regulatory mechanisms in muscle

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Muscle fatigue: physiology lab 7 part A
Laboratory objectives
At the end of this laboratory, you should be able to:
Describe the regulatory mechanisms in muscle
Use practical techniques to investigate contractile properties of muscle
Plan experimental procedures using information provided
Manipulate data and use appropriate mathematical analysis
Present data to show significance of experimental results
Background information and pre-work
It is well established that contraction of striated muscle is due to the cyclic interaction of myosin in the thick filaments of the sarcomere and actin in the thin filaments.  Myosin heads attach to actin and the resultant structural change leads to filament sliding and the subsequent detachment of the heads.  This is thought to occur asynchronously many times during contraction.  Adenosine triphosphate (ATP) provides the immediate source of energy for muscle contraction.  Hydrolysis of the bonds between the phosphate residues of ATP are associated with the release of a large amount of energy.  In muscle the hydrolysis of ATP to adenosine diphosphate (ADP) is catalysed by the contractile protein myosin, thus adenosine triphosphatase (ATPase) activity is found in the heads of the myosin molecules where they are in contact with actin.
In preparation for this practical, and to help give you some further background to muscle fatigue, please read the following review article:
Place, N., Yamada, T., Bruton, J.D. et al. 2010. Muscle fatigue: from observations in humans to underlying mechanisms studied in intact single muscle fibres. Eur J Appl Physiol (2010) 110: 1 (https://doi.org/10.1007/s00421-010-1480-0)
This review will also be available on Blackboard
Plan of the session
In humans, the psoas major muscle joins the upper body and the lower body, the axial to the appendicular skeleton. It forms part of a group of muscles called the hip flexors, whose action is primarily to lift the upper leg towards the body when the body is fixed or to pull the body towards the leg when the leg is fixed.
In this practical class you will use glycerinated fibres of a rabbit psoas muscle to study the ultrastructure and the mechanism of contraction. The rabbit psoas muscle is a
muscle composed of both fast and slow twitch muscle fibers, and assists
during rotation of the hip.
A Glycerinated muscle fibre is obtained by taking muscle fibres from a freshly killed animal and immersing in equal volumes of water and glycerol at 0C and subsequently storing at -20C.  This treatment leaves the contractile structure intact whilst perforating the cell membrane (sarcolemma) and removing other factors such as ATP and destroying the ATP-restoring systems. 
By the use of appropriate chemicals, the fibre can be made to relax, to contract or be made extensible.  It cannot, however, be stimulated electrically. In this experiment you will investigate what components are required for effective muscle contraction, and in particular, study what effect the changes in pH and phosphate concentration have on muscle contraction in a model of muscle fatigue.
Protocol
Table 1: Solutions used in this experiment – vials (1 ml) containing the following prepared solutions:
 
Distilled deionised water
0.25% ATP (@4mM)
MgCl2 (1mM )
KCl (100mM)
Buffered at pH 7.1
A
ü
 
 
 
 
B
ü
ü
 
 
 
C
ü
ü
ü
 
 
D
ü
ü
ü
ü
 
E
ü
ü
ü
ü
ü
Solution X: 50% Glycerol in distilled deionised water
NB.     ATP and salt solutions should be kept in ice at all times and discarded at the end of each practical.
Make Glass teasing needles.  These should be made from pipettes manufactured from heavy wall precision soda glass tubing.  The narrow tip of the pipette should be held over the flame of a Bunsen burner (using forceps).  As the glass heats, pull gently apart, this should result in a short and a long needle.  It is not intended that the end should be sharp, rather that the tip is rounded to avoid damage to the tissue.  If the needle tip breaks during use discard immediately. The demonstrator will show you how to make the teasing needles.
Collect a sample of the psoas muscle on a petri dish
Under a dissecting microscope, using your glass teasing needles, tease the segment of muscle into very thin groups of myofibres.  Single fibres if obtained will demonstrate the greatest contraction.  Strands of muscle exceeding 0.2mm in cross sectional diameter must not be used.
Take care not to allow the tissue dry out or remain at room temperature for extended periods this may prove detrimental.  Tissue not immediately required should be placed back in ice; this will also assist teasing of fibres.
Using the fine brush, transfer one of the thinnest strands onto a slide and add a cover slip.  Examine under low and high power magnification, note the striations in the fibres, the smooth walls, and nuclei. Draw a diagram of what you can see down the microscope in the box below (don’t forget to label it).
Select a second strand with a minimal amount of glycerol and transfer onto a second slide, position the strand straight and parallel to the slide. The amount of glycerol used depends on the heat of the microscope lamp, and the length of exposure to this heat.  With no appreciable heat, the glycerol which adheres to the strands of fibres is sufficient.  The fibres are easier to measure with minimal glycerol.
Place the slide under the dissecting microscope and measure the length of the fibre with the scale provided held beneath the slide.  Record the length of the fibre as number of squares in table 2. Cover slips should not be used with this microscope, as over-lying pressure may decrease contraction.
Flood the myofibre with several drops of solution A (see table 1).  Observe the reaction of the fibres. The speed and extent of the reaction are influenced by the amount of the glycerol on the slide, the concentration of active ATP, the ions present, and the width of the muscle strand.  Under favourable conditions a myofibre can be expected to contract fully within 10s.
After 30s remeasure the fibre, record the result and calculate the degree of contraction.  To calculate the percentage contraction use the following simple equation:
Original fibre length – final fibre length          x100
Original fibre length
 Add cover slip and examine under the compound microscope, what differences can you see? Has the width of the fibres changed?
Repeat the experimental stages 6 – 10, using a clean slide, and new myofibrils for each of the solutions (A – E).
NB.     Care must be taken to avoid any possibility of cross-contamination between the ATP and salt solutions.  Such contamination will lead to ambiguous experimental results.
Results
Table 2: Results from the Psoas muscle experiment
Solution tested
Length before application (Number of squares)
Length after application (Number of squares)
Percentage contraction
A    
 
 
 
B    
 
 
 
C    
 
 
 
D    
 
 
 
E    
 
 
 
1. What effect did ATP have on muscle contraction? Why do you think this was the case?
________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
2. What effect did buffering the solution at pH 7.1 have on contraction? Why do you think this was the case?
________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
End of practical
Once you have completed your practical work, please clear everything away as instructed.
Make sure a member of staff signs your completed
workbook before you leave
Muscle fatigue: physiology lab 7 part B
Laboratory objectives
At the end of this laboratory, you should be able to:
Describe the regulatory mechanisms in muscle
Use practical techniques to investigate contractile properties of muscle
Plan experimental procedures using information provided
Manipulate data and use appropriate mathematical analysis
Present data to show significance of experimental results
Plan of the session
In this practical class you will conduct the same experiment as last time but you will use a number of different solutions to investigate the effect of pH and phosphate concentration on muscle fiber contraction. This practical will be written up as a mini lab report for assignment 4. After this class you should:
Collect the class data
Calculate Mean and SD for each of the conditions
Plot a graph of the results
Compare the results through the use of statistical analysis
Protocol
Table 1: Solutions used in the pH experiment – vials (1 ml) containing the following prepared solutions:
pH of solution
Distilled deionized water
0.25% ATP (@4mM)
MgCl2 (1mM )
KCl (100mM)
pH 7.1
ü
ü
ü
ü
pH 6.7
ü
ü
ü
ü
pH 6.3
ü
ü
ü
ü
pH 5.9
ü
ü
ü
ü
Table 2: Solutions used in the phosphate experiment – vials (1 ml) containing the following prepared solutions:
[Phosphate]  (mM)
Distilled deionized water
0.25% ATP (@4mM)
MgCl2 (1mM )
KCl (mM)
pH 7.1
10
ü
ü
ü
90
ü
20
ü
ü
ü
80
ü
30
ü
ü
ü
70
ü
40
ü
ü
ü
60
ü
Solution x         50% Glycerol in distilled deionised water
Protocol Week Two
Make glass teasing needles (see practical 7A), place the muscle to be teased into a glass petri dish and under a dissecting microscope and using glass needles tease the segment of muscle into very thin groups of myofibres.
Using the fine brush, transfer one of the thinnest strands onto a slide. 
Place the slide under the dissecting microscope and measure the length of the fibre with the scale provided held beneath the slide.  The fibres are easier to measure with minimal glycerol. Record the length as number of squares.
Flood the myofibre with a drop of one of the test solutions and observe the reaction of the fibres.
After 30s remeasure the fibre, record the result and calculate the degree of contraction.  To calculate the percentage contraction use the following simple equation:
Original fibre length – final fibre length    x100
Original fibre length
Repeat the experimental stages 1 – 5, using a clean slide, and new myofibres for each of the test solutions.
Repeat the experimental stages 3-7 using the remaining solutions (see tables 1 and 2), record your data in tables 3 and 4 and and plot 2 graphs showing the effect of pH against percentage contraction and the effect of phosphate concentration against the level of contraction.
Results
Table 3: Results of the pH experiment
pH tested
Length before application (Number of squares)
Length after application (Number of squares)
Percentage contraction
     
 
 
 
     
 
 
 
     
 
 
 
     
 
 
 
Table 4: Results of the Pi experiment
[Phosphate] mM
Length before application (Number of squares)
Length after application (Number of squares)
Percentage contraction
     
 
 
 
     
 
 
 
     
 
 
 
     
 
 
 
Questions
1. From your data what can you conclude about the effect of increasing pH on muscle contractility?
________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
2. From your data what can you conclude about the effect of increasing phosphate concentration on muscle contractility?
________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
End of practical
Once you have completed your practical work, please clear everything away as instructed.
Make sure a member of staff signs your completed
workbook before you leave

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