Kagramian 1

Using ATP to Trigger Satellite Cell Activity for Muscle Regeneration Therapy in Patients
with Facioscapulohumeral Muscular Dystrophy
Michelle Kagramian

Abstract:
For individuals diagnosed with muscular dystrophies or other muscle related diseases, there is
often no remedy for their muscular atrophy. As a form of therapy, ATP can be used to create
large enough contractions to cause damage to the muscle for stem cells to repair to become
stronger than before. Satellite cells, the stem cells within skeletal muscle, are triggered by
damaged muscle and work with fibroblasts to repair and rebuild the muscle larger and stronger.
Muscle damage has shown to be caused by strong enough muscle contraction. The factors which
heavily influence the degree of muscle contraction were tested in this research by comparing the
effects of various ratios of ATP and KCl-MgCl2 solutions had on a rabbit psoas muscle. A thin
fiber of muscle was first measured for its initial length. Then, after its submergence under the
solution being tested, the resulting length was measured once again and the degree of contraction
was calculated. Statistically, there was a significant difference amongst the six different solutions
tested; meaning, one ratio of ATP to salts created the largest contraction (ANOVA, P<0.05). ATP
has shown to be a key factor in causing strong contractions, however only within the correct
balance with its corresponding salts (R2=0.2191). Electrical muscle stimulation (E-stim) holds
the potential to become a treatment for a variety of disabilities for thousands of people as a way
to work against the progressive factor of muscular dystrophy. The method of using E-stim works
by sending currents throughout the body to stimulate the release of Adenosine triphosphate
(ATP) as well as increase the activity of satellite cells and fibroblasts. This muscular therapy
could supply disabled patients with enough strength and hope to further fight their disease.

Introduction:
Individuals diagnosed with Facioscapulohumeral muscular dystrophy (FSHD) experience
progressive muscle degeneration with muscle weakness and atrophy. It is a genetic disorder that
asymmetrically affects skeletal, facial, upper body, and leg muscles. FSHD is caused by a
dominant genetic mutation that leads to inappropriate expression of the DUX4 gene on
chromosome 4 (Muscular Dystrophy Association, 2016). As the disease progresses, one of the
results is expressed in the muscles meant to stabilize the shoulder blades getting weaker, making
raising the arms an arduous task; walking, as well as other common tasks, become increasingly
more difficult to perform. The muscles affected, and to what degree muscles have been
weakened, varies for each patient (NORD, 2022). In some cases, the disease could even affect
the heart, vision, and hearing. Aside from FSHD, there are eight other muscular dystrophies:
Becker, Congenital, Congenital, Distal, Emery-Dreifuss, Limb-Girdle, Myotonic, and
Using ATP to Trigger Satellite Cells Kagramian 2

Oculopharyngeal. Although each dystrophy differs in which muscles are affected, the age of
onset, and its rate of progression, they are all alike in their relation to muscles (Johns Hopkins
Medicine, 2022). Animal muscles are similar to human muscles. Studies have shown that rabbit
muscles can be used as a predictive model for human muscles (Percy et al., 1990).
Human satellite cells have the regenerative ability to treat muscular dystrophies through
gene therapy. The specialized cells are found in the ganglia of the peripheral nervous system (Get
Body Smart, 2017; Figure 1). Satellite cells are the stem cells within skeletal muscle. They are
an essential component of muscle growth, hypertrophy, and regeneration (Kao et al., 2007). Stem
cells are the body’s raw material responsible for generating all other specialized cells (Mayo
Clinic, 2022). Pax7, a satellite cell marker, interacts with Tcf4, a fibroblast marker, to effectively
repair damaged muscle tissue. The regeneration of muscles is a complicated process that can not
be achieved without the involvement of other cells. Fibroblasts are cells used to produce
connective tissue which attaches other tissue or organs to the body (National Human Genome
Research Institute, 2022; Figure 1). Pax7 and Tcf4 were found to proliferate near each other
when the muscle underwent damage. A study showed that without Pax7 present, the fibroblasts
were unable to restore the damaged tissue (Figure 2). Later, when a muscle was tested without
Tcf4, satellite cells were shown to restore the muscle less effectively. A lack of fibroblasts causes
the satellite cells to prematurely divide and differentiate which results in smaller muscles after
regeneration (Figure 2). Fibroblasts were shown to regulate the expansion of satellite cells
(Murphy et al., 2011). Satellite cells surround myofibers and function as myogenic precursors for
muscle growth. Myofibers make up muscular tissue used for excitability and contractility
(Cretoiu et al., 2018). Seven satellite cells are able to generate over one hundred new myofibers.
After muscles were experimentally induced to muscle injury, cells proliferated and regenerated
clusters of myofibers (Collins et al., 2005). In the event that a muscle contraction causes damage
to the fibers, satellite cells will repair the myofibrils and increase their thickness as well as
number (Leyva, 2013).
Skeletal muscles are an important part of locomotion; nearly every bodily movement
requires muscle contraction. They have also been shown to continue functioning consistently
despite human aging. Functioning satellite cells had been obtained from a 96-year-old woman
who had been dead for 17 days (Latil et al., 2012). Muscles can be easily and acutely damaged
through harsh and rapid movements and exercise. For this reason, satellite cells are vital for
Using ATP to Trigger Satellite Cells Kagramian 3

repairing accumulated damage (Lepper et al., 2011). Another cause of muscle damage is muscle
contraction (Peake et al., 2017). Contractions can be initiated by ATP.
Adenosine triphosphate (ATP) is a molecule that transfers energy to cells (ACS, 2021). A
muscle contraction is the binding and releasing of 2 strands of the sarcomere. ATP prepares
myosin for binding (LibreTexts, 2018). Myosin is a protein that converts energy to the movement
of the muscle cells, such as contractions (Cooper, 2000). ATP activates P2X7 receptors in
satellite cells which enwraps each DRG neuron and increases communication between neurons
and satellite glial cells (Zhang et al., 2007). Satellite glial cells (satellite cells) help to provide
nutrients to neurons and prevent pain by initiating muscle regeneration.
With enough ATP energy, a contraction should be able to trigger satellite cells to restore
muscle. These cells hold the power to be used as treatments for muscular dystrophy, heart failure,
and urological dysfunction (Motohashi et al., 2014). FSHD allows for irregular regenerative
ability; even with 76% of FSHD muscle biopsies from quadriceps having regenerative qualities,
it is not enough to hold back the progression (Banerji et al., 2020). ATP can be used to trigger
satellite cells into restoring the withering of the muscle caused by muscular dystrophy.

Figure 1: A diagram showing the location of satellite cells in relation to

fibroblasts.
Using ATP to Trigger Satellite Cells Kagramian 4

Figure 2: Model of the Pax7 satellite cells and Tcf4 fibroblast interaction
during a regenerative process (A-C). A normal interaction between Pax7 and
Tcf4 where both components are present and able to restore the muscle
efficiently (A). An interaction lacking Pax7 where the muscle fails to
regenerate (B). An interaction lacking Tcf4 where the muscle resources less
efficiently and with less muscle mass in the restored tissue (C).
Using ATP to Trigger Satellite Cells Kagramian 5

Methodology:

Study site:
The solution of strips of rabbit psoas muscle (a muscle that helps to flex the hip) in a tube
of 50% glycerol was stored at -4 °C until ready for use. The ATP-KCl-MgCl2 solutions were
stored in the fridge. ATP-induced contractions will be tested in the MATES Research Laboratory.

Prepare the rabbit muscle sample:

a. The first muscle sample and glycerol liquid were transferred into a petri dish (Figure 3).
b. The length of the muscle sample was measured to be 11cm. The length was cut into 10
pieces, 2cm each, using dissecting scissors. The thin myofibers were separated from the
larger bundle of muscle fibers using a needle (Figure 6). The thinner the myofiber is, the
greater the contraction will be visible (Figure 4).
c. A wet mound was prepared to contain one 2cm slice of the muscle without any glycerol
liquid

Initiating contractions:
Using the Ward's® ATP Muscle Kit procedure, the correlation of chemicals to the
degree of contractions will be determined (Figure 5)

d. A dissecting and compound microscope were prepared for viewing the striation of the
skeletal muscle before and after the added chemicals.
e. The muscle was flooded with either the ATP-only solution (Solution A), the metal
salts-only solution (Solution B), the ATP and metal salts solution (Solution C), a 4:1 ratio
of Solution A and B (Solution D), a 1:4 ratio of Solution A and B (Solution E). Distilled
water was used as the control (Solution F).
f. After about 30-45 seconds, the fiber was remeasured. The measured length will be used
to calculate the degree of contraction.
g. The entire process of the muscle reacting to the solutions was observed through the
microscopes and observations were recorded (Figure 7).
h. Steps a-g were repeated for each new 2cm fiber with various combinations of solutions
(Figure 5)

Recording data and results:

Using ATP to Trigger Satellite Cells Kagramian 6

i. A table was kept to record the various combinations of solutions tested as well as their
corresponding, resulting fiber lengths.
j. ​Each trial of testing different amounts of ATP on rabbit psoas muscle tissue was recorded
and compared at the end for the highest degree of muscle contraction.

Statistical analysis:
An analysis of the collected data using the ANOVA method (⍺ = 0.05) to find out which
amount of chemicals will produce the strongest contractions. Linear regression will be used to
determine whether there is a correlation between the amount of ATP and the degree of
contractions.

Figure 3: The 11cm strip of rabbit psoas

muscle was removed along with the liquid
was transferred into a petri dish. Figure 4: The long strip of muscle was cut
into 2cms and separated into thinner fibers.
Using ATP to Trigger Satellite Cells Kagramian 7

Figure 5: The Ward's® ATP Muscle Kit used

in the methodology of this study. It comes
equipped with twenty-four muscle samples
Figure 6: The dissecting kit used contains the
(one strip of psoas muscle per tube of 50%
scissors and needle.
glycerol), ATP, salt solutions, and chemical
instructions.

Figure 7: The skeletal muscle viewed under the dissecting microscope (left) and the
compound microscope (right)
Using ATP to Trigger Satellite Cells Kagramian 8

Results:
The results of this experiment showed evidence that the strength of a muscle contraction
is directly related to the amount of ATP present. The degree of contraction produced ranged from
<0.1 to 0.225. An ANOVA test was run with a p-value of P<0.00001, meaning there was a
significant difference among the data. The ANOVA test showed that Solution C, 0.25% ATP plus
0.05M KCl plus 0.001M MgCl2, produced the greatest degree of contraction: 0.225; Solution B
and F both averaged a 0 degree of contraction, meaning the presence of salt alone or water alone
will not cause a contraction. The trials with the greatest amount of ATP, Solution C and D,
exhibited the greatest contractions (Figure 8). As the amount of ATP present increased, the
degree of contraction did as well as supported by the R2 value calculated (Figure 9).
Furthermore, a combination of solutions where the presence of salts dominated ATP resulted in
fewer contractions as supported by the R2 value calculated (Figure 10). While the presence of
salts is necessary for strong contractions to occur, they are not the driving source of the
movement.

Figure 8: Each of the 6 solutions (n=6) were put through 5 trials; they were
compared against each other to determine if muscle contractions vary greatly
with different concentrations of ATP and salts. An ANOVA was conducted to
determine significance (⍺ = 0.05; p<0.00001).
Using ATP to Trigger Satellite Cells Kagramian 9

Figure 9: The positive correlation between the % concentration of ATP within

the solution to the degree of contraction. A linear regression test showed a strong
correlation between the two variables (R2=0.2191).

Figure 10: The negative correlation between the % concentration of salts within
the solution to the degree of contraction. A linear regression test showed a strong
correlation between the two variables (R2=0.2191).
Using ATP to Trigger Satellite Cells Kagramian 10

Discussion:
In general, a greater concentration of ATP will increase the degree of contractions within
skeletal muscle. However, a 1:1 ratio of ATP solution to KCl-MgCl2 yields the greatest degree of
contraction. Muscle contraction is caused by the interaction of actin and myosin when the
hydrolysis of ATP takes place (Cooke & Bialek, 1979). When a muscle is relaxed, a lack of
calcium ions causes the Tropomyosin protein to block the cross-bridge bind sides for myosin and
actin. After calcium ions bind to the troponin structures on actin, tropomyosin binding sites
become exposed for myosin to interact with actin. The heads which extend from myosin, a thick
filament protein, interact with actin, the thin filament (Figure 11). The cross-bridges of the
myosin filaments attach to the actin filaments and move them forward which is called the sliding
filament mechanism of muscle contraction. The contraction begins when the ATP hydrolyzes
into ADP and Pi and triggers the myosin to attach to a binding site on the actin known as a cross
bridge. The myosin is triggered by the power stroke to move the actin closer toward the m line of
the sarcomere, a unit of muscle fiber (Iron Pharaoh, 2017; Figure 12). An example of ATP being
a key factor of muscle contraction is in Electrical muscle stimulation (E-stim) which works by
sending micro-size currents throughout the body to stimulate the release of ATP as well as
increase blood circulation (Pro Healthcare Products, 2016). E-stim can enhance stem cells as
well as fibroblast activity (Du et al., 2018; Zeisberg et al., 2000).
The muscles used in this research were glycerinated muscles, which are different from
muscle from living tissue. The purpose of glycerol is to retain the muscle's contractile properties
without the membrane or other systems aiding the muscle tissue (BENSON et al., 1958).
Glycerination removes ions and ATP from the tissue which disrupts the tropomyosin and
troponin complex and replaces the need for calcium to unlock the myosin binding sites
(Contraction of Glycerinated, 2023). In the event that ATP is added, contraction of the muscle
will be triggered because myosin requires the ATP binding for activation (Cooke & Bialek,
1979). However, in the presence of other ions such as magnesium (Mg+2) or potassium (K+), the
binding of the ATP becomes stronger thus resulting in a greater degree of contraction (Dragomir
et al., 1975). ATP prepares myosin to bind with actin through a cross-bridge by increasing its
energy state. The phosphate ion (Pi) dissociates and the sarcomere shortens, lowering the energy
level (“Muscle Contraction and Locomotion,” 2018). The process of removing a phosphate
group from ATP is called dephosphorylation. The presence of salts stimulates the
Using ATP to Trigger Satellite Cells Kagramian 11

dephosphorylation of ATP. The addition of salt alone will not cause any muscle contraction due
to the fact that there is no unit of energy allowing the myosin to move the actin forward.

Conclusions:
Muscular dystrophy can manifest itself within an individual in many ways. The specific
muscular dystrophy by the name of Facioscapulohumeral dystrophy (FSHD) causes atrophy to
the skeletal muscles, weakening every muscle making every physical task an arduous one. In a
living organism where the tissue is not glycerinated, the presence of more ATP will cause a
greater degree of contraction as long as there is enough calcium to allow for the reaction. ATP
can be introduced into an organism through glycolysis and mitochondrial respiration. The act of
working a muscle will trigger the use of ATP during those muscle contractions; an increase in
ATP levels correlates with greater contractions. Due to this fact, ATP can be used as a way of
therapy for patients with muscular dystrophy because in the event that a contraction large enough
were to cause muscle trauma, satellite cells operate to not only repair the damage, but to rebuild
it larger and stronger. A method such as Electrical muscle stimulation causes contractions large
enough to create damage, only to be repaired by the stem cells and fibroblasts. Such a form of
therapy could be used for treatment of many muscular dystrophy diseases. As further research
however, the stem cells in individuals with specific dystrophies should be studied to determine
the rate at which they function and whether or not their speed is lower than that of a healthy
muscle. Although the application of this method as a form of therapy for muscular disabilities is
not a substitute for a cure, it holds the potential to be extremely beneficial to thousands of people
as a way to work against the progressive factor of muscular dystrophy.
Using ATP to Trigger Satellite Cells Kagramian 12

Figure 11: A diagram showing the location of the globular head of

the myosin and its relative location to actin. The troponin and
tropomyosin are also located on the actin.

Figure 12: A diagram showing the location of the m line on the sarcomere. The
M line, in the center of the H zone, consists of proteins that hold the myosin
filaments in place.
Using ATP to Trigger Satellite Cells Kagramian 13

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