RAPID COMMUNICATION
Equibiaxial strain and strain rate stimulate early activation of G proteins in cardiac fibroblasts

Siva R. P. Gudi, Ann A. Lee, Craig B. Clark, and John A. Frangos

Department of Bioengineering, University of California, San Diego, La Jolla, California 92093

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 [alpha -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 Galpha q and Galpha 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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 Galpha q and Galpha i1, but not Galpha i3, as the Galpha subunits activated rapidly by mechanical strain.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 Galpha 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 Galpha subunits: Galpha q, Galpha i1, and Galpha 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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Fig. 1.   Stretch-induced GTP binding in adult rat cardiac fibroblasts cultured on collagen-coated silicone elastic membranes. Cells were incubated with azidoanalido [alpha -32P]GTP (AAGTP) and then subjected to 1 of 3 strain protocols (top), followed by ultraviolet (UV) irradiation at 1 min. Autoradiograph (bottom) illustrates AAGTP binding in unstretched sham controls (lanes A and B) or equibiaxial strain for 1 min at 3% maximum strain for 6 cycles (protocol 3, lane C), 3% maximum strain for 12 cycles (protocol 2, lane D), and 6% maximum strain for 6 cycles (protocol 1, lanes E and F). Treatment with ANG II (lanes G and H) and bradykinin (lane I) served as positive controls. AAGTP binding to 42-kDa protein was detected in cells stretched at higher strain rate (lanes D-F) and in positive controls but not in cells stretched at lower strain rate (lane C) or in unstretched cells. Two experiments with n = 3 minimum for each data point; representative radiograph shown.

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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Fig. 2.   Effect of strain rate for 1 cycle on GTP binding in adult cardiac fibroblasts. Cells were incubated with AAGTP and subjected to 1 cycle of loading and unloading at 6% maximum equibiaxial strain over 10 or 60 s (top). Autoradiograph (bottom) illustrates AAGTP binding at 42 kDa for high strain rate (lanes A-C), but binding was negligible for lower rate (lanes D and E). Unstretched cells were treated with ANG II as a positive control (lanes F and G). Two experiments with n = 3 minimum for each data point; representative radiograph shown.

Immunoprecipitation of G proteins for specific Galpha subunits. Immunoprecipitation was used to identify the specific Galpha 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 Galpha q and Galpha i1 identified AAGTP-labeled proteins in stretched cells (S), compared with no detectable binding for these subunits in unstretched controls (C). Antibodies for Galpha i3 did not bind to 32P-labeled Galpha subunits in either unstretched or stretched cells. Antibodies for Galpha i3 and Galpha i1 formed immunocomplexes with radiolabeled G proteins in cells treated with either BK or ANG II.


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Fig. 3.   Immunoprecipitation of stretch-stimulated G proteins. After growth to confluency in equibiaxial stretch device, cardiac fibroblasts were incubated with AAGTP, subjected to 6% maximum strain for 1 cycle for 10 s, and UV irradiated. Galpha subunits were immunoprecipitated by adding polyclonal antibodies to Galpha . In stretched cells (S), Galpha q and Galpha i1 were identified, whereas antibodies to Galpha i3 did not yield immunoprecipitated protein. None of the 3 Galpha subunits were detected in immunoprecipitates from unstretched controls (C). As positive controls, Galpha i3 was immunoprecipitated in cells incubated with either bradykinin (BK) or ANG II, whereas Galpha i1 was activated only in BK-treated cells. Radiograph represents n = 2 with samples pooled for immunoprecipitation.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study shows that G proteins are rapidly activated in adult cardiac fibroblasts subjected to uniform and planar equibiaxial strain. Immunoprecipitation identified Galpha q and Galpha i1, but not Galpha i3, as the heterotrimeric G protein subunits specifically activated by mechanical strain. Moreover, these results demonstrate that activation of specific Galpha 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-beta 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 PGF2alpha 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 Galpha 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. Galpha q and Galpha i1, but not Galpha 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.

    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-40696.

    FOOTNOTES

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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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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