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NEBULIN’S EFFECT ON THIN
FILAMENT LENGTH
Department of Cell and Molecular Medicine, Tucson, Arizona
Yaeren Hernandez, Ambjorn Brynnel, Frank Li,
Hendrikus Granzier
BACKGROUND
A sarcomere is the basic contractile unit of a muscle
What is a sarcomere?
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
NEBULIN
What is it? Where is it?
• Thin Filament Protein
•154 Actin-binding modules
•Wrapped around actin
•22 highly homologous groups
(Super-Repeats)
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.
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
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.
MATERIALS AND METHODS
Stationary Hook
Metal electrodes
conduct
electricity,
stimulate
contraction
Extensor Digitorum Muscle
(EDL) is dissected
EDL is mounted.1. 2.
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.
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
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 )
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 )
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.
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.
WHAT I HAVE LEARNED
• Practice / Training
• Time
• Collaboration
• Persistence
• Rewarding
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:
QUESTIONS

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v2. Nebulin PP

  • 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:

Editor's Notes

  1. Sarcomeres are only found in skeletal muscle, not smooth Primary based of actin and myosing proteins Works at a micrometer scale.
  2. Nebulin has many functions,
  3. A specialized technique I needed to measure thin filament length, and 10 weeks is not enough time to train and perform this technique, therefore sarcomere length was measured which should reflect thing-filament length.