Heart Muscle

CARDIAC MUSCLE: THE FROG HEART

PURPOSE:

The purpose of this laboratory exercise is to study the physiology of cardiac muscle by observing characteristics of the frog heart and several influences on its control.

THEORY/INTRODUCTION:

Cardiac muscle has properties similar to skeletal and smooth muscle. Like skeletal muscle, cardiac muscle has striation (i.e. microscopic "stripes"). Certain types of smooth muscle (i.e. intestinal and uterine) are "autorhythmic". That is, they contract spontaneously (without external stimuli) or, more specifically, they have their "own rhythm". Heart muscle too is autorhythmic. Individual cells of cardiac muscle, if isolated in a saline solution, would beat spontaneously at random rates.

A specialized bundle of tissue known as the "pacemaker" (i.e. sinoatrial node) is responsible for determining the normal heart rhythm. Since the normal rate at which the pacemaker "fires" is inherently faster than the spontaneous rate of other heart cells, the pacemaker "dictates" the heartbeat rate.

Overall, the heartbeat rate and cardiac output of blood is affected by nervous and hormonal activity. The primary cardiovascular control center is located in the medulla oblongata of the brain, and the heart is innervated by sympathetic and parasympathetic nerve fibers which terminate at the sino-atrial node.

The heart rate is regulated precisely by balancing the slowing effect (i.e. bradycardia) of the parasympathetic nerve activity against the accelerating effect (i.e. tachycardia) of the sympathetic nerve activity. Other functions such as temperature, ion concentration, and hormones other than epinephrine also influence the heart rate.

This experiment is divided into two parts. The first experiment deals with the frog electrocardiogram (EKG) (taken before the body is opened to expose the heart). The second experiment involves recording the mechanical contractions of the exposed heart.

Before the frog EKG experiment can begin, it will be necessary to prepare the frog and the electronic recording apparatus. One part of each lab group should begin by preparing the frog (discussed below) while the others should turn to the section on FROG EKG.

FROG PREPARATION: (see lab manual on each bench for details)

The TA will supply each lab group with a freshly pithed frog. A frog is pithed by running a needle into the brain and spinal column, effectively destroying the central nervous system. This procedure renders the frog insensitive to stimuli. While the frog is "dead" in the conventional sense, the heart will beat, and the normal metabolic functions will continue for a matter of hours. There may some reflex movements but the frog feels nothing.

Place the frog, ventral (belly) side up, in a wax-filled dissecting pan. Insert a clean dissecting pin through each limb. Be sure that the pins hold securely but do not penetrate the wax far enough to touch the metal pan below. Attach the EKG cable, as shown in the diagram, using the alligator clips plugged into the ends of the 3-Lead EKG Cable.

FROG ELECTROCARDIOGRAM:

The lead arrangement used here corresponds to the LEAD I configuration used for human. Hence, the frog EKG can be compared with the human EKG taken with a LEAD I configuration.

Record the frog EKG for 15-30 seconds. Record at a fast paper speed and calculate the HR. Compare the rate and EKG with the human EKG that we have discussed in class. (Why is the heart rate so slow?) The frog heartbeat rate can be calculated by measuring the elapsed time between 5 successive R wave intervals and expressing the value in beats/min. Try leads II and III. How are the leads placed for leads II and III? Does the heart rate change?

One of the most noticeable differences between the frog and human EKG is the difference in the R wave. Because of the way the frog heart is positioned in the body and because a frog has only has 1 ventricle, its electrical polarity causes R waves to register upside down from that of the human. The (+) and (-) lead wires can be reversed if it is desired to record the frog EKG with the R waves pointing upward like that of the human EKG.

MECHANICAL ACTIVITY OF THE FROG HEART:

Having recorded the electrical activity of the frog heart, attention is now turned to recording the mechanical motion of the beating heart. By setting up the mechanical recording apparatus, it will be possible to observe some of the chemical, physical, and electrical influences which affect the heart contraction cycle.

Before the experiment can proceed, it will be necessary to expose the frog heart and prepare the electronic apparatus.

EXPOSING THE FROG HEART:

Almost any surgical technique can be used to expose the heart. Remember to keep the open area of the body small to prevent tissue drying, to hold a pool of Ringers’ solution and to prevent blood loss.

With the frog ventral side up, begin by removing the skin over the heart. Use forceps to lift the skin and small scissors to cut it away. Tweezers can now be used to hold up a small part of the muscle over the heart (at the tip of the bony girdle) while scissors are used to start an incision. Continue to cut away a small circular piece of muscle in the region of the heart. Do not cut too deeply. The heart should now be visible inside the white pericardial sac. Further expose the heart by cutting away the part of the bony girdle over the heart.

The frenulum, a small segment of connective tissue, holds the apex (bottom) of the heart to the body cavity. Carefully cut away the frenulum to free the heart from the body cavity. Next, expose the apex by cutting a small slit in the pericardium at the apex. Using tweezers, slide the pericardium down the heart ventricle toward the auricles.

Ask the TA to tie a ligature around the tip of the apex. The beating heart should now be ready to attach to the Transducer.

APPARATUS FOR RECORDING HEART CONTRACTION:

Either the Displacement Transducer or the Force Transducer can be used for recording the mechanical events of the heart cycle. See diagrams for the setups. Note that the displacement transducer is much more sensitive and should be used if you have a weak frog heart. Much of this will be prepared by the TA. The ECG is not recorded at this time because the heart is pulled from the chest cavity and thus electrical activity is not conducted to the limbs.

A typical tracing may resemble:

EFFECT OF TEMPERATURE ON HEARTBEAT:

This section demonstrates that changes in the local temperature of the heart affect its rate of beating. Begin by recording the heartbeat with the heart moistened with Ringer’s solution at room temperature. Find the heartbeat rate. After a stable recording at room temperature, apply several drops of ice-cold Ringer’s solution to the heart and note any changes (amplitude and frequency) in the tracing of the heartbeat over the period of 1 min. Return heart to control using room temperature Ringer’s. Always allow a control period for conditions to return to control. A control tracing should be obtained because with time the control conditions may change. Next, apply several drops of warm Ringer’s solution to the heart while recording the beat for 1 min. Again, note changes in amplitude and frequency. How does the heart rate compare for each of the three trials? Can the temperature effects on heartbeat rate be explained in terms of metabolic effects? What happens to gates and channels?

NERVOUS AND HORMONAL CONTROL OF HEARTBEAT:

This section demonstrates the effects of acetylcholine (we will use 1% Pilocarpine which is similar to acetylcholine) and epinephrine (1%) on the heartbeat rate. Acetylcholine, released by parasympathetic nerve fibers, tends to inhibit the pacemaker and slow down (bradycardia) the heartbeat rate. Epinephrine (adrenalin), a hormone released from the adrenal medulla into the blood stream, causes the heart to accelerate (tachycardia) its heartbeat rate.

First, obtain the heart rate from the recording of the frog heart moistened with Ringer’s solution.

Apply several drops (it may take a full pipette) of 1% pilocarpine (acetylcholine) in Ringer’s to the frog heart while recording the heartbeat. Note the depressive effect on the heart rate over a period of about 2 min. You can conserve chart paper by running at the 5 mm/sec speed. What about the force of contraction?

Rinse off the heart with Ringer’s. Note the heartbeat. Next, apply several drops 1% epinephrine in Ringer’s to the heart. The heartbeat should accelerate. What happens to the force of contraction?

Perform similar experiments with 1% Norepinephrine. Next combine pilocarpine and 1% Atropine Sulfate (blocks Acetlycholine receptors). Record what happens to HR and heart contractility.

The last experiment of the day is to add 5% KCl to the heart. Why should this be saved until the last experiment?

Note that all chemicals are dissolved in dilute HCl and then added to Ringer solution in the appropriate concentration. The solution is then pH to 7.4.

REFRACTORY PERIOD OF THE HEART:

This section demonstrates the presence of a relatively long refractory period in the heart cycle during which the heart will not respond to stimuli. Apply electric shocks to the beating heart while attached to the Displacement Transducer. The Force Transducer can be used equally well. Position the Stimulating Electrodes gently against the heart ventricle. Be sure to keep the heart moist with Ringer’s solution.

The stimulator should be setup as follows.

Connect the event terminal of the stimulator to the other channel of the recorder. Connect the stimulating electrodes to the 2 output jack on the stimulator. Set the stimulator to deliver a single volt pulse 100 ms in width each time the manual button is pressed.

If the shock (you must gradually increase the voltage) fails to produce a premature beat, the heart must be in the refractory period. If a premature beat is observed the extra contraction will be followed by a pause. Why? Note that the heart is only irritable during the period of relaxation. The relatively long refractory period of the heart prevents fusion and summation.

Return to Physiology Syllabus

House's Home | Biology Department | Elon University