Principles

     The rate of contraction of the vertebrate heart is controlled by both intrinsic and extrinsic factors. The basal rate of contraction is established by specialized cardiac muscle cells located in a small area of the wall of the sinus venosus called the sinoatrial node.  A characteristic feature of these so-called "pacemaker" cells is the absence of a stable resting membrane potential.  Thus these cells undergo steady, spontaneous depolarizations which lead to a rhythmic pattern of cardiac action potentials. Action potentials within the pacemaker region are communicated over the entire heart as a wave of excitation, spreading first across the atria and then, through a specialized region called the atrioventricular node, to the ventricles. Atrial contractions thus precede ventricular contraction, a crucial feature for the efficient movement of blood.

     This intrinsic pattern of electrical and contractile activity also is affected by extrinsic inputs from both the parasympathetic and sympathetic branches of the autonomic nervous system. Parasympathetic cholinergic fibers from the vagus nerve innervate the sinus venosus. Acetylcholine and its analogs normally slow heart rate by reducing the rate of membrane depolarization in the pacemaker cells. The catecholamine, norepinephrine, which is released from sympathetic nerve terminals within the sinus venosus, atrioventricular node, and ventricles, has the opposite effect. The rate and force of contraction are both increased as a result of increases in pacemaker rhythm and in conduction velocity through the atrioventricular node.

     In the current laboratory exercise, you will examine in some detail autonomic control of cardiac contractility using several pharmacological agents.

Preparations

A. Living Material

 Red-eared slider turtles Trachemys scripta elegans of both sexes will be used. Turtles will be anesthetized with ketamine (1 ml/ turtle, i.m.) and pithed according to instructions given by your Instructor.

B. Solutions

1. Reptilian Ringer's Solution (137 mM NaCl, 2.7 mM KCl, 1.4 mM
CaCl2, 11.9 mM NaHCO3, 0.5 mM MgCl2, 0.4 mM NaH2PO4, 2.8 mM glucose)

2. Acetylcholine (0.1 mg/ml) in reptilian's Ringer's Solution.  Make fresh.

3. Epinephrine (1:1000) in reptilian Ringer's Solution.  Make fresh.

4. Nicotine 0.1% in reptilian Ringer's Solution.  Make fresh

5. 0.5% Atropine in reptilian Ringer's Solution

Procedures

A. Preparation of the Heart

 1. Anesthetize and pith the turtle. Tie the turtle's head and four legs to the support board, making sure that the neck is in a fully extended position. Remove the lower shell (plastron), being careful to keep the scissors close to the ventral plate to avoid excess bleeding while cutting the skin and connective tissue. After you have removed the plastron, you should see the heart beating within the pericardial cavity. Slit the pericardium and expose the ventral surface of the heart. Take a moment to identify its various parts and to observe the sequence of blood flow during each cardiac cycle.  Now, attach the heart to the force transducer, using the S-shaped hook suspended from it. Push the hook through the ventricular muscle just superior to the frenulum, being careful not to puncture the ventricular lumen, which will result in extensive hemorrhaging.

2.  Calibrate the force transducer.  Using the directions at your workstation calibrate the force transducer using the Biopac Student Lab Pro software as you did in the skeletal muscle lab.

 3. Adjust the position of the force transducer so that there is a slight upward tension on the heart. Click on start and make sure you have a trace that you can work with.  You may need to adjust the gain to obtain a good trace.

B. Autonomic Control of the Turtle Heart

 1. Action of Acetylcholine:  As its name implies, acetylcholine binds to cholinergic receptors. These are located on dendrites of the postganglionic nerve fibers (nicotinic) that innervate pacemaker cells of the sinoatrial node and at the sinus venosus itself (muscarinic). The parasympathetic ganglion where these receptors are located lies dorsal to the heart in the wall of the pericardial cavity. Remove any blood or other fluids from the pericardial space and apply 1.0 ml of 0.1 mg/ml ACh under the heart. Wait a few minutes and observe any effects. Remove the ACh solution, wash the heart with reptilian Ringer's.  Allow the heart rate to return to a stable baseline before continuing.

 2. Action of Epinephrine: Epinephrine will, at high enough concentrations, act as an agonist of norepinephrine the postganglionic neurotransmitter of the sympathetic nervous system.  It binds to adrenergic receptors on the sinus venosus and sinoatrial node. Remove any fluids from the pericardial space and apply 1.0 ml of 1:1000 epinephrine to the heart.  Wait a few minutes and record the heart rate and contractility. Remove the epinephrine solution, wash the heart with reptilian Ringer's.  Allow the heart rate to return to a stable baseline before continuing.

 3. Action of Nicotine:  Apply 1.0 ml of 0.1% nicotine to the pericardial cavity.   Record the effect on heart rate.   Remove the nicotine solution, wash the heart with reptilian Ringer's.

C. Effects of Temperature on Heart Rate and Contractility

1. Normal Isotonic Recording: Record two to three minutes of normal contractions, noting heart rate, and force of contraction (amplitude).  Determine the percentage of time in the cardiac cycle that is spent in ventricular systole (contraction) and in diastole (relaxation).

2.  Remove heart from the pericardial cavity after cutting all the major vessels and place in a beaker of cold Ringer's.  Place the beaker on a slide warmer and suspend the heart in the Ringer's using the clamp provided.  The slide warmer is adjusted such that temperature should rise about 1 degree C/ min.  Monitor the temperature using a thermometer and record the heart rate at 5, 10, 15, 20, 25 and 30 C.

Optional Exercise

Vagal Stimulation:  Carefully cut through the skin along the ventral midline of the neck and expose the underlying musculature. Cut through this muscle layer, again making your cut along the ventral midline, and expose the trachea (identifiable by its many cartilaginous rings) and the esophagus. Using a blunt probe, separate the connective tissue on each side of the trachea and locate the vagus nerves. These are quite large and appear as white strands running along each side of the neck adjacent to the carotid arteries. Loop a piece of surgical thread under each vagus nerve, making sure to separate it first from the carotid artery. Do not cut or stretch the nerves during the dissection and keep them and surrounding tissues moist with reptilian Ringer's Solution at all times.

 1. Stimulation of the Right Vagus: Set the stimulator to deliver 1.0 volt pulses at 30 pps. Support the right vagus nerve on the metal prongs of the hand electrode and begin stimulation in the continuous mode. Increase the voltage in 1.0 volt increments at 20 - 30 second intervals until a significant change in heart rate is observed. This is the threshold voltage. Note that the amplitude of contraction increases as the heart rate slows, a demonstration of the Law of Starling, which states that increased contractile force is generated when ventricular muscle is more stretched.

 2. Stimulation of the Left Vagus:  Repeat the above procedure and determine the threshold voltage for the left vagus.