TITIN, A GIANT ELASTIC PROTEIN IN MUSCLE: ROLE IN ELASTIC RECOIL IN TENDON CELLS?
+ Banes Albert J1,2,3,4, Jie Qi4, Henk Granzier5, Seigfried LaBeit6, Carol Otey7, Xi Yang1, Donald Bynum1, Michael Chua8, Leo Alexopoulos9, Greg Williams9,
and Farshid Guilak9.
+Orthopaedics1, Biomedical Engineering2, Applied and Materials Sciences3, University of North Carolina, Chapel Hill, NC, Flexcell International Corp4.; Dept
of Veterinary and Comparative Anatomy5, Washington State University, Pullman, WA; Institute for Anesthesiology and Operative Medicine6, University of
Mannheim, AEG; Department of Cell and Molecular Physiology7, UNC, Chapel Hill, NC; Michael Hooker Confocal Facility8, UNC, Chapel Hill, NC;
Department of Orthopaedics9, Duke University, Durham, NC.
Introduction: Striated skeletal muscle cells use the 3.2 MDa elastic
protein titin to passively return energy from the contracted state to the
steady state distance in the inter-Z band length of a sarcomere (1). Titin
can absorb and return energy through its multiple spring elements and
help the cell keep pace with the mechanical requirements of the tissue
during cyclic loading. We hypothesize that some connective tissue cells,
such as tendon cells, use titin to return their shape to a steady state to
coordinate extension and relaxation phases of the resident tissue. If cells
lack an organized recoil response and lose synchrony with the elastic
return of the tissue, they will be inordinately deformed on the return
phase and sustain damage. We further hypothesize that connective tissue
cells that are more stress shielded by their surrounding matrix, such as
chondrocytes in a pericellular matrix, may express or organize less titin
and have reduced stiffness. One prediction is that the more titin and
elastic recoil within a cell, the less mechanosensitive it may be because
the elastic element can stress-shield the cell. Conversely, cells with less
titin and less elastic recoil, will be more mechanosensitive because of
reduced internal stress shielding.
Methods: Titin protein was visualized in frozen sections of flexor
digitorum profundus (FDP), Achilles tendon (AT) or tibial, condylar
articular cartilage after reaction with primary anti-titin antibodies
recognizing the I/A junction region, amino or carboxy ends or the PEVK
spring region. Secondary antibodies to titin were Alexa 488 labeled.
Sections were also stained with antibodies to pan myosin, -actinin or
rhodamine phalloidin for F-actin. Nuclei were stained with DAPI.
Cultured cells from avian or discarded, human surgical specimens were
also stained as above. A Zeiss confocal microscope with an oil
immersion 63X objective was used to obtain differential interference
contrast as well as fluorescence pictures of tissue and cells. Lasers were
tuned to each Alexa dye (488nM for titin or 568nm for pan myosin, actinin) and wavelength channels and checked for lack of crossover.
mRNA expression patterns of titin in avian and human tissues were
measured by quantitative RT-PCR with an internal rRNA target. Cell
stiffness was measured by aspirating a cell into the bore of a calibrated
micropipette with a calibrated vacuum source (2) The cell-aspiration
process was videotaped for subsequent data analysis to calculate the pipet
bore size, the steady state pressure required to aspirate a segment of a cell
into the pipet bore and the time constant for aspiration.
Results: Data in Figure 1 show the presence of titin antigen stained with
anti-I/A
junction
antibody in avian
flexor tendon tissue
fibroblasts (panel a)
as well as isolated
cells (panel b).
Titin was detected
throughout the cell
from the cell body
into the extensive
pseudopods amidst
collagen
fibrils.
Titin was readily
discernible as a
diffuse
network
throughout the cell
in isolated human
cultured
internal
Figure 1. Immunostaining of tendon (a-c) or
tendon fibroblasts
cartilage (d) titin with an anti-I/A junction
(panels c). The anti
monoclonal antibody
I/A
junction
antibody
reacted
with the mid-portion of titin and stained a continuous network from
pseudopods at one cellular pole, over the nucleus to the other cell end.
Titin co-localized with myosin, actin (even cytoplasmic distribution and
-actinin, peripheral and cytoplasmic) though in slightly different
patterns. Cartilage tissue cells gave a weak, diffuse, structureless stain for
titin (panel d) that required a 3 fold greater gain setting than tenocytes,
whereas -actinin and myosin were present in chondrocytes (not shown).
Titin mRNA expression was robust in control muscle tissues as well as in
fibrous connective tissues such as flexor tendon, ligament and meniscus ,
but less so in
cartilage (Figure
2a). RNA from
tendons
of
osteoarthritic (OA)
or Charcot-Marie
Tooth
patients
expressed
an
aberrant form of
titin that contained
Figure 2. Expression of titin in avian tissues and
human tendon tissue.
a splice variant
with a 102 bp
intron
of
an
important N2A
spring
element
domain
(N2A
402bp) that binds
accessory
proteins (Figure
2b, 3). The elastic
modulus
for
tenocytes
from
fresh,
whole
avian tendon was
2-4 times greater
than that for
chondrocytes
Figure 3. Measurement of cell elasticity.
from
tibial
cartilage (Figure
3, aspiration of a tenocyte into a micropipet; Young’s Modulus (Pa):
tendon tissue cells 1630, cartilage tissue cells 330). Cultured cells were
less stiff than freshly isolated cells from either tissue.
Discussion: Titin was expressed in each connective tissue examined and
was verified by message as well as protein. Cartilage cells expressed less
titin; anti-titin antibody detection was less robust, indicating that the
protein was not highly expressed in chondrocytes. Titin in tenocytes, colocalized with actin, -actinin and myosin as it does in skeletal and
cardiac muscle, but in a less organized pattern than in a muscle
sarcomere. Preliminary results indicated that the elastic modulus of
tenocytes was greater than that of cartilage cells, supporting the thought
that externally stress shielded cells may require less internal stress
shielding. Human mutations in the N2A spring element region may result
in a phenotype that results in an altered ability to stress shield tenocytes
from load and result in pathology.
References:
1. Bang M. et al., The complete gene sequence of titin, expression of an
unusual 700 kDa titin isoform and its interaction with obscuring identify a
novel Z-line to I-band linking system. Circ, Res. 89:1065-1072, 2001. 2.
Jones W. et al., Alterations in the Young’s modulus and volumetric
properties of chondrocytes isolated from normal and osteoarthritic human
cartilage. J Biomech. 32: 119-127, 1999.
3. Clark K. et al., Striated muscle architecture: An intricate web of form
and function. Ann Rev Cell Dev Biol. 18: 637-706, 2002.
50th Annual Meeting of the Orthopaedic Research Society
Paper No: 0050