SPECIAL LAB: CARDIAC PHYSIOLOGY

PROCDURE:
You will be given an anesthetized frog (double pithed). The following procedures will be demonstrated before you have to do them on your own. Place the frog ventral side up on the board, and secure all four legs to the board with string. Cut the skin away from the area of the sternum and just below. Then, carefully lift the muscular layer using a forceps, and make a cut through the muscles. Be careful not to cut too deep, as the heart and other organs lie just beneath. Make the cut extend from the sternum about one inch along the abdomen. Then cut laterally to expose the pericardial area. You should be able to see the heart beating at this point. Now cut away the bottom half-inch protrusion of the sternum using the large scissors. This should expose the entire heart. Carefully lift the pericardial membrane and cut it away from the heart using the fine scissors. Be careful not to puncture the heart or associated blood vessels. Get help if you are uncertain about this step.

Balance and calibrate (using the internal calibration button on the myograph) an F-60 Myograph and recording channel such that 5 gm of force equals 10 cm at a sensitivity of 50 mV/cm. Position the pen near the bottom of its range. Grasp the apex (bottom) of the ventricle gently with a forceps, and insert the myograph hook through the muscular wall. Try to get the hook as low as possible and through the muscle only, not through the blood cavity, to avoid bleeding. Adjust the myograph and stand so that the heart, string, and myograph are in a straight line, or as close as possible. With the recording channel on, adjust the tension on the string so that the ventricular contractions give a peak of about 3-4 blocks. The vernier tension adjuster (the screw that raises or lowers the myograph) should be turned almost all the way "down" (clockwise). Adjust the tension by gently moving the myograph stand itself.

Record a page of normal contractions using a relatively slow paper speed. You will want to be able to calculate heart rate and strength of contraction. Then, change the paper speed to either 50 or 100 mm/sec, and record one good page of contractions. From this record you will measure the duration of contraction and relaxation.

STARLING'S LAW
Record about a half-page of normal contractions. The height should be about 3 blocks. While the record is running, hold the myograph stand steady with one hand, and GENTLY turn the vernier counterclockwise ("up") one-tenth of a turn for each contraction (or, think of it as turning the knob on the vernier about 2-3 mm each time). Do this for about 10 - 20 contractions, making the turn in the vernier at the point when the ventricle is relaxed. This adds a slight bit of extra tension to the ventricular muscle at the beginning of each new contraction. You should see a change in the strength of the contraction. Then, turn the vernier back down in the same stepwise manner, until the contractions have returned to normal.

EFFECT OF TEMPERATURE
Record about a half-page of normal contractions. Stop the record. Obtain some cold saline, and apply it to the heart using a 5 ml syringe. Apply about 5 ml slowly. Then record about 10-20 seconds of contractions. Stop the record and make another application of saline, and record another 10-20 seconds. Repeat this procedure until you see a change in the rate and strength of contraction. Then wash with room-temperature saline to warm the heart back up.

CONTROL OF HEART RATE BY NEUROTRANSMITTERS
Use a relatively slow paper speed, and adjust the tension so the ventricular contraction gives a peak height of about 3-4 blocks.

EFFECT OF Ach
Record about a half-page of normal contractions. Stop the record. Identify the sinus venosus below the right atrium (gently lift the atrium to see the sinus venosus). Fill a 1 cc syringe with acetylcholine (0.5 mg/ml Ach). While the record is still running, apply half directly to the sinus venosus and the other half to the general area of the atria. Keep the record going until you see a measurable change in the rate of contractions. This may take a couple of minutes, or it may happen sooner. Measure the rate vs that of the control; you should see a decrease. If not, add another 0.5 cc to the sinus venosus and keep recording. If Ach doesn't have an effect, get one of the TAs to help you. Wash the SV and atria thoroughly with saline until the rate returns to normal.

EFFECT OF PILOCARPINE
Record about a half-page of normal contractions. Stop the record, and add 1.0 cc of pilocarpine (0.5 mg/ml) to the SV. Wait about 2 min and record 30 seconds of contractions. Did the rate slow down? If not, wait another 2 min and record again. Repeat this until you see a decrease in the heart rate. It may take a while for the drug to permeate enough into the tissue to have an effect. Do not add more pilocarpine unless the TA says so. Immediately upon seeing a decrease in rate, stop the record and wash thoroughly with saline. Pilocarpine can be hard to wash out, so as soon as you see an effect, wash thoroughly. Pilocarpine is said to be an analog of Ach. That should tell you the mechanism of its effects.

EFFECT OF PHYSOSTIGMINE FALLOWED BY Ach
Record about a half-page of normal contractions. Stop the record, and add 0.2 cc of physostigmine. Keep the record going and watch closely for a decrease in heart rate. As soon as you see it, immediately add 1 cc of Ach to the SV and surrounding atria and keep recording. The rate should slow down even more. Compare this decrease with the one seen for the addition of Ach alone.

If the rate does not slow down within 2 min after you add the physostigmine, stop the record and wait 2 min and then try the record again. Again, it may take a while for the drug to take effect. Add more physostigmine only after consulting with the TA. Physostigmine is an inhibitor of the enzyme acetylcholine esterase, the enzyme that breaks down Ach in the synaptic cleft.

Wash the SV and atria thoroughly with saline until the contractions return to their normal rate.

EFFECT OF EPINIPHRINE
Record about a half-page of normal contractions. Continue recording and add 0.5 cc of epinephrine (1 mg/ml) to the ventricle (why do we add it to the ventricle and not the SV or atria?). Keep recording until you see a change in the rate and/or strength of contraction.

EFFECT OF ATROPINE
Record about a half-page of normal contractions. While the record is still going, add 1 cc of atropine (1 mg/ml) to the SV and surrounding atria. What happens to the heart rate? Then, add 1 cc of Ach to the SV and atria. Record for about 30 sec, stop the record for 2 min, and record another 30 sec. Get at least 4 records, each at 2 min intervals, to see if the rate changes. The rate should not change. Atropine is a blocker of muscarinic receptors.