From Human Physiology & Pathology, the basic knowledge about cardiac function & the generation of the heart beat was known from last year. There are four auricles, the atria is placed on the on the two upper auricles (known as the left & right atrium). The role of the right atrium is to receive de-oxygenated blood from the superior vena cava, inferior vena cava & coronary sinus. The left atrium receives the oxygenated blood from the left & right pulmonary veins. The ventricles are placed on the bottom two auricles (known as the left & right ventricle). The role of the right ventricle is to pump blood into the pulmonary circulation for the lungs. The left ventricle pumps blood into the systematic circulation through the aorta into the rest of the body.
The heart has a pacemaker activity, which is initiated through the sinoatrial (SA) node. The SA node is the pacemaker tissue located in the wall of the right atrium of the heart, near the entrance of the superior vena cava & thus the generator of the sinus rhythm. They do not contract, even though they possess contractile filaments. They send action potentials to the atrioventricular (AV) node, causing the ventricles to contract, pushing the blood through the aorta into the rest of the body.
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The atrium of the guinea pig is able to survive in vitro & it is able to beat spontaneously as long as the SA node isn't damaged. In comparison to the atria, the ventricle has no pacemaker activity.
Before entering the laboratory room, the pharmacology lecturers (Dr. Javid & Dr. Airley) had dissected the heart in to the components (atria & ventricles) needed. This was all done in the Ringer-Locke solution (isotonic electrolytic infusion solution) to maintain the heart was at optimum level. The dissection of the atria was done by trimming away the fat & exposing the atrioventricular junction (appears as a pale yellow line). The left side of the heart can be distinguished by positioning it, so both the auricles can be seen. After the atria had been isolated, a vertical cut between the auricles along the atrial septum was made. The strip of atrial tissue was then connected to the tissue holder & transducer. The tissue was then placed under a light amount of tension initially to avoid stretching & damaging the tissue' thus leading to impairing of the sinoatrial node (SA). If the SA node was impaired it would have been a problem, as the cells within the SA are the primary pacemaker site/activity which generates electrical impulses (or action potential) which control the heart. The cells have no true resting potential, but generate regular action potentials.
For the dissection of the ventricle, while the heart was in the Ringer-Locke solution a vertical cut was made into the ventricle. A parallel cut was done to isolate the strip of heart muscle. Finally it was connected to the tissue holder & transducer. As the ventricle has no pacemaker activity, an electrode was connected to the stimulator with the assist of some crocodile clips. The stimulator was needed to pace the stripped ventricle.
Both the atria & ventricles were placed into organ bath (separate), with a final bath volume of 50ml. The LabTutor setup had two channels. The first channel was measuring the output from the transducer & this was totally dependent on the preparation. The second channel was calculating the variation of the contraction rate with time.
The LabTutor was used to establish the baseline contractile activity. The atria should be beating spontaneously due to the SA node. The ventricles however will need pacing using the stimulator setup.
There were 4 parts to this experiment. Which were:
Exp1 1a:- Effects of isoprenaline
To observe the response to the ventricle with the addition of isoprenaline.
The first step was to calculate the volume of isoprenaline needed for the Final Bath Concentration (FBC) of 10-10 M, using equation 1 (from practical sheets). The volume calculated was 0.005µl, & but was too small to measure using the Gilson pipette. Therefore an addition of 10µl was added to the ventricular tissue. The response was observed on the LabTutor & before the next dose of isoprenaline, the response signal had to be stabilised. Unfortunately there was no response, so the ventricle tissue was washed before the dose was doubled to 20µl, then 40µl. In both cases there was no response observed, so the concentration was again doubled to 80µl. There was still no response, so using the stimulator the tissue was shocked. Even with the shocking there was still no response as the tissue was unable to pace itself. There was a further & final addition of 160µl, but there was still no response. Therefore no results or data were collected.
Expt 1b:- Effects of Isoprenaline after Propranolol
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To observe the response of both the atria & ventricle, with the addition of the EC50 dose of isoprenaline in the presence of propranolol.
The next step was to add propranolol to the FBC of 5-10-6M, & a comment was added on the LabTutor so it was known when it was added. The preparation was left for 10 minutes so it could equilibrate. Then the EC50 dose of isoprenaline was added & another comment was added. After the addition isoprenaline the response of the heart tissue was measured using the LabTutor. In the next 15 minutes the heart tissue was washed out every 5 minutes, so results of the heart tissue could be seen (3 times). Again there was no response, so no data or results collected.
Expt 2a:- Effects of Acetylcholine
To observe the response of both the atria & ventricle to acetylcholine.
The dilution & volumes were again calculated using Equation 1. As the heart needed pacing, there was an addition of 10µl of isoprenaline. There was no response observed, therefore 20µl of isoprenaline was added again. There was no signal, therefore any response. There was no further addition, as it would have been a waste!
Expt 2b:- Effects of Acetylcholine after Atropine
The final part of the experiment was to observe the response on both the atria & ventricle with the dose of IC50 of acetylcholine, in the presence of atropine.
There was no signal, as the heart tissue didn't respond from the previous part.
Figure 1 shows the effect as the concentration of isoprenaline (Expt 1a) is increased on the heart tissue.
The inotropic & chronotropic effects were increased, as the concentration of isoprenaline was increased. During the wash out of the heart tissue (to clean the previous dose), the inotropic effect dropped to a minimum of 0.7mN. There was a reduction of chronotropic effect, as the signals were less frequent. However after the addition of isoprenaline, the contraction force returned higher going up to 1.5mN. Therefore in the presence of isoprenaline there is a positive increase in both inotropic & chronotropic effect.
Figure 2 shows the effect of isoprenaline in the presence of propranolol on the heart tissue.
It shows the baseline as the tissue is at normal activity & being paced with isoprenaline. Once the tissue was stabilised, propranolol was added to see the response. It shows the chronotropic effect had decreased, as the heart rate of the heart tissue had slowed down, reducing the frequency. When isoprenaline was added it can be seen the inotropic effect of the heart tissue being decreased thus decreasing the volume of blood pumped from one ventricle of the heart with each beat (stroke volume). This reduced the cardiac output as there was less blood being pumped around the body; thus increasing the end-systolic volume. As the EC50 dose of isoprenaline was added to the heart tissue, it can be clearly seen that both the inotropic & chronotropic effects increased. Unfortunately the contraction force & frequency of the response weren't as vigorous in the absence of propranolol. The clinical side to a decrease in inotropic could be bad for patients whose heart is not beating at the normal rate (lower). If there is a negative inotropic effect, then the contraction force will not be forceful, resulting in a low heart rate (Bradycardia).
Figure 3 shows the effect of isoprenaline in both the presence & absence of propranolol on the heart tissue.
The inotropic effect increased as there was an increase of concentration of isoprenaline. The EC50 (horizontal line) is the concentration of the isoprenaline that provokes a 50% response between the baseline & maximum response. The EC50 is higher when in the presence of propranolol, this shows a greater concentration of isoprenaline is needed to produce the same response (compared to isoprenaline alone). Both propranolol & isoprenaline were competing for the ß2-adrenergic receptors located on the heart, & it can be seen that propranolol has the higher affinity.
Figure 4 shows isoprenaline added to the cardiac tissue, for the pacing.
Before the addition of isoprenaline the force was relatively stable between 0.6-0.7mN, after it raised to a maximum of 0.8mN.
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Figure 5 shows the effect as the concentration of acetylcholine is increased on the heart tissue.
Both the inotropic & chronotropic effect had decreased of the heart tissue, as the concentration of acetylcholine had increased. As the heart tissue was washed out, the inotropic & chronotropic effect had increased. This is due to the previous dose of acetylcholine being washed out. After each dose of acetylcholine (after being washed out & as the concentration was increasing), the inotropic & chronotropic effect had dropped dramatically. The contraction force goes to a minimum of 0.6mN.
Figure 6 shows the effect of acetylcholine in the presence of atropine in the heart tissue.
It shows with the addition of atropine there was a large increase in both the inotropic & chronotropic effect. With acetylcholine, both the inotropic & chronotropic effect had dropped significantly, with the contraction force dropping to a minimum of 0.8mN. Negative inotropic effect shows a decrease in myocardial contractility & cardiac workload.
Figure 7 shows the effect of acetylcholine in both the presence & absence of atropine on the heart tissue.
The inotropic effect was greater when in the absence of atropine. The IC50 (horizontal line) is the measure of the effectiveness of a compound in inhibiting biological or biochemical function. The IC50 is higher when in the presence of atropine, this shows a greater concentration of acetylcholine is needed to produce the same response (compared to acetylcholine by it itself).
Isoprenaline is a potent non-selective beta-adrenergic agonist (activates ß1 & ß2 equally) & it has a low affinity for a-adrenergic receptors. It is a sympathomimetic agent; therefore you should see an increase in the heart rate as the contraction force is going to be more powerful & more frequent.
From Figure 1 & 3 it can be clearly seen that there was an increase in inotropic, chronotropic & dromotropic effect. This is because there are ß1-adrenergic receptors on the heart which bind to isoprenaline; thus leading to a higher cardiac output & oxygen supply (greater stroke volume & heart rate). The ß-adrenergic receptors are a type of G protein-coupled receptors that are targets of catecholamines. Isoprenaline is a catecholamines. The G protein-coupled receptor is linked to adenylate cyclase, which is an enzyme. Isoprenaline binding causes a rise in the intracellular concentration of the second messenger cAMP (cyclic Adenosine MonoPhosphate). Downstream effectors of cAMP include cAMP-dependant protein kinase (PKA), which causes phosphorylation & activates the calcium ion channels. Thus release of calcium ions by the sarcoplasmic reticulum (SR) in the heart makes the cardiac muscle contract.
It was predicted as the concentration of isoprenaline increased, there would be more response from the cardiac tissue. Both the inotropic & chronotropic effect would increase, as the contraction would be more vigorous & the signal was more frequent (higher heart rate). This is because more of the unoccupied receptors would become occupied forming a complex.
From Figure 1 & Figure 3, it can be seen as the concentration of isoprenaline was increased there were more vigorous contraction & the signal was more frequent. Thus increasing the inotropic & chronotropic effect, so the prediction was right.
In both the presence & absence of propranolol there were differences with the response. Comparing the two, it is expected in the presence of propranolol there is going to be a negative inotropic, chronotropic & dromotropic effect. In the absence of propranolol, there is going to be a positive inotropic & chronotropic, because there is going to be strong contraction force as isoprenaline is a direct cardiac stimulant.
From Figure 2, it can be seen in the presence of propranolol, both the inotropic & chronotropic effects decreased. From Figure 3, the EC50 dose of isoprenaline is greater when in the presence of propranolol. The response is pretty insignificant, as there is very little occurring.
From Figure 1, after each dose of isoprenaline (after the wash out & with increasing concentration), the contraction force was large. The contraction force reached a maximum of 1.5mN. There was a lot of frequency & signals, they were rapid (therefore high heart rate). There were significant changes in the response of the cardiac tissue.
Isoprenaline is used in bradycardia. It works by activating the ß1-adrenergic receptors located on the heart, which induces an increase in inotropic, dromotropic & chronotropic effects. This leads to an increase in the stroke volume & heart rate, thus greater cardiac output (stroke volume - heart rate). Therefore the blood is pumped through the aorta at a high pace. It is also used for Adams-Stokes attack, COPD etc.
A high dose of isoprenaline helps in bronchoconstriction, and it is used by relaxing the bronchial smooth muscle, thus relaxing the airways to increase airflow in the lungs. There is also an increase in end-diastolic volume & sympathetic volume.
Isoprenaline isn't used much, because it leads to too many adverse reactions for example dyspnoea. This causes the muscle spindles in the chest wall to signal the stretch/tension of the respiratory uscles. The poor ventilation leads to left heart failure, leading to interstitial oedema, asthma causing bronchoconstriction.
However, isoprenaline main adverse reaction is tachycardia. This is because heart is beating above its normal rate, the heart doesn't pump at its optimum level, and therefore less blood is being supplied to the heart/body. The increased heart rate leads to increased work & myocardial oxygen demand, which can lead to myocardial infarction. This occurs due to myocardial oxygen supply not meeting the demand causing myocardial cells to die off. This leads to angina & to ischemic heart disease.
Side effects include headache, sweating, chest pain etc, due to its non-selective behaviour. It can't be administered with other direct cardiac stimulants because it increases the chance of serious arrhythmias. It can't be given to patients with tachycardia as the heart rate is above normal rate. Isoprenaline will bind to the ß1-adrenergic receptor which is located in the heart, making the contractile force & frequency greater; thus an increase in inotropic, chronotropic & dromotropic effects. This will lead to a larger stroke volume, heart rate & the myocardial oxygen supply; hence an increase in cardiac outputà inducing a heart attack. There's no change in the blood pressure. There are many drugs that are newer & work better than isoprenaline. Such drugs include Dobutamine, which is a sympathomimetic drug used in the treatment of heart failure and cardiogenic shock. Its primary mechanism is direct stimulation of ß1-adrenergic receptors of the sympathetic nervous system.
Propranolol is used for in the treatment of hypertension, angina & atrial fibrillation. When hypertension occurs there is a rise in Cardiac Output (CO), with the Total Peripheral Resistance normal (TPR). There is a large force pushing the blood via the aorta to the body. Therefore there is a positive inotropic & chronotropic effect. With the addition of propranolol, it will bind to the M2-receptors located in the heart. They will occupy the receptor, but won't induce an effect. This will lower the force of contraction; thus reducing the inotropic effect.
Propranolol reduces the amount of blood flowing through the body, therefore it has an effect on the skin, muscle etc. Patients will begin to feel drowsy & tired.
It is best to avoid when injecting with adrenaline. It will cause a severe allergic reaction. By using them simultaneously it will result in a high blood pressure & marked slowing of the heart beat.
Some of the adverse reactions include bradycardia, hypotension & heart failure.
New drugs are being used because of its non-selective characteristics. It is unpredictable; therefore there are some substitutetable drugs. For example acebutolol is selective for ß1 receptors only.
Acetylcholine is a neurotransmitter (NT) in the peripheral & central nervous system. It is the only NT used in the motor division of somatic nervous system. It is the principal NT in all autonomic ganglia. It binds to the acetylcholine Muscranic receptors (e.g. M2-receptors located in the heart). The M2-receptors slow down the heart rate down to normal sinus rhythm, by slowing the speed of depolarisation. Also by reducing the contraction forces of the atrial cardiac muscle & conduction velocity of the AV node. They have no effect on the contraction forces on the ventricle muscle. Muscranic receptors are G protein-coupled acetylcholine receptors found in cells. By acting via G-protein type receptor, there is a decrease in cAMP, leading to inhibitory effects (on the heart). The effect acetylcholine has on the heart is it reduces both the inotropic & chronotropic effect, by modulating the muscarinic potassium channels.
Atropine acts as a competitive antagonist on muscranic-acetylcholine receptors. However atropine has no efficacy at the M2-receptor therefore it blocks the effect of acetylcholine. Then acetylcholine begins to slow down the heart. Therefore atropine has no cholinergic effect & therefore it is unable to slow down the heart. But due to neuromodulation, it will allow the sympathetic innervations to dominate. Noradrenaline increases the heart rate, so the net effect of atropine on the heart rate is to increase the rate of firing from the SA node. The heart has both cholinergic & noradrenergic receptors in itself, but it's also controlled through the cardiac & vagus nerve.
It was predicted as the concentration of acetylcholine increased, there would be less response from the heart tissue. Both the inotropic & chronotropic effect would decrease, as the contraction force won't be forceful & the signal & frequency would be less (lower heart rate). This is because more of the unoccupied receptors would become occupied, but it wouldn't be initiated.
However from Figure 5 & Figure 7, it can be seen as the concentration of acetylcholine was increased there were more less contraction & the signal/frequency was less moderate. Thus decreasing the inotropic & chronotropic effect.
In both the presence & absence of atropine there were differences with the response. Comparing the two, it is expected in the presence of atropine there is going to be a negative inotropic & chronotropic effect. In the absence of atropine, there is going to be a positive inotropic & chronotropic, because there is going to be strong contraction force as it is a competitive antagonist for the muscarinic acetylcholine receptor. It binds to the M2-receptors located on the heart.
From Figure 6, it can be seen in the presence of atropine; both the inotropic & chronotropic effects were increased. From Figure 7, the IC50 dose of acetylcholine is greater when in the presence of atropine. The response is pretty insignificant, as there is very little occurring. This is because they compete for the same receptors & as acetylcholine occupies the receptors, it shows that it has higher affinity than atropine.
Atropine binds to muscarinic receptors so it stops acetylcholine from binding to and activating the receptor. By blocking the actions of Acetylcholine, atropine blocks the effect of vagal nerve activity on the heart. Thus reducing the inotropic & chronotropic effect. In a clinical situation, the effect of atropine would also be mediated by its inhibitory effect on the vagus nerve.
From Figure 5, after each dose of acetylcholine (after the wash out & with increasing concentration), the contraction force dropped. The contraction force reached a maximum of 0.8mN, which was below the contraction force at the baseline period. There was a low heart rate as the signals were not frequent. There were significant changes in the response of the cardiac tissue in both the presence & absence of atropine.
Acetylcholine is used for Ocular peri-operatives drug. It is instilled into the anterior chamber of the eye during surgery, quickly producing miosis which lasts for 20 minutes.
Contraindication include pregnancy & breast-feeding, but it's not advisable.
Adverse reactions include hypotension, Shortness Of Breath (SOB), sweating etc.
Atropine is used for many things, bradycardia is one option. Atropine binds to the ß2 adrenergic receptor found on the heart. It increases its inotropic & chronotropic effect; thus getting a higher heart rate & stroke volume. Therefore the cardiac output increases; meaning the blood (with oxygen) was travelling via the aorta with more conviction. The dose should be started titrating from a lower dose. Initial dose of 500µggiven intravenously; the dose can be repeated every 3-5 minutes as long as you don't exceed 3mg.
Contraindication include an increase in risk in antimuscranic side effect, if given with disopyramide.
Adverse reactions include paradoxical heart rate slowing when given at very low doses, presumably as a result of central action in the Central Nervous System (CNS)
Errors & Improvements
Errors with the practical include the SA node might have been dissected/damaged, as this is where the pacemaker activity occurs. As there was no response, it could mean the SA node might not have been functioning.
As there was no result as the heart tissue died, need to have a fresh heart at disposal.
I learnt the different type's sympathomimetic & parasympathomimetic agents & how they affect the cardiac muscle.
The effect of isoprenaline alone increases the inotropic, chronotropic & dromotropic effects. In the presence of propranolol, the inotropic, chronotropic & dromotropic effects were reduced. Also I have gained a clinical perspective on both isoprenaline & propranolol.
The effect of acetylcholine alone decreases the inotropic & chronotropic effects. In the presence of atropine, the inotropic & chronotropic effects were positive. Also I have gained a clinical perspective on both acetylcholine & atropine.
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