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

NEBULINWhat 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 50 100 150 200 2500

200

400

600

800

Force Frequency Relationship

Frequency (Hz)

Stre

ss (m

N/m

m2 ) WT (n=9)

Het (n=8)Hom (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 4.00.0

0.5

1.0

Length-Tension Relationship

Sarcomere Length (m)

Fra

ctio

n o

f L

0 T

etan

ic F

orc

e

WT Active (n=4)

Het Active (n=6)

Hom Active (n=5)

Optimal length of Sarcomeres.

2.0

2.5

3.0

3.5

Sarcomere Length at L0Sa

rcom

ere

Leng

th (

m) p = 0.29

p = 0.50

* (p = 0.02)

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