Name: Nisal Karawita Student I.

D: 27822427 Authcate:nlkar1 Prac day: Thursday AM

Tutor: Marshall Mrocki Cell type: Myocytes
MYOCYTES

INTRODUCTION

After the sperm fertilises the ovum, the blastocyte with the inner cell mass is formed.
Gastrulation from the inner cell mass creates the epiblast, which gives rise to the ectoderm,
endoderm and mesoderm. Myocytes of the human body are derived from the mesoderm. The
primitive streak allows the cells of the epiblast to migrate to the blastocoel. The mesoderm is
formed by the cells that settle between the epiblast and the endoderm. Fibroblast growth
factor signalling controls the migration of mesodermal cells through the primitive streak.
FGF8 protein pushes migrating cells to move away from the streak. When the mesodermal
cells enter the membryo, lateral movement is controlled by platelet derived growth factor.
Later on, Wnt5a helps the cell to become the lateral plate mesoderm. Wnt3a acts against
Wnt5a and catalyses the formation of the paraxial mesoderm. Alongside the lateral plate
mesoderm and the paraxial mesoderm, the Intermediate Mesoderm and the Chordamesoderm
is formed. However, these structures have a minimal contribution to myogenesis. The
chordamesoderm forms the notochord, a temporary structure which will help to create
anterior-posterior patterning. Covering the notochord from both axis, the paraxial mesoderm
forms somites, which will produce the majority of skeletal muscle. A larger distance away
from the notochord is the lateral plate mesoderm, this gives rise to the splanchnic mesoderm,
where the cardiac and smooth muscle originates.(1)

Figure 1: The mesoderm differentiates into its respectful subgroups. Paraxial and Lateral Plate
Mesoderm plays the largest role in muscle formation. Neural tube directs this growth process. (1)
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes
Paraxial Mesoderm

Due to ethical and technological difficulties, the study of mesodermal development has been
mainly conducted in avian embryos. However, many common features are found in humans.
For example, the presence of somitomeres (segments of the paraxial mesoderm) is found in
both mammals and birds. The formation of somitomeres, results in the regression of the
primitive node towards the caudal region. When about 20 somitomeres are created, the initial
somites form. After this, the development of somites occurs. Nevertheless, the first 7
somitomere pairs, are not subjected to further changes. These will form the skeletal muscles
in the cranial region. The formed somites eventually compartmentalise. The myotome is the
major region where skeletal muscle is formed. The Dorsomedial section of the myotome
forms the back muscles (epaxial)and the ventrolateral part forms the limb muscles, body wall
and tongue (hypaxial). (2) The myotome is formed by two waves of cells. The first wave
forms through the medial epithelial somite and the second wave is released from the
dorsomedial lip, which encompasses the rostral and caudal areas of the dermomyotome. The
intercostal muscles originate from ventrolateral lip (in combination with fibres from the
dermamyotome). (3) During the formation of muscle fibre, fast myosin heavy chains are
displayed first, slow myosin heavy chains are found next. Initially, the mRNA for slow
myosin isoforms are limited to central myotomal fiber nuclei. Despite this, fast myosin heavy
chains are distributed throughout the fibres. During maturation, the slow myosin fibres spread
throughout the muscle fibres. (4) Somite formation starts at the rostral segment of the
embryo, thus the rostral end is more mature than the caudal end. Moreover, the neural tube
(notochord) is an essential aspect for myotome development. In the dorsal portion, Wnt-1
growth factor catalyses myogenesis in the paraxial mesoderm. The ventral tube is thought to
play a smaller, but still important role. (5)

Lateral Plate Mesoderm (LPM)

The lateral plate mesoderm divides itself into the somatic and splanchnic mesoderms.
Moreover, the body cavity (Coelom) originates from the lateral plate mesoderm. The coelom
is made by the combined development of somatic and splanchnic mesoderms. The ectoderm
controls this process by directing the specification and migration of the lateral plate
mesoderm. The differentiation of the LPM from the paraxial mesoderm is governed by the
secretion of different concentrations of BMP-4. (6) Initially, the LPM is divided up into the
cardiac mesoderm (CM) and the posterior lateral plate mesoderm (PLPM). The CM produces
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes
cardiac progenitor cells via the splanchnic mesoderm and creates the myocardium. The
PLPM is responsible for the formation of limbs. The formation of CM is limited by retinoic
acid, to allow for the induction of forelimbs. Hox genes direct the patterning of the PLPM
along the anterior-posterior axis. Eventually, the cells of the PLPM proliferate and form the
somatic and splanchnic layers. Foxf1 is a growth factor that promotes the separation of the
PLPM to two layers. In the somatic layer, Tbx5 promotes Fgf10 to form the limb buds. (7)
The occipital lateral plate mesoderm is also thought to give rise to skeletal muscles in the
neck and thorax. Research has shown that the majority of cucullaris muscle in the neck has
originated from the LPM. (8) Meanwhile, the splanchnic mesoderm forms the cardiac and
brachiomeric muscles. Pitx2 catalyses the formation of brachiomeric muscle. (9) The anterior
ectoderm is thought to provide the factors necessary for cellular differentiation of the
splanchnic mesoderm. An example is BMP2, which is found in the ectoderm. It initialises
the expression of Csx/Nkx-25 (cardiac specific homeobox/neurenberg kim homeobox) in the
cardiogenic mesoderm. The fgf factor heartless (htl) is thought to induce the developmental
cascade of the heart. It activates Ras 1, which has started heart formation, even when the
animal specimen has a mutation in the htl factor. Cell migration is also directed by
transcription factors such as GATA, which ensure that the cardiac tube is formed properly.
Furthermore, retinoic acid controls apoptosis, so that the fusion of the endocardial tubes
occurs accurately. (10)

Skeletal muscle development

Skeletal muscle is needed to produce skeletal movement through contraction and stretching.
On average, males tend to have a slightly higher body percentage of skeletal muscle than
females (42% vs 36%). Skeletal muscles assist in the generation of heat, storage of protein
and the maintenance of posture. The development of Skeletal muscle consists of 3 key steps.
Firstly, myogenic progenitor cells proliferate and differentiate from the myotome into
myoblasts. Following this, the myoblasts fuse into myotubes. Finally, the myotubes form
myofibers. The recently disovered microRNA (miRNA) regulates gene expression through
miRNA-mrNA inteactions to control myogenesis. (Specialised miRNA named myoMiRS are
available in muscle fibers) (11) In addition to miRNA, muscle regulatory factors (MRFs) of
the basic helix-loop-helix type were shown to be important regulators of myogenesis. MyoD,
MRF4, Myogenein and MRF 5 are some MRFs that have been identified. MRFs are regulated
by Paired- Domain containing transcription factors (Pax 3 and 7). Pax 3 directs MyoD to
initiate the differentiation of skeletal muscle while Pax 7 assists satellite cells. In the paraxial
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes
mesoderm, Wnt 5b and Frizzled 7 are factors that signal cells to intiate myogenesis.
Inhibitory molecules such as Notch and Twist slows down myogenesis, thus preventing
premature muscle development. (12) The aforementioned satellite cells conduct the growth
and repair of skeletal muscle. Caspase 3 induces apoptosis by inactivating Pax 7 and limiting
satellite cell self renewal. Researchers believe that skeletal muscle regeneration after injury
can be accelerated by regulating the activity of caspase 3. (13)

Figure 2: A transverse section of a chick embryo, showing the growth factors that direct mesodermal
growth. The paraxial mesoderm receives signals such as Wnt6 (ectoderm), BMP4 (the neural tube),
FGF8 (Notochord) and BMP2 (Lateral plate). (12)

Types of skeletal muscle

Skeletal muscles can be broadly categorised into fast and slow twitch fibers. Research done
on zebrafish has demonstrated adaxial cells (precursor to slow fibres), are found in the depths
of the somite, adjacent to the midline tissue. On the other hand, fast twitch fibers fuse
together and arrange themselves into syncytial fibers while occupying a large part of the
myotome. The growth factor, Blimp1 is responsible for the specification of slow twitch
muscles. It also represses the growth of fast twitch fibers by binding to the promoter of the
myosin light chain (fast twitch muscle) gene. Thus, Blimp1 is the master regulator in the
differentiation of skeletal muscle. (14)

Nervous innervation of skeletal muscle

Through neurogenesis, the spinal nerve bifurcates to the ventral ramus (to the ventral trunk
and limbs) and to the dorsal ramus (muscles of the back). In the ventral ramus, SEM3A-
neuropilin-1 and Sema3F-neuropilin-2 conduct chemorepulsive guidance signalling and
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes
contact mediated repulsive guidance signalling is directed by eprinA-EphA4. These pathways
control the spread of motor axons towards the limb regions. Recent research has shown that
SEMA3A and FGF8 molecules are possibly involved in the development of the dorsal ramus.
FGF8 was found to direct the primitive stages of development, while SEM3A prevented these
immature motor axons from entering the myotome. If SEM3A is unavailable, the dorsal rami
fibres will spread themselves amongst the muscle fibres in an unregulated manner. (15)

Figure 3: SEMA3A is labelled with a blue/ magenta colour cRNA probe, in this chick embryo. It is
observable in the lateral myotome (MF20), dermatome, epidermis and notochord during stage 20 of
development. However it is no longer observable in stage 29, when its function as a regulator of
development is no longer required. (15)
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes
RESULTS

Figure 4:Macroscopic Haematoxylin-eosin and

alcian blue stained sagittal section of mouse
embryo head (4x magnification)

In this image, the development of the

neurocranium and viscerocranium of the skull
is observable. Divided by the
intramembranous area, the meckels cartilage
is beginning to form the mandible, which will
make up the viscerocranium. (facial
bones)The cranial bones make up the
neurocranium, which is divided into the
cranial and endochondral parts. A sliver of
cartilage is observed on the top of the skull,
while the chondrocytes that will ossify into
the vertebral column help to form the skull base.

Figure 5: Microscopic Haematoxylin-eosin

and alcian blue stained sagittal section of
mouse embryo tongue (10x magnification)

This section shows a developing tongue.

The striations in the skeletal muscle are
not observable yet. This might be due to
the muscles being in the early stages of
development. Blood vessel that supply
the tongue can be observed at the tip. The
lingual papillae which assist in
mastication and the manipulation of food
are in the early stages of development.
Mesenchymal cells will direct the growth
and proliferation of epithelial tissues in
the oral cavity. Cartilage can be observed
in the upper and lower jaws.
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes

Figure 6: Microscopic Haematoxylin-eosin and alcian blue stained

sagittal section of the nose and oral cavity of a mouse embryo (4x
magnification)

The cartilage primordium that forms the upper half of the

nasal cavity is visible. Cartilage primordium of the septum is
positioned below this. The nasal bone provides stability to the
nasal cavity, while septum directs airlow to the lungs. The
olfactory epithelium will develop into ciliated

pseudostratified columnar epithelia that will secrete mucous,

remove dust particles and protect the nasal cavity. Interesting
facial features such as the philtrum and lips are present in this specimen.

Figure 7 : Microscopic Haematoxylin-eosin

stained sagittal section of skeletal muscle in the lower jaw of a mouse embryo (20x magnification)
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes

The striations and peripheral nuclei of skeletal muscle are clearly observable. These muscles
will most likely be developed to be used for mastication (can possibly be the masseter
muscle). The presence of blood vessels around the muscle indicates that this muscle will need
a large oxygen and nutrient supply through blood in order to facilitate the chewing of food.

Figure 8: Microscopic Haematoxylin-eosin and alcian blue stained sagittal section of Meckel’s
Cartilage in the lower jaw of a mouse embryo (40x magnification)

In humans, the posterior and anterior aspects of the Meckels Cartilage will be subjected to
endochondral ossification to produce the mandible and ear bones. The medial area will create
the sphenomandibular ligament. The age of specimen can be estimated to be at least 13 days
as the mesenchymal cells have congregated in the meckels cartilage. During the later periods
of gestation, the mesenchymal cartilage will disappear as it forms components of the jaw and
teeth.
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes

DISCUSSION – RHABDOMYOLYSIS IN CROSSFIT

The causes, symptoms, effects and mechanism of rhabdomyolysis

During intense exercise, the breakdown of skeletal muscle can occur. This results in the
release of myoglobin from the myofibres. The increased levels of myoglobin will cause
myoglobinaemia (myoglobin in blood) which will eventually lead to myoglobinuria
(myoglobin in urine). Often, rhabdomyolysis is diagnosed by the tea like brown colour in
urine (due to myoglobin). The danger of this situation that myoglobin can induce acute renal
failure. Myoglobin will block renal tubes by forming a cast with uric acid. The distal
nephrons can be blocked by a gel of myogolobin. This will result in the necrosis of renal
tissue. Furthermore, myoglobin can reduce the Glomerular filtration rate. If not treated, the
combination of these factors can bring about renal failure within a week. (16) Many causes of
rhabdomyolysis have been identified. They are broadly divided into Hypoxic, Physical,
Chemical and Biological causes. Hypoxic causes are subdivided into external and internal
causes. Internal causes consist of exposure to environmental factors such as cyanide, while
internal factors include developmental and genetic defects. Since skeletal muscles are formed
peripherally during embryogenesis, their oxygen supply is more likely to be compromised,
which can lead to injury. Physical factors constitute trauma and burns. The benign exertional
rhabdomyolysis found in crossfitters belong to this category. Chemical causes are made up
external factors like alcohol and illicit drugs and internal chemical imbalances like
hypokalemia. Hormonal and developmental defects are included in internal biological causes.
Insect bites,bacteria and parasites are some external biological causes. For the purposes of
this report, a larger focus will be placed on rhabdomyolysis induced by trauma. Injury to
muscles will disrupt the cellular membrane and cause Calcium to accumulate in the
cytoplasm. The resultant lysis of the cell will release electrolytes, enzymes alongside
myoglobin. A prominent enzyme that can be used to diagnose rhabdomyolysis is creatine
kinase. If the serum of a patient has a creatine kinase level of more than 1000IUL,
rhabdomyolysis is diagnosed. An approach for treating rhabdomyolysis has not been defined
yet. However, treatment is centered around rescuing skeletal muscle from damage, avoiding
renal failure and saving the patients life. Acute kidney injuries are treated by rescuciating the
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes
urine volume, restoring renal perfusion and increasing the urine flow. If this strategy fails,
renal replacement therapy and/or haemodialysis will be used to remove myoglobin and
reverse negative side effects (hyperkalemia, metabolic acidosis). The risk of mortality in
rhabdomyolysis significantly increases if acute renal failure occurs. Thus an early diagnosis
and the prevention of renal failure is very likely to yield the best outcome.(17)

Do high intensity exercises of crossfit pose a risk factor for rhabdomyolysis?

Recently, the US military has adapted crossfit training. Since traditional military training
mainly involved aerobic endurance, crossfit was used to add variety to the training
regime via the use of gymnastics and strength training. However, the risk of injury
could potentially derail the career of servicemen. Traditional training usually has an
injury rate of 32%-63%. These injuries consist mostly of muscle strains and sprains.
However, an exercise program with a low injury rate is also considered to be
ineffective. Furthermore, research has shown that many extreme conditioning
programmes have a high rate of injury. Rhabdomyolysis has been linked to high heat,
high humidity and less fit individuals overexerting themselves. Moreover, 39.2% of
marines who underwent traditional training displayed symptoms of rhabdomyolysis
and marathon runs (which are endorsed by the army) has a high rate of
rhabdomyolysis. (18) Thus, it can be hypothesised that the risk of rhabdomyolysis in
Crossfit can be greatly reduced by taking the necessary precautions and scaling
workouts to suit an individuals level of fitness.

Risk factors for rhabdomyolysis in crossfit

In order to decrease the risk of rhabdomyolysis occurring, the risk factors need to be
determined. In a study done at Crossfit Inc in Santa Cruz, California, the factors that
contribute to injury in crossfit were analysed. Individuals who have sustained injuries
before doing crossfit, were more likely to reinjure themselves. Surprisingly, age and
crossfit experience had no association with the likelihood of injuries. However,
participants who reported that they rarely or never train with instructors had a 66.6%
chance of injuring themselves. In addition to that, participants with joint injuries were
3.75 times more likely to be injured by Crossfit. (19) Once again, this data
demonstrates that proper guidance can greatly reduce the chance of injury. It is also
advisable for people with a history of sports injuries to approach crossfit with caution.
Males, Unconditoned athletes and black people were found to be more likely to suffer
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes
from exertional rhabdomyolysis. New evidence shows that genetic factors such as,
single nucleotide polymorphisms in the CK muscle isoform, alpha-actin-3 and myosin
light chain kinase can increase the probability of rhabdomyolysis. Nevertheless, the
cause cannot be identified in many cases leading to the suggestion that there is an
unidentified genetic influence. (20)

Strategies to minimise the chance of rhabdomyolysis occurring and possible

modifications to suit each individual

Having the knowledge that doing crossfit without proper guidance can have serious
ramifications, prospective crossfitters should seek out a reliable coach. Since anyone
who completes the crossfit level 1 course over two days is qualified to coach, it is
advisable to look for supplementary qualifications. The community aspect of crossfit
is inspiring and enjoyable in many ways, but participants should not refrain from
modifying their workouts due to peer pressure. Even though crossfit workouts are
timed, participants should strive to execute the movements with accurate form.
Rhabdomyolysis results from attempting to do as many repetiitons as possible with
unsuitable form. Concomitantly, beginners should use light weights and perfect their
technique with echnical movements such as the snatch and overhead squat. Certain
movements can be extremely damaging for crossfitters with pre-existing disabilities.
A person with a spinal cord dysfunction having to do box jumps is an example. When
box jumps are done repeatedly obese individuals will experience a larger stress in
their joints. Instead of pushing through and injuring oneself, the box jump can be
modified to a step up. Diet is another important issue. Many crossfitters follow a
paleolithic diet in which they consume high amounts of protein, with very low
amounts of carbohydrates and processed foods. During this fad diet craze, many
athletes forget recommended dietary intake of nutrients, resulting in a reduction of the
ability to perform well. Poor nutrition can indirectly contribute to rhabdomyolysis as
muscles will weaken with a reduced glycogen storage. This in turn will make them
more susceptible to be broken down. (21) Crossfit is an intense training programme. It
does not have an exceptionally high risk of rhabdomyolysis when compared to other
training methods. Nevertheless, overexertion and poor nutrition can increase the risk
of muscle breakdown. Thus, it is imperative that participants understand the limits of
their body while doing crossfit.
Name: Nisal Karawita Student I.D: 27822427 Authcate:nlkar1 Prac day: Thursday AM
Tutor: Marshall Mrocki Cell type: Myocytes
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