Introduction to Section II - Creighton University



Biology 450 - Animal Physiology |Fall 2007 | |Lab 8 – Cardiovascular Physiology of the Frog

In this lab, you will expose a frog heart in situ in order to observe the cardiac cycle in an active heart, investigate the regulatory effects of two neurotransmitters on heart rate and contractile strength, and attempt to identify the nature of two unknown compounds through their effects on the frog heart. In addition, you will look for a relationship between the stretching of the ventricle and the force of contraction (Starling’s “Law of the Heart”).

Background

Cardiac cycle

Individual heart muscle cells, if isolated in saline solution, will contract spontaneously at a fairly regular rate. Spontaneous contraction in muscle is sometimes referred to as myogenic activation, and regular myogenic activity is referred to as “autorhythmicity.” A specialized bundle of tissue, known as the “pacemaker” (usually the sinoatrial, or SA, node), is responsible for setting the heartbeat rate since it fires inherently faster than the spontaneous rate of other heart cells. The activity of the SA node ultimately stimulates the contractile cells of the myocardium.

When the cardiac muscle fibers in the atria or ventricles are stimulated, contraction occurs. Since the cells are united by gap junctions, the excitation producing contraction is spread from cell to cell and the muscle contracts as a single unit, ejecting blood from the chamber. After contraction, the heart chambers relax and refill. A complete heart beat or cardiac cycle consists of atrial contraction and relaxation followed by ventricular contraction and relaxation. These ventricular events are typically referred to as systole and diastole. The atria are normally contracted for about 0.1 seconds and relaxed for 0.7 seconds. Ventricular systole lasts for about 0.3 seconds with diastole taking 0.5 seconds. A single cardiac cycle thus takes about 0.8 seconds when the heart rate is 75 beats per minute. The lag time between the contraction of the atria and ventricles results from a delay imposed on the conduction system at the atrioventricular node.

One additional aspect of the cardiac cycle to consider is ventricular ejection, which occurs when ventricular pressure exceeds that within the arteries and blood is ejected from the heart. The sudden increase in volume and pressure in the arteries causes them to expand elastically; they decrease in size more slowly during diastole.

In today's lab you will be examining the cardiac cycle of the frog, Rana catesbeiana, and measuring how certain parameters affect it. This organism is an amphibian (obviously), which means that its heart is three-chambered rather than four-chambered like an avian or mammalian heart. It has a single ventricle from which all blood exits. The large dorsal sinus venosus is the location of the pacemaker.

Regulation of heart activity

The control of cardiac contractility involves the activity of both intrinsic (within the heart) and extrinsic (from outside the heart) factors. The pacemaker cells act as an intrinsic control of heart rate, since an excised heart will still beat at a regular rate. Extrinsic controls include factors such as nervous and hormonal inputs, which can influence both heart rate and cardiac output.

With regard to nervous control of the heart, the primary regulating center is located in the medulla oblongata of the brain, and the heart is innervated by both sympathetic and parasympathetic nerve fibers that terminate at the SA node. The neurotransmitters released by these nerves affect both heart rate (chronotropic effects; chronos = time) and the strength of contraction (inotropic effects), by influencing the timing and magnitude of ion currents across the cell membrane.

The vagus nerves contain parasympathetic cholinergic neurons that release acetylcholine at their terminals. This neurotransmitter binds with muscarinic receptors and initiates many effects, including a cascade that increases the number of K+ channels in the open position; thus keeping the membrane near the equilibrium potential for K+ and making depolarization more difficult.

The sympathetic cardiac nerves contain adrenergic neurons that release epinephrine or norepinephrine, depending on the species. (Amphibians release both of these neurotransmitters.) These neurotransmitters bind β1 adrenergic receptors at both the SA node and in the myocardium. This binding can cause a variety of effects, including an increased inward flux of Ca++ into the cell.

The function of the muscarinic and adrenergic receptors in the heart can be revealed by examining how the heart responds when these receptors are activated or inactivated. Compounds that activate a receptor are called agonists, while compounds that inactivate a receptors by binding to it are called antagonists. Most medications used in the treatment of heart disorders are antagonists for either muscarinic or (-adrenergic receptors.

In this lab you will examine the chronotropic and inotropic effects of two agonists, epinephrine and acetylcholine. You will also quantify the effects of an unknown antagonist and, based on your observations, identify the type of receptor to which it binds.

Lab Procedures

You will use a 10g force transducer in combination with Chart to measure heart rate and contractility. Although the frog heart is quite hardy, you must avoid damaging it unnecessarily and keep it wetted during the course of your experiments.

Setup

The hardware setup for these experiments is fairly simple.

• Arrange the force transducer so that it is 10-15 cm off the benchtop with the hooks facing towards you (in other words, to record a horizontal rather than a vertical force).

• Make the appropriate connections to the bridge amp and the PowerLab, then start Chart and make sure all hardware and software settings are appropriate.

Dissection

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|Ventral view |

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|Dorsal view |

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|Internal view (ventral) |

|Anatomy of the frog heart |

You need to expose the heart of a frog in order to measure its activity. You will be supplied with a pithed or decapitated frog by your instructor.

During the dissection, be very careful not to damage the heart or its major vessels. Throughout your experiments, keep the heart and internal organs wet with frog Ringer’s.

1. Place the frog in dissecting pan and use dissection pins to hold the frog in place.

2. Using a scalpel or sharp scissors, make a lateral incision through the skin across the chest of the frog (from armpit to armpit). The first incision should expose the muscle and connective tissue. Carefully cut through this layer of tissue to expose the heart. Gently remove the pericardium from around the heart. The frog heart has a single ventricle, which pumps blood to both the lungs and the rest of the body. Remove other tissue as necessary to get a clear view of the heart.

3. Using forceps, insert a fishing hook into the apex of the heart, being careful not to puncture the chamber of the ventricle.

4. Attach the line at the other end of the fishhook to the force transducer, then gently move the frog away from the transducer so the heart generates slight tension on the line. The transducer should be low enough so that the heart pulls against it primarily in the horizontal plane. When properly positioned, the voltage output of the force transducer will be proportional to the force of contraction of the heart without overstretching the heart. Adjust the voltage range in Chart to maximize the visibility of the heartbeat.

Exercise 1 – Observation of the cardiac cycle

Although not an experiment per se, you may find it interesting to observe the cardiac cycle in the frog heart. You can see the relative timing and force of atrial and ventricular contraction, and it is usually possible to perceive the blood moving through the chambers and blood vessels. You should also be able to observe the expansion of the major arteries during systole.With careful adjustment, both atrial and ventricular contractions may register on the force transducer.

Exercise 2 – Starling’s “Law of the Heart”

Starling observed that increased filling of the ventricle leads to more forceful contractions, and deduced that this was because of the increased stretching of the muscles prior to contraction. In this exercise, you will apply increasing tension to the ventricle, thus stretching it more and more, and examine the resulting force production of the muscle.

1. Adjust the position of the frog prep so that the tension on the line is just sufficient to provide reliable readings from the force transducer. Begin recording in Chart.

2. Slowly move the prep further from the force transducer, watching the force production. (Note that the baseline reading will increase as tension increases. You are interested in the additional force generated by the ventricle.) Continue increasing tension until force production begins to decrease or there is danger of damaging the prep.

3. Return the prep to its original configuration.

Exercise 3 – Effects of known agonists

In this exercise you will examine the response of the heart’s activity to the cholinergic agonist acetylcholine, and to the adrenergic agonist epinephrine.

1. Obtain small amounts of the epinephrine and acetylcholine solutions, and be sure your setup is still healthy. Keep the heart wetted with Ringer’s solution.

2. Record the activity of the heart for one minute to provide baseline data.

3. While still recording, drip epinephrine solution onto the heart at about one drip per second for about 10 seconds. Use the comment feature of Chart to mark your recording. Watch for changes in heart activity over the next five to ten minutes. If you are not seeing any changes, try repeating the dose.

4. When the heart’s activity no longer appears to be changing in response to the epinephrine, rinse the heart and surrounding areas well with Ringer’s, then wait five minutes to make sure the epinephrine is no longer having an effect. You can stop the recording to collect data on the epinephrine at this point.

5. Repeat steps 2-4 for acetylcholine.

Exercise 4 – Effects of an unknown antagonist

In this exercise you will examine the effect of an antagonist on the activity of the heart and on the effects of your two agonists. Often, antagonists block the effects of agonists on a particular class of receptor.

1. Again, record the activity of the heart for one minute to provide baseline data.

2. Drip the antagonist onto the heart and watch for any changes in heart activity over the next five to ten minutes.

3. Do not rinse the heart, but instead apply one of the agonists from exercise 2. Compare the response to that seen earlier.

4. Rinse the heart well with Ringer’s

5. Repeat steps 2-4 for the other agonist.

Exercise 5 – Effects of an additional agonist

The heart has additional classes of receptors in addition to the cholinergic and adrenergic ones. In this experiment, you will examine the effects of another neurotransmitter, serotonin, on heart function. To do this, follow the protocol used for the agonists in exercise 2.

Additional experiments

If time allows, you may want to try additional experiments, such as obtaining an EKG from the frog, or blocking the pacemaker signals between the atria and ventricle by applying a ligature. Ask your instructor for assistance if you get this far.

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