BIOEN 3102 Bioengineering Lab II

Laboratory VIII handout: 2 weeks

Cardiac Activation

 

Goal

·        To introduce basics of the cardiac action potential

·        Effects of ionic concentration

·        Role of different channels

·        Strength-duration curves

·        To introduce techniques for measuring cardiac ECG responses

·        Near-field vs Far-field responses

·        To acquaint with the operational properties of heart contraction

·        To observe and record atrial and ventricular systole and diastole in the frog heart.

·        To examine changes as a function of temperature

 

Assignment

 

Experiment 1

 

Cardiac action potential simulations

The goal of this assignment is to experiment with a numerical simulation of the cardiac action potential. The form of this simulation is just as described in class, using the Hodgkin-Huxley formalism and differential equations to reproduce the currents responsible for the action potential. In addition to varying ionic concentrations, there are six major ionic currents present in cardiac ventricular cell membrane that are used in these simulations: INa, a fast sodium current; Isi, a slow inward (largely) calcium current; IK, a time-dependent potassium current; IK1, a second time-independent potassium current; IKp, a plateau potassium current; and Ib, a time-independent background current. Simulations allow one to conduct several “virtual” experiments to determine the effect of different parameters on the model performance. For instance, we can turn off different channels by changing the “Gate inactivation” parameters.

1.       Action potentials only fire if the stimulus strength above a threshold. Determine at least ten combinations of stimulus strength and duration that will just barely elicit a normal action potential and plot these value pairs (strength versus duration) in an X-Y graph. What do these results tell you about how the (simulated) cell responds to stimulation? How does this compare to neurons?

2.     In order to simulate the experiments performed out by Hodgkin and Huxley on the nerve axon, carry out a set of simulations in which you systematically alter the concentration gradient of sodium (use reasonable values, holding the intracellular concentration constant) and observe the effects on action potential shape and amplitude. Why does the action potential not disappear completely even when the sodium gradient approaches zero? What is providing the inward current in this case?

3.     During real cardiac ischemia, the extracellular potassium concentration rises from its normal value of 5 mM to the range of 12-15 mM. In a set of simulations, adjust the external potassium concentration gradually and document the resulting effect on potential and currents.

4.    Perform at least 3 other modifications to the simulation of your choosing and document the results. Feel free to alter any parameter provided in the front panel of the model (not just ion concentrations), but justify your choices in terms of either normal or pathophysiology. Try and explain the results in terms of your qualitative knowledge of cellular electrophysiology.

Experiment 2

Introduction

The heart's primary function is simply to act as a pump that provides pressure to move blood to its ultimate destination - the tissues. The control of cardiac contractility is complex and represents a balance of intrinsic (within the heart) and extrinsic (from outside the heart) factors. In the following experiment you will examine some of these intrinsic and extrinsic factors that make the heart such a unique and versatile pump. Cardiac muscle differs from skeletal muscle both morphologically and functionally. Probably the most striking and fascinating feature of its contractility is that it is able to initiate its own rhythmic contractions without requiring a stimulation from outside the heart. This is due to "leaky" cell membranes, in which calcium and sodium ions slowly leak into the cells. This leaking causes a slow depolarization to threshold, thus firing an action potential and initiating contractions of cardiac muscle. The cells that are most "leaky" to ions and that depolarize fastest control the rate of contraction of all other cardiac cells; thus, they act as pacemakers for the rest of the heart. In the mammalian heart, the pacemaker is the sinoatrial (SA) node, a group of specialized cells near the junction of the vena cava and the right atrium. In the frog heart, the pacemaker is the sinus venosus, an enlarged region between the vena cava and the right atrium. (The mammalian SA node is believed to be an evolutionary remnant of the sinus venosus.)

Anatomy of Amphibian Heart

In this experiment as well as the one next week, you will use a frog heart because it functions well at room temperature and will continue to beat even when excised from the body. Mammalian hearts have the same contractile characteristics, but must be supplied with a constant flow of warm, oxygenated blood to maintain their contractility. The frog heart differs from the mammalian heart anatomically in that they are three chambered rather than four chambered. The pacemaker in the amphibian heart is the sinus venosus, a thin-walled sac that receives blood from the anterior and posterior caval veins and empties blood into the right atrium. The single ventricle receives blood from both atria and pumps blood out through the large artery called the truncus arteriosus. In contrast, the mammalian ventricle has separate left and right chambers, which prevent mixing of the venous and arterial blood.

Dissection Procedure

Double pith a frog and fasten it to a frog board, ventral side up. Use scissors to make a longitudinal incision through the skin and body wall of the thoracic region to expose the heart. Note the pericardial sac surrounding the heart. Hold the pericardium with forceps and carefully cut away the sac from the heart, using scissors. From this point on make sure that the heart is periodically moistened with from Ringer's solution. Using forceps, gently lift the apex of the heart upward. Insert a suture needle through the tip of the ventricle, being careful not to damage the ventricle. Tie a thin thread to the hook and connect the ventricle to the transducer and adjust the tension on the ventricle to record the contraction of the heart.

Cut the skin from the groin to the throat of the frog.

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Cut through the pectoral girdle to expose the heart in the pericardial sac.

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Attach a small hook tied with thread through the frog heart

 

Loop the thread from the frog heart to the force transducer. Slowly adjust the transducer stand height and position so the connection between the heart and the transducer is relatively taut — be careful not to tear the heart out!  Place a recording and/or stimulating electrode on the surface of the heart with distant return and reference electrodes.

Experimental Procedure

1. Normal Heartbeat

Obtain ECG and force recordings of the normal cardiac rhythm. Distinguish the atrial and ventricular contractions. The duration of systole and diastole of the ventricle can be determined. Measure the delay between the electrical and mechanical activation of the heart. Attach a labeled portion of your record to the Laboratory Report. In a 10-second segment, measure the heart rate.

2. Temperature Effects

Record the heart contractions at room temperature. Then drop warm (40C) frog Ringer's solution on the heart until significant changes are seen in rate and contractility. Record contractions at this time and measure heart-rate. Finally, drop cold (5C) Ringer's on the heart and record when changes are observed. Then rinse the heart with room-temperature Ringer's to return the beat to normal before continuing the experiments. Can you see why amphibians becomes so sluggish when the temperature drops? Determine the heart rate at each temperature.

3. Refractory Period of Heart

Position the transducer to eliminate as much as possible the atrial contraction in the recording. Arrange for electrical stimulation of the ventricle by clamping the stimulating electrode so that the points touch the ventricle gently and constantly during the contraction cycle. Record the ventricular contractions. Using single stimuli of 100mV and 1-msec duration, stimulate the ventricle at different times in the cardiac cycle. It is often a tricky procedure to obtain an extra systole using single stimuli, because it is difficult to catch the ventricle immediately after its refractory period. If you have trouble, try using repeated stimulation so that one of the stimuli can catch the ventricle at the proper time to produce an extra systole. What is the result of stimulating during the systolic phase of the cycle? During the diastolic phase? Can a second contraction be elicited before the normal rhythmic contraction occurs? Look for the appearance of an extra systole followed by a compensatory pause.

4. Tetanization of Heart

Record the contraction of the heart while it is being stimulated with a tetanizing frequency (10 to 15 stimuli per second) and 20 V. How do your results compare with tetanization of skeletal muscle? How is this response related to the refractory period?

5.    Drug Effects (see update next week!)

Using a syringe, apply the following drugs to the heart until you see significant changes in rate and/or contractility. Best results are seen if the drug is dropped on the sinus venosus region of the heart. Be very cautious in applying these drugs, because they are very potent and may stop the heart completely if an overdose is given. After the effect is recorded, rinse the heart with frog Ringer's and allow the heart to return to normal before the next drug is applied. You will be expected to measure the dose-response curve for 2 of these drugs.

 

Before application of any drug, record 10 seconds of a baseline response. Record 10 seconds of response every minute following drug application. Finally, record a return to baseline after washing the drug out with Ringer’s solution. Perform the same procedure for different concentrations of a particular drug or for other drugs. Report the effect of each drug on heart-rate and contractility. Also report the dose-response curves, i.e. the drug-effects for different concentrations.