1. NEBULIN’S EFFECT ON THIN
FILAMENT LENGTH
Department of Cell and Molecular Medicine, Tucson, Arizona
Yaeren Hernandez, Ambjorn Brynnel, Frank Li,
Hendrikus Granzier

2. BACKGROUND
A sarcomere is the basic contractile unit of a muscle
What is a sarcomere?

3. The force a muscle produces is proportional to the amount of
overlap there is between thin and thick filaments in the sarcomere.
Length – Tension Relationship

4. NEBULIN
What is it? Where is it?
• Thin Filament Protein
•154 Actin-binding modules
•Wrapped around actin
•22 highly homologous groups
(Super-Repeats)

5. Why is it important?
Nebulin mutations is the most common cause of nemaline
myopathy (NM).
NM hereditary neuromuscular disorders characterized by
severe muscle weakness.
Ottenheijm et al. and Granzier, Hum Mol Genet. 2009;18:2359, Ottenheijm et al. and Granzier, Brain. 2013;136:1718.

6. Function of nebulin?
Molecular ruler that regulated thin filament length.
In Theory:
• Nebulin changes
• Thin filament length changes
• Descending limb of the Force
Sarcomere Length relation
changes

7. HYPOTHESIS
Through genetic engineering nebulin was shortened
(by deleting Super Repeats 9 through 11), will
shorten the thin filament and this causes a shift in
the descending limb muscle of the force-sarcomere
length relationship to the left.

8. MATERIALS AND METHODS
Stationary Hook
Metal electrodes
conduct
electricity,
stimulate
contraction
Extensor Digitorum Muscle
(EDL) is dissected
EDL is mounted.1. 2.

9. 3. 4.Muscle is fixed at L0
Sarcomere length is
measured.
A Brief Introduction to Muscle. Digital image.Http://www1.imperial.ac.uk/resources/57B0BE7A-6851-4E1C-
9388-38D229D022EA/he1_081.jpg. Imperial College of London, 2013. Web. 7 Aug. 2014.

10. 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
0
2 0 0
4 0 0
6 0 0
8 0 0
F o rc e F re q u e n c y R e la tio n s h ip
F re q u e n c y (H z)
Stress(mN/mm
2
)
W T (n = 9 )
H e t (n = 8 )
H o m (n = 5 )
Force Frequency Relationship for extensor EDL. No statistical difference in
active stress at all frequencies.
RESULTS

11. Length-Tension Relationship : Homozygous mice display a shift to the left
1 .5 2 .0 2 .5 3 .0 3 .5 4 .0
0 .0
0 .5
1 .0
L e n g th -T e n s io n R e la tio n s h ip
S a rc o m e re L e n g th ( m )
FractionofL0TetanicForce
W T A c tiv e (n = 4 )
H e t A ctive (n = 6 )
H o m A ctive (n = 5 )

12. Optimal length of Sarcomeres.
W
T
H
e
t
H
o
m
2 .0
2 .5
3 .0
3 .5
S a rc o m e re L e n g th a t L 0
SarcomereLength(m)
p = 0 .2 9
p = 0 .5 0
* (p = 0 .0 2 )

13. CONCLUSION
• All mice appear to be healthy and exhibit no distinct weakness.
• There was a 0.2 micrometer shift to the left in the length-tension
relationship in the homozygous mice. Also a right shift in
heterozygous mice.
• Future : There needs to be an increase in sample number.
• Specific analysis of the thin-filament lengths through electron
microscopy or immunofluorescence.

14. In summary:
We tested the role of nebulin in thin filament length
regulation by shortening nebulin and measuring how
this affects the force-sarcomere length relation of
muscle. The results support that nebulin plays an
important role in determining the thin filament length.

15. WHAT I HAVE LEARNED
• Practice / Training
• Time
• Collaboration
• Persistence
• Rewarding

16. THANK YOU
•The University of Arizona
• BIO5 Institute
• Hendrikus Granzier Laboratory
• Border Latino & American
Indian Summer Exposure to
Research program
Special thanks to program coordinators, Lori Vargo, Maria
Teresa Velez, PhD student Frank Li, and Ambjorn Brynnell,
and Hendrikus Granzier for their time, motivation, hard work
and patience.
This research is supported by:

17. QUESTIONS