The effect of electric current on protein biosynthesis in mammalian fbroblasts was investigated with neonatal bovine fibroblastpopulated collagen matrices. The fidd strength dependence ofelectric field modulation ofproline incorporation into extracelllar and intracellular protein was measurd ove a frequency range from 0.1 to 1000 herz A frequency- and amplitude-dependent reduction in the rate of incorporation was observed. In tissues containing cels aligned either parallel or perpendicular to the dectric field, this response was dependent on the orientation ofthe cells relative to the direction of the applied electric field. This study demonstrates that currents of physiological strength can stimulate alterations in biosynthesis and thereby may influence tissue growth, remodeling, and repair. ELCS WITHIN MAMMALIN CONnective and skeletal tissues are regularly exposed to time-varying electric currents. These currents are produced endogenously, arising predominantly from the spatial and temporal integration of currents from excitable cells (1), and through cur- K. J. McLeod and R. C. Lee, Continuum Electromechanics Group, Laboratory for Electomagnetic and Electronic Systems, Department of Electrical Eneering and Computer Scence, Massachuset nstitute of Technology Cambridge, MA 02139. H. P. Erlich, Shriners Bums Institute, Massachusetts General Hospital, Boston, MA 02139. *Present address: Deartment of Orthopaedic Surgery, State University of New York, Health Sciences Center, Stony Brook, NY 11790. I2 JUNE I987 rents generated by mechanical strain in glycosaminoglycan- rich connective tissues (2) and bone (3). These currents may well regulate the growth and remodeling of tissues (4) and alter cellular function. Physiological electric currents (5) can modulate the behavior of nonexcitable cells. For example, the rate ofDNA synthesis by pelleted chondrocytes was enhanced by applied current densities of less than 10 pVA/cm2 (6). Glycosaminoglycan synthesis by chondrocytes in monolayer culture was enhanced by current densities as low as 1 Ia.A/cm2 (7). In organ culture, current densities of 1 to 5 iAcm2 have been shown to alter calcium metabolism in chick tibiae (8). We have measured the rate of incorporation of proline into protein by bovine fibroblasts cultured within collagen matrices and found that it is sensitive to sinusoidal electric currents in the frequency range from 0.1 to 1000 Hz. This response manifests an abrupt current density threshold that is frequency- dependent. In addition, we found this threshold of the current density to be dependent on the orientation ofthe cell with respect to the direction of the applied current. The remodeling of connective tissue is regulated by physical stresses (9). Because fibroblasts are primarily responsible for this remodeling in soft connective tissue, they were selected for these experiments. Neonatal bovine fibroblasts were obtained by disaggregating superficial fascial tissue from the thigh of 2-week-old calves by serial typsin and collagenase digestions. The cells obtained were plated in flasks, maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum, and transferred to new flasks to avoid overcrowding two to five times prior to incorporation into gels. To control the extracellular matrix composition and cell density, we fabricated tissues of constant composition by incorporating fibroblasts in collagen matrices usethe technique of Bell et al. (10). Native type I collagen (2 mg/ml) was obtained through extraction of the tail tendons of young. For nonspherical cells, a maximum imposed membrane potential occurs when the major axis of the cell is aligned in the direction of the imposed electric field. This alteration in transmembrane potential may mediate the cellular response to low-frequency extracellular electric fields. One test of this hypothesis makes use of the nonspherical shape of the cells used in this study. Fibroblasts, in the collagen lattices, took on a bipolar morphology that has been previously described (17). The maximum length ofthese cells was approximately 150 ,um, which is seven to ten times the length of their minor dimension. If the depression in incorporation is mediated by a change in the membrane potential, then cells exposed to fields parallel to their major axes should exhibit a different threshold intensity than cells with their major axes perpendicular to the applied electric field. Of course, other, probably significant, parameter changes occur with changes in the cells' orientation. The plasma membrane area over which the maximum transmembrane potential alteration occurs is reduced when cells are aligned with the field. Also, the interaction of the electric field tangential to the plasma membrane with the cell surface will change with cell reorientation. Despite these complicating factors, such an orientation- dependent effect provides further evidence of a response dependent only on the electric field. We investigated the role of cell orientation by constructing FPCMs with cells predominantly oriented in one direction. To uniformly align cells in the collagen matrix, the FPCMs were allowed to contract over 3 days around two porous polyethylene posts held at a fixed separation distance of 2 cm. The FPCMs contracted with the cells and collagen aligned along an axis defined by the line passing through the porous posts (Fig. 4). The electrical conductivity of the mediafilled posts was within 10% of that of the free media solution. Three days after casting, the FPCMs were placed in the exposure chamber illustrated in Fig. 4. In each chamber, half of the FPCMs were installed with the major axes of the cells parallel to the applied electric field, while the other half were installed with cells oriented perpendicular to the direction of the electric field. Current was passed through the experimental samples for 12 hours. After the exposure period, the center sections between the posts were removed and analyzed for proline incorporation with the same protocol as used for the FPCMs with random cell orientation. Because the cells were most sensitive to 10-Hz fields, we used this frequency to examine the effect oforientation on the field 0.4i ° 0.2 S 0 0. O -0.2 3 0 -0.4 0. S -0.6 -T -O0 -T--I TP-c0.004 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Current density (gAIm2) Fig. 5. Normalized extracellular proline incorporation (dosed symbols) and intracellular proline incorporation (open symbols) plotted against current density for oriented FPCMs at 10 Hz. Samples with cells oriented parallel to field (diamonds) demonstrate a threshold current density below that seen in the samples of randomly orented cells, whereas samples with cells perpendicular (cirdes) show no depression of proline incorporation at a current intensity above the threshold level for randomly oriented samples. Data points (n = 6), dashed lines, and probabilities are as defined in the legend to Fig. 3A. NS, not significant. intensity threshold. For the randomly oriented FPCMs, a current density of 0.3 p.A/cm2 produced no significant effect on the rate of proline incorporation. However, when cells aligned with the electric field were exposed to the same current density, a significant reduction in proline incorporation was detected. In contrast, the cells oriented perpendicular to the field did not respond. Cell alignment with respect to the electric field modulated the intensity threshold (Fig. 5). Cells parallel to the field responded at 0.3 pA/cm2, whereas cells perpendicular to the field did not show a significant depression in protein secretion at 0.6 pA/cm2. Therefore, at 10 Hz cells with their major axes aligned with the field detected a field intensity as low as 20 p,V/cm. This corresponds to a maximum membrane potential perturbation of less than 0.5 ,uV. Our study has demonstrated that protein production in FPCMs is more sensitive to electric fields over the physiological frequency range than was previously shown for connective tissue cells. Ifthe cells are equally sensitive in vivo, mechanically induced fields in connective tissue may be able to trigger cell-mediated changes in tissue repair and remodeling as proposed by Bassett (18). Similarly, fibroblasts in vivo may also be sensitive to other endogenously or exogenously generated electric currents. The strong frequency dependence of the response suggests two modes of electrically mediated control of tissue composition. In the presence ofa constant frequency current, a change in biosynthetic activity could follow an increase in local current density. 468 Alternatively, if the frequency of local electrical currents were altered, for example, through a change in the mechanical loading rate on the tissue, then a biosynthetic response could be triggered even at constant current amplitude. Through either pathway, endogenously generated currents might be used as a feedback signal for tissue remodeling and repair. Other cell types have been shown to have frequency-dependent responses to electric fields. The action potential firing rate of Aplysia ganglion cells was modified by tissue current densities as low as 2 P,A/cm2, and a distinct frequency sensitivity was established with a peak sensitivity near 0.5 Hz (19). The heart rate offrogs was found to be depressed at current densities above 500 pA/cm2 with a peak sensitivity near 0.5 Hz (20), and the respiration rate of cats decreases at current densities as low as 1 pA/cm2, with a peak sensitivity at 2 Hz (21). The molecular mechanisms through which weak electric fields trigger a biosynthetic response are unknown. Because a variety of cell types (22) also exhibit extreme sensitivity to electric fields, this capability may be a primitive one and may serve a fundamental role in the interaction of living systems with their environment.
Xanya Sofra Weiss
Xanya Sofra Weiss
