone elastomer in the dish. Using long Vannas scissors, cut from the right ventricular outflow tract to the apex along the septum.
Next, cut from the right ventricular outflow tract to the right atrium near the aorta, and then cut through the right atrium. Using forceps, carefully pull open the right ventricular free wall from the outflow tract without stretching the tissue. White thin connective tissue strands, if found, can be cut, but not the larger pink tissue strands, as they may be trabeculae.
Pin the free wall of the right ventricle triangle to the dish to expose the right ventricle. Using a thin blunt-end melted glass pipette, search the exposed endocardium for freestanding trabeculae without applying pressure. Avoid the triangular papillary muscles and select trabeculae with parallel sides, which are often found near the base of the right ventricular free wall and along the septum.
Dissect the trabecula using small Vannas scissors, leaving a one millimeter-cubed piece of tissue at each end of the trabecula to allow for attachment. Next, cut approximately two inches off the end of a seven-milliliter transfer pipette, slowly draw the trabecula into the pipette, and transfer it into a new weigh dish, containing 50%perfusion solution and 50%modified Tyrode's solution. Allow the muscle to equilibrate to the increase in extracellular calcium within the mixed solution For several minutes.
Turn off the pump supplying superfusion or suction to the experimental chamber. Using the large bore transfer pipette, move the trabecula into the experimental chamber filled with Tyrode's solution. Pin one cubed piece of tissue at the end of the trabecula to a hook on the force transducer, then pin the second cube to the motor.
Restart the superfusion and begin pacing the muscle to determine the threshold voltage. Pace at 20%above the threshold voltage for approximately one hour. At the end of this equilibrium period, slowly stretch the muscle using the micrometer connected to the motor, until optimal developed stress generation is achieved by observing the developed tension.
Stop increasing the muscle length when the passive diastolic tension rises faster than the peak tension, indicating that the optimal length has been passed. Turn off the transmitted microscope illumination and illuminate the trabecula using a gooseneck illuminator at a steep angle. Using a previously calibrated camera connected through the microscope optics, capture an image of the trabecula during diastole into the experimental folder.
Average the diameter measures and convert the diameter and length from pixels to millimeters using a previously obtained calibration. Calculate the cross-sectional area and the muscle length in micrometers. In the data acquisition software, acquire load clamped data by defining the afterload in the dap file, and iterate values of proportional gain and integration parameters by saving the file in the text editor, then press Run Experiment"in the interface.
Control the end of the load clamp by changing the mode. Repeat the acquisition while changing the end of the load clamp from zero to complete, relengthening back to the starting length. To increase the length, incrementally increase the threshold for ending the load clamp from zero until the muscle nearly relengthens back to its original length.
If desired, modify the afterload and repeat the acquisition immediately. If desired, modify the preload by stretching or shortening the muscle, or treat the muscle by adding compounds to the Tyrode's solution, but wait a minimum of 20 minutes to ensure that the slow force response has stabilized and for the compound to fully penetrate the muscle. Once the data acquisition is completed, remove the trabecula and clean the experimental system.
Quantify relaxation by ensuring that the data analysis program analyzes the clamped beat, and that the program correctly acquires the start of the load clamp. After quantifying all traces of a given condition, plot the relationship between the relaxation rate and strain rate, limiting the maximum data to a physiological strain rate of less than one per second. Exclude data at low strain rates, as the relaxation phase may not reflect the exponential decay.
Obtain the slope of the line between the relaxation rate and the strain rate, and record the slope as the index of mechanical control of relaxation. A single cardiac trabecula illuminated using a gooseneck LED light at a steep angle of 75 degrees from the axis of the microscope lens is shown here. Stress time and strain time curves for the same trabecula showed an isometric twitch and three load clamp twitches at increasing and systolic strain rates.
The strain rate is calculated from the derivative of the strain just prior to isometric relaxation. The data are plotted on a relaxation rate versus strain rate graph, where the slope of the line provides the mechanical control of relaxation, or MCR index. The most difficult component in this protocol is a careful isolation of the cardiac trabecula.
This protocol can be combined with other measurements of muscle, including intracellular calcium, contractility, and sarcomere length. Also, this method is being used to identify molecular mechanisms and better understand the pathophysiology of impaired cardiac relaxation.
