Stretch activation of GTP-binding proteins in C2C12 myoblasts
Craig B. Clark,
a
Nathan L. McKnight,
a,b
and John A. Frangos
a,b,
*
a
Department of Bioengineering, University of California San Diego, La Jolla, CA 92093-0142, USA
b
La Jolla Bioengineering Institute, La Jolla, CA 92037, USA
Received 31 December 2002, revised version received 24 September 2003
Abstract
Mechanical stimulation has been proposed as a fundamental determinant of muscle physiology. The mechanotransduction of strain and
strain rate in C2C12 myoblasts were investigated utilizing a radiolabeled GTP analogue to detect stretch-induced GTP-binding protein
activation. Cyclic uniaxial strains of 10% and 20% at a strain rate of 20% s
À1
rapidly (within 1 min) activated a 25-kDa GTPase (183 F 17%
and 186 F 19%, respectively), while 2% strain failed to elicit a response (109 F 11%) relative to controls. One, five, and sixty cycles of 10%
strain elicited 187 F 20%, 183 F 17%, and 276 F 38% increases in activation. A single 10% stretch at 20% s
À1
, but not 0.3% s
À1
, resulted
in activation. Insulin activated the same 25-kDa band in a dose-dependent manner. Western blot analysis revealed a panel of GTP-binding
proteins in C2C12 myoblasts, and tentatively identified the 25-kDa GTPase as rab5. In separate experiments, a 40-kDa protein tentatively
identified as Ga
i
was activated (240 F 16%) by 10% strain at 1 Hz for 15 min. These results demonstrate the rapid activation of GTP-binding
proteins by mechanical strain in myoblasts in both a strain magnitude- and strain rate-dependent manner.
D 2003 Elsevier Inc. All rights reserved.
Keywords: C2C12; Myoblast; Uniaxial strain; Strain rate; Mechanotransduction; Photoaffinity
Introduction
Mechanical loading and deformation play an important
role in the physiology of a variety of tissues, with extensive
research in both animal and cell culture models identifying a
rapidly expanding list of cellular responses. Mechanotrans-
duction—translation of a mechanical stimulus into an intra-
cellular biochemical processes—has been the subject of
intensive study, yet many questions remain as to the
underlying cellular mechanisms and the physical stimuli
which drive them. Muscle provides an ideal tissue for the
study of mechanotransduction, as it experiences a wide
range of strains during normal use. In vivo, muscle is
affected by a variety of stimuli including neural interactions,
membrane depolarization, intracellular calcium flux, metab-
olite transport or depletion, hormonal influences, as well as
the stresses and mechanical strains due to both external
loading and myosin-generated tension. While each of these
stimuli influences muscle performance and adaptation, me-
chanical strain has been proposed as a fundamental deter-
minant of muscle physiology [16].
The strains under loading (strain being a normalized
measure of stretch; where Strain = Deformation / Initial
Length) and the cyclic strains during locomotion impose a
spectrum of mechanical stimuli to direct the muscular phe-
notype, with strains during normal locomotory movements
typically F10% of resting length, at strain rates of 0–700%
[17,18,23,47]. Strain rate (the rate at which the deformation is
applied; where Strain Rate = Strain / Duration) varies by
orders of magnitude and may therefore be an important
parameter in modulating the mechanoresponse of cells and
tissues. A host of candidates have been proposed for the
mechanotransduction of physical forces including adhesion
molecules, kinases, cytoskeletal elements, ion channels, and
guanine nucleotide-binding regulatory proteins (GTP-bind-
ing proteins) [3,11,22]. The diverse family of GTP-binding
proteins play a central role in signal transduction [27,36], and
are also sensitive to physical forces, being activated by both
fluid shear and mechanical strain [3,19,20]. GTP-binding
proteins therefore provide an ideal candidate for the study of
the mechanical stimulation of skeletal muscle.
In the present study, the mechanotransduction of strain
and the sensitivity to strain rate were investigated using
0014-4827/$ - see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2003.09.017
* Corresponding author. La Jolla Bioengineering Institute, 505 Coast
Boulevard South, La Jolla, CA 92037-4616. Fax: +1-858-456-7540.
E-mail address: frangos@ljbi.org (J.A. Frangos).
www.elsevier.com/locate/yexcr
Experimental Cell Research 292 (2004) 265–273