Frog Heart Experiment

Rob MacLeod, Brian Birchler, and Cris Lapierre

February 14, 2008


1 Purpose and Background

Purpose:

To examine the effects of temperature and various drugs on the frog heart muscle, specifically contraction strength and heart rate.

Background

There are a number of external influences that can affect cardiac output. Some of these are mediated by the autonomic nervous system and others are a response to changes in temperature and ionic concentrations. For background, look in your textbook for information on the effects of neurotransmitters on cardiac rate and contraction (pages 477-479 and 512-523). We will use drugs in the lab that either are neurotransmitters or act through other mechanisms to alter the physiology of the whole heart.

Frog anatomy/physiology

There are a number of excellent web sites you can peruse to find background information relevant to this lab. We will cover some basics in class, but please at least go through the virtual dissection site at curry.edschool.virginia.edu/go/frog/Frog1/menu.html.

The full list of web sites to visit include:

2 Procedure


2.1 Materials

The equipment required consists of:

2.2 The instruments

Figure 1: Photograph of the front and back panels of the ETH-256, dual channel amplifier.
 
photo of amplifier

The photos in Figures 1 show the dual channel amplifier that we will use in this lab (and others to follow). Dual channel means it can individually amplify two independent input signals. Note the paired input connectors and the fact that either channel can take either a force transducer or just measured biopotentials. When used with the force transducer, the amplifier provides the power to drive a bridge circuit for accurate readings of deflection of the transducer blade.

Note also that the outputs from the amplifier are on the back of the unit, as are the power switch and power cable connector.

Figure 2: Photograph of the force transducer and bipolar measurement electrode.
 
photos of sensors

Figure 2 shows the force transducer and bipolar electrodes that are the main sensors we will use. Resistors connected to the force transducer blade change resistance proportional to the amount of deflection. These resistors, in turn, are part of a bridge circuit, which produces a change in output voltage proportional to blade deflection. The bipolar electrode consists of two wires that we will place in contact with the heart (or other electrically active tissues) and detect the potential difference between the wires (a lead).

Note that both instruments are fragile so treat them with care.

2.3 Preparing the force transducer and circuit

Figure 3: Circuit diagram for the recording of contraction and electrograms from the frog heart.
 
calibration of transducer

Please carry out the following steps (Note Do not start the frog dissection until you have completed all the setup steps!):

  1. Setting up the measurement circuit according to Figure 3:
    1. Connect the force transducer to the CH 1 8 pin DIN input of the ETH-256 dual channel bioamplifier.
    2. Place a T-connector on the corresponding output of the bioamplifier and then connect one end to the input (CH 1) of the oscilloscope and the other to the input (0) for the computer A/D converter using BNC cables.
    3. Adjust the settings on the bioamplifier to get a clean signal on the oscilloscope in which you can see the response to gentle bending of the force transducer. Start with the following settings on the bioamplifier:
      • Low pass filter (LPF) at lowest frequency setting
      • High pass filter (HPF) at DC
      • Gain at x5
      On the oscilloscope, try the following settings (make sure all settings are in calibrated mode, i.e., latched into fixed settings):
      • DC coupling
      • 200 mV/div
      • mathend000# 0.5 s/div

    4. Launch the acquisition program (C:\bioen\CB8ChanScop) computers for acquiring the signals. Then select sampling parameters from the program (sampling rate of 100-200 is adequate) and run it to make sure it acquires a signal. Note: Once you have obtained a clean signal, record all settings (amplifier and CB8ChanScope) in your lab notebook. If you ever change a setting, make a note of the new setting and when you changed it in you notebook as well.

    Figure 4: Calibration of the force transducer.
     
    calibration of transducer

  2. Calibration of the force transducer (see Figure 4):
    1. Mount the transducer in the magnetic stand and rotate the transducer so the flat side of the blade is parallel (horizontal) to the table; deflections in the up and down direction should cause the signal on the oscilloscope to change.
    2. The zero-offset is controlled via the offset knob on the bioamplifier. Adjust the offset accordingly to make maximum use of your recording range. Because we do not anticipate bi-directional movement of the transducer blade (the heart only tugs in one direction), setting the offset to zero will waste half our recording range. Therefore set the offset such that the entire voltage range displayed on CB-Scope (and the oscilloscope) is utilized.
    3. Weigh a set of 3-4 paper clips of different sizes and then hang them alone and in combination from the transducer blade and note the total weight and the associated deflection of the signal on the oscilloscope (or CB8ChanScope). The resulting table of values will be the basis for calibration of the transducer.
    4. When you are done, rotate the blade of the transducer back into the vertical orientation and, if necessary, reposition the offset. You should now be ready to perform the measurements of the contracting heart.

2.4 Frog Preparation

Once you have everything set up and the force transducer calibrated, you can move on to the frog preparation as follows (see Figure 5)

Figure 5: Dissection of the frog showing the open skin flaps, the removal of the sternum and, underneath, the exposed heart inside the pericardial sack.
 
frog-disection

  1. Obtain a pithed frog from the lab TA/Instructor and fix the frog on its back using the big needles in the pan. Open the thorax of the frog with a central incision and two flaps, which is also shown nicely through a series of images in the web site
    curry.edschool.virginia.edu/go/frog/Frog1/menu.html. Go to the point of the Layer One section and focus on the heart. The point here is not to perform a detailed dissection but to make you familiar with the general anatomy and comfortable with the preparation of the animal. Do not cut or remove any organs other than the skin and some of the ribs covering the heart.

  2. To expose the heart, make sure to remove the lower and middle sections of the rib cage as they will interfere with the transducer you will use to measure contraction. Cut low enough so that added drugs are able to drain from the thorax. The heart of the bullfrog is quite large and red and should be slowly beating. Figure5 shows the process of removing the ribs and sternum with the exposed heart below. If the frog is still cold, the rate may be very slow so run some Ringer's solution over the heart to help it warm up. Observe the atria and single ventricle of the heart and note the sequence of contraction of each.

  3. Once the heart is open, regular apply a few drops of Ringer's solution to keep is moist.

  4. If you have a camera available, take photos of the thorax and mark organs on them. If you do not have a camera, find images from a classmate and label them for your lab report.

    Figure 6: Photo of the complete frog preparation including the thread connecting the heart to the transducer.
     
    Frog in the pan

  5. Attaching transducer to the frog (See Figure 6):
    1. Very carefully, cut open and remove the pericardium from the heart so you can see it fully exposed.

    2. Using the curved needle and suture provided, run the needle through the lower part of the ventricle, about 3-5 mm from the apex of the heart, and tie a loop with the suture thread. Then clip off the needle and discard it carefully in the sharps container (red plastic). Run the other end of the suture through the hole in the transducer blade and tie a knot there as well. Make sure there is at least 30 cm of suture available between the heart and the force transducer.

    3. Place the transducer at the end of the pan, elevated about about 20 cm above the table surface with the blade oriented perpendicular to the thread. The thread from the frog heart to the transducer should be quit flat (horizontal) so that you apply tension to the long axis of the heart. See Figure 6 for reference.

    4. Use alligator clips to attach a wire between the metal dissection tray and the large metal plate on which you are working. This can reduce the electrical noise levels substantially when we start to perform electrocardiographic measurements.

    5. Now apply enough tension to the thread such that you see a signal on the oscilloscope that reflects the contraction of the heart. Sensitivity of the oscilloscope should be in the range of 200-500 mV/div. Adjust location and tension so as to generate as clean a signal as possible, ideally one that reveals the separate components of atrial and ventricular contractions. Make sure the tension of the thread is just enough to pull the thread taught and lift the heart slightly. Check also that there is no obstruction from the side of the pan or any other object. Place the pan and the stand well away from the edge of the lab bench and always be careful not to touch the post or the thread accidently. Otherwise, any change in orientation will alter the resting tension and the reference signals, which will add error to subsequent measurements.

2.5 Experimental protocol

There are a set of interventions that you should carry out to see how the heart responds to external stimuli, both mechanical and chemical.

2.5.1 Response to stretch

Now try and replicate the effect of the Frank Starling mechanism with the preparation by measuring contraction and progressively stretching the heart to simulate the effect of increased venous return.

  1. Arrange the pan and transducer so that there is just enough tension for the thread to clear the edge of the pan (adjust the height of the transducer if necessary) and for you to get a contraction signal. Take this as the baseline value and make a 5-10-second recording of the force signal on the computer. Note: the signal processing after the experiment will consist of extracting both the tension (force) before contraction and the peak force of the contraction so make sure the signal quality is adequate for these measurements.
  2. Move the pan a few millimeters further away from the transducer so that it increases tension on the heart slightly. Again, record a 5-10-second sample of the force signal on the computer.
  3. Repeat this process in 5-10 small steps until the heart looks dangerously stretched, at each step recording the force signal on the computer. Check with the TA or instructor if in doubt about how far to stretch the heart.
  4. For the report, construct a plot of peak contraction force versus pre-tension force and explain which mechanism(s) explains the results. The pre-tension is the background tension, before (and after) the time-varying signal from the heartbeat. The peak contraction is the difference between the peak tension during the contraction and the pre-tension before the contraction. The resulting plot of peak contraction versus pre-tension should look something like the Frank-Starling curves from the text (or class).
  5. Note: make sure to apply the calibration curve to all the data you acquire from the lab so that units are in grams. While force is measured in Newtons not grams, the latter (a unit of mass not force) is typically used in practice.

2.6 ECG and Data acquisition

Now, to visualize and acquire the electrical signals, carry out the following steps.

  1. Attach a T-connector to the second output of the bioamplifier and split the output between the second channel of the oscilloscope and channel 1 of the A/D converter.
  2. Try the following settings on the bioamplifier:

Figure 7: Exposed heart with applied bipolar electrodes. The electrodes should touch the exposed heart lightly.
 
frog-electrodes

Set up an electrogram, the signal recorded directly from the heart surface, as follows (see Figure 7):

  1. Take a bipolar electrode holder, attach it to a magnetic stand that can lift up and down, and place the electrodes in contact with the heart surface.
  2. Connect the wire from the electrode to the second input (use the BNC connection) of the bioamplifier. Connect the reference lead to one of the pins that hold down the feet of the frog. Adjust the electrode location so as to get a clean signal of both atrial and ventricular ``electrograms''.
  3. Record the electrogram together with the contraction signal on the computer.

Note: Once you have obtained a clean signal, record all settings (amplifier and CB8ChanScope) in your lab notebook. If you ever change a setting, make a note in your notebook of the new setting and when you changed it.

2.6.1 Thermal and chemical stimulants

  1. First obtain and save a record of the normal heart contractions in normal Ringer's solution. Save it as a reference on the oscilloscope display so that you will be able to observe the changes in heart rate and contraction strength directly from there. Repeat this reference recording before each application of a drug!
  2. Apply a few drops of the following solutions directly on the heart and wait long enough to observe their effectsThis may take several minutes so be patient!!
  3. Record and save the tension and the electrogram signals on the computer. Wash with normal Ringer's solution after each solution and record again. For each intervention, save 10-20 seconds of data. If necessary, record several times--you can delete extra files later, however, you cannot come back to re-record signals.
    1. 2o mathend000#C Ringer's solution.
    2. 30 mM of Caffeine.
    3. 0.5 mM of Cadmium chloride (CdCl2).
    4. 50 mathend000#M of Epinephrine.
    5. 1, 5, or 10 mM ACh (start small and only move to a higher dosage only if there is no effect).
    6. 1 mg/ml atropine (apply this immediately if the heart stops beating after the ACh)
    7. 1 M of Potassium chloride (KCl).

3 Expectations

Observe the effect of each intervention on the heart rate and contraction strength. Record and compare the ECG and contraction signals between the normal and intervention states. Save the data from the experiment in Matlab and generate time signal plots to include in your report. Describe the possible mechanisms of the individual effects based either on class room material or a literature search.

The lab report this time should include the traditional components: Introduction, Methods, Results, and Discussion. Do not spend too much time repeating the methods but focus more on the results and discussion. Keep in mind the overall goal of characterizing the response of the heart to external interventions so that for each one, you report what happened and what the mechanism(s) might have been. As an integrative step, try to tie these responses to the natural state of the heart and the frog's interactions with its environment.

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Frog Heart Experiment

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