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Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling
Chapters
Summary August 1st, 2016
This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.
Transcript
The overall goal of this procedure is to access the myofilament calcium sensitivity in order to enhance our understanding of the mechanisms that underlie the contraction in healthy and diseased hearts. This method can help answer to questions related to heart congestion and heart failure. The main advantage of this technique is that together with ions channels, ion transporters and inter-cellular calcium handling it provides a comprehensive understanding of the mechanism that underlies mouse heart contractility in healthy and disease of the heart.
To begin this procedure, heat two water baths to 37 degrees Celsius. Add five milliliters of calcium free BSA solution to 25 milliliters of collagenase solution one and 3.3 milliliters of calcium free BSA solution to 16.7 milliliters of collagenase solution two. Then, add 100 milliliters of isolation solution to column one and 30 milliliters of collagenase solution one to column two in the Langendorff perfusion system.
Oxygenate the isolation solution and collagenase solution one in column one and column two via the oxygen connection tube in the Langendorff perfusion system. Similarly, oxygenate collagenase solution two and the BSA solution in the shaking water bath with 100%oxygen via the oxygen connection tube. In this step, after anesthetizing an SD rat via inter-peritoneal injection of pentobarbital sodium, confirm the anesthetic status by toe pinching and the absence of withdrawal reflex.
Afterward, transfer the rat to a dissection tray. In the supine position, secure four legs to the sides of the body with tape. Then, clamp the aorta with fine forceps and immediately mount the cannula of the Langendorff perfusion system.
After that, tie the suture thread tightly over the aorta. Turn on the valve on column one and perfuse the isolated heart with pre-warmed, oxygenated isolation solution for 10 minutes. Subsequently, turn on the valve on column two and perfuse the heart with collagenase solution one for eight to 10 minutes.
Afterward, remove the digested heart by cutting the aorta and transfer it to the flask containing fresh isolation solution. Using fine scissors and forceps, cut most of the LV free wall, including the septum, into smaller pieces. Next, transfer the tissues into a flask containing pre-warmed and oxygenated, fresh collagenase solution two.
Then, shake them for 10 minutes and keep the myocyte containing collagenase solution two oxygenated. Subsequently, transfer the myocyte suspension to a 10 milliliter centrifuge tube and add calcium containing BSA solution. Centrifuge it for two minutes.
After two minutes, discard the supernatant and re-suspend the myocyte pellet in five milliliters of BSA solution. Then, centrifuge and discard the supernatant again. Next disperse the myocyte pellet and keep the myocytes in 10 milliliters of pre-oxygenated storage solution at room temperature until the end of the experiment.
Now, load LV myocytes with two micro-molar acetoxymethyl ester Fura-2AM. Centrifuge one milliliter of myocyte suspension for 10 seconds. Then, discard the supernatant and re-suspend the myocyte pellet in one milliliter of Tyrode solution with a low calcium concentration.
Subsequently, add Fura-2AM and two microliters of poloxamer 407 to the myocytes. Gently disperse the myocyte suspension and keep the mixture at room temperature for 15 minutes. After 15 minutes, centrifuge the mixture for five seconds.
Discard the supernatant and disperse the myocyte pellet in one milliliter of perfusion solution containing 500 micro-molar calcium. After 10 minutes, centrifuge the mixture for five seconds and discard the supernatant. Next, add fresh perfusion solution and keep the Fura-2AM loaded myocyte pellet in this solution for recordings.
Before recording, fill the perfusion tube that runs through a water jacket with the 36 degree Celsius Tyrode solution. Subsequently, place a few drops of the Fura-2AM loaded LV myocyte suspension on the chamber of an inverted florescence microscope and wait for five to eight minutes. Slowly perfuse it with the Tyrode solution at 2.2 milliliters per minute.
Then, press the start button on the front panel of the digital stimulator to start field stimulation at two hertz. Select the myocytes that contract stably for recording. Shown here are the raw traces of sarcomere shortening and Fura-2 ratio measurement in LV myocytes from sham and hypertensive rats.
Here are the average traces of the sarcomere length and the Fura-2 signals. And here are the phase plane plots of the Fura-2 ratio versus sarcomere length in the two groups. The trajectory loop is shifted to the right and EC50 tends to be higher in hypertension, suggesting myofilament calcium desensitization.
Prior attempting this procedure, it's important to remember to test the cortium, the isolated myocytes, by detecting myocyte contraction without Fura-2AM loading. In addition, always check myocyte contraction after Fura-2AM loading to avoid overload. Following this procedure, other methods like patch clamp techniques can be performed in order to answer additional questions.
For example, A type ca channel activity, that is, in verb, the myocyte excitation-contraction coupling. After its development, this technique have paved the way for researchers in the future of cardiac physiology to explore more insights into heart diseases in humans. After routine experience, you should have a good understanding of how to analyze the myofilament calcium channel activity.
In order to better understand the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.
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