Skeletal Muscle Physiology: Experimental Analysis of Toad Muscle

This report investigates the physiological properties of skeletal muscle using the isolated toad gastrocnemius muscle. The experiments cover the single twitch, graded response, muscle length and tension relationship, muscle tetanus, and muscle fatigue. The introduction provides background the...Read More

Muscle physiology
EXPERIMENTAL INVESTIGATION OF THE PHYSILOGICAL PROPERTIES OF SKELETAL
MUSCLE
AIMS
• To understand the properties of skeletal muscle including ; the structure,
nervous distribution, response to excitation or contraction and control of activity
• To measure and grade physiologic responses of skeletal muscle
• To gain experience in the use of tissue isolated from organs
• To develop biomedical research skills, analysis and representation
Of data
INTRODUCTION
The body integumentary system is composed of the skeletal and muscular systems.
Muscles are divided into skeletal, cardiac and smooth muscle. The skeletal muscle anchors on
the skeletal tissue.3,6 Skeletal muscle is voluntarily controlled. The muscle has contractile
proteins; actin and myosin. Muscle fibres make up the muscular system. Each fibre is a
unicellular model of a multinucleated, long, cylindrical cell surrounded by a sarcolemma.
Contractile proteins are invested in the fibres forming myofibrils. 7The sarcomere is the
functional unit of skeletal muscle and contracts and generates force when electrically
stimulated.7 Sliding of the thick and thin filaments during contraction constitute the sliding
filament theory. Non-contractile portions (troponin and tropomyosin) remain static. The

Muscle physiology
sarcolemma has tubular invaginations called Transverse tubules which are associated with the
sarcoplasmic reticulum. The sarcoplasm is the cytoplasm of the muscle cells and excludes the
myofibrils.5,8
Skeletal muscle receives innervation of the Central Nervous System (CNS). The axons
from the CNS form multiple synapses on the sarcolemma forming a motor end plate. The
neuromuscular junction synapses have a presynaptic and a post synaptic knob.9The presynaptic
knob has synaptic vesicles with Acetylcholine (Ach). The nervous stimulus cause acetylcholine in
vesicles to be released into the synaptic gap. The action potential initiates an electric action
potential in the sarcolemma.10

Muscle physiology
The Ach released from the initiation of the action potential binds the nicotinic
receptors at the motor end plate allowing Na= influx this depolarizing the sarcolemma. Impulse
from the resultant action potential spread rapidly through the transverse tubules especially on
the terminal cisternae where dihydropyridine (DHP) receptors are triggered.6,8,10 The
sarcoplasmic reticulum releases Calcium ions stored in the sarcoplasm. Calcium ions bind
troponin changing conformation and allowing movement of tropomyosin from the active site
on actin. Exposed actin results in myosin binding on its active site.5 The binding of actin to
myosin forms a cross bridge which stimulates ATPase activity thus power stroke occurs, this is
the ‘pulling’ of actin towards the M line by pivoting of the myosin head. In the power stroke the
myosin head moved from a high energy state to a low energy state.7 ATP serves to break the
cross bridge and repositioning of myosin. The repeat of the contraction occurs until the impulse
is stopped of depletion of ATP occurs; this called fatigue.

Muscle physiology
Muscle fatigue after progressive contraction are caused by depletion of ATP stores,
glucose, and buildup of lactic acid and exhaustion motor plate Ach. On the other hand, muscle
twitch occurs due to rapid muscle contraction in response to a single stimulus. The mechanism
involves progressive latency, contraction and relaxation. Muscles also obey the ‘all or none law’
of graded responses. The law indicates that an impulse can either generate an action potential
or not at all. In temporal summation an increase in frequency of stimulatory impulse results in
more contraction. Tetany may occur during contraction. Incomplete tetanus involve partial
relaxation between contractions while complete tetany has no relaxation between contractions
thus maximum sustained contraction. Varying numbers of muscle cells may also contract
depending on motor units in spatial summation. Sometimes additional motor units in a single
muscle are activated thus an increase in the contractile strength of the muscle.6
MATERIALS AND METHODS
Materials;
• LabChart 7 software – Toad Muscle Settings 2012
• PowerLab Data Acquisition Unit
• Bridge Pod
• Force Transducer
• Small weight between 5–50 grams
• Safety glasses
• Retort Stand
• Micropositioner and clamps

Muscle physiology
• One toad (Bufo marinus), double pithed, per 2 groups of student
• Wash bottle with Ringer’s solution
• Dissection tools. Muscle Stimulation Equipment: Animal Nerve Stimulating
Electrode ,Stand clamp for the electrode ,Femur clamp
Procedure
The power lab device is turned off and USB connected to a computer. The force
transducer cables are connected and electrodes placed on outward panel. The force transducer
and micro positioner and then set on a stand.
The force transfer is then calibrated from millivolts to force in Newtons (N). The bridge
pod is then opened of the lab chart and recordings made. Known weights are placed after five
second recording intervals. The data is recorded on the lab chart software.
The toad is then dissected and the leg removed from the hip joint. A thread is attached
to the gastrocnemius tendon, the tibiofibular bone is cut and clamping is done. The setup is
then placed as follows;

Muscle physiology
The exercises on Twitch recruitment, stretch and contraction, summation, tetany and
fatigue are done and results obtained.
RESULTS
Appendix 1

Muscle physiology
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1
0
0.05
0.1
0.15
0.2
0.25
stimulus amplitude(v)
contractile force(N)
The contractile force increases with increased stimulus to peak at around 0.6V then
decreases gradually. This is because of maximum contraction and resultant fatigue.8
0 0.04 0.09 0.1 0.12 0.16 0.18 0.2 0.21 0.16
0
0.05
0.1
0.15
0.2
0.25
Appendix 2
PRELOAD(N)
NET TWITCH FORCE(N)
The force of the twitches increase with an increase in the force of the preload. This
because of the recruitment of muscle and cross bridge formation.5

Muscle physiology
400 200 100 50 20
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
Appendix 3
Force of First Peak (N) Force of Second Peak (N)**
Stimlus interval(mS)
force of peak(N)
The force of the second peak increases with the increased stimulation interval. This is
due to frequency summation.
400 200 100 20
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Appendix 4
Contractile Force (N)
stimulation time (mS)
contractile force(N)
The force of the second peak increases with decrease in stimulation time. An increase in
frequency results in increased contractile force. More motor plates are recruited.3

Muscle physiology
(s) 1 5 10 15 20 25 30
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Appendix 5
Time Since Stimulation(S)
CONTRACTILE FORCE (N)
The contractile force increases and peaks at 1s, the contraction reduces gradually to
baseline due to muscle fatigue from substrate exhaustion.
Discussion
1. An increase in the stimulus intensity results in an increase in the threshold
of the action potential thus an increase in the number of recruited motor end
plates.2,5,8,10
2. The increase in the preload results in an increase in the stretch, on
isometric contraction, the tension is directly proportional to the numerical value of all
cross-bridges between the myosin and the actin myofilaments. When as stretching force
is applied, the overlap between the myofilaments decrease and in turn the number of
cross bridges reduce. The resting length also decreases and the distance the actin
filaments move is shortened resulting in a stronger contraction.3,7,9

Muscle physiology
3. There is a hyperbolic relationship between velocity and load. This indicates
that the smaller the load the higher the velocity and the heavier the load the lower the
velocity of the muscle fibres shortening. A heavy preload would result in engagement of
numerus bridges this clearly seen in power output of muscle under tension.7
4. Summation occurs after continuous muscle summation or frequency
summation. With multiple action potential on the given muscle fibres, the muscles twitch
and summate increasing the tension developed .The higher the frequency of stimulation,
the greater the tension. Close contractions result in tetany.4
5.Fatigue is the progressive loss of muscle contraction from prolonged use caused by
ATP shortage and glucose depletion Smooth muscles unlike skeletal; muscle do not fatigue
hence continuous contraction. The muscle evades fatigue through selective recruitment of
muscle fibres and calcium storage.6,8
CONCLUSION
Muscle is a crucial component of the human body. Skeletal muscle is the basis behind
movements and spatial orientation in space. 3This is the basis from which the experiment was
done. The sole aim of the experiment was to establish the dynamics of muscle contraction and
the physiological adaptation to the functioning.5 It was established that muscle tissue is
contractile, excitable, extensible and elastic. The mechanism of action of muscle that facilitates
movement was also established. The objectives of the experiment were achieved and the
understanding of experimentation, research and integration of knowledge made lucid.10

Muscle physiology
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physiology. 2008 Nov 1;586(21):5047-61
3. Boron, Walter F., and Emile L. Boulpaep. Medical Physiology: A Cellular and
Molecular Approach. 2012.
4. Horowitz A, Menice CB, Laporte R, Morgan KG: Mechanisms of
smooth muscle contraction. Physiol Rev 2012; 76:967-1003
5. Devasahayam SR. Skeletal Muscle Contraction. InSignals and Systems in
Biomedical Engineering 2013 (pp. 225-252). Springer, Boston, MA
6. Hall, John E., and Arthur C. Guyton. Guyton and Hall Textbook of Medical
Physiology. Philadelphia, PA: Saunders Elsevier, 2011.
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(editors): Principles of Neural Science, 5th ed. McGraw-Hill, 2013
8. Linari M, Caremani M, Lombardi V. A kinetic model that explains the effect of
inorganic phosphate on the mechanics and energetics of isometric contraction of
fast skeletal muscle. Proceedings of the Royal Society of London B: Biological
Sciences. 2010 Jan 7;277(1678):19-27.
9. Winegrad S. The intracellular site of calcium activation of contraction in frog
skeletal muscle. The Journal of general physiology. 1970 Jan 1;55(1):77-88.
10. Gillies AR, Lieber RL. Structure and function of the skeletal muscle extracellular
matrix. Muscle & nerve. 2011 Sep 1;44(3):318-31.

Muscle physiology