The purpose of this experiment was to subject a rat cardiac muscle to different physiological situations and determine their effect on the twitch force and duration. Force-frequency relation show a positive trend suggesting that as frequency of stimulation increases (for the range of 0.2-0.8 Hz), the force increases. Also the extra cellular calcium concentration effects on the peak force were measured, showed an increasing trend when the [Ca2+ ]o was incrementally increased from 0.2 to 3mmolL-1. The rat cardiac muscle was also subjected to cardiac glycoside such as ouabain, which resulted in an increase in the diastolic force, inotropic agents such as isoprenaline, which increased the velocity of contractility, and finally the effects of caffeine were considered.
Experimental set up:
We were provided with a ventricular strip of a rat heart muscle; it had the dimensions of (~20mm resting length, 5mm wide and 1mm thick). The strip was embedded in an oxygenated 1.5mM Ca2+ Tyrode solution (constant PH=7.4) with constant O2 partial pressure of oxygen is 760 mmHg, because the cell needs oxygen in order to survive, the strip mounted using threads to a force transducer and a stimulator (REF1), these were connected to power LAB (amplifier) which was connected to a computer, the Chart software was used to record the data.
Note that the temperature the experiment was performed at was 14 C, and specimen was field stimulate at a voltage of 60V in order to make the whole muscle contract.
Field stimulation is the stimulation of the specimen through a medium, so that the whole tissue is electrically stimulated, point stimulation is stimulating a single muscle cell.
Measurements of frequency-force relation:
We applied different stimulus frequencies to the muscle (ranges 0.1-0.8 Hz) and recorded the force response, each frequency stimulated the muscle for a short period of time (around 1 minute) until the force response seemed to be steady then 1 twitch was recorded at high recording speed.
Dose response relation for Ca2+:
To measure the dose response relation for Ca2+, the muscle was stimulated at 0.4Hz, with the solution containing a starting amount of 0.2 mmolL-1 of Ca2+ , a single twitch was captured at steady state, successive amounts of 0.4 mmolL-1 of Ca2+ were added to the solution and steady state force of a single twitch was captured at each new added amount.
Effect of ouabain:
After flushing the organ bath a few times, an aliquot containing ouabain was used to determine its effect on the rat cardiac muscle at 0.4Hz. Steady state force measurements were taken at 0.25 ml of the aliquot but no change was noticed, so we double the amount and measured steady state, again no change in steady state was noticed, finally a total amount of 1.25 ml was added to measure a noticeable force response.
Effect of Isoprenaline:
After flushing the organ bath a few times, 0.05ml aliquot that contains isoprenaline was added to the solution; no change of force or duration was noticed. Then a total of 0.2 ml of the aliquot (containing 2.0 ìmolL-1 of isoprenaline) was added to see a noticeable change in the force response.
Effects of caffeine:
A 0.4Hz stimulus was applied to the muscle at control conditions then, the bath was filled with the provided caffeine solution (containing 10 mmolL-1 caffeine and 1.5 mmolL-1 Ca2+). A single stimulus was recorded at that frequency then another using a 1s burst stimuli of 2Hz (the suggested 10 Hz stimuli did not show any noticeable effect, the cell was fatigued)
• The 50Hz noise from the main power supply was being recorded in chart, made it more difficult to analyse, we should have repeated every part of the experiment many times and averaged them out to reduce the signal to noise ratio, but we didn’t due to time limitations.
• The time given for the muscle to reach an assumed steady state force response may not have been sufficient enough (~1 minute), in this case the data might not have been representative.
• The oxygenation process was causing movements in the muscle set up which might have been recorded in Chart along with the signal.
• Flushing the organ bath did not fully return the muscle to control conditions.
• There could have been diffusion limitations because of the size of the specimen.
• The muscle was subjected to different condition during the course of the experiment, which might have affected its mechanical performance.
• Muscle values are usually expressed in units of stress to make comparisons with different muscle types (different sizes) possible.
• Calculating the stress would also produce errors, depending on the accuracy of measuring the specimen dimensions especially the length because it will vary during contraction, weight, the assumptions made regarding the shape of its cross sectional area, which will also vary during contraction thereby change the stress value.
Frequency force relation:
• From the graphed data points of the twitch force (frequency ranges from 0.2-0.8 Hz steps of 0.1 Hz) one can see that an increase in frequency of stimulation would results in an increase in the twitch force of the rat muscle at 14C, i.e. (positive stair case). See figure1.
• Also as the stimulation frequency increase, half contraction time increase and half relaxation time decrease. We define half width as the difference between half contraction and relaxation times (the time to reduce 50% from the peak force minus the time it takes to increase to 50% of the peak force), as frequency increases half width decreases. See figure2.
• Note that half width is just an indication of the twitch duration; the data also shows that peak force is inversely proportional to half width. See figure3.
• Increasing the temperature would lower the stress on the muscle with a given frequency, and widen the range of the possible frequency intervals. And vice versa when decreasing the temperature (physiological temperature is 37 C.) 
Figure 1: Frequency-force relation in rat cardiac muscle
Figure 2: stimulation frequency against measures of force duration.
Figure 3: Peak force against half width
Results from the Ca2+ - force experiment:
• As the extra cellular calcium concentration is increased from 0.2 mmolL-1 to 3 mmolL-1 the peak force shows an increasing trend, See figure4, with a large increase from 0.2 to 0.6 mmolL-1, then almost a steady increase at the other proceeding concentrations. Note that the extra cellular concentration of calcium in the rat heart muscle is about 1.5 mmolL-1. These values suggest that an increase in the extra cellular calcium concentration would result in an increase in peak force, at lease at the tested range, the peak force has increased by (~60%) when the [Ca2+ ]o was increased by 140%, from 0.2-3 mmolL-1.
Figure 4: Peak force as a function of extra cellular calcium
Figure 5: duration as a function of extra cellular Calcium concentration.
Results from the ouabain experiment:
• There is a small change in half width (from 0.1625sec at control to 0.1725sec at 1.25mmolL-1 of ouabain), suggesting longer relaxation period, but a very large increase in the diastolic force (force before the start of a twitch, increase = 2.5mN). Also a decrease in the peak force comparing to control (decrease of 2.91mN). This resulted in a total amplitude decrease of 5.41mN.
Results from the isoprenaline experiment:
• Adding 1.0 ìmolL-1 of isoprenaline has resulted in a force reduction of 0.77mN (from 3.72mN control after the ouabain experiment to 2.95 upon adding the aliquot).
• Adding this aliquot has caused a reduction in half width i.e. duration of 0.04 seconds (from 0.1425 at control to 0.1025 upon adding the aliquot).
The frequency force relation suggests that an increase in stimulation frequency (from 0.2-0.8Hz) would cause an increase in contraction force ; this is the case in most species other than rats. Studies have usually showed that the force frequency relation in rats shows a decrease in contraction force as frequency increases (negative staircase) (REF1), but our study shows quite the opposite. Note that the resting heart rate of rats is around 5-7 Hz; our specimen was not tested under that physiological frequency. A possible hypotheses could be that as frequency increases, more action potentials travel through the transverse tubule (T-tubule) causing voltage gated calcium channels to open more frequently, this calcium entry will cause the sacroplasmic reticulim (SR) to release calcium from the cell as a result of calcium induced calcium release (CICR). Calcium released from the SR will then bind to troponinC and contraction will take place. The more frequent opening of the calcium channels might cause more calcium will be released from the SR causing a larger twitch response, the half width of the twitch decreases with increasing frequency which suggests that the uptake of the SR increases with frequency i.e. muscle relaxes faster. This is also seen from the inversely proportional relation ship between peak force and half width. Note that this hypothesis was not specifically testing and is just an interpretation of the data (Ref1).
With respect to skeletal muscle increasing frequency would also increase twitch force.
Force and extra cellular calcium concentration relation:
The force - [Ca2+ ]o relation indicate in increase of twitch force when the [Ca2+ ]o is increased (at least up to 3mmolL-1) for a given frequency, a possible reason could be that as the [Ca2+ ]o increases there will be more calcium entering the cell up on stimulation because the concentration gradient has increased, causing the SR to release more calcium and increasing contraction force (as discussed above).
The half width indicates that as more calcium enters the cell, more calcium up taking mechanisms start to operate to permit relaxation, this is seen from the decreased half width as [Ca2+ ]o is increased.
Increasing [Ca2+ ]o in skeletal muscle would not affect the force, because it does not depend on the extra cellular [Ca2+ ].
Effects of ouabain on Rat cardiac muscle:
Ouabain is a cardiac glycoside that exerts a positive inotropic effect on cardiomyocytes; it inhibits the plasma membrane sodium pump (Na, K-ATPase), which will result in a decreased Ca-extrusion by the sarcolemmal cardiac sodium/calcium exchanger (one of the calcium extrusion mechanisms). This will result in an increase in the intracellular calcium concentration, thereby increasing the diastolic force . From the experiment results the increase in intracellular calcium is seen from the increase in diastolic force (increase of about 2.5mN, when 1.25mmolL-1 of ouabain was added).
This will not affect skeletal muscle (at least not with respect to an increase in diastolic force) because [Ca2+ ]o does not affect the contractility of skeletal muscle.
Effects of isoprenaline on Rat cardiac muscle:
It is a beta-adrenergic agonist that affects the sympathetic nerve activity and increases contraction velocity , as seen from the results the half duration has decreased by 0.04 seconds up on adding 1.0 ìmolL-1.
Isoprenaline would not affect skeletal muscle because sympathetic nerves do not innervate skeletal muscle, and it is a voluntary muscle.
Nor adrenaline and adrenaline affects the beta-adrenergic receptor and results in an increase in heart rate and twitch force, but only increases heart rate upon acting on alpha-adrenergic receptors, in whole heart isoprenaline will add to the effect of these two.
Propranalol is a beta-adrenergic antagonist, which has the opposite effect of isoprenaline.
Effects of caffeine on Rat cardiac muscle:
Adding 10 mmolL-1 of caffeine resulted in tetanus when the muscle was subjected to 2Hz frequency stimulation.
Adding caffeine and isoprenaline together might result in an accelerated tetanus, i.e. a cycle of short tetanus contractions.
If a one-off 600mg of caffeine were ingested, nothing would happen to the person because this amount will be diluted with body fluids.
If more than 600mg of caffeine were ingested daily, long term users may suffer from chronic insomnia, persistent anxiety and depression, and stomach ulcers, also
Caffeine use appears to be associated with irregular heartbeat 
• We have concluded from our experiment that twitch force increases with frequency, at least for the range tested
• Twitch force increases with an increase of external calcium concentration.
• Ouabain increases the diastolic force.
• Isoprenaline increases the velocity of contraction, and
• Caffeine caused the rat cardiac muscle to go into tetanus.