Frog Heart Lab, Animal Physiology

Chemical and Environmental Effects on the Heart Introduction The heart is the centerpiece of the circulatory system, its muscular contractions allow for the timely delivery of essential gases and nutrients to virtually all cells of the body. The pressure created by the heart also plays a vital role in eliminating wastes through organs such as the kidney, thus the heart delivers and helps maintain nutrient and waste composition throughout the body. The heart, like all muscle cells, releases ionic calcium when stimulated which binds to troponin which in turn causes tropomyosin to uncover the myosin-actin binding sites on the muscle.

Temperature has effects on the metabolism and activity of all cells. Warmer temperatures increase the kinetic energy of molecules in cells, providing more energy which allows metabolic processes to proceed more quickly. Cooler temperatures, on the other hand, decrease molecular kinetic energy and cause slower metabolic rates in cells and tissues, hence when a bear hibernates, its body temperature is some degree lower than it is during the bear’s active periods.

The heart is also susceptible to certain molecules for which are able to bind to its receptors or diffuse across its membrane and affect intracellular activity and consequently have effects on the overall homeostatic condition of the organism. The Sinoatrial Node (SA Node) acts as the pacemaker of the heart by providing a small, autorhythmic electrical pulses that travel to the atrioventriclar node (AV node) and through the Bundle of His and Purkinje Fibers through gap junctions at the intercalated disks which stimulate the cells of the heart to contract via calcium release.

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This contraction is similar to a neuron in the sense that a threshold stimulus is needed to cause a contraction, a refractory period follows contraction at which time a new contraction cannot occur. Drugs that have an effect on the tissues of the heart, especially those where the SA Node resides can have an effect on the frequency and strength of muscular contraction via causing a stimulus to occur and lowering the threshold needed to cause a contraction. The heart is under both nervous and hormonal control.

The brain is constantly receiving information from the body such as pH, CO2 levels, and many others that the hypothalamus and medulla play a role in translating and reacting to via the release of hormones such as epinephrine which affects the SA node, either by stimulating or inhibiting contraction rate. Removal of the heart from the body would result in eventual cessation of beating as these sources are depleted from the immediate environment, not to mention the absence of the appropriate ion levels needed to maintain resting cellular electrochemical gradients.

All of the aforementioned aspects of heart control coordinate with Starling’s Law of the Heart, which relates to stroke volume, contractions strength, and frequency of heart contraction. This paper is interested in investigating what the effects of the alteration of temperature, chemical environments, and physical obtrusion have upon the strength and frequency of cardiac muscle contractions. Decreasing the temperature of the heart’s environment should hypothetically result in a decrease in both frequency and strength of contractions due to the decreased ability of calcium ion channels to open and cause contraction.

Various chemicals such as epinephrine and calcium ion solutions should correlate to both and increase in frequency and strength of the resultant contractions due to direct effects on the hearts mode of activation (SA node stimulus) and increasing the levels of available calcium needed to cause a contraction. Other chemicals such as Atropine should indirectly increase heart rate via the blocking of the effects of the parasympathetic system resulting in a predomination of sympathetic activity.

Acetylcholine, which acts on the muscarinic receptors of the heart, should display inhibitory effects on the heart by decreasing available cAMP levels, which results in fewer phosphorylated Protein Kinases which are needed to open the calcium channels which result in contractions of all muscles of the body. Additionally, chemicals such as nicotine should have little to no effect on the effects of muscle contraction due to lack of receptors on the heart for such substrates as well as lack of nicotinic receptors on any body tissues that indirectly affect heart rate such as the brain.

Methods Procedure 1: The Heart Rate The dissected frog, whose heart was left attached and embedded in the frog, was connected to a string at the most basal aspect of the heart, and wrapped around an electrical stress sensor located 15cm above the heart to detect changes in pressure on the apparatus caused by heart contractions. Unless otherwise stated, all subsequent procedures will have the same setup to minimize variability in the results obtained. The resting heartbeat was then recorded via the described instrumentation. Procedure 2: Effects of Cold Temperature

Initially, 10mL of room temperature Ringer’s solution was applied directly to the heart and allowed to contract freely for 15 seconds. The data obtained from the contractions was recorded. The heart was allowed 1 minute to recover from exposure to the solution. Next, 10mL of chilled Ringer's solution was applied directly to the heart and allowed to contract freely for 15 seconds. This data was recorded. Procedure 3: Effects of Drugs Thirty seconds of normal heart contractions were recorded at which time 2mL of epinephrine was dropped onto the heart itself.

Contractions were allowed to proceed for 60 seconds during which time data was recorded. Following exposure to epinephrine, the heart was allowed to return to its resting state determined in procedure 1. This same procedure was repeated with the following chemicals: 1) Acetylcholine, 2) Atropine, 3) Calcium solution, 4) Nicotine solution, and 5) Caffeine solution. Procedure 4: The Refractory Period of the Heart Resting heart contractions were recorded for thirty seconds until the heart rate was less than 60 beats per minute. A stimulator electrode to be used was set to the following states: Amplitude of 4. 0 Volts, a stimulus delay of 50ms, stimulus duration of 10ms, a frequency of 1. 0Hz, and a pulse number of 30. The electrode was then placed in direct contact with the heart for 30 seconds at which time the data was observed and recorded. Procedure 5: Effects of a Ligature on the Heart A 30cm piece of thread was placed around the heart at the Atrioventricular groove (AV groove) and tied in a knot but left loose so as to not interrupt the normal function of the heart. The heart was allowed to beat for about 15 seconds with no pressure.

After 15 seconds the knot was slowly tightened while taking care to stay on the AV groove while tightening. Data was observed and recorded. Results Procedure 1: The Heart Rate This experiment was carried out as noted about in Procedure 1. The resting heart rate was established and used as a baseline value from which to compare all future deviations. While data could not be exported from the computer to be definitively known, the relative rate and strength of the contractions were noted on a visual basis from which to compare the following experiments.

Procedure 2: Effects of Cold Temperature As noted above in Procedure 1, technical data could not be obtained from this experiment and visual analysis had to suffice for data. Upon addition of room temperature Ringer’s solution, no notable change in contraction strength or frequency could be noted. Time was allowed for the heart to recover from the effects of the initial exposure. The application of cold Ringer’s solution resulted in a clear and observable slowing of the heart rate, though no change in strength of the contractions could be detected.

Procedure 3: Effects of Drugs Upon addition of epinephrine directly to the heart, the contraction rate showed a considerable increase in frequency. The strength or magnitude of each contraction also significantly increased as the heart actually was lifting itself off of its resting place. Exposure of the heart to acetylcholine had clear effect on the heart as well. A substantial decrease in heart rate was noticed upon exposure; however the magnitude of contraction seemed to remain somewhat constant.

Addition of Atropine to the heart resulted in an increase in heart rate. The magnitude of each contraction showed a minor, but noticeable, increase in strength. A calcium solution was applied to the heart and showed a mild increase in contraction rate with the magnitude of each contraction seemingly remaining constant. The addition of both nicotine and caffeine had negligible effects on the rate or strength of heart contraction. Table [ 1 ]. Applied Chemical and Its Effect on Heart Contraction Rate and Strength Chemical| Heart Rate| Contraction Strength|

Normal Ringers| Control Rate| Control Magnitude| Cold Ringers| Decrease| No change| Epinephrine| Increase| Increase| Acetylcholine| Decrease| Slight Decrease| Atropine| Increase| Increase| Calcium solution| Increase| No change| Caffeine| No change| No change| Nicotine| No change| No change| Procedure 4: The Refractory Period of the Heart Upon exposure to a mild electric current, the heart rate was altered from the normal resting heart rate. While it definitely slowed, the contractions were sporadic at best.

The heart lost its regularity and showed random contraction intervals, some ranging shorter than normal while others had greater time gaps between contractions. Procedure 5: Effects of a Ligature on the Heart After the knot had been tied around the AV groove, no noticeable alterations were noticed to heart contraction. Upon tightening of the thread however, the heart appeared in clear distress. Beat irregularities ensued with an increase in magnitude of each pulse. The heart ceased functioning before the allotted time period had elapsed.

Discussion Procedure 1: The Heart Rate The resting heart rate of the frog was perfectly normal. Steady, rhythmic contractions around 60 beats per minute were observed and used as a baseline for other experiments. The frog appeared healthy, showing no signs of beat irregularities or any other defects that may have impacted the experiment’s validity. Procedure 2: Effects of Cold Temperature The slowing of the heart in the presence of chilled Ringer’s solution was to be expected.

All metabolic processes decrease in all cells upon exposure to cold due to the nature of chemical interactions. In the case of the frog heart, the cold solution probably decreased the rate at which calcium channels were able to open and thus, decrease the rate at which contractions were likely to occur since calcium entry to the cytosol initiates the cascade of reactions that leads to muscle contraction. Thus, the experimental hypothesis was correct given that the rate at which the heart contracted decreased and the magnitude of each contraction also lowered.

Procedure 3: Effects of Drugs Exposure to epinephrine increased both the rate and strength of each muscular contraction as was expected. The heart contains many adrenergic receptors which are responsive to epinephrine, especially near the SA node, which initiates the contractions of the heart. Epinephrine acts to increase the release time of calcium from the sarcoplasmic reticulum via a cascade of reactions. The fact that epinephrine had a positive impact on the heart indicates that the heart was healthy and responsive to normal physiological chemicals and pathways.

Addition of acetylcholine predictably lowered the heart rate of the frog. Acetylcholine blocks the cAMP cascade pathway that ultimately leads to calcium release, thus the frequency at which calcium is released is lowered and as a result, the contraction rate follows suit and lowers as well which is what you would expect from a fully functional heart. This part of the experiment was a success. The presence of Atropine, a parasympathetic system inhibitor, acted appropriately and increased the heart rate of the frog.

The parasympathetic and sympathetic nervous system act antagonistically to one another and as a result, decreased activity in one serves to act as if an increase in the other had occurred. Addition of Atropine, in effect, should have had similar effects as the addition of epinephrine which it did. Exposure resulted in an increase in the rate of contraction and a mild but noticeable increase in the strength of contraction compared to the resting heart rate and magnitude which was what the response was predicted to have been.

Neither caffeine nor nicotine had any visual effect on the hearts rate or strength of contraction. This was expected as both of these chemicals exert their effects by activating the release of neurotransmitters and hormones in the hypothalamus, specifically epinephrine, which then affects the heart. Because these tested chemicals were applied directly to the heart and not placed in the bloodstream where they could produce an indirect effect, it is reasonable that no effect was noticed from the heart upon exposure to these chemicals. Procedure 4: The Refractory Period of the Heart The SA node is the pacemaker of heart.

It is produces electrical currents that travel to the AV node and through the Bundles of His and the Purkinje fibers and stimulates the cells of the atrium and ventricles to contract. This is electrical conduction that is carried through gap junction of the intercalated disks separating heart cells and thus it is reasonable to infer that applying an electric current directly to the heart would interfere with the steady, rhythmic contractions normally observed in a healthy, undisturbed heart. Accordingly, as we applied a current to the heart, the cyclic contractions of the heart became erratic and unpredictable.

No steady pattern was detectable in the muscular contractions. Some had longer periods between contractions while others had shorter time gaps between beats. This was expected as the heart would be receiving constant signals to contract along with the rhythmic electrical signals from the SA node itself and would result in interference and overlap of contraction signals which is exactly what was observed. This leads us to believe that both the electrical apparatus and the heart were working exactly as designed. Procedure 5: Effects of a Ligature on the Heart

After placing the thread around the AV groove and tightening, the heart was clearly under a great deal of strength. Both the anterior and posterior segments of the heart swelled considerably, no doubt as a result of restriction of blood flow and buildup of pressure within the atriums and ventricles themselves. The AV node is a particularly sensitive portion of the heart to constriction as blood flow through the heart itself occurs at this junction. The heart contraction magnitude increased considerably while the contraction rate decreased substantially.

Over the course of time when the thread was tightened, the heart appeared to get weaker and weaker until it finally gave out itself. Using the electrical apparatus, we tried to revive the frog and succeeded, however, a steady and consistent heartbeat was never again established and was much weaker and slower than before the thread was utilized. This indicated that heart damage had occurred and it was unlikely that any more significant and reliable data could be obtained from the frog’s heart and the experiment was finished as a result.

Overall, the experiment can be considered a success as the appropriate responses to all the varying conditions were observed. While the experiment can be considered a success, the conditions with which the experiments were carried out were far from ideal. The experimental apparatus used was sufficient but hardly the equipment of choice. Far from accurate and precision, as well as the lack of ability to export numbered data from the labs computers, it is difficult to really analyze the data and produce concrete results that reflect the true magnitudes of effect each experimental variable had on the heart.

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