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Transcript
- Heart Muscle - Background The heart is composed of myocardial cells, which contract in a coordinated fashion to pump blood around the body. The pacemaker of the heart is the sinoatrial (SA) node, located in the right atrium. The SA node contains modified muscle cells that are autorhythmic (have a pacemaker potential). An action potential from the SA node travels via gap junctions to adjacent cells in the atria. The gap junctions, which are part of the intercalated disks between adjacent atrial cells, allow the action potential to move around both atria like a wave, causing the atria to contract. The action potential also spreads to the atrioventricular (AV) node, which is also composed of specialized muscle fibers. The action potential moves slowly along the electrical pathway in the AV node, and then travels rapidly along the Bundle of His and Purkinje Fibers to the fibers of the ventricle. The slow transmission of the action potential through the AV node insures that the ventricles contract after the atria. This delay allows the ventricles to fill with blood from the atria before the ventricles contract. The contraction of the heart cells follows the sliding filament model as described for skeletal muscle. Contraction of the atrial cells force blood into the ventricle, and contraction of the ventricle force blood out to the body. Mammals have two ventricles, which pump blood to the lungs and rest of body separately. The term “systole” refers to the contraction phase of the heart, and “diastole” refers to the relaxation phase of the heart cycle. Unless otherwise indicated, these terms refer to the action of the ventricle. Change number of channels (upper right hand corner) to 4 Change Function to Stim 1 Change Name to Stimulator 6 Once you go back to the Main Screen, you will have to minimize Channels 1 & 2 The Dissection Hints 1 Keep the exposed heart moist with Ringer's solution until the experiments have been completed. 2 Be prepared to do your experiments quickly and efficiently, And then perform your data analysis after all of the exercises have been completed. Equipment Setup 1 Plug the DIN connector on the cable of the FT-100 force transducer into Channel 3 of the iWorx unit (Figure 1). 2 Plug the BNC-double banana adapter into the positive (red) and negative (black) sockets of the stimulator of the iWorx unit. Check the side of the double banana adapter for a tab. This is the side of adapter that goes into the negative (black) socket of the stimulator. 3 Do not connect the stimulator cable to the adapter on the stimulator of the iWorx unit until you reach Exercise #4 . Figure 1: The equipment used to record heart mechanical contractions. Pretend this is a picture of turtle. Procedure 4 Arrange the clamps on the ring stand so that the string from the heart will be at as low an angle as possible without the pin/string hitting the sides of the hole. The blade of the transducer should be perpendicular to the sting. 1 Place the recently-pithed turtle ventral surface up, in the dissection tray. A hole saw was used to make a circular cut in the plastron (ventral bony plate). Start the Software 2 Lift up gently on the disk of bone, and use the scissors to cut the connective tissue and remove this disk. 1 Click on the LabScribe icon on the Desktop. 2 Click on the Settings menu again and select the HeartMuscle settings file. 3 After a short time, LabScribe will appear on the computer screen as configured by the HeartMuscle settings. 4 Close the pdf file that opened automatically. 5 We need to also display the stimulator output. To do this: Click on Edit From the pull down menu, chose Preferences 3 Locate the heart, which is covered by the pericardial sac. Use forceps to grasp and lift up on the pericardial sac, not the heart. Dissect away the pericardial sac from the heart, trying to avoid the cutting of blood vessels in this sac. Do not cut any of the vessels attached to the heart. 4 Use masking tape to secure the turtle to the tray, without blocking the hole (Figure 2). Force transducer Choose a destination on the computer in which to save the file(e.g. the iWorx or class folder). Click the Save button to save the file (as an *.iwd file). Exercise 2: Effects of Temperature thread Aim: To record changes in heart rate and force of contraction after the heart is bathed in warm and cold Ringer’s solution. Procedure 1 Click Start to begin recording. Click AutoScale to increase the size of the deflection on the Main window. Figure 2: Set up for turtle in dissecting tray ready for testing.. 5 Use forceps to grasp the frenulum (a tendon at the apex of the ventricle) and insert the heart pin thought the heart near the frenulum, penetrating about 2 mm deep (Figure 3). The cut the frenulum. 6 Tie the thread of the heart pin to the force transducer (the thread should be as short as possible), and gently move the ring stand so that the thread is taut and the heart is slightly stretched. 2 Type “Room Temp Ringer’s” on the comment line to the right of the Mark button. After recording about 15 seconds of heart contractions, use a Pasteur pipet to apply about 20 drops of frog Ringer's solution (at room temperature) to the heart. Press the Enter key on the keyboard when the solution is dropped on the heart, and record for about another 45 seconds before clicking Stop. 3 Type “Cold Ringer's” on the comment line, and repeat this 60-second procedure with cold Ringers. 4 Repeat the procedure with warm Ringers. 5 Select Save in the File menu. 6 Flush the heart with room temperature Ringer’s solution. A A Exercise 3: Effects of Drugs Vent. Aim: To monitor the effects of natural and pharmaceutical chemicals on the amplitude and rate of heart contraction. Procedure 1 Click Start to begin recording. Click AutoScale to increase the size of the deflection on the Main window. frenulum Figure 3: The turtle heart with pin in place Exercise 1: The Heart Rate and Force Aim: To record the mechanical trace produced by the contraction of a resting heart, and to determine the resting heart rate. Procedure 1 Click Start to begin recording. Click AutoScale to increase the size of the deflection on the Main window. 2 Type “Resting” on the comment line to the right of the Mark button. Press the Enter key on the keyboard to attach the comment to the record. 2 Type “Epinephrine” on the comment line to the right of the Mark button. 3 Record about 15 seconds of heart contractions, and then use a Pasteur pipet to apply 10 drops of room temperature epinephrine solution to the heart. Press the Enter key on the keyboard when the solution is dropped on the heart. Record the effects of epinephrine for anther 45 seconds. Then, flush the heart with fresh, room temperature Ringer’s solution until the heart rate returns to its resting value. 4 Click Stop to halt recording. Select Save in the File menu. 5 Repeat this 60-second procedure for acetylcholine. Note: If the heart “arrests,” rinse the Acetylcholine solution off the heart with fresh, room temperature Ringer’s solution. If the heart is still “arrested” after 10 seconds, add two drops of Epinephrine solution to the heart. 3 Record about 15 seconds of heart contractions. Then click Stop to halt recording. Remember to moisten the heart with frog Ringer’s solution at all times. 6 Repeat this 60-second procedure with atropine, but do not rinse the heart with Ringers when this is over. Immediately repeat the acetylcholine 60-second procedure. Then rinse with room temperature Ringer’s solution until the heart rate returns to its resting value. 4 Select Save As in the File menu, type a name for the file. 7 Finally repeat this 60-second procedure for nicotine. Exercise 4: The Refractory Period of the Heart Aim: To stimulate the ventricle to produce extra ventricular contractions (extra-systoles), and to determine when the heart is in its refractory period and unable to create extrasystoles. Procedure 1 Adjust the bipolar stimulating electrodes on the ring stand so the tips are touching either side of the ventricle, and the ventricle is able to move up and down as it contracts, or hold it steady by hand so that both prongs are touching the ventricle. 2 Attach the BNC connector of the stimulator cable to the adapter on the stimulator of the iWorx unit. 3 The stimulator has already been turned on and programmed by the HeartMuscle setting file. The values of the following parameters should be seen in the stimulator control panel under the LabScribe toolbar: • the stimulus amplitude is 4V. • the stimulus delay is 50ms. • the stimulus duration is 10ms. • the frequency is 1.0Hz. • the number of pulses is 30. 4 Click Start, and then AutoScale. Type “Refractory” on the comment line. Press the Enter key on the keyboard. You should see a rhythmic deflection on the trace. 5 Click Stop after recording for the 30 seconds that the heart was stimulated. Examine your recording. Look for extra ventricular beats (Figure 3). If you don’t see an extrasystole, do the following, and repeat steps 4 and 5: • Decrease the stimulus frequency. 6 Select Save in the File menu. 2 Click Start, and then AutoScale. Type “Tetanus” on the comment line. After 10-15 seconds of recording, press the Enter key on the keyboard. After the pulses stop, continue recording for another 10-15 seconds. Exercise 6: Effects of Stretching the Heart Aim: To determine the relationship between length of cardiac muscle and the force it produces (Starling’s Law of the heart). Procedure 1 Remove the stimulating electrode for now. Make sure that the thread from the heart to the force transducer is taunt and the heart is only slightly stretched. 2 Start the recording and after 15 seconds, slide the turtle (and tray) about 2 mm away from the force transducer, stretching the heart. Record at this heart muscle length for about 15 second. 3 Slide the tray another 2mm, and repeat this every 15 seconds until you have slid it at least 5 times. The heart is quite flexible, so this should not tear it. 4 After you complete the stretching, slide the tray back to reduce the stretch on the heart Exercise 7: Effects of a Ligature on the Heart Aim: To observe the effects of blocking action potentials from passing from the atria to the ventricle. Procedure 1 Take a piece of thread about 12 inches long. Place the center of the thread around the AV groove that separates the ventricle from the atria. 2 Tie a single overhand knot in the thread, to form a loop around the AV groove. Don’t tighten the loop at this time! 3 On the left and right sides of the heart, insert other thread pieces through the ligature loop (Figure 4). On each side, you will later bring the two ends together, and pull outward to undo the ligature. A A Vent. Ligature release threads Figure 3: Stimulation of the ventricle produced an extra contraction of the ventricle when delivered at the appropriate time during the cardiac cycle. Exercise 5: Effects of a Tetanizing Stimulation Aim: To determine whether tetanus can be induced in cardiac muscle, as seen in skeletal muscle. Procedure 1 Adjust the stimulator settings so that the frequency is 10 Hz and the number of pulses is 100 (10 seconds of pulses). ligature Figure 4: A ligature (thread) around the AV groove, and two ligature release threads inserted BEFORE the ligature is gradually tightened. 4 Click Start, and then AutoScale. Type “Normal” on the comment line. Press the Enter key. Record contractions for about 15 seconds. Click Stop to halt recording. 5 Slowly tighten the knot a bit, making sure that the thread stays in the AV groove. Then repeat Step 3, typing “Ligature #1” on the comment line. 6 Continue to tighten and record for 15 seconds several more time, labeling the traces “Ligature #2”, #3, etc. Examine the recording each time. If the atria and ventricle still contract in a coordinated fashion, but the time between the arial and ventricular contractions are farther apart, this is a first-degree block. If there are several (2-6) atrial contractions between each ventricular contraction, in a very regular pattern, this is a second-degree block (Figure 5). A third-degree block is when the ventricle stops beating completely, or its beats are far apart and not synchronized with any atrial contraction. 7 Select Save in the File menu, but do NOT remove the ligature until completing the next exercise. Exercise 9: Independent Contractility Aim: To determine which portions of the heart are capable of beating on their own (have cells with pacemaker potentials). Procedure 1 Remove the heart pin, wash it carefully, and get it back to your instructor. It’s very easy to loose track of these, and then someone gets stuck or the pin gets lost. 2 Carefully cut the attached blood vessels so that you can remove the entire heart and put it in a Petri dish with Ringers. 3 Determine the heart rate of the atria and the ventricle (beats/min.). 4 Carefully separate the three chambers of the heart in the Petri dish. Determine the heart rate of each. Data Analysis Temperature and Drugs In these sections of the experiment, you recorded cardiac activity before and after the application of the test solution. To extract the pertinent information from the data set, you should: 1 Scroll to the section of data that is relevant to the solution being tested. Click AutoScale to maximize the size of the response on the window. 2 Scroll to the Mark indicating the point when the application of the solution was made. The GoTo command in the Marks window can be used to locate the marks in the recording. Figure 5: A ligature causes the atria (smaller peaks) and ventricle to contract independently. 3 Measure the heart rate and the contraction amplitude at 30, 20 and 10 seconds before the mark (the control values), and at the mark and every 10 seconds after the mark, for the duration of the treatment (the experimental values). Exercise 8: Threshold 4 At each time point, use the Display Time (Half or Double) icons in the LabScribe toolbar to adjust the Main window, so that a couple of contractions appear. Aim: To determine whether the contraction of the heart (ventricle) is graded or all-or-none. Procedure 1 With the ligature still in place, use the stimulator to shock the ventricle with a single pulse (still at 4V). If the ventricle is not contracting at all, you can do this at any time, but if it is contracting on its own occasionally, stimulate it between these. Any contraction that occurs other than when you stimulate should be ignored. You can “mark” this first stimulation “4V”. 2 Reduce the voltage and repeat this procedure, marking with the appropriate voltage, until you do not get a contraction (i.e., the voltage is below the heart’s “threshold”). 5 Click the 2-Cursor icon (Figure 6), so that two blue vertical lines appear over the Muscle channel on the Main window. Move the cursors to positions on the recording window to measure the following parameters: • The amplitude (= force) of the heart contraction, which is the voltage difference between the cursors placed on the baseline and the peak of the ventricular contraction (Figure 7). The amplitude is the value for V2-V1, displayed in the upper right corner of the channel window. • The period of one heart cycle, which is time difference between cursors placed on adjacent peaks of ventricular contractions. The period is the value for T2-T1, displayed on the upper left side of the Main window. 3 Examine your record and decide if the heart’s contraction is a graded or an all-or-none event. 4 Now, use the ligature-release threads to remove the ligature, and wait until you get the heart back to normal beating (as much as possible) before proceeding to the last exercise. You may need to poke it (another form of “stimulation”) regularly for a few minutes, and/or add some epinephrine to help it return to a normal rhythm again. Figure 6: The LabScribe toolbar. considering the initial amount of stretch to be zero (0mm), and each subsequent heart stretch to be 2 mm more. 3 Graph this force (y-axis) for each length (X-axis). Ligature 1 Scroll to the appropriate section of data, and first go to the initial 15 seconds before the ligature was tightened (first degree block). Figure 7: Using two cursors to measure the amplitude of a contraction. 6 Data can be entered in the Journal, by clicking on the Journal icon in the LabScribe toolbar, and typing the values for the amplitudes and periods and corresponding times in the Journal window. 7 Convert heart cycle periods to heart rates by using the following equation: 60 / one heart cycle period = heart rate (sec/min) (sec/beat) (beats/min) 8 Graph heart rates and contraction amplitudes as a function of time before and after the application of the solution, for each experiment. Refractory Period of the Heart 1 Scroll to the appropriate section of data. 2 Click the 2-Cursor icon (Figure 6), so that two blue vertical lines appear over the Muscle channel on the Main window. Move the cursors to positions on the recording window to measure the period of time from the beginning of an atrial contraction to the beginning of its ventricular contraction. The period is the value for T2-T1, displayed on the upper left side of the Main window. 3 Repeat this measurement in each 15 second recording to see if/when a first-degree heart block occurs. 4 Visually inspect the rest of the 15 second recording to look for second-degree blocks (Figure 5), and third-degree blocks. Thought Questions 1 What are the effects of cold and warm Ringer's solution on the rate and the amplitude of the ventricular contraction? What Mechanisms are involved in producing these effects? 2 How do Acetylcholine and Epinephrine produce their effects on the heart rate? On the amplitude of ventricle contraction? 2 Find the first extrasystole (Figure 3). 3 What effect does Atropine have on the heart? How does Atropine work? 3 Click the 2-Cursor icon (Figure 6), so that two blue vertical lines appear over the Muscle channel on the Main window. 4 When, in the cardiac cycle, does the refractory period for the ventricle occur? 4 Place one cursor at the beginning of a naturally occurring AP and at the location of the stimulus that created the extrasystole. Record the time difference, T2-T1, between these dips. 5 What is the significance of the long refractory period to the function of the heart? What kind of action potential is this? 5 Examine the complete record of the refractory period exercise. For all additional extrasystoles, make the same measurement. The shortest time between a normal contraction and an extrasystole should be close to the refractory period of the ventricle. 7 How does the force of ventricular contraction change as it is stretched? How does this compare with skeletal muscle? 6 Now look at all the contractions that did NOT have an extrasystole. If a stimulus occurred during a contraction, and a extrasystole was not produced, record the time. 7 If the heart rate was not the same as the stimulation frequency, it can be assumed that the pulses were applied at different times during the heart beat cycle. Examine the complete refractory recording and determine: • During which phases of the cardiac contraction cycle were extra ventricular contractions recorded? • During which phases of the cardiac contraction cycle were extra ventricular contractions not recorded? Stretching the Heart 1 Scroll to the appropriate section of data. 2 For each level of stretching, determine the force of contraction (amplitude). Quantify the stretch on the heart by 6 Does the heart go into tetanus like a skeletal muscle? 8 In the ligatured heart in third-degree block, or when the chambers are physically separated, the atria and ventricle should beat at their own rates. Which rate is closest to the heart rate seen in the turtle’s normal heart beat? Where are the pacemakers for the atrial and ventricular rhythms?