Aging Skeletal Muscle and Exercise

An Annotated Bibliography Submitted in

Partial Fulfillment of the Requirements for

HES 474 Physiology of Exercise

Professor: Anil Joseph, MA

By

Gracie Cabana

April 22, 2020

Table of Contents

Description of Topic
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Additional Readings

Narrative Summary

Description of the Topic Researched:

As humans age, our skeletal muscles decrease in strength, size, power, and ability to
perform everyday functional tasks. Our decline in strength in muscles is thought to be one of the
reasons for the decline in the elderlys motor skills and loss of independence. The main causes of
neuromuscular junction dysfunction are remodeling endplate morphology, an increase of
inflammation, mitochondrial dysfunction, and neurodegeneration. My research is specifically on
the structural changes on the aging neuromuscular junction and the functional alterations that
come from the structural changes, mainly the excitation-contraction uncoupling process.
The Neuromuscular junction (NMJ) is the chemical synapse between a motor neuron and
a muscle fiber, which allows the motor neuron to transmit signals to the muscle fiber, which
causes the muscle to contract. The impairment of the NMJ can be caused by several different
factors including: nerve terminals, postsynaptic folds. Synaptic vesicles, neurotransmitters, and
the mitochondria.
Excitation-contraction coupling is the process that causes a muscle to contract. It begins
with an action potential from the brain or spinal cord, then arrives at the alpha motor neurons
dendrites, then travels down the axon, then to the axon terminals where the nerve endings
releases neurotransmitters (Ach), then the chemical neurotransmitter crosses the synaptic cleft
and binds to the receptors on the plasmalemma. If there is enough Ach it causes the action
potential to be transmitted full length of the muscle fiber which causes the sodium ion channels
to open and a rush of sodium enters the cell which causes the cell to depolarize. The uncoupling
of this process, also known as excitation-contraction uncoupling is one of the main functional
alterations which can impact several factors including: the impairment of the activation of
specific muscles, the RyRs and DHPR, the schwann cells, reinnervation, motor neurons, calcium,
and AChr.
There has been a lot of research and studies done to figure out the specifics with the
neuromuscular junction as it ages. Specifically, the structural changes and the functional
alterations. However, for as much research and studies that have been done there are still many
questions left unanswered and a lot of mechanisms left unquestioned.

Annotation #1

Source: Neuromuscular Junction Transmission Failure is a Late Phenotype in Aging Mice

 Chugh D, Iyer CC, Wang X, Bobbili P, Rich MM, Arnold WD. Neuromuscular junction
transmission failure is a late phenotype in aging mice. Neurobiology of Aging. 2020;86:182-190.
doi:10.1016/j.neurobiolaging.2019.10.022

Purpose of the Article:

The purpose of this article is to investigate SFEMG jitter and blocking across the lifespan of
mice. This study was performed to address if age mice have features of failed NMJ transmission
furing SFEMG recordings, when do NMJ transmission defects show in the lifespan of mice, and
how NMJ transmission failure relate to loss of muscle contractility, motor unit numbers, and
clinical frailty? This article hypothesized that NMJ transmission failure would occur with the
onset of loss of muscle contractility and motor unit loss in mice as they age and that the mice
with increased clinical frailty and loss of muscle contractility would have more findings of NMJ
transmission failure and motor unit loss.

Conclusions by Author:

This author concluded that their findings of late NMK transmission defects imply that
modulating NMJ at earlier stages of life may not be beneficial. They agreed that future studies
that involve the NMJ modulation at different stages are needed to better understand how
neuromuscular transmission defects affect the loss of physical function in the older population.

Bulleted Notes from Article:

1. Measurements of frailty scores were done to understand the relationship between

clinical deterioration, motor unit connectivity, and NMJ transmission failure. They tested
the frailty of the mice by evaluation of the integument, musculoskeletal system,
vestibulocochlear and auditory system, ocular and nasal systems, digestive system,
urogenital system, respiratory system, signs of discomfort in the mice, body weight, and
surface body temperature. It was tested on a scale of 0-1.
2. Postsynaptic alterations cause the adaption of the enhanced presynaptic transmitter
release.
3. Reduction of muscle fiber excitability may be the mechanism for NMJ transmission
and action potential failure in old mice (on SFEMG).
4. In other studies in humans and rodents, have shown different effects aging has on
muscles with different muscle fibers.
5. The study showed that motor unit loss started to occur by about 20 months of age in
the mice. SFEMG findings of increased jitter and blocking were not demonstrated until
27 months of age in the mice.
6. Denervated muscles do not contribute to the changes on SFEMG in 27-29 month old
mice.
 7. At about 20 month of age in the mice, loss of motor units may initiate the response of
reinnervation to compensate. The process of reinnervation happens through sprouting
which causes the absence of NMJ transmission defects until after 24 months of age in the
mice. At older ages, the motor unit degeneration may happen instead of the regenerative
response, which causes abnormal remodeling of the NMJ and abnormal NMJ
transmission at about 27 months of age in the mice.
8. This study suggests that early loss of muscle strength is not due to failure of NMJ
transmission. However, it may be related to other factors like, denervation or
subsarcolemmal dysfunction. These factors could affect the mechanisms of calcium
release from intracellular calcium stores. Which means, excitation-contraction
uncoupling is important in the decline in specific muscle force as humans age.
9. Studies show that age-dependent changes in voltage-gated calcium channels, result in
the loss of specific muscle force as humans age.
10. In another study it showed that older men with sarcopenia had reduced reinnervation
capacity, but that the motor unit number estimation (MUNE) was not different compared
to older men without sarcopenia. Which means, motor unit loss may not be the reason for
sarcopenia but that the failure of reinnervation that may cause sarcopenia phenotype in
the older humans.

Thoughts and Comments:

I liked this article and the study they performed. However, they mentioned a lot of other studies,
almost more so than their own. It was hard to follow and understand when they were talking
about the experiment they conducted versus other studies other people did. It did have good
information pertaining to my research topic.
Annotation #2

Source: Expression and Regulation of Excitation-Contraction Coupling Proteins in Aging

Skeletal Muscle
 Delbono O. Expression and Regulation of Excitation-Contraction Coupling Proteins in
Aging Skeletal Muscle. Current Aging Sciencee. 2011;4(3):248-259.
doi:10.2174/1874609811104030248

Purpose of the Article:

The purpose of this article is to review the proteins mediating excitation-contraction coupling
and their expression and regulation in humans and rodent models of skeletal muscle as its
function declines with age. This article also reviews the mechanisms that cause the impairment
of skeletal muscle functions humans age. These mechanisms include: external calcium-
dependent skeletal muscle contraction, increased voltage-sensitive calcium channel subunit and
junctional face protein, and decreased Ca 1.1 expression. Also, the potential role of these
mechanisms and other molecules in the muscle T-tubule and Sarcoplasmic reticulum junction in
excitation-contraction uncoupling. This article also examines neural influences and trophic
factors like IGF-1.

Conclusions by Author:

The author concludes that the decrease in production of IGF-1 and neurotrophins and tissue
resistance can result in spinal cord motor neurons loss and atrophy. The reduced synthesis of
IGF-1 causes the failure of an IGF-1 mediated pathway to decrease CREB phosphorylation,
which causes the reduced DHPR-alpha transcription, excitation-contraction uncoupling, and
decreased muscle force. Finally, there are a number of triad proteins that show the molecular
signaling that is involved in excitation-gene expression and excitation-contraction coupling.
Therefore, neural factors are important in preventing excitation-contraction uncoupling in
humans as we age.

Bulleted Notes from Article:

1. The changes in sarcolemma potential to increased intracellular calcium is key to

successful muscle contraction. The DHPR, located at the sarcolemma transverse T-tubule
and the Ca1.1 activation evokes calcium release from the SR through the RyR1 channels
into the myoplasm. The reduced number, function, or interaction with these receptors
causes reduced intracellular calcium mobilization and fore development.
2. A significant decrease in DHPR expression causes denervation and alterations of the
excitation-contraction coupling in the skeletal muscle of adult rats. Nerve crush causes
reduced levels of mRNAencoding DHPR subunits and RyR1 in muscle (DHPR and
RyR1 expression depend on innervation of skeletal muscle). DHPR and mRNA levels
change during development in relation to fiber innervation. Myotube depolarization
triggers the appearance of binding sites. Exercise and chronic stimulation increased
DHPR expression in the soleus and extensor digitorum longus muscles. Fiber-type
 composition, DHPR, RyR1, and excitation-contraction coupling depend on nerve
stimulation and muscle activity.
3. Excitation-contraction uncoupling causes a decline in muscle specific force, the loss
of muscle mass associated with a decrease in muscle fibers and muscle atrophy, changes
in fiber type, decrease in force, slower sliding speed of actin and myosin, and impaired
recovery after eccentric contraction.
4. Alterations in the neuromuscular junction causes the instability of the neuromuscular
junction. After the synapse, the terminals of the same axon show heterogeneity with
acetylcholine release. This causes nerve terminal selection in the developmental
transition from innervation of each muscle fiber by multiple nerve endings, which results
in the adult one-on-one pattern.
5. Physical activity is important for a successful neuromuscular junction.
6. Experimental denervation in rodents leads to changes such as, reorientation of
costameres, proliferation of triadic membranes, decrease in the functional expression of
the DHPR voltage sensor and alterations in the SR calcium release channel. Age-related
denervation may induce structural and functional changes in mammals.
7. Target derived neurotrophic factors, neurotrophin and nerve growth factor are
important in regulating the survival of developing neurons in peripheral and CNS. NT-4
appears to act as a neurotrophic signal for the growth and remodeling of the
neuromuscular junction.
8. Muscle IGF-1 has trophic effects on motor neurons, which means the overexpression
of IGF-1 is effective in delaying or preventing the decline aging has in tissues.
9. External calcium is needed to maintain fiber force with repeated stimulation.
Depressed SR calcium release causes a decline in fiber force. The failure to generate an
AP causes a decrease in intracellular calcium mobilization and tetanic force in aging
muscles.

Thoughts and Comments:

I thought this article was a very good article with a lot of good information pertaining my
research topic. However, a lot of the studies used in this article have been used in many other
articles. I liked rereading the studies done on mice because it helped me understand it a little bit
better but it was also very repetitive.
Annotation #3

Source: Neural Control of Aging Skeletal Muscle

Delbono O. Neural control of aging skeletal muscle. Aging Cell. 2003;2(1):21-29.

doi:10.1046/j.1474-9728.2003.00011.x

Purpose of the Article:

The purpose of this article is to explain the mechanisms that underlie the neuromuscular
impairments in old age. The mechanisms include: neural influences, the effect of age on the
neuromuscular junction, excitation-contraction uncoupling, and the effects of IGF-1 in
excitation-contraction uncoupling and on neurons.

Conclusions by Author:

The author concludes that a decrease in local production of IGF-1 and neurotrophins in addition
to tissue resistance to these factors may result in loss and atrophy of spinal cord motor neurons.
The impairment in motor neuron function may lead to reduced synthesis of muscle-derived IGF-
1, which leads to neurodegeneration. Reduced synthesis of IGF-1 may contribute to decreased
CREB phosphorylation, which may result in reduced DHPR transcription with consequent
excitation-contraction uncoupling and decreased muscle force. He hypothesized that neural
factors play an important role in preventing age-related excitation-contraction uncoupling and
therefore, interventions aimed at counteracting nerve loss will reverse the effects and the loss of
force able to produce in animal models of aging and aging humans.

Bulleted Notes from Article:

1. As humans age, after the synapse formation, the terminals of the same axon release
different amounts of acetylcholine, which contributes to nerve terminal selection, which
will make the transition from innervation of each muscle fibre go from multiple nerve
endings to the adult one-to-one pattern.
2. The Level of physical activity in the elderly is very important for a successful
neuromuscular junction.
3. The study they performed on rats that made the synaptic terminals occupying the rats
motor endplates electrically silent by the sodium channel blocker tetrodotoxin were
frequently displaced by regenerating axons that were inactive and ineffective concluded
that evoked or spontaneous activation of acetylcholine receptors is required for
competitive reoccupation of neuromuscular synaptic sites by regenerating motor axons in
adult rats.
4. As humans age, excitation-contraction uncoupling is one of the major factors for the
decline in the capacity of individual cells to produce force.
5. As nerves get damaged, the calcium channels (DHPR and RyR1) levels get reduced,
which leads to the impairment of muscle contraction.
6. Both the decline of neural influence on skeletal muscle which leads to changes in the
muscle composition, results in the uncoupling of the motor neuron and muscles fiber,
which causes the impairment of the excitation-contraction coupling process.

Thoughts and Comments:

I thought this article was a really well written article and had a lot of good explanations and a lot
of good information specifically pertaining to my research topic. However, there were a lot of
things that were not completely explained and if would have been explained in more detail
would have made the information make more sense.
Annotation #4

Source: The Neuromuscular Junction: Aging at the Crossroad Between Nerves and Muscle

Gonzalez-Freire M, Cabo RD, Studenski SA, Ferrucci L. The Neuromuscular Junction:

Aging at the Crossroad between Nerves and Muscle. Frontiers in Aging Neuroscience. 2014;6.
doi:10.3389/fnagi.2014.00208

Purpose of the Article:

The purpose of this article is to review the current events that are believed to lead to
Neuromuscular Junction (NMJ) dysfunction. Including: the aged NMJ, biomarkers of NMJ in
aging and signaling pathways, the study of aging NMJ in animals models, and interventions to
improve NMJ dysfunction in aging.

Conclusions by Author:

The author concludes that morphological and physiological changes cause the remodeling of the
motor unit and a decline in the number of motor neurons. This leads to excitation-contraction
uncoupling and a loss of communication between the nervous and muscular system. Which
causes a decrease in muscular strength and size. However, we still don’t know if denervation
causes sarcopenia or if sarcopenia causes denervation.

Bulleted Notes from Article:

1. In the NMJ the nerve terminal area and the number of postsynaptic folds are reduced,
which leads to the functional impairment in the postsynaptic response of the NMJ. The
denervation of muscle fibers and muscle atrophy are correlated with presynaptic plaque
changes, such as high levels of oxidative damage, decreased number of synaptic vesicles,
and lower number of neurotransmitters released during depolarization.
2. Increased levels of oxidation and nitrosylation products, and decreased enzyme activity
are characteristics of mitochondrial dysfunction. This causes the effect on neurons and
muscle fibers because they are metabolically active. A lot of mitochondria in the axon
provide energy and buffer the large calcium ion loads needed for excitation-contraction
coupling. The reduction of ATP production and imapired calcium buffering in
subsarcolemmal mitochondria (near NMJ) may impair neurotransmission and vesicular
recycling.
3. With aging, motor neurons are imapried to sprout and reinnervate denervated fibers,
because of this motor units become smaller and more fatigable and muscle fibers atrophy.
Atrophy of muscle as humans age are from individual muscle fiber atrophy and a
decrease in type 2 fibers. The changes in NMJ cause more dispersed ACh with greater
uncoupling between ACh vesicles and receptors. Impairment to Schwann cells are
increased fragmentation, damage, or denervation which could contribute to the
ineffective re-innervation and neuromuscular dysfunction with aging. Muscle excitation-
contraction uncoupling is due to a mismatch between DHPR and RyR (RyR more
numerous). This causes the reduction of calcium release after an AP, which causes a less
efficient muscle contraction. IGF-1 may prevent the age decline in the number of DHPR,
which prevents the changes in nerve terminal and NMJ.
4. High levels of inflammatory markers (IL-6, IL-7, TNF-alpha, CRP) in blood and
tissues. Inflammation is a risk factor for the decline of muscle mass and strength as
humans age and down regulates the production of IGF-1. Inflammation is associated with
impaired amino acid utilization and protein anabolism (after meal or after exercise).
 5. Changes in production and response to neurotrophins cause reduced axonal
regeneration and dysfunction in motor axons, postsynaptic membrane, and schwann cells.
BDNF plays a critical role in neural plasticity, facilitates synaptic function by increasing
pre-synaptic depolarization at NMJ, maintains AChR clustering in the NMJ. GDNF
declines with aging, denervation leads to an up regulation of GDNF. GDNF is important
for motor neuron survival and regulator of pre and post synaptic plasticity. It is also
activity dependent. IGF-1 circulating declines with age, which causes NMJ degeneration
and motor unit denervation. The overexpression of IGF-1 reverses sarcopenia, prevents
age dependent decrease in type 2B and increases type 2A fibers, and leads to improved
nerve regeneration by acting on axons, schwann cells, and NJM. Volted-gated sodium
channels are essential for the initiation and propagation of action potentials in nerve and
muscle, Nav1.5 is most relevant to aging because it is mainly expressed in adult muscle
after denervation, which means it could be a biomarker for muscle denervation in aging
muscles.
6. Caloric resistance and exercise are innervations to delay the onset of age-related
Neuromuscular junction dysfunction and sarcopenia. Caloric restriction is the most
effective way to extend lifespan and delay the onset of age-related diseases. In mice, the
frequencies of fragmented and denervated postsynaptic sites were lower in the caloric
restricted mice than the normal mice, which the authors concluded that caloric restriction
reduced the incidence of axonal atrophy and reduce the force of the effects age has on the
NMJ. It is also proposed that caloric restriction increases the expression of DHPRs which
preserves the mechanical properties of the aging skeletal muscles. The reduction in
exercise with age leads to reduced resistance fatigue, decreased muscle strength, and
increased risk of frailty. Exercise induces NMJ hypertrophy and improves recovery from
peripheral nerve injuries.

Thoughts and Comments:

This article was a great article about the neuromuscular junction dysfunction as a whole. It has
great information. However, it did not go into as much detail as I hoped about how it affects
excitation-contraction uncoupling.
Annotation #5

Source: The Aging Neuromuscular System and Motor Performance

Hunter SK, Pereira HM, Keenan KG. The aging neuromuscular system and motor
performance. Journal of Applied Physiology. 2016;121(4):982-995.
doi:10.1152/japplphysiol.00475.2016

Purpose of the Article:

The purpose of this article is to review the evidence that as humans age there are changes in
motor unit morphology and properties that lead to impaired motor performance. These changes
reduce maximal strength and power, decrease contraction velocity, increased fatigability,
increases variability during and between motor tasks, which decreases force steadiness, increased
variability of contraction velocity and torque over repeat contractions. The increase of variability
of motor performance causes reduced and more variable synaptic inputs, fewer and larger motor
units, less stable neuromuscular junctions, lower and more variable motor unit action potential
discharge rates, and smaller and slower muscular fibers. The purpose of this article is also to
focus on areas that are directly influenced by changes in the motor unit and the input it receives
from the spinal and supraspinal sources including: maximal strength and power, velocity of
contraction, fatigability, and steadiness during submaximal contractions.

Conclusions by Author:

The author concludes that practice and physical activity can potentially improve some of the
differences in variability of structure and function of the motor unit. As we age, motor
performance declines.

Bulleted Notes from Article:

1. Loss of muscle fiber innervation by alpha motor neurons (caused by spinal motor
neuron apoptosis and distal axon retraction) causes age-related loss of muscle mass, also
known as sarcopenia. Age-related changes in alpha motor neurons are caused by
oxidative stress and inflammation and a less stable neuromuscular junction, which alter
the normal cycle of denervation and reinnervation of muscle fibers. As this process
accelerates with age it leads to fewer but larger surviving motor units.
2. Regular physical activity may modify the age-related changes in motor unit structure
and function across the lifespan. However, other processes can also modify the aging
neuromuscular junction including: genetics, nutrition (decreased calories and protein
intake), altered hormonal status, inflammatory mediators, and factors that result in altered
protein synthesis and sarcopenia.
3. Age-associated changes on the input can change the output of motor neurons, which
can also change the gain, the ratio of the output to the input. Age-associated changes in
the function and structure of cortical neurons contribute to neuromuscular junction
dysfunction with aging. Structural: Neural atrophy, 40% reduction in size of motor
cortical cell bodies, reductions in grey and white matter. Functional: decrease in
inhibition and increased activity in cortical areas in old adults, this causes the inability to
inhibit appropriate cortical areas during focused and targeted motor tasks which can lead
to activation of not needed motor units. In reflex response, the decrease of response is
due to the reduction in the process of afferent and efferent processes. Timing and
magnitude of synaptic inputs onto motor neurons changes from aging and changes in the
common synaptic inputs across many motor neurons both help in coordinating muscle
activation.
 4. As we age the neuromuscular junction undergoes remodeling and inpaired
transmission. The typical 1:1 transmission of the AP from the motor neuron to the muscle
fiber decreases with aging. In animals, morphological changes include: increased
presynaptic branching, larger and more spaced postsynaptic endplates, and reduction in
the relationship of presynaptic vesicles and postsynaptic receptors.
5. A net loss of muscle fibers and reduction in the size of muscle fibers occurs as
humans age. Each fiber is smaller, which is typically from lower protein synthesis and
less satellite cells in type 2 fibers (needed for muscle growth and repair). Lower
intracellular calcium in skeletal muscle fibers and a lower calcium sensitivity to
activation causes a reduction in specific tension in skeletal muscle. Reduced contraction
speed is a result of slower cross-bridge mechanics and slower rates of calcium uptake into
the SR.
6. Muscle strength is reduced and correlates to the loss of muscle mass. Power reduction
is greater than reduction in strength, power is associated with stair climbing, and raising
time from a chair. The muscles of the older adults exhibit lower rates of force
development and slower relaxation rates during voluntary muscle contractions.
Inadequate activation of motor units contributes to age related loss of strength and power.
7. Fatigue is the reduction in force or power of a muscle after exercise. As we age, the
consequences of more fatigue further increases the loss of strength and power. It also
further impairs performance of daily activities. As humans age, we become less steady in
everyday tasks, especially during light-load tasks that are often required during those
everyday tasks.

Thoughts and Comments:

This article was a very good article. I enjoyed reading it, the information all pertained to my
topic and made a lot of sense. The way it was written, the words or phrases I didn't understand
right away were easy to break down and figure out. The only thing about this article that was
inconvenient was that it was 29 pages long.
Annotation #6

Source: Rejuvenation of the Aged Neuromuscular Junction by Exercise

Kreko-Pierce T, Eaton BA. Rejuvenation of the aged neuromuscular junction by

exercise. Cell Stress. 2018;2(2):25-33. doi:10.15698/cst2018.02.123

Purpose of the Article:

The purpose of this article is to review the mechanisms of the neuromuscular junction and how
exercise induces the expression of many of the neurotrophins which provide an explanation for
the rejuvenation of the structure and function of the neuromuscular junction that comes from
exercise.

Conclusions by Author:
 The author concludes that changes in muscle-derived neurotrophin signaling during aging are
responsible for the changes in synapses function and morphology and that neurotrophin
expressions are sensitive to muscle activity. Exercise increases neurotrophin expression in
skeletal muscles which is important and is a main contributor to the positive effects exercise has
on the neuromuscular junction function and structure.

Bulleted Notes from Article:

1. Muscle growth and synaptic input produces the muscle-derived retrograde trophic
signaling which supports the growth of the synaptic innervation.
2. From the studies it was found that the change in neurotransmission was due to an
increase in the total number of synaptic vesicles that are released. Because of the
preciseness of the adjustments in neurotransmitter release it is thought that there is a
signal that originates from the muscle and instructs the motor neuron and the nerve
terminal about the growing muscle which causes
3. A number of neurotrophins are required for the proper development of the
neuromuscular junction. GDNF, which are NT’s known for motor neuron survival and
maintenance support the existence of retrograde trophic signaling at the adult NMJ.
Through studies it was shown that GDNF has the ability to drive synapse growth and
increased presynaptic release by increasing the expression of frequenin, which increases
N-type Calcium channel activation, which leads to an increase of the calcium influx into
the nerve terminal. Calcium influx into the presynaptic nerve terminal is a mechanism of
retrograde control of presynaptic neurotransmitter release.
4. Studies have shown that endurance and resistance training both stimulate extensive
morphological adaptations of the presynaptic nerve terminal of the neuromuscular
junction. The increased strenuous physical training stimulates NMJ hypertrophy which
leads to an increase in the degree and length of nerve terminal branching. “Existence of
an exercise-induced muscle-derived retrograde signal(s) that can modify the
morphological and functional properties of the NMJ to adapt to the demands on
neurotransmission release.”
5. There is a relationship between exercise-induced GDNF muscle expression level and
exercise-induced NMJ remodeling, which means exercise and the increased expression of
neurotrophins have similar responses at the NMJ at synaptic morphology and
neurotransmission. Muscle-derived retrograde neurotrophins signaling causes the
adaptation of the neuromuscular junction to exercise.
6. Studies with rodents shown that the loss of motor function with age results from
changes in the structural integrity of the NMJ, including morphologies: increased
branching of nerve terminals, progressive fragmentation of nerve terminals (postsynaptic
Ach), increased extrajunctional AChR’s , and myofiber denervation.
 7. It is thought that the increase in the release of neurotransmitters from the presynaptic
nerve terminal is compensating for the decrease in muscle contraction and/or changes in
how the muscle and neurotransmitter work.
8. Two possibilities why there is an increase in the release of neurotransmitters: impaired
homeostatic control mechanisms and decline in muscle function.

Thoughts and Comments:

I thought this article did a very good job at reviewing the potential causes for a decline in
function and structure of the neuromuscular junction. It also reviews the benefits and physiology
behind how exercise can help rejuvenate the neuromuscular junction as we age. Overall, I
enjoyed the material. However, it could have been more defined and not as many of the theories
having a question mark.
Annotation #7

Source: Mechanisms of Excitation-Contraction Uncoupling Relevant to Activity-Induced

Muscle Fatigue

Lamb GD. Mechanisms of excitation-contraction uncoupling relevant to activity-induced

muscle fatigueThis paper is one of a selection of papers published in this Special Issue, entitled
14th International Biochemistry of Exercise Conference – Muscles as Molecular and Metabolic
Machines, and has undergone the Journal’s usual peer review process. Applied Physiology,
Nutrition, and Metabolism. 2009;34(3):368-372. doi:10.1139/h09-032

Purpose of the Article:

The purpose of this article is to review how excitation-contraction uncoupling, which disrupts the
cytoplasmic calcium within the muscle fiber is raised for a period of time. This occurs at the
coupling step between the voltage sensors and the release channels, which causes inadequate SR
calcium release with stimulation. This uncoupling process could be responsible for the decreased
calcium release that underlies the long-lasting fatigue occuring after eccentric contractions.
Conclusions by Author:

The author concludes that if cytoplasmic calcium near the triad junctions reaches above normal
levels for a relatively short period of time can cause long-term disruption of calcium release,
which causes low-lasting low-frequency fatigue. The above normal levels of calcium at the triad
junctions would be because of excessive or continual release of calcium from the SR, and also
could be from the influx of extracellular calcium from the t-tubules. The excitation-contraction
uncoupling can also be viewed as a beneficial aspect. It can be a self-limiting mechanism that
can reduce excessive rises in intracellular calcium, which if goes unnoticed could lead to more
significant widespread calcium mediated damage through a muscle fiber.

Bulleted Notes from Article:

1. Excitation-contraction uncoupling is when the normal process of excitation-contraction

coupling process in skeletal muscle fiber is disrupted when the cytoplasmic calcium
within the fiber is raised for periods of seconds to minutes.
2. Normal excitation-contraction coupling mechanism is retained and fully functional in
skinned fibers.
3. If the cytoplasmic free calcium in a skinned fiber was raised from normal resting
levels, there was irreversible reduction in responsiveness to t-system depolarization.

4. The process of excitation-contraction uncoupling occurs at, or before, the step where
voltage sensors-DHPRs trigger the opening of the calcium release channels. The resting
potential of the t-system has to be more negative than -70mV for the sensor voltage
sensors to be in an activatable state.
5. The effects of chronic depolarization on voltage-sensor function are dependent on
membrane potential, which means any increase in resting level of depolarization in
partially uncoupled fibers should increase the extent of voltage-sensor dysfunction and
further reduce calcium release.
6. Voltage-dependent sodium channels (needed to move AP) in the t-system are more
sensitive to chronic depolarization than the voltage-sensors.
7. Uncoupling occurs at the step where the voltage-sensors normally physically interact
with and open the SR calcium release channels. Highly suggestive that electron
micrographic examination of the triad junction (where the t-system normally abuts the
terminal cisternae of the SR) showed that the junctions were frequently distorted in
uncoupled fibers, in some cases were completely severed.
8. When there is a higher level of calcium than normal for an extended period of time it
causes the excitation-contraction uncoupling process. However, the experiment that took
place used calcium concentrations that were 40-100 times higher than in the normal body.
 Which infers the calcium in the normal body would need to reach 40-100 times higher
than normal conditions in order for uncoupling to occur at this rate.
9. From the experiments it showed that the excitation-contraction uncoupling can be
caused by excessive calcium release from the SR to the muscle fiber and that the site of
action is likely to be close to the release channels in the triad junction, which goes with
how uncoupling appears to be due to the disruption in communication between the
voltage sensors and the release channels at the triad junction.
10. Inhibitors of calcium dependent proteases known as calpains, a specific one called
leupeptin, slowed or prevented the uncoupling in toad fibers but only if it was present in
high concentrations. This effect is suggestive that calpains might be involved in the
excitation-contraction uncoupling. Skeletal muscle contains lots of amounts of calpain-1
and calpain-2. Calpain-3 does not have an affect on uncoupling.

Thoughts and Comments:

I really enjoyed this article, it explained the excitation-contraction uncoupling process very well.
The only part that would have been more beneficial was if the article could have done a better
job at explaining the roles of calpains in the process. Otherwise, this article had a lot of useful
information for my research topic.
Annotation #8

Source: Long-term Low-Intensity Endurance Exercise along with Blood-Flow Restriction

Improves Muscle Mass and Neuromuscular Junction Compartments in Old Rats

Pour MB, Joukar S, Hovanloo F, Najafipour H. Long-term Low-Intensity Endurance

Exercise along with Blood-Flow Restriction Improves Muscle Mass and Neuromuscular Junction
Compartments in Old Rats. IJMS Vol. 42. 2017 November; 6

Purpose of the Article:

The purpose of this article is to clarify if low-intensity aerobic exercise with limb blood-flow
restriction can improve muscle atrophy and nicotinic acetylcholine receptors at the
neuromuscular junction. The article hypothesized that ”low intensity aerobic training combined
with BFR might recover the clustering of nAChRs and improve the performance of the NMJ.”

Conclusions by Author:
The author concluded that BFR with low-intensity exercise caused beneficial effects on muscle
mass and nAChR clustering at the NMJ in old rats. Therefore, a combination of BFR and low-
intensity exercise may be a safe method to improve muscle mass and motor skills in the elderly.

Bulleted Notes from Article:

1.The denervation of motor units happens, then the neuromuscular junction remodeling
as we age, causes reductions in the number of presynaptic vesicles, nerve terminals,
nicotinic acetylcholine receptors with an increase in the space at the end of the axon
terminal. Then, the type 2 fibers conversion into type 1 fibers, which causes an increase
in type 1 fibers.
2. Resistance training and endurance training both increase the number of presynaptic
and postsynaptic components of the neuromuscular junction. Aerobic training improves
muscle function, neuromuscular junction function, metabolism, and motor unit
adaptability.
3.When a low-intensity exercise is combined with BFR, it significantly augments muscle
size, strength, and endurance. Aerobic training improves cardiopulmonary endurance.
4. The results of the experiment showed that the model of exercise (KAATSU)
significantly upregulated nAChRs at the NMJ and induced hypertrophy in extensor
digitorum longus and soleus when compared to the other groups.
5. As humans age, there is the remodeling of NMJ and dispersion of nAChR clusters.
Changes in cytosolic calcium homeostasis from alterations in dihydropyridine receptors
and calcium pump proteins and increase in free cytosolic causes the age-dependent
dispersion of nAChR clusters. Increased cytosolic calcium activates calcium dependent
proteases, such as calpain, which can interact with the nAChR clustering protein rapsyn,
which disrupts the nAChRs complex.
6. Mitochondria released by SR may be depolarized when there is a calcium overload in
muscle fibers.
7. Exercise alone was unable to nAChR clustering at NMJ in olf rats. However, 10 weeks
of low-intensity running WITH BFR upregulated nAChR clustering at the NMJ.
8. The number and function of mitochondria and frequent modifications of the
mitochondrial morphological structure with aging is an important factor in the reduction
of NJ responsiveness to exercise. In this study, the combination of low-intensity exercise
and BFR reversed the negative effects aging has on the NMJ responsiveness to exercise
and recovered the nAChR clusters in the EDL and soleus.
9. Nitric oxide (NO) and nitric oxide synthase (NOS) are both other factors involved in
nAChR clustering. The expression of neural NOS is decreased with aging and endurance
exercise can upregulate its production in both gastrocnemius and soleus. Reinforcement
of NO production with exercise and BFR may promote nAChR clustering at the NMJ in
the study.
10. 10 weeks of BFR with mild exercise caused hypertrophy in the EDL and soleus.
 11. BFR with exercise is associated with the production of lactic acid, which causes
muscle swelling. Which may reduce proteolysis and activate mTOR and MAPK
pathways. Which increases muscle protein synthesis, which causes muscle hypertrophy.
12. The trained animals in this study lost weight. This is explained by the reduction in
adipose tissue and increased mitochondrial production.

Thoughts and Comments:

I really enjoyed this article. The experiment that was done was interesting and for the most part
easy to follow and understand. I thought the results concluded nicely to my research topic. Very
good article in my opinion.
 Additional Readings

9. Excitation-Contraction Coupling. Excitation-Contraction Coupling - an overview |

ScienceDirect Topics. https://www.sciencedirect.com/topics/veterinary-science-and-veterinary-
medicine/excitation-contraction-coupling. Accessed April 22, 2020.

10. Marta G-F, Cabo Rde, Studenski SA, et al. Changes in the neuromuscular junction
with aging. Structural changes... ResearchGate. https://www.researchgate.net/figure/Changes-in-
the-neuromuscular-junction-with-aging-Structural-changes-together-with_fig3_265056822.
Published October 10, 2018. Accessed April 22, 2020.
 Narrative Summary

As humans age, our skeletal muscles decrease in strength, size, power, and ability to
perform everyday functional tasks. Our decline in strength in muscles is thought to be one of the
reasons for the decline in the elderlys motor skills and loss of independence. This research
pertains to the dysfunction of the neuromuscular junction including: the structural changes of
NMJ, and the functional changes, which mainly pertain to the process of excitation-contraction
coupling, and finally, how exercise impacts or does not impact the aging decline of motor
performance.
The neuromuscular junction (NMJ) is the chemical synapse between a motor neuron and
a muscle fiber, which allows the motor neuron to transmit signals to the muscle fiber, which
causes the muscle to contract. NMJ dysfunction is caused by inflammation, mitochondrial
dysfunction, and neurodegeneration. It is divided into two different categories: structural and
functional. Where the structural changes typically cause the functional alterations (10).
The structural changes in the NMJ with aging are derived from the remodeling of the
endplate morphology. As we age the neuromuscular junction undergoes remodeling and
impaired transmission. The typical 1:1 transmission of the AP from the motor neuron to the
muscle fiber decreases with aging (5). In the NMJ, the nerve terminal area and the number of
postsynaptic folds are reduced. This leads to the functional impairment in the postsynaptic
response of the NMJ (4). The postsynaptic alterations cause the adaption of the enhanced
presynaptic transmitter release (1). it was found that the change in neurotransmission was due to
an increase in the total number of synaptic vesicles that are released (6). It is thought that the
increase in the release of neurotransmitters from the presynaptic nerve terminal is compensating
for the decrease in muscle contraction and/or changes in how the muscle and neurotransmitter
work (6).
Increased levels of oxidation and nitrosylation and decreased enzyme activity are characteristics
of mitochondrial dysfunction, which causes the effect on neurons and muscle fibers. Since there
are a lot of mitochondria in the axon that provide energy and buffer the large calcium ion loads
(needed for excitation-contraction coupling). This causes the reduction of ATP production and
impaired calcium buffering in subsarcolemmal mitochondria, near NMJ, may impair the
neurotransmission (4). The number and function of mitochondria and frequent modifications of
the mitochondrial morphological structure with aging is an important factor in the reduction of
NJ responsiveness to exercise (8).
Excitation-contraction coupling is the process that causes a muscle to contract. It begins
with an action potential from the brain or spinal cord, then arrives at the alpha motor neurons
dendrites, then travels down the axon, then to the axon terminals where the nerve endings
releases neurotransmitters (Ach), then the chemical neurotransmitter crosses the synaptic cleft
and binds to the receptors on the plasmalemma. If there is enough Ach it causes the action
potential to be transmitted full length of the muscle fiber which causes the sodium ion channels
to open and a rush of sodium enters the cell which causes the cell to depolarize (9). The decline
of neural influence of skeletal muscle leads to changes in muscle composition, which leads to the
uncoupling of motor neurons and muscle fibers, which then causes the impairment of the
excitation-contraction coupling process. One of the main functional alterations in the NMJ with
aging is the process of excitation-contraction uncoupling. As humans age, excitation-contraction
uncoupling is one of the major factors for the decline in the capacity of individual cells to
produce force, which leads to the decline in muscular force (3). This process is when the normal
process of excitation-contraction coupling process in skeletal muscle fiber is disrupted when the
cytoplasmic calcium within the fiber is raised for periods of seconds to minutes (7). Uncoupling
occurs at the step where the voltage sensors normally physically interact with and open the SR
calcium release channels. It is also said that when there is a higher level of calcium than normal
for an extended period of time it causes the excitation-contraction uncoupling process (7). It is
found that excitation-contraction uncoupling causes a decline in muscle specific force, the loss of
muscle mass associated with a decrease in muscle fibers and muscle atrophy, changes in fiber
type, decrease in force, slower sliding speed of actin and myosin, and impaired recovery after
eccentric contraction (2).The dihydropyridine receptor (DHPR), which is the voltage gated
calcium channel located at the sarcolemma transverse T-tubule and the Ca1.1 activation evokes
calcium release from the sarcoplasmic reticulum (SR) through the ryanodine-sensitive calcium
channels (RyR1) into the myoplasm. The reduced number, function, or interaction with these
receptors causes reduced intracellular calcium mobilization and fore development which causes
the impairment of the calcium release. A decrease in the expression of DHPR can cause
denervation and alterations of the excitation-contraction coupling (2). As humans age, after the
synapse formation, the terminals of the same axon release different amounts of acetylcholine,
which contributes to nerve terminal selection, which will make the transition from innervation of
each muscle fibre go from multiple nerve endings to the adult one-to-one pattern (3). As nerves
get damaged, the calcium channels (DHPR and RyR1) levels get reduced, which leads to the
impairment of muscle contraction (3). It is shown that older men with sarcopenia had reduced
reinnervation capacity, but that the motor unit number estimation (MUNE) was not different
compared to older men without sarcopenia. Which means, motor unit loss may not be the reason
for sarcopenia but that the failure of reinnervation that may cause sarcopenia phenotype in the
older humans (1). The motor neurons, as we age, are impaired to sprout and reinnervate
denervated fibers, which causes the motor units to become smaller, more fatigable, and atrophy
of muscle fibers (4). High levels of inflammation markers (IL-6, IL-7, TNF-alpha, and CRP) in
blood and tissues , which causes the decline of muscle mass and strength more likely (4). Loss of
muscle fiber innervation by alpha motor neurons (caused by spinal motor neuron apoptosis and
distal axon retraction) causes age-related loss of muscle mass, also known as sarcopenia. Age-
related changes in alpha motor neurons are caused by oxidative stress and inflammation and a
less stable neuromuscular junction, which alter the normal cycle of denervation and
reinnervation of muscle fibers. As this process accelerates with age it leads to fewer but larger
surviving motor units (5). External calcium is needed to maintain fiber force with repeated
stimulation. Depressed SR calcium release causes a decline in fiber force. The failure to generate
an AP causes a decrease in intracellular calcium mobilization and tetanic force in aging muscles
(2). it showed that the excitation-contraction uncoupling can be caused by excessive calcium
release from the SR to the muscle fiber and that the site of action is likely to be close to the
release channels in the triad junction, which goes with how uncoupling appears to be due to the
disruption in communication between the voltage sensors and the release channels at the triad
junction (7). As humans age, there is the remodeling of NMJ and dispersion of nAChR clusters.
Changes in cytosolic calcium homeostasis from alterations in dihydropyridine receptors and
calcium pump proteins and increase in free cytosolic causes the age-dependent dispersion of
nAChR clusters. Increased cytosolic calcium activates calcium dependent proteases, such as
calpain, which can interact with the nAChR clustering protein rapsyn, which disrupts the
nAChRs complex (7). It is also suggestive that some calpains might be involved in the
excitation-contraction uncoupling process (7). Another factor that can impact aging skeletal
muscle is the muscle insulin-like growth factor 1 (IGF-1). It has many fucntions such as
neurotrophic effect, promotion of motor neuron survival, maintenance of muscle mass and
strength, and protextion from oxidative stress (4). It has trophic effects on motor neurons, which
means the overexpression of IGF-1 is effective in delaying or preventing the decline aging has in
tissues (2). IGF-1 circulating declines with age, which causes the degeneration of the NMJ and
motor unit denervation. The overexpression of IGF-1 reverses sarcopenia, prevents age
dependent decrease in type 2B fibers and increases type 2A fibers, and leads to improved nerve
regeneration by acting on axons, schwann cells, and NMJ. Another reason for NMJ transmission
failure and action potential failure (in old mice) was found to be due to the reduction of muscle
fiber excitability (1).
 Exercise is the activity that requires physical effort to maintain or improve health and
fitness. As we age, our body declines in responsiveness to the exercise, more specifically the
neuromuscular junction. One of the important factors in the reduction of NMJ responsiveness to
exercise with age is the number and function of mitochondria and frequent modifications of the
mitochondrial morphological structure with aging (8). Regular physical activity may modify the
age-related changes in motor unit structure and function across the lifespan. However, other
processes can also modify the aging neuromuscular junction including: genetics, nutrition
(decreased calories and protein intake), altered hormonal status, inflammatory mediators, and
factors that result in altered protein synthesis and sarcopenia (5). Muscle strength is reduced and
correlates to the loss of muscle mass. Power reduction is greater than reduction in strength.
Power is associated with stair climbing, and raising time from a chair. The muscles of the older
adults exhibit lower rates of force development and slower relaxation rates during voluntary
muscle contractions. Inadequate activation of motor units contributes to age related loss of
strength and power (5). Studies have shown that endurance and resistance training both stimulate
extensive morphological adaptations of the presynaptic nerve terminal of the neuromuscular
junction. The increased strenuous physical training stimulates NMJ hypertrophy which leads to
an increase in the degree and length of nerve terminal branching (6). It is found that glia cell-
derived neurotrophic factor (GDNF) is a potent trophic factor for motor neuron survuval and a
potent regulator of pre- and post-syanptic plasticity (4). There is a relationship between exercise-
induced GDNF muscle expression level and exercise-induced NMJ remodeling, which means
exercise and the increased expression of neurotrophins have similar responses at the NMJ at
synaptic morphology and neurotransmission. Muscle-derived retrograde neurotrophins signaling
causes the adaptation of the neuromuscular junction to exercise (6). As humans age, motor units
go through denervation, the NMJ goes through remodeling. This causes a reduction in the
number of presynaptic vesicles, nerve terminals, nicotinic acetylcholine receptors with an
increase in the space at the end of the axon terminal. Resistance and endurance training is
thought to increase the number of presynaptic and postsynaptic components of the NMJ, while
aerobic training improves muscle function, NMJ function, metabolism, and motor unit
adaptability (8). When a low-intensity exercise is combined with blood-flow restriction (BFR), it
significantly augments muscle size, strength, and endurance. BFR with exercise is associated
with the production of lactic acid, which causes muscle swelling. Which may reduce proteolysis
and activate mTOR and MAPK pathways. Which increases muscle protein synthesis, which
causes muscle hypertrophy (8).
In conclusion, as humans age our skeletal muscle strength, mass, power, and ability to
perform everyday functional tasks decrease. The neuromuscular junction is the synapse for the
action potential to transmit from a motor neuron to a muscle fiber that causes the skeletal muscle
to contract. As we age, the stability of structure and function of the NMJ declines and the
dysfunction of the NMJ is caused by inflammation, mitochondrial dysfunction, and
neurodegeneration. The structural changes in the NMJ with aging cause the functional alterations
of the NMJ, specifically the excitation-contraction uncoupling process. The structural changes of
the NMJ with aging, which includes the remodeling of endplate morphology. This impacts the
nerve terminals, postsynaptic folds, synaptic vesicles, neurotransmitters, and the mitochondria.
These structural changes lead to functional alterations, mainly the excitation-contraction
uncoupling process which includes the impact of: the impairment of the activation of specific
muscles, the RyRs and DHPR, the schwann cells, reinnervation, motor neurons, calcium, and
AChr. Some of the research in the articles conflicted with research found in other articles or my
additional reading information.