1. Effect of Erk1/2 on Muscle Fiber Morphology and Differentiation
Gabrielle Henslee, Bonnie Seaberg, Ximena Paez, Mendell Rimer
Department of Neuroscience & Experimental Therapeutics, Texas A&M Health Science Center College of Medicine, Bryan, TX, USA
Materials and Methods
Fiber-typing: Sternomastoid (STN) and tibialis anterior (TA) muscles from 14-week-old mice (three animals from each of the
four above-mentioned genotypes) were dissected and flash frozen with OCT medium (tissue tek) in an isopentane/liquid N2
bath. 14μm-thick cross-sections were cut in a cryostat and mounted on a slide. Sections were not fixed at any time. Sections
were washed in PBS for 5min, then washed three times in PBS-T for 5min each. The cross sections were blocked in PBS-T +
10% normal goat serum for 20min at room temperature and then incubated overnight in a myosin 2B antibody (undiluted,
DSHB 10F5) or a myosin slow antibody (undiluted, DSHB A4.840) plus dystrophin (1:300, AbCam 15277). After three 15min
washes in PBS-T the following morning, the muscle sections were incubated at room temperature for 2h with a fluorescein-
conjugated goat anti-mouse (1:128) and rhodamine-conjugated goat anti-rabbit (1:400) secondary antibody. After washes in
PBS-T and PBS, slides were mounted using VectaShield. A 10X, 0.3 NA objective (Nikon Eclipse 1000) was used to acquire
widefield images of myofibers with MetaMorph Software, taking as many images needed to encompass the entire area of a
single whole muscle section with overlapping myofibers. The images were then compiled into a single picture. Total fibers were
counted on the dystrophin channel, and unstained fibers were counted on the color merge. The number and percentage of
stained fibers were calculated from these values.
Fiber Area: The dystrophin-stained images for control and Cre+Erk1-/-Erk2f/f from the above-described compilations were
analyzed for myofiber area using the integrated morphometric analysis tool in MetaMorph. Calibrated images were thresholded
for dark objects and manually adjusted to fill the entire interior core surrounded by dystrophin staining for as many myofibers
as possible per image. The entire section was analyzed and myofibers overlapped by multiple images were only analyzed a
single time.
Introduction
Fiber Types: Muscle fibers can be classified into two distinct types. Type 1 fibers, also called slow fibers, are characterized by
small size and great resistance to fatigue due to rich myoglobin content and mitochondria. Type 1 fibers contract slowly and do
not generate much force. Type 2 fibers can be subdivided into three separate categories. Type 2B fibers are generally the
largest in size and can generate a great amount of force, but due to low mitochondrial count, they fatigue easily, and thus are
also known as fast fatigable units. Type 2A units are an intermediary between slow and 2B. They are not as fast as 2B,
generate more force than slow, and are moderately sized. While they fatigue faster than type 1, they have more endurance
than 2B. These characteristics have led to them being known as fast fatigue-resistant units. The third type is designated 2X,
and its characteristics fall midway between 2A and 2B.
Extracellular-signal Regulated Kinase: Extracellular-signal regulated kinase 1 and 2 (Erk1/2) are part of a signaling
cascade involved in many different cellular processes. Erk1/2 can act as an inhibitor or an activator during skeletal muscle
differentiation. It has also been implicated as playing a role in the regulation of fiber type differentiation. However, literature
reports conflicting results regarding the need of Erk1/2 signaling for slow or fast fiber differentiation. Currently no one has fully
investigated the effect of Erk1/2 on fiber type distribution in vivo.
Hypothesis: Elimination of Erk1/2 (separately and concurrently) will affect muscle fiber morphology and development.
Animal Model: The mice used in this study are C57BL/6 background. Erk1 knockouts were generated by inserting a Neo
cassette, resulting in germline Erk1 elimination and are noted as Erk1-/-. Erk2 knockouts were generated conditionally making
use of the Cre/loxP system. Cre excises sequences flanked by loxP sites; in this way, a gene’s elimination can be controlled by
either pharmacologically or constitutively supplying Cre. In this experiment, Cre expression was limited to skeletal muscle
fibers by using a human alpha-skeletal actin (HSA) promoter to drive Cre expression. Erk2 conditional knockouts are noted as
Cre+Erk2f/f. Control animals were Cre- siblings, as Erk2 expression is unchanged in the absence of Cre. Double knockouts
were generated by crossing Erk1-/- and Cre+Erk2f/f animals and backcrossing to reestablish homozygocity. These animals are
noted as Cre+Erk1-/-Erk2f/f.
Conclusions and Future Studies
Area: There is an observed shift in distribution in myofiber area between the control and Cre+Erk1-/-Erk2f/f that, together with
total fiber count, likely contributes to both the smaller muscle size and lower observed weights.
Fast 2B Fibers: The 2B fiber type comprises 50% or more of the total fibers in both STN and TA for all genotypes and are
visually observed to be larger than unstained (non-2B) fibers. No statistical significance was found between the controls and
various mutants.
Slow Fibers: Neither STN nor TA has many slow fibers. In STN there is a slight reduction in slow fibers in all mutant
genotypes as compared to control with significance observed for Erk1-/- only. On the other hand, there is an increase in slow
fibers in all mutant genotypes with significance in Cre+Erk1-/-Erk2f/f as compared to control in the TA.
Future Directions: Future work will include continuing fiber type analysis on 2A and 2X in STN and TA of the four genotypes
and looking for correlation to observed neuromuscular junction phenotypes (data not shown). The necessity of Erk1/2 signaling
for slow fiber differentiation will be studied in the soleus muscle.
Figure 1: Comparison of fiber area and count between control and Cre+Erk1-/-Erk2f/f in STN.
(A) Area distribution of control and Cre+Erk1-/-Erk2f/f. Measured area from MetaMorph thresholding was grouped into “bins” of 250μm2. Values were normalized by using percentages of the total number of fibers. Both the
control and the Cre+Erk1-/-Erk2f/f have the majority of their fibers fall in the 500-2250μm2 range. However, the control has a second smaller grouping of large fibers in the 2500-3500μm2 range that Cre+Erk1-/-Erk2f/f lacks. In
addition, Cre+Erk1-/-Erk2f/f has more <500μm2 fibers than the control. (B) Average fiber count (±SEM) for controls and Cre+Erk1-/-Erk2f/f. Total fiber counts were obtained from control and Cre+Erk1-/-Erk2f/f animals
(n=3/genotype) during the course of the fiber type analysis. A reduction in total number of fibers can be observed for Cre+Erk1-/-Erk2f/f. The results from the myofiber area distribution, together with the fiber count, account for
the observation that Cre+Erk1-/-Erk2f/f has smaller muscle sections than the controls (Fig 3).
Results, Continued
Results
Figure 3: Myosin and dystrophin fluorescent images.
TA sections for control (A), Erk1-/- (B), Cre+Erk2f/f (C), Cre+Erk1-/-Erk2f/f (D) and
STN sections for control (E), Erk1-/- (F), Cre+Erk2f/f (G), Cre+Erk1-/-Erk2f/f (H)
stained with myosin 2B (green) and dystrophin (red) antibodies, imaged at 10X and
compiled into single images. 2B fibers in both muscles are observed to be larger
than the unstained non-2B fibers, which is consistent with referenced literature
descriptions of fiber types. Scale bar: TA=300μm; STN=200μm. TA sections for
control (I), Erk1-/- (J), Cre+Erk2f/f (K), Cre+Erk1-/-Erk2f/f (L) and STN sections for
control (M), Erk1-/- (N), Cre+Erk2f/f (O), Cre+Erk1-/-Erk2f/f (P) stained with myosin
slow (green) and dystrophin (red) antibodies, imaged at 20X. Slow fibers were
spread throughout the muscle and thus could not be visualized in a single image.
The low number of slow fibers is consistent with fiber composition for these
muscles. Soleus muscle (Q) was used as a staining control, as this muscle has a
large number of slow fibers. Scale bar=100μm.
Figure 4: Quantification of fiber type expression.
Average percentage of fast 2B fiber expression (A) and slow fiber expression (B) in TA and STN was obtained for each of the three animals for each genotype and averaged. 2B comprises roughly half of the total fibers in
both muscles, and no statistical difference was observed within each muscle type. Statistical significance was observed in some of the slow fiber samples. In TA, there is a significant increase in Cre+Erk1-/-Erk2f/f compared to
the control (p=0.007). In the STN, there is a significant decrease in slow fibers in Erk1-/- (p=0.03). Loss of Erk1/2 does not seem to have a major affect on 2B expression, and while there is some impact on slow fibers, these
are such a small portion of the total that it likely does not account for any of the observed phenotypic changes in these animals (data not shown).
0%
2%
4%
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16%
PercentFibers
Fiber Area (μm2)
Fiber Area Distribution
Control
Double KO
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1,400
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TotalNumberofFibers
Genotype
STN Fiber Count
Control
Double KO
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10%
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TA STN
Percent2BFibers
Muscle Type
Fast 2B Fiber Expression
Control
Erk1
Erk2
Double KO
0.00%
0.05%
0.10%
0.15%
0.20%
0.25%
TA STN
PercentSlowFibers
Genotype
Slow Fiber Expression
Control
Erk1
Erk2
Erk Double KO
Figure 2: Animal weights corresponding to age.
Male (A) and female (B) weights for all four
genotypes were taken every week from 4wks through
16wks of age, then taken once every two weeks until
40wks. Little difference is observed between the
controls and Erk1-/-. Cre+Erk2f/f animals were slightly
heavier than the controls, but significance was only
observed in the females at 13, 15, 20, 24, and 28wks
(p=0.004). Cre+Erk1-/-Erk2f/f demonstrates significant
weight difference starting at 6wks for males
(p=0.002) and 10wks for females (p=0.3 at 10wks,
p=0.003 at 11-24wks). Considering the evidence
provided by the area distribution and fiber count,
some of the weight difference observed in Cre+Erk1-/-
Erk2f/f may be attributed to these factors.
10
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4 5 6 7 8 9 10 11 12 13 14 15 16 18 20 22 24 26 28 30 32 34 36 38 40
Weight(g)
Age (weeks)
Males
Controls
ERK1 Mutants
ERK2 Mutants
ERK1/2 Mutants
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4 5 6 7 8 9 10 11 12 13 14 15 16 18 20 22 24 26 28 30 32 34 36 38 40
Weight(g)
Age (weeks)
Females
Controls
ERK1 Mutants
ERK2 Mutants
ERK1/2 Mutants
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C D
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G H
I J K
L M N
O P Q
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Erk2
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Erk2
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Erk2
Erk Double KO
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Erk Double KO
Control
Erk Double KO
Control Control
TA Control
STN Control STN Erk1 STN Erk2
STN Double KO
TA Double KO
Double KODouble KO
SOL Control
TA Erk1
TA Erk2
Erk2Erk2
Erk1 Erk1
Supported by NIH grant NS077177 to MR
Schiaffino S and Reggiani C (2011) Fiber types in mammalian skeletal muscles. Physiol Rev 91: 1447-1531.
Purves, Augustine, Fitzpatrick, Hall, LaMantia, and White. Neuroscience. 5th ed. 2012. Print.