MUSCLE
BioC/Phsl/BMEn 5444
1-24-2013
Skeletal Muscle Development
and Growth
Dawn Lowe, PhD
Associate Professor
Program in Physical Therapy and Rehabilitation Science
University of Minnesota
lowex017@umn.edu
Muscle Development and Growth
Questions to be addressed:
• How do undifferentiated cells become
muscle fibers? I.e., How do muscles and
muscle fibers develop?
• How does muscle growth occur and how is it
regulated?
• How is injured muscle repaired?
How do skeletal muscles develop?
• All multicellular organisms start as one cell
• Embryogenesis: The processes leading to the
development of an embryo from an egg
Diagram of a cross-section thru an early vertebrate embryo
**
Germ layers : the 3 layers of cells comprising the early embryo
Diagram of a cross-section thru an early vertebrate embryo

Somite : segmentally arranged blocks of mesoderm
How does a muscle
fiber develop?
A
A : Mesodermal
cell
How do muscle
fibers develop?
A
A : Mesodermal
cell
- undifferentiated
-“presumptive
myoblast” or
“muscle precursor
cell”
-proliferative
(mitotic)
How do muscle
fibers develop?
A
B : Myoblast
B
- spindle-shaped
- microtubules and
filaments
-  ribosomes
http://www.fbs.leeds.ac.uk
/research/confocal/assets/
ephb2.tif.jpg
http://www.fbs.leeds.
ac.uk/research/conf
ocal/Gallery.htm
How do muscle
fibers develop?
C : Myotube
- myoblasts fuse A
- multinucleated
B
- myofibrils
- immature fiber
- non-mitotic
C
How do muscle
fibers develop?
A
B
A : Mesodermal
cell
C
B : myoblast
C : myotube
D : fiber
-sarcomeres
-peripheral nuclei
D
What controls
these steps?
Myogenic differentiation pathway involves:
(1) Determination of mesodermal precursors as myoblasts,
(2) Differentiation of myoblasts into multinucleated myotubes, and
(3) Maturation of myotubes into fibers is completed by innervation
Muscle Determination and Differentiation:
Myogenic Regulatory Factors (MRFs)
• MRFs
– Transcription factors : nuclear proteins that bind DNA
– All contain a basic helix-loop-helix (sequence homology)
– Bind to E-box sequence at 5’ end of many muscle genes
(desmin, myosin light chain, troponin, creatine kinase, etc)
•
•
•
•
MyoD Family
E-proteins
Myocyte enhancer factors (MEFs)
Inhibition of differentiation (Id) factors
Muscle Determination and Differentiation:
Myogenic Regulatory Factors (MRFs)
• MyoD Family
–
–
–
–
Myf5 expressed in early somites
Myogenin expressed in myoblasts
MyoD expressed in myoblasts later than myogenin
MRF4 expressed throughout fetal and adult life
• 2 approaches for studying MRFs
1. Transgenic mice
2. Cell culture
1. Null (knock out) mutants for MRFs
• Myf5 null
– Mice survive until birth then die due to abnormal rib
development
– MyoD appears to substitute for Myf5 in early myoblast
development
– MyoD or myogenin can not substitute for myf5 in rib
development
1. Null (knock out) mutants for MRFs
Biochem Cell Biol 73: 723, 1995
Muscle Determination and Differentiation:
Myogenic Regulatory Factors (MRFs)
• MyoD Family
–
–
–
–
Myf5 expressed in early somites
Myogenin expressed in myoblasts
MyoD expressed in myoblasts later than myogenin
MRF4 expressed throughout fetal and adult life
• 2 approaches for studying MRFs
1. Transgenic mice
2. Cell culture
2.
MRF proteins exhibit the ability of converting nonmuscle cells
into skeletal muscle cells in culture, but only under reduced
serum conditions
From website of Dr. Steven Konieczkny, Purdue Univ.
MyoD family MRF Summary
1) Not all MRFs are required for normal muscle formation
2) Individual MRFs can not completely substitute for each other
3) Each MRF appears to have some unique function
4) Most all combination nulls die at birth
5) MyoD and myf5 have roles in early myoblast determination while
myogenin and MRF4 are required for differentiation
6) MRFs can convert nonmuscle cells in muscle cells given the right
conditions
Muscle Determination and Differentiation:
Myogenic Regulatory Factors (MRFs)
• MyoD Family
• E-proteins
– Ubiquitous transcription factors found in most cells (i.e., not
just muscle cells)
– For MRFs to bind E-boxes they must first dimerize; often
MRFs form a heterodimer with an E protein
Muscle Determination and Differentiation:
Myogenic Regulatory Factors (MRFs)
• MyoD Family
• E-proteins
• Myocyte enhancer factors (MEFs)
– MEF 2A, B, C, D
– Transcription factors expressed in many cells
– MEF2C is one of the earliest markers of the cardiocytes
(MEF2C nulls do not form normal hearts)
– MyoD MRFs have MEF2 binding sites; feedback loops and
cooperative interactions between many MRF proteins
Muscle Determination and Differentiation:
Myogenic Regulatory Factors (MRFs)
• MyoD Family
• E-proteins
• Myocyte enhancer factors (MEFs)
• Inhibition of differentiation (Id) factors
– HLH proteins but lack the basic region and therefore cannot
bind to DNA
– Heterodimerize with E proteins inhibiting them from binding
to MRFs
– Negative regulators of MRFs: keeps some mesodermal
cells from differentiating into myoblasts
Muscle Determination and Differentiation:
Control of muscle genes by MRFs
Hormones
Cytokines
Growth factors
Transcription
factors (+ and -)
Dev Biol 154: 261, 1992
Myogenic differentiation pathway involves:
(1) Determination of mesodermal precursors as myoblasts,
(2) Differentiation of myoblasts into multinucleated myotubes, and
(3) Maturation of myotubes into fibers is completed by innervation
Maturation of fibers involves
gene expression and regulation
• Protein synthesis
– Cytoskeletal proteins expressed first, “framework”
– Soon after, myofibrillar proteins are expressed and
organized, striated appearance
• Different isoforms of many muscle proteins arise via
gene duplication and alternative splicing
e.g., Myosin heavy chain
• Embryonic
• Neonatal
• Type 1
• Type 2A, 2X, 2B (more in Fiber Types lecture)
Muscle Development and Growth
Questions to be addressed:
• How do undifferentiated cells become
muscle fibers? I.e., How do muscles and
muscle fibers develop?
• How does muscle growth occur and how
is it regulated?
• How is injured muscle repaired?
How do skeletal muscles grow?
• Hypertrophy
– Increase in fiber diameter (and/or fiber length)
– Primary method for muscles to increase in size
• Hyperplasia
– Increase in number of fibers
– Occurs to a very limited extent
– Basically, the number of muscle fibers at birth
does not increase
Regulation of muscle growth
• Hormones
–
–
–
–
Growth Hormone (Somatotropin)
Testosterone
Insulin
Estrogens
“Anabolic steroids” work primarily by increasing
protein accretion
Regulation of muscle growth
• Hormones
• Growth Factors
– Includes cytokines and hormones
– Produced locally in differentiated muscle (influenced by
hormones) and external to muscle
– Regulate proliferation, differentiation, and growth (+, -)
in muscle
• IGF
• FGF
• HGF
• TGF-ß (myostatin aka GDF-8)
IGF is a positive regulator of muscle growth
“Growth” is partnered with the regulation of atrophy
MuRF1 and Atrogin-1 are “atrogens”
(atrophy-related genes)
J Appl Physiol 84: 1716-1722, 1998
Am J Physiol Endocrinol Metab 287:
E591-E601, 2004
TGF-ß is a negative regulator of muscle growth
Double-muscled cattle:
mutation in myostatin gene
Whippets: one copy of mutated
myostatin makes dog fast but two bad
copies makes an over-muscled “Bully”
Myostatin knock-out mice
Increased number and size of muscle fibers
Myostatin mutation associated with gross
muscle hypertrophy in a child
patient
6 days old
NEJM 350;2682, 2004
control
7 months old
patient
Regulation of muscle growth
• Hormones
• Growth Factors
• Passive Stretch*
• Work (Exercise)*
• Nutrition
Main events leading to muscle growth
Muscle Development and Growth
Questions to be addressed:
• How do undifferentiated cells become
muscle fibers? I.e., How do muscles and
muscle fibers develop?
• How does muscle growth occur and how is it
regulated?
• How is injured muscle repaired?
How is injured muscle repaired?
Regeneration
• Inflammatory response triggered
– Intrinsic cytokines are activated (IL-6, TNF, etc)
– Infiltration of phagocytic cells
How is injured muscle repaired?
Regeneration - Infiltration of phagocytic cells
How is injured muscle repaired?
Regeneration
• Inflammatory response triggered
–
–
–
–
–
Intrinsic cytokines are activated (IL-6, TNF)
Infiltration of phagocytic cells
Macrophage secrete growth factors
MRFs are increased and upregulate muscle genes
Protein synthesis stimulated
How is injured muscle repaired?
Regeneration
• Inflammatory response triggered
–
–
–
–
–
Intrinsic cytokines are activated (IL-6, TNF)
Infiltration of phagocytic cells
Macrophage secrete growth factors
MRFs are increased and upregulate muscle genes
Protein synthesis stimulated
• Satellite cells activated
Satellite Cells
• Remnant myoblasts (~ muscle stem cells)
• Located beneath the basal lamina of myofibers
• About 2% of myofiber nuclei are satellite cells
Satellite Cells
• Remnant myoblasts
• Located beneath the basal lamina of myofibers
• Dormant until needed for growth or repair of myofiber
Quiescent satellite cell
Active satellite cell
Satellite cell
Myf5, myogenin detected
Myogenin, MyoD detected
MyoD, MRF4
detected
Satellite cells are restored
How is injured muscle repaired?
Regeneration
• Inflammatory response triggered
• Satellite cells are activated and recapitulate
embryogenesis of muscle fibers
How is injured muscle repaired?
Regeneration
• Inflammatory response triggered
• Satellite cells are activated and recapitulate
embryogenesis of muscle fibers
• Are satellite cells the only muscle precursor
cells that contribute to repair and lead to
myogenesis in mature skeletal muscle?
Possible cells contributing to muscle
regeneration
*
*
J Histochem Cytochem 50: 589, 2002
fibroblasts
adipocytes
chondroblasts
osteoblasts
Regeneration and growth (hypertrophy)
are not exactly the same
Major events in muscle regeneration
How is injured muscle repaired?
Regeneration
• Inflammatory response triggered; “clean-up” and stimulate
protein synthesis
• Satellite cells activate and proliferate; protein synthesis
• Non-myogenic sources of myoblasts are minimal in vivo
– Agents to induce non-myogenic cells to become muscle is
promising
– Agents to increase the # of non-myogenic cells to become muscle
possible
– Done in non-mammals (e.g., newt can grow new limbs)
Muscle Development and Growth
Questions answered:
• How do undifferentiated cells become muscle fibers? I.e.,
How do muscles and muscle fibers develop?
• How does muscle growth occur and how is it regulated?
• How is injured muscle repaired?
Muscle cells are very unique and complex
References (review articles):
Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells.
Tedesco et al. The Journal of Clinical Investigation 120: 11-19, 2010.
Regeneration of mammalian skeletal muscle: Basic mechanisms and clinical
implications. Ciciliot and Schiaffino. Current Pharmaceutical Design 16: 906-914,
2010.
Molecular signatures define myogenic stem cell populations. Martin, Russell,
Ferdous and Garry. Stem Cell Reviews 2: March 2006
The Skeletal Muscle Satellite Cell: The Stem Cell That Came in From the Cold.
Zammit, Partridge and Yablonka-Reuveni. Journal of Histochemistry and
Cytochemistry 54 (11): 1177-1191, 2006.
Control of the size of the human muscle mass. Rennie et al. Annual Review of
Physiology 66: 799-828, 2004.