Department of Bioengineering, University of California, San Diego, La Jolla, California 92093
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ABSTRACT |
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Cardiac
fibroblasts are responsible for the production of the extracellular
matrix of the heart, with alterations of fibroblast function implicated
in myocardial infarction and cardiac hypertrophy. Here the role of
heterotrimeric GTP-binding proteins (G proteins) in the
mechanotransduction of strain in rat cardiac fibroblasts was
investigated. Cells in an equibiaxial stretch device were incubated
with the photoreactive GTP analog azidoanalido
[-32P]GTP (AAGTP)
and were subjected to various regimens of strain. Autoradiographic
analysis showed a 42-kDa protein labeled for cells exposed to 12 cycles
of 3% strain or 6 cycles of 6% strain over 60 s (strain rate of
1.2%/s), whereas 6 cycles of 3% strain (0.6%/s) elicited no
measurable response. To further investigate the role of strain rate, a
single 6% cycle over 10 or 60 s (1.2% and 0.2%/s, respectively) was
applied, with the more rapid cycle stimulating AAGTP binding, whereas
the lower strain rate showed no response. In cells subjected to a
single 6% cycle/10 s, immunoprecipitation identified the AAGTP-labeled
42-kDa band as the G protein subunits G
q and
G
i1. These results demonstrate
that G protein activation represents one of the early
mechanotransduction events in cardiac fibroblasts subjected to
mechanical strain, with the rate at which the strain is applied
modulating this response.
cell mechanics; mechanical stretch; mechanotransduction
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INTRODUCTION |
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CELLS IN THE HEART are subjected to a complex physical environment that is dynamic, nonuniform, and multidimensional (27). Studies have shown that changes in mechanical loading may regulate the synthesis and deposition of multiple components of the cardiac extracellular matrix (ECM), which include fibrillar collagens, fibronectin, proteoglycans, and numerous growth factors (5, 6). Because the cardiac fibroblast is the cell type primarily responsible for the production of ECM components in the heart (14), recent studies have examined the potential effects of mechanical strain on its cellular and molecular function. However, while mechanical stretch has been reported to stimulate cell proliferation (28) and collagen synthesis (9, 10) in neonatal rat cardiac fibroblasts, these studies did not provide quantitative analysis of cellular strain or elucidation of the mechanotransduction mechanisms that may be the important determinants of stretch-induced responses in these cells.
One candidate mechanism of mechanical signaling in cells involves heterotrimeric guanine nucleotide binding proteins (G proteins) (4, 12, 15, 30). In neonatal rat cardiac fibroblasts, G protein subunits have been recently shown to be localized at sites of focal adhesions (19), which are widely considered potential sites of mechanical signal transduction (8, 20, 25). In the vasculature, G protein signaling has been strongly implicated as a primary mechanism of mechanotransduction in endothelial cells subjected to fluid shear (3, 12). More recently, this laboratory has reported that fluid shear stress stimulated the rapid activation of G proteins in human endothelial cells (18). The results suggested that the potential for early activation and the distinct roles of subunits in G protein signaling may provide important mechanisms for signaling specificity in cells that are subjected to different types of mechanical forces.
In this study, we report, for the first time, the early stimulation of
G protein activation by mechanical strain within 1 min of loading in
adult rat cardiac fibroblasts. Uniform and equibiaxial strains,
verified previously by digital imaging and finite strain analysis (23),
were applied to the fibroblasts, and the results of this study
indicated that the stimulation of GTP binding at a selected strain
magnitude was dependent on the rate of applied strain. The specificity
of GTP binding was verified by immunoprecipitation, which clearly
identified Gq and
G
i1, but not
G
i3, as the G
subunits
activated rapidly by mechanical strain.
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MATERIALS AND METHODS |
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Materials. Transparent silicone
elastic membrane (0.25 mm thick, gloss finish) was purchased from
Specialty Manufacturing (Saginaw, MI), and 0.01% collagen type I
solution (Sigma Chemical, St. Louis, MO) was used for coating membranes
by airbrushing (Badger Air-brush, Franklin Park, IL). Cell culture
media and supplements were purchased from GIBCO BRL. Azidoanilido
[32P]GTP (AAGTP; sp
act 6.1 Ci/mmol) was purchased from ICN Radiochemicals. Rabbit
polyclonal antibodies to G were obtained from Calbiochem (La Jolla,
CA). ATP-free medium 199 and fetal bovine serum (FBS) were from
HyClone, and protein Sepharose CL-4B was from Schleicher & Schuell. All
other reagents were purchased from Sigma Chemical.
Cell culture. Adult rat cardiac fibroblasts were prepared following a previously described protocol (24). In brief, three to four hearts from 7- to 8-wk-old male Sprague-Dawley rats were minced and digested in an enzyme solution containing collagenase (100 Mandl units/ml) and pancreatin (0.6 mg/ml). Cell suspensions were pooled, centrifuged, and resuspended in DMEM with 10% FBS and antibiotics (penicillin, streptomycin, and fungizone; PSF). The resuspended mixture was subsequently plated onto cell culture dishes for 30 min for preferential attachment of fibroblasts. Identification and purity of the adult rat cardiac fibroblast population have been previously characterized by immunocytochemical staining (33). Cardiac fibroblasts were grown to confluence on either glass slides or the silicone elastic substrates of the equibiaxial stretch devices in DMEM/PSF with 10% FBS. Cultures on glass slides were used as agonist positive controls, whereas unstretched cultures on silicone membranes (identical to stretched cultures) served as static "sham" controls. The media were replaced with incubation medium (growth medium without FBS) at least 3 h before the treatment by growth factors or stretch.
Equibiaxial strain system and cell strain analysis. The design, use, and calibration of an equibiaxial strain system for cultured cells have been recently reported (23). Briefly, cells are cultured in a stretch device that applies planar, homogeneous, and equibiaxial strains to a transparent silicone elastic substrate. In situ visualization of fluorescent markers attached to both substrate and cells allowed for the quantification of cellular strain by digital imaging and two-dimensional finite strain analysis (17, 34). For this study, the threads of the stretch device were modified to allow for the selection of higher equibiaxial strain at lower rotation angles compared with a previously reported calibration curve (23).
Photoaffinity labeling of G proteins. Confluent monolayers of cardiac fibroblasts grown in the stretch devices or on glass slides were incubated in medium containing digitonin (20 µM) and AAGTP [10 µCi (1.64 nM)/106 cells] for 3 min at 37°C. After the appropriate stretch or agonist protocol, the cells were immediately irradiated for 1 min with ultraviolet (UV) light (254 nm) on ice. UV serves to covalently bind the radiolabeled GTP. As a detergent, digitonin may act not only to permeabilize the membrane but to activate membrane-bound G proteins. Data are normalized to identically treated sham controls, which serve to account for these effects, as well as any activation due to handling of cultures during the experiment. For agonist-stimulated positive controls, ANG II (1 nM) or bradykinin (BK; 1 µM) was applied to fibroblast cultures on glass slides before incubation with AAGTP. After a 5-min agonist stimulation, AAGTP was added for 3 min of further incubation. After UV exposure, cells were rinsed with ice-cold PBS with 4 mM dithiothreitol and were scraped from the slides or silicone membranes. Cell suspensions were dissolved in SDS-PAGE sample buffer and analyzed by SDS-PAGE autoradiography.
SDS-PAGE and autoradiography. SDS-PAGE
was performed on a discontinuous slab gel system with a 4%
acrylamide stacking and 10% acrylamide separating gel. After
electrophoresis, gels were dried and then exposed to Kodak XR-OMAT film
with an intensifying screen for 2-5 days at 80°C.
Autoradiographs were quantified using an image analyzer (Alpha
Innotech, model IS-1000).
Immunoprecipitation of labeled G
proteins. For the identification of the AAGTP-labeled G
protein subunits by immunoprecipitation, cells were lysed for 30 min in
buffer containing 50 mM Tris · HCl, pH 7.4, 100 mM
NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 0.2 mM sodium
vanadate, 10 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml
leupeptin, 2 µg/ml pepstatin A, and 2 µg/ml aprotinin on ice.
Lysates were ultracentrifuged at 100,000 g for 30 min. The clear supernatants
were incubated at 4°C for 6 h with polyclonal antibodies specific
for the following G subunits:
G
q,
G
i1, and
G
i3. Mixtures were incubated
with protein A-Sepharose CL-4B for 4-5 h at 4°C and washed
four times with NET buffer (in mM: 150 NaCl, 0.5 EDTA, 50 Tris · HCl, pH 8.0). Immunoprecipitates were
solubilized in electrophoresis sample buffer and were analyzed by
SDS-PAGE autoradiography.
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RESULTS |
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Uniform equibiaxial strain stimulates GTP binding in adult cardiac fibroblasts within 1 min. Adult rat cardiac fibroblasts were incubated with AAGTP and subjected to one of the following equibiaxial loading regimens for the duration of 1 min (Fig. 1, top): 1) 6 cycles of 6% maximum strain (1.2%/s); 2) 12 cycles of 3% maximum strain (1.2%/s); and 3) 6 cycles of 3% maximum strain (strain rate of 0.6%/s). Autoradiography showed that a 42-kDa protein was labeled by GTP in fibroblasts stretched at protocols 1 and 2, as shown in Fig. 1, bottom (lanes E and F and lane D, respectively). In the stretched cells, relative GTP binding for the 42-kDa protein increased 4.7-fold for protocol 1 and 5.5-fold for protocol 2 compared with unstretched controls, whereas no difference was detected between protocol 3 and controls as determined by densitometry. Both treatment with ANG II (2.8-fold) and BK (3.0-fold) showed similar GTP binding (lanes G and H and lane I, respectively).
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GTP binding in cardiac fibroblasts is strain rate dependent. Next, we examined the effect of strain rate for one loading cycle on the AAGTP binding in these cells. Confluent monolayers of adult cardiac fibroblasts were subjected to a single cycle of loading and unloading at 6% maximum equibiaxial strain for either 10 or 60 s total duration (1.2 and 0.2%/s, respectively, Fig. 2, top). The lower strain rate regimen allowed further confirmation of the strain rate (0.2% vs. 1.2%) rather than strain magnitude (6% for each) effect. Fibroblast cultures loaded at the higher strain rate showed significant GTP binding for a 42-kDa protein (Fig. 2, bottom, lanes A-C) compared with no detectable binding for cells loaded at the lower strain rate (lanes D and E). Monolayers on glass slides were treated with ANG II as a positive control (lanes F and G).
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Immunoprecipitation of G proteins for specific
G subunits. Immunoprecipitation was
used to identify the specific G
subunits that were rapidly activated
in cardiac fibroblasts subjected to a single cycle of 6% maximum
strain for 10 s. As shown in Fig. 3,
polyclonal antibodies specific to
G
q and
G
i1 identified AAGTP-labeled proteins in stretched cells (S), compared with no detectable binding for these subunits in unstretched controls (C). Antibodies for G
i3 did not bind to
32P-labeled G
subunits in
either unstretched or stretched cells. Antibodies for
G
i3 and
G
i1 formed immunocomplexes with
radiolabeled G proteins in cells treated with either BK or ANG II.
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DISCUSSION |
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This study shows that G proteins are rapidly activated in adult cardiac
fibroblasts subjected to uniform and planar equibiaxial strain.
Immunoprecipitation identified
Gq and
G
i1, but not
G
i3, as the heterotrimeric G
protein subunits specifically activated by mechanical strain. Moreover,
these results demonstrate that activation of specific G
subunits may
be dependent not only on strain magnitude but on the rate of the
applied strain.
The application of calibrated uniform and isotropic strain allowed for the quantitative correlation between the external mechanical signal and the subsequent rapid and specific response in G protein activation. In contrast to nonuniform experimental systems applying uniaxial or biaxial strain (9, 10, 28), cells in the equibiaxial system are subjected to pure tension or compression at any point within a loading cycle, with no variations in cellular deformation arising from plating orientation or location on the substrate (23). The ability to control the mechanical strain applied uniformly to cultured cells was critical for the elucidation of the differential effects of strain magnitude or rate on G protein signaling.
Changes in the myocardial environment due to acute and chronic loading,
exercise and conditioning, altered myocardial stiffness, pacing, and
ischemia alter cellular function and response within the
myocardium (26). Pathophysiological remodeling of the cardiac ECM has
clinical significance in the adult heart, as processes such as fibrosis
during cardiac hypertrophy and scarring after infarction are thought to
be significantly influenced by the regional mechanical environment (5).
Cardiac fibroblasts constitute the majority (>90%) of the nonmyocyte
cells in the myocardium and are responsible for producing ECM
components such as collagens types I, III, and IV, fibronectin, and
laminin (14). They respond to a variety of agents such as ANG II (23)
and transforming growth factor-1 (14), as well as to mechanical
strain (9, 10, 28). Static stretch has been utilized in vitro to induce proliferation in fibroblasts as well as hypertrophy in myocytes (28),
whereas cyclic stretch modulated ratios of collagens types I/III (10)
and overall collagen synthesis (9). Elucidating the details of cardiac
fibroblast mechanoregulation is an important step toward understanding
remodeling of the myocardium.
In the myocardium and blood vessels, activation of G protein-linked
receptors for a number of extracellular stimuli, including ANG II, BK,
and endothelin, initiates second messenger cascades that include the
mobilization of intracellular Ca2+
through production of inositol trisphosphate (5, 7, 21). The
localization of G proteins at the membranes of cardiac fibroblasts in
the adult rat heart (29) and at sites of focal adhesions in neonatal
rat cardiac fibroblasts (18) support the potential importance of G
protein signaling mechanisms in transducing mechanical strain in these
cells. G protein activation as a stretch-induced response has been
demonstrated indirectly in cultured skeletal muscle subjected to
mechanical strain (32), in which the
Gi subclass-specific inhibitor
pertussis toxin attenuated cyclooxygenase activation and
PGF2 production, as well as
cell growth. Stretch-induced immediate early gene expression in
cultured cardiac myocytes has been linked to protein kinase C
activation and was shown to be pertussis toxin insensitive (22),
implicating Gq activation in the
stretch response. A link between
Gq activation and cardiac hypertrophy has also been provided by two recent transgenic mouse models. Akhter et al. (1) reported an attenuation of left ventricular hypertrophy in pressure-overloaded transgenic mice expressing a
Gq inhibitor peptide in a
cardiac-specific manner. Conversely, cardiac-specific
Gq overexpression (11) induced
marked increases in fetal cardiac gene expression, ventricular
hypertrophy, and contractile dysfunction relative to controls. These
studies demonstrate a central role for G proteins in the
mechanotransduction of stretch and myocardial remodeling.
Although previous studies indirectly assessed G protein activation by
stretch through the use of inhibitors (32) or measurement of subunit
expression levels (35), the photolabile GTP analog utilized here
allowed the direct detection of G protein activation levels in response
to defined mechanical strain. The photoaffinity technique allows
resolution of the signal transduction time course (1 min), indicating
their rapid activation. When compared with BK and ANG II treatment,
which elicited a different profile of G subunit activation,
immunoprecipitation also indicated that the G protein-mediated
mechanotransduction mechanism may be conducted through signaling
pathways distinct from that of growth factors in cardiac fibroblasts.
In vascular endothelial cells, fluid shear stress has been shown to
rapidly activate G proteins belonging to
Gq and
Gi3 subtypes within 1 s of flow
onset (18), implicating G protein signaling as a primary mechanism in
mechanotransduction.
Cellular response to mechanical forces such as fluid shear (13) and mechanical strain (2, 31) have been well established. Cell culture systems such as the one presented here allow stimuli such as agonists and mechanical strain to be applied to an isolated cell population in a distinct and defined manner. Recent studies of the mechanotransduction of fluid shear have begun to dissect out the differential response of cells to flow transients at the onset of flow from the sustained response of the steady flow component (16). The rate of application of flow is perceived by the cell as a signal separate from that of the flow itself, allowing the identification of unique shear rate and shear responses. Similar mechanisms may exist in the transduction of mechanical strain, with strain rate presenting a stimulus separate from the strain itself, triggering an intracellular cascade of events, possibly originating at the activation of membrane-bound G proteins. The results presented here indicate that this may indeed be the case, as increasing the rate of the applied strain elicited increased activation of G proteins. Further quantitative studies of mechanical strain and signaling mechanisms are planned to elucidate the essential questions of the thresholds and specificity of the cell response to its physical environment.
In summary, the present study demonstrates that G proteins are rapidly
activated in adult cardiac fibroblasts in response to stretch.
Gq and
G
i1, but not
G
i3, are activated rapidly by
mechanical strain, identifying important early events in the mechanotransduction of strain. The results further suggest that the
activation G proteins by strain may be strain rate dependent.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-40696.
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FOOTNOTES |
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Address for reprint requests: J. A. Frangos, Dept. of Bioengineering, Univ. of California, San Diego, La Jolla, CA 92093-0412.
Received 22 December 1997; accepted in final form 26 February 1998.
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