Forbes, Jeffrey G.

Wang, Kuan

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Wednesday, March 9, 2011 589a3185-Pos Board B290
A Novel Assay of Mechano-Transduction in Single Muscle Cells
Christopher W. Ward, Benjamin L. Prosser, Maura Greiser,
Ha˚kan Westerblad, Ramzi Khairallah, W.J. Lederer.
In striated muscle the tight integration of contractility and biomechanical prop-
erties with electrical, metabolic and hormonal signaling are defined as
mechano-transduction signaling pathways. The great interest to define the mo-
lecular basis for mechano-transduction is underpinned by the growing number
of loss-of-function disorders and diseases in which altered mechano-
transduction has been identified.
Intact, single cell preparations are attractive for studying mechano-
transduction, however the mechanical loading of single cells requires techni-
cally challenging methods. We have recently developed MyoTak, a tenacious
biological adhesive, which enables us to attach single dissociated striated my-
ocytes (skeletal and cardiac) to force transducer and length controller units. The
myocytes thus prepared can be readily imaged using an inverted microscope
equipped with confocal, widefield and multiphoton fluorescence imaging sys-
tems. With this preparation we have been able to control myocyte length while
assaying passive tension and electrically evoked contractility. In recent exper-
iments we have explored the interaction of myocyte stretch and contractility
with Ca2þ signaling, mitochondrial function and ROS production. We will
present these new findings, the novel methods and additional experiments ex-
amining the role of microtubules as mechano-transducer elements that contrib-
ute to these interactions.
Supported by RC2 NR011968 to CWW, and to WJL (NHLBI P01 HL67849
and R01-HL36974; Leducq North American-European Atrial Fibrillation Re-
search Alliance; European Union Seventh Framework Program (FP7) ‘‘Identi-
fication and therapeutic targeting of common arrhythmia trigger mechanisms’’
and support from the Maryland Stem Cell Commission).
3186-Pos Board B291
Dynamic Interactions between the Myocyte and Extracellular Matrix
Promote Myocyte Differentiation and Myofibril Assembly
Maide O. Raeker, Mark W. Russell.
During development, skeletal myoblasts differentiate into myocytes and skel-
etal myotubes with mature contractile structures that are precisely oriented
with respect to surrounding cells and tissues. Establishment of this highly or-
dered structure requires reciprocal interactions between the differentiating
myocytes and the surrounding extracellular matrix to form correctly posi-
tioned and well organized connective tissue attachments from the skeletal
muscle to the bony skeleton. Using the developing zebrafish embryo as
a model, we examined the relationship between new myofibril assembly
and the organization of the membrane domains involved in cell-
extracellular matrix interactions. We determined that apical clustering of
integrins was associated with changes in cell morphology which included
myoblast elongation along the body axis. Through the analysis of zebrafish
embryos depleted of obscurin A that have impaired formation of the myoten-
dinous junctions, we determined that cell elongation and, as a result, new
myofibril formation was delayed in regions that lacked integrin clustering
and fibronectin matrix organization. In addition, it was noted that striated
myofibrils first form at the cell periphery and are associated with the early pat-
terning of the lateral sarcolemma. These specialized membrane domains will
ultimately form mature lateral connections between the myofibril and the ex-
tracellular matrix at the costameres. We have also determined that the giant
cytoskeletal and sarcomeric protein obscurin has an important role in the or-
ganization and maturation of the MTJ and in the maturation of the costamere.
As myocyte-extracellular matrix connections have critical roles in force
transduction and tension-mediated signaling defining the processes that pro-
mote and sustain these connections will have important implications for the
understanding the pathogenesis of, and developing new treatment strategies
for, a range of myopathies and muscular dystrophies.
3187-Pos Board B292
Muscle Giants Create Order from Chaos with Force
Kuan Wang, Jeffrey G. Forbes.
Titin and nebulin possess perhaps the longest span of intrinsically disordered
protein segments in the human proteome. We have implemented an integrated
platform of nanomechanics to investigate the elasticity of intrinsically disor-
dered segments and its functional manifestations. Our titin studies have indi-
cated that the titin PEVK region is an elastic spring, with charge pairing as
a new mechanism of creating diversity and modulating molecular elasticity.
Significantly, PEVK also serves a dual role as a giant scaffold for SH3-
containing signaling proteins. Many novel hybrid and overlapping motifs for
SH3 domains are embedded in the seemingly random proline-rich sequencesthroughout the titin molecule. We now propose that titin PEVK directly couples
the force sensing and response, by controlling the accessibility of SH3 receptor
proteins via the opening and closing the access to binding sites upon mechan-
ical stress, reversibly and elastically. In other words, titin’s elastic PEVK ap-
pears to act both as an analog force sensor and as a transducer that converts
the force input directly into biochemical signals of the SH3 pathways. Our neb-
ulin studies indicated that native full length nebulin and nebulin modules are
intrinsically disordered and elastic. We proposed that that nebulin is an "elastic
or adjustable ruler" that has to be stretched and lengthened properly to interact
and stabilize actin filaments. The continued presence of compressive force ex-
erted by stretched nebulin may well be a requirement for thin filament assem-
bly, integrity and maintenance. Additionally, since skeletal muscle thin
filaments is thought to be "dually-regulated" where calcium activates contrac-
tion by targeting both troponin and nebulin-bound calcium sensor proteins
(Root and Wang 1994), we envisage that nebulin tethers elastically myosin
heads to actin in the inactivated state and then releases and de-inhibits myosin
heads upon calcium activation.Excitation Contraction Coupling II
3188-Pos Board B293
Electron Microscopy of Cryo-Sectioned Skeletal Muscle by Focused ion
Beam Milling
Terence Wagenknecht, Chyong-ere Hsieh, Michael Marko,
Clara Franzini-Armstrong.
Knowledge of the detailed three-dimensional architecture of the couplon, the
fundamental functional unit of excitation-contraction coupling (ECC), is essen-
tial for understanding the mechanism of ECC in striated muscle. Transmission
electron microscopy of conventional thin-sectioned skeletal muscle resolves
some structural features of the couplon: (1) the sarcoplasmic reticulum (SR)
-associated ryanodine receptors whose cytoplasmic regions (‘‘foot’’ structures)
bridge the junctional gap between the transverse (T-) tubule, and (2) density in
the SR lumen attributable to calsequestrin. However, many structures are surely
not resolved due to resolution and specimen preparation limitations. To ad-
vance further, we propose to image the native ECC machinery by cryo-
electron microscopy and tomography of vitreously frozen thin sections of mus-
cle. Obtaining suitable vitreous sections by ultramicrotomy is difficult and
artifact-prone, and so we are investigating the application of focused ion
beam (FIB) milling to obtain cryo-sections of 200 nm or less in thickness.
The first cryo-micrographs of FIB-milled skeletal muscle from toadfish swim-
bladder (glutaraldehyde fixed) have been obtained, and they show structural
features seen previously, as well as some additional ones (e.g., mass densities
within the T-tubule lumen, densities associated with the T-tubule in the vicinity
of RyRs). The micrographs obtained from FIB-milled specimens appear com-
parable to those obtained by cryo-ultramicrotomy, but without the defects, such
as specimen compression, associated with the latter technique. Our goal is to
extract many RyR-containing sub-volumes from tomograms of cryo-
sectioned muscle, and then to average them so as to reproducibly resolve addi-
tional fine structure within the couplon.
3189-Pos Board B294
Inducible Silencing of Junctophilins in Skeletal Muscle Leads to Reversible
Remodeling of the Triad Junction Structure and Compromised Store-
Operated Calcium Entry
Jae-Kyun Ko, Kyoung-Han Choi, Xiaoli Zhao, Shinji Komazaki, Zui Pan,
Noah Weisleder, Jianjie Ma.
Junctophilins (JPs) play an essential role in muscle excitation-contraction (E-C)
coupling by contributing to the formation of junctional membrane complexes
(JMCs). However, the lethality associated with germ-line ablation of either
JP1 or JP2 prevents physiological evaluation of their function in the mainte-
nance of calcium homeostasis in adult muscle fibers. To investigate the phys-
iological role of JP genes, we developed a novel transgenic system for tissue-
specific and inducible control of gene expression in mouse models. This system
employs a Tet-response CMV promoter that controls expression of a small-
hairpin (sh) RNA against JP1 and JP2, which is non-functional until an inter-
rupting reporter gene cassette is excised by the Cre recombinase. Insertion of
the natural Dicer and Drosha-RNAse processing sites within the shRNA se-
quence allows for generation of specific siRNA probe for efficient knockdown
of JP1 and JP2. Under the tight control of doxycycline (Dox), tissue- or lineage-
specific expression of siRNA is achieved by the use of inducer mice that ex-
presss Cre in a given tissue. Transgenic mice with muscle-specific expression
of shRNA against JP showed no apparent change of JP expression before treat-
ment with Dox; inducible and reversible knockdown of JP in skeletal muscle


Muscle Giants Create Order from Chaos with Force 

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Muscle Giants Create Order from Chaos with Force

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