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MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Department of Pathology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Shadyside Hospital, Pittsburgh; and Departments of 2Cardiothoracic Surgery, 3Genomic Sciences, 4Pathology, and 5Pediatrics, Drexel University School of Medicine, Allegheny General Hospital, Pittsburgh, Pennsylvania
Submitted 10 April 2006 ; accepted in final form 11 January 2007
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ABSTRACT |
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72 h by cardiac fibroblasts. Serum concentrations were held constant under all media conditions, and complete medium exchanges were performed daily. Cardiomyocytes began contracting within 24 h at clonal or mass densities with <5% of cells expressing vimentin. Immunocytochemical analysis revealed progressive expression of 
-smooth muscle actin in cardiomyocytes after 24 h in all conditions. Only cardiomyocytes in fibroblast-conditioned medium stopped contracting by 72 h. There was a significant, sustained increase in vimentin expression specific to these cultures (means ± SD: conditioned 46.3 ± 6.0 vs. control 5.3 ± 2.9%, P < 0.00025) typically with cardiac myosin heavy chain coexpression. Proteomics assays revealed 10 cytokines (VEGF, GRO/KC, monocyte chemoattractant protein-1, leptin, macrophage inflammatory protein-1, IL-6, IL-10, IL-12p70, IL-17, and tumor necrosis factor-) at or below detection levels in unconditioned medium that were significantly elevated in fibroblast-conditioned medium. Latent transforming growth factor-
and RANTES were present in unconditioned medium but rose to higher levels in conditioned medium. Only granulocyte-macrophage colony-stimulating factor was present above threshold levels in standard medium but decreased with fibroblast conditioning. These data indicated that under the influence of fibroblast-conditioned medium, cardiomyocytes exhibited marked hypertrophy, diminished contractile capacity, and phenotype plasticity distinct from the dedifferentiation program present under standard culture conditions. proteomics; myosin heavy chain; vimentin; myofibroblast; primary culture; dedifferentiation; plasticity
In vitro studies have established that signals induced by myocardial injury affect fibroblasts and cardiomyocytes and may regulate the degree of fibrosis and hypertrophy associated with the recovery process (16, 17, 20, 32). For example, complex cytokine-mediated interactions dissected in vitro revealed activation of mitosis in cardiomyocytes with concomitant suppression of fibroblast proliferation. These intercellular signals, e.g., interleukin (IL)-1, IL-6, and transforming growth factor (TGF)-, as well as various undefined soluble factors, may be of nonmyocardial, epicardial, or myocardial in vivo origin and act through paracrine or autocrine pathways (16, 17, 25, 32, 37, 41–43). Important cardiac fibroblast-cardiomyocyte interactions have been demonstrated in appositional cocultures involving direct membrane contact and/or cell fusion as well as through the use of noncontact cell culture paradigms to assess the role of soluble factors (12, 13, 17, 42).
The present study examined the effect of medium containing factors derived from cardiac fibroblasts on the behavior of highly purified, isolated ventricular cardiomyocytes. The findings demonstrated that cardiac fibroblasts induced changes in cardiomyocyte phenotype different from or in addition to the in vitro dedifferentiation process. These changes included alteration of myocyte structural and functional characteristics including hypertrophy, intracellular expression of vimentin, and reduction of chronotropic contractile activity. High-sensitivity protein analysis of cytokines and chemokines implicated in this response revealed 10 factors at negligible levels in standard medium but present at significantly higher levels in the conditioned medium including vascular endothelial growth factor (VEGF), growth-regulated -protein (GRO/KC), monocyte chemoattractant protein-1 (MCP-1), leptin, macrophage inflammatory protein-1 (MIP-1), IL-6, IL-10, IL-12p70, and IL-17, and tumor necrosis factor (TNF)-. Furthermore, two factors present in standard medium were significantly elevated after cardiac fibroblast conditioning including latent TGF- and RANTES (regulated on activation of normal T cell expressed and secreted). Only one factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), fell significantly below concentrations obtained in standard medium as an effect of the conditioning process.
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MATERIALS AND METHODS |
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Cardiac myocyte cell culture model.
Cardiomyocytes were seeded at clonal (25–100 cells/60-mm plate) or subconfluent densities (5–10 x 104 cells/100-mm plate) in parallel plates so that immunocytochemical analysis and precise population counts or doubling times could be calculated to determine the growth rates and purity of the preparation throughout the study. All cardiac cells were grown on collagen in medium comprising 10% horse serum (AMG16770; Hyclone, Logan, UT) and 5% fetal bovine serum in Dulbecco's modified Eagle's medium (DMEM)-medium 199 (4:1) at 37°C (10 ml/plate). A complete medium exchange was performed every 24 h to minimize conditioning effects by the cardiomyocytes. Cells from the fibroblast band were plated at high densities (2 x 105 cells/100-mm plate) to achieve confluence, at which time the medium was left unchanged for a minimum period of 3 days. At that time, the conditioned medium was removed and filtered to remove cells and particulate matter (0.22-µm syringe filter). Conditioned medium was used immediately after collection and filtration or frozen for subsequent use. All media were allowed to undergo only one freeze-thaw cycle for inclusion in this study. Bead-based multianalyte proteomics analysis and enzyme-linked immunosorbent assays (ELISA) were performed on media utilized in this study before their use in cell cultures, including determination of the acid-activated form of TGF-1 (R&D Systems, Minneapolis, MN).
Experimental paradigm. Preliminary studies indicated that plating cells from the second digest after Percoll gradient centrifugation provided a cardiomyocyte purity ranging from 95 to 99.1% of the total cells present after 24 h. Thus all experiments were performed only on cardiomyocytes from this subset of the preparatory protocol and meeting this criterion. Cell density was determined every 24 h for 144 h by counting a minimum of 20 random fields per plate and extrapolating the average population to the area of the entire plate. Parallel serial plates were fixed each day and double stained for myosin heavy chain (MyHC) and vimentin to confirm cardiomyocyte and fibroblast composition, respectively, based on cell counts and immunochemical phenotype. Cardiomyocytes were often binucleate, and the number of cells falling into this classification was also monitored. The fibroblast cell band obtained from the Percoll gradient was examined after serial clonal and mass population platings and selected to contain predominantly fibroblasts (>99%). In rare cases, endothelial cells were identifiable by formation of a cobblestone colony phenotype, and differential preplating was repeated to purify the fibroblast population.
To analyze potential paracrine effects of fibroblasts on cardiomyocyte behavior, we mixed fibroblast-conditioned medium (72 h of conditioning) or PBS 1:1 with standard medium containing twofold concentrations of horse and fetal bovine serum to maintain a standard serum concentration. This medium was used to feed cardiomyocytes 24 h after plating and was changed daily for 144 h. The assignment of cells to receive standard, conditioned, or PBS control medium was performed randomly on plates seeded with equivalent amounts of cardiomyocytes derived from the second digest. The experimental paradigm meeting the criterion for cardiomyocyte culture purification (95%) was successfully repeated on five independent ventricular harvests performed at different times as well as an expanded experiment in which ventricles derived from six litters of neonatal rats were concomitantly harvested, pooled, purified, and subjected to the identical experimental protocol comparing the effects of standard and conditioned media.
Electron microscopy-ultrastructural analysis.
Cultured cells from each experimental condition were detached with trypsin-EDTA (GIBCO Invitrogen) and collected by serial rinse in 1x PBS as previously described (22). The suspended cells were centrifuged in a 1.5-ml tube (1,200 rpm; Beckman TJ-6) to obtain a pellet, and the supernatant was replaced with 3% glutaraldehyde for overnight fixation at 4°C. Postfixation was performed in 1% osmium tetroxide for 90 min, followed by dehydration with ethanol (50%, 30 min; 70%, 30 min; 95%, 2x 30 min; 100%, 2x 30 min) and acetonitrile (2x 30 min). The pellet was then slowly infiltrated with epoxy resins (EMbed-812; Electron Microscopy Sciences, Hatfield, PA), embedded, and heated overnight at 65°C. Thin sections (
90 nm) were cut, stained with uranium and lead salts, and examined in a Philips CM 10 electron microscope.
Immunocytochemical specification of cell phenotype.
All plates were rinsed twice in cold PBS and fixed in AFA for immunocytochemical analysis as previously described (22). Expression of MyHC was determined by immunolabeling with MF20, a pan-MyHC monoclonal antibody mixed 1:1 with BA-G5 (provided by Dr. S. Schiaffino, Universita degli Studi di Padua, Padua, Italy), an antibody specific for -cardiac MyHC (1, 38). An indirect immunostaining procedure was used, incorporating an alkaline phosphatase-conjugated secondary antibody (1:500 dilution, IgG, goat anti-mouse; Sigma) followed by Western blue stabilized substrate (Promega, Madison, WI) as a chromogen in levamisole (125 mM) to block endogenous phosphatase activity. Similarly, a monoclonal antibody specific to connexin 43 associated with gap junction formation was employed (1:100; Sigma), utilizing a horseradish peroxidase-conjugated secondary antibody (1:500 dilution, goat anti-mouse; Sigma) and visualized using diaminobenzidine tetrahydrochloride staining (22, 38). The presence of vimentin was determined using monoclonal antibody (clone V9) (1:250 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) with a biotinylated secondary antibody (1:500 dilution, goat anti-mouse, Sigma). A streptavidin-alkaline phosphatase tertiary reagent (1:500 dilution; Zymed Labs, San Francisco, CA) was subsequently employed and visualized through the use of Fast red substrate in naphthol phosphate buffer (Sigma). An -smooth muscle actin specific monoclonal antibody, clone 1A4 (1:100 dilution; Sigma) conjugated to alkaline phosphatase was utilized, incorporating an indirect immunostaining procedure with Vulcan red (Biocae Medical, San Diego, CA) serving as the chromogen. Initial platings of purified but untreated fibroblasts (V9+, MF20/BAG5–) and cardiomyocytes (V9–, MF20/BAG5+) were analyzed in parallel with antibodies specific to cardiomyocytes (MF-20/BAG5: MyHC) or fibroblasts (V9: vimentin) as exclusionary controls for the immunocytochemical assay as well as a check for cell purity (4, 22).
Images of individual cardiomyocytes positive for MyHC expression were acquired randomly from 60-mm plates seeded at clonal density containing 1) untreated controls in standard cardiac medium and 2) parallel cultures treated with fibroblast-conditioned medium. Each untreated plate was paired with a treated plate obtained from the same cell harvest and an identical lot of conditioned medium. Five different cell expansions and unique lots of conditioned medium were studied for 144 h. Two plates represented each condition and time point, including the "zero" time point, while nine plates represented each condition and time point in the pooled cardiomyocyte study. A minimum of 25 cardiomyocyte images was obtained from each plate and imported into the NIH Image analysis package for calculation of cell surface area (http://rsb.info.nih.gov/nih-image). The outer membrane border of each cardiomyocyte image was manually inscribed with a digital cursor, and the area demarcated by the tracing was calculated with a macrosubroutine (Image J) calibrated before use against a micrometer slide image captured at the same magnification as the cell image. Surface area was determined for cells from control and conditioned media at each time point.
Confocal imaging for determination of protein coexpression. Confocal images of cultured cardiomyocytes were obtained after immunofluorescent labeling with both myosin-and vimentin (rabbit polyclonal H84; Santa Cruz Biotechnology)-specific antibodies to ascertain coexpression of these proteins. A Leica DM RXE upright microscope attached to a TCS SP2 AOBS confocal system (Leica Microsystems, Nussloch, Germany) was employed for the analysis. Images were obtained directly from cells fixed with AFA in their respective culture plates by using a long working distance x63, 0.90 numerical aperture, water-immersion objective. The secondary antibody for myosin was an IgG-fluorescein-5-isothiocyanate (FITC)-conjugated goat anti-mouse antibody (Sigma), whereas the vimentin secondary was a goat anti-rabbit phycoerythrin (PE)-conjugated antibody (Sigma). FITC (maximum excitation/maximum emission 495/519 nm)-labeled myosin and PE (maximum excitation/maximum emission 565/576 nm)-labeled vimentin were visualized using excitation wavelengths of 488 and 594 nm, respectively. The corresponding emission detector widths were set at 498–568 nm and 608–710 nm. Laser settings were adjusted to remove overlap between detector channels. In addition, the cells were imaged with standard transmitted light.
Multiplex bead and ELISA analysis of standard and conditioned media.
Fluorokine MultiAnalyte Profiling (xMAP) using the Luminex 100 platform (Luminex, Austin, TX) was employed to measure levels of 24 defined small proteins in conditioned and unconditioned media samples. The technology incorporated polystyrene microspheres dyed internally with differing ratios of two spectrally distinct fluorophores to create a family of different spectrally addressed bead sets (catalog no. RCYTO-80K-PMX, Lincoplex kit; Linco Research, St. Charles, MO). Each bead set utilized in this study was conjugated with a biotinylated capture antibody specific for a unique rat cytokine/chemokine target, including eotaxin (eosinophil chemotactic protein: small inducible cytokine A11: CCL11), interleukins (IL-1, IL-1, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17, IL-18), IP-10 (small inducible cytokine B10: Cxcl10), TNF-, interferon (IFN)-
, GM-CSF (Csf2), GRO/KC (growth-regulated -protein: Cxcl1), VEGF, G-CSF (granulocyte colony-stimulating factor), leptin, RANTES, MIP-1, and MCP-1 (small inducible cytokine A2: CCL2). The assay was performed without antecedent serum depletion with a sensitivity 
0.1 pg/ml. Analysis was performed on five samples of medium conditioned by fibroblasts derived from separate ventricular harvests. Results were compared with standard cardiac medium placed on gelatin-coated plates in the absence of cells and incubated under identical culture conditions for 72 h. The assays utilized a 96-well microplate format and were processed according to the manufacturer's protocol, including generation of a standard curve for each target prepared in background medium diluent (DMEM) over a fourfold range of dilution from 4.9 to 20,000 pg/ml, except for leptin, which was calibrated from 24.41 to 100,000 pg/ml. A value of 3.8 pg/ml was accepted as the lowest threshold for sensitivity, because this value fell within the linear range of the calibration assays. Standards and test media were pipetted at 25 µl per well in duplicate and mixed with 25 µl of the bead mixture. The microplate was incubated overnight at 4°C on a microtiter shaker. Wells were then washed with buffer (3 times) using a vacuum manifold. A secondary antibody cocktail was added to all wells and incubated for 2 h with agitation at room temperature. Streptavidin-PE was added to the wells and incubated for 30 min with constant shaking at room temperature. Wells were washed twice, assay buffer was added to each well, and samples were analyzed using the Bio-Plex suspension array system and Bio-Plex Manager software 4.0 (Bio-Rad Laboratories, Hercules, CA). Quantities were determined by comparison to standard curves obtained for each analyte.
An ELISA was performed to assay TGF- levels via a quantitative sandwich immunotechnique with the target monoclonal antibody precoated on a 96-well microplate (Invitrogen, Carlsbad, CA). Standards, controls, and experimental samples were added to wells according to the manufacturer's protocol, and the data calculated were compared with a standard calibration curve that also defined the dynamic range of the assay. The presence of TGF- in control and test samples was determined by its binding to the immobilized antibody. Unbound substances were washed away, and an enzyme-linked polyclonal antibody specific for the cytokine of interest was added to the wells to sandwich the immobilized cytokine from the primary incubation step. Any unbound antibody enzyme reagent was then washed away. A chromogen solution was then added to the wells, and color developed in proportion to the amount of bound cytokine. The optical density was determined using an automated plate reader at 450 nm (Biotech, Winooski, VT) with a KC junior program set to ±450 nm.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-smooth muscle actin expression emerges in a subset of adult and neonatal cardiomyocytes in culture (9, 14). Immunocytochemical analysis demonstrated that this phenomenon occurred in the present study as well (Fig. 1B). Within the context of the pooled study, the number of cardiomyocytes expressing smooth muscle actin was significantly higher at each time point in conditioned medium compared with controls, affecting 60% of the total population by 144 h. Cells positive for MyHC and connexin 43 grown in fibroblast-conditioned medium displayed a large diversity of cell diameters and shape characteristics by 72 h that persisted for the duration of the study (Figs. 5 and 6).
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Immunocytochemical staining for myosin often exhibited refractive birefringence consistent with an organized myosin macromolecular structure despite significant structural changes including development of cellular holes, cavities, and vacuoles (Fig. 6A). This was seen in fibroblast-conditioned medium as well, where few cells displayed contractions. The shape of the cardiomyocyte depicted in Fig. 6B was typical of each culture; however, the presence of vimentin in binucleate cells like this specimen was predominantly restricted to fibroblast-conditioned medium. Thus conditioned medium induced a unique vimentin expression pattern in cardiomyocytes atypical of the other culture conditions employed in this study. By serial immunocytochemical analysis, it was established that the actin-positive cells also expressed connexin 43 and retained their capacity to form gap junctions with other cardiomyocytes regardless of the media conditions (Fig. 6C).
Confocal image analysis confirmed that myosin and vimentin were coexpressed within individual cells grown in fibroblast-conditioned medium (Fig. 7C). Myosin was typically organized into filamentous structures among the cells that retained a sarcomeric organization (Fig. 7A). Vimentin displayed this birefringent pattern in those cells as well but was typically distributed in an amorphous pattern throughout the cytoplasm, except in the nucleus (7B). It is important to note that a few cardiomyocytes retained their original phenotype of exclusionary myosin expression throughout the 6-day exposure to the fibroblast-conditioned medium. At the same time, rare cells were detected under standard cardiomyocyte growth conditions in which myosin and vimentin were coexpressed. Nevertheless, a dominant phenotype emerged among those cardiomyocytes exposed to fibroblast-conditioned medium where both myosin and vimentin were routinely present. Thus medium conditioned by factors specific to cardiac fibroblasts appeared to markedly amplify and/or select for this phenotype compared with the other conditions employed in this study.
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, IL-6, IL-10, IL-12p70, IL-17, and TNF-) were at or below threshold levels of detection in the unconditioned medium but were significantly elevated in the fibroblast-conditioned medium. RANTES was detectable above threshold levels in the unconditioned medium but rose to significantly higher levels in the fibroblast-conditioned medium. Of the 25 cytokines assayed, only GM-CSF was decreased in medium after exposure to cardiac fibroblasts compared with the unconditioned medium. ELISA analysis indicated that the conditioned medium had comparable levels of active TGF-1 compared with the unconditioned medium conditions antecedent to use in culture (Table 1). However, ELISA performed after acid activation (pH 4) revealed that a significantly higher level of latent TGF-1 was present in the conditioned medium compared with the standard (P < 0.01) or PBS control medium (P < 0.01).
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DISCUSSION |
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-smooth muscle actin across all culture conditions was consistent with the dedifferentiation paradigm in contrast to vimentin expression specific to the fibroblast-conditioned medium (9, 14). These data suggest that cardiac fibroblasts introduced a soluble factor or factors into the medium that altered cardiomyocyte function and phenotype by either 1) a direct cell membrane or intracellular interaction or 2) sequestration, consumption, or modification of factors in the standard medium, leading to an indirect biological effect. Further studies are required to dissect the specific factors and pathways underlying the in vitro cardiomyocyte response and to determine whether the paradigm applies to the complex in vivo environment. Recent studies have emphasized the complexity of cardiac intercellular interactions where heart cells may be both source and target of signals such as cytokines and growth factors (16, 17, 31, 36). Contact-specific and density-related effects have been previously described for cardiomyocytes cocultured with fibroblasts, but the contribution of each cell fraction was difficult to interpret, particularly in light of recent evidence that cardiomyocytes can fuse with fibroblasts in vitro (12, 13, 27, 42). Therefore, we optimized our purification methods and immunocytochemical assays to restrict and monitor the presence of fibroblasts vs. cardiomyocytes by eliminating those cultures and conditions where multiple cell types were present. Based on 1) the randomized treatment paradigm, 2) the immunocytochemical results obtained on parallel control plates, and 3) the daily serial cell counts including binucleate cells, effects of the conditioned medium were not explained by selective overgrowth of fibroblasts or amplification of other cell types. Furthermore, confocal analysis confirmed that typical exclusionary expression of myosin in cardiomyocytes and vimentin in fibroblasts under standard culture conditions was replaced by coexpression within a substantial number of cardiomyocytes treated with conditioned medium.
Vimentin expression has rarely been assayed in neonatal and adult cardiomyocytes in vitro and has been implicated as part of a dedifferentiation or developmental program largely because embryonic myocytes in vivo and in vitro express vimentin during a fetal transition period (12, 21, 28, 29, 31, 46, 52). The present study is the first to directly demonstrate the emergence of coexpression of vimentin and MyHC in a highly purified postnatal cardiomyocyte culture. The delayed in vitro onset of vimentin expression in myosin-containing cells under the influence of conditioned medium differed in pattern from the developmental myogenic program where vimentin expression preceded mature myosin expression. It is interesting to note that coexpression of myosin, smooth muscle actin, and vimentin is a defining characteristic of the myofibroblast cell type (8, 19, 39, 47–49). Myofibroblasts derive from fibroblasts, monocytes, or circulating progenitor cells under the influence of TGF- and form granulation tissue, produce extracellular matrix molecules, and contribute to wound contraction (7, 19, 50). Although TGF- was not elevated in the conditioned medium before use, latent TGF- was significantly increased. Thus the latent form may have been proteolytically activated after cardiomyocyte binding inducing the canonical TGF- signal pathway, or latent TGF- may have directly activated NF-
B in a pathway associated with cellular transformation (2, 7, 19, 26, 50). Respecification of endothelial, epithelial, and muscle cell phenotype has been reported previously in vitro but has not been confirmed in vivo (18, 24, 35). Thus it is intriguing to consider that cardiomyocytes might be a source of myofibroblasts and participate in the scarification process if the plasticity demonstrated in the present study extends to the in vivo domain. Robust induction of myofibroblasts is known to occur after myocardial injury, so determination of whether these cells may derive from cardiomyocytes in a manner analogous to the present study will require precise in vivo lineage studies.
Cardiac fibroblasts are known to play a critical role in scar formation based on their prolific proliferative capacity as well as through production of extracellular matrix glycoproteins. The present study adds to a growing literature that cardiac fibroblasts also generate paracrine signals that may directly influence cardiomyocyte behavior. In an analogous approach, a cardiomyocyte cell-survival signal pathway activated in vitro by the secreted protein thymosin 4 has been identified to ameliorate the effects of myocardial infarct in mice (6). The present data also reinforce in vitro cardiomyocyte-fibroblast coculture studies delineating IL-6 as an activator of cardiomyocyte hypertrophy (16, 17). Although we were able to maintain equivalent pH, temperature, and serum conditions across all media conditions, we cannot rule out changes in ionic composition and osmolality associated with the conditioning protocol that could account for physiological changes in highly sensitive processes such as spontaneous contractility, nor can we rule out the possibility that fibroblasts consumed or altered critical molecules during medium conditioning, thereby engendering a cardiomyocyte response by their absence or modification. The proteomics analysis revealed minimal signs of substrate depletion, but this likely reflected selection of an assay targeting cytokines and chemokines secreted by fibroblasts rather than growth factors, ions, and metabolites consumed by these cells. Among the 26 proteins assayed, only GM-CSF was significantly decreased in the medium after exposure to fibroblasts. No direct effect of GM-CSF administration or diminution has been reported on cardiomyocyte phenotype or function to date, but GM-CSF is important in hematopoiesis, the immune response, and arteriogenesis, and knockout mice exhibit evidence of cardiac amyloidosis (40). Thus we recognize the importance of efforts directed at determining molecules that, by their elimination, could account for changes in cardiomyocyte phenotype.
It was interesting to note that the capacity for fibroblasts to produce the cytokine protein profile detected in the cardiac fibroblast-conditioned medium has previously been demonstrated at the gene expression level in synovial fibroblasts in both primary tissue and serial culture associated with inflammatory joint disease (36). Results from those studies provided evidence of a persistent proinflammatory or immunopathogenic response originating in or enhanced by fibroblasts that produced a cytokine and chemokine profile very similar to that of the present study. When the proteins in the present study were analyzed for correlation with canonical cell signal transduction networks (Ingenuity Pathway Analysis 4.0; Redwood City, CA,), we were surprised to note that the proteomics profile fit equally well into four other important pathways with the same high statistical significance (P < 0.001) as the inflammatory/immune response pathway. These included signal networks associated with 1) cellular proliferation, 2) cell-to-cell activation, 3) cellular movement, and 4) apoptosis. The fact that these pathways are fundamental to in vivo cardiac remodeling lends support to the hypothesis that cardiac fibroblasts play an important effector role in modulating the repair process apart from activation of an acute phase response.
These data reinforce the use of well-defined, highly purified cardiomyocyte preparations in vitro for investigating intercellular paracrine interactions and cell plasticity. The effects of fibroblast conditioning—hypertrophy, loss of spontaneous beating, coexpression of MyHC and vimentin, and glycogen accumulation—could be separated from global dedifferentiation changes attributable to the in vitro model based on the carefully controlled experimental paradigm. We recognize that a reduced culture preparation places greater interpretive distance from organismal biology, thereby requiring translational studies to determine whether the findings apply to adult cardiomyocytes both in vitro and in vivo. Nevertheless, the data add to recent findings demonstrating both autocrine and paracrine effects of nonmyocardial cells on the structure and function of cardiomyocytes. Furthermore, they suggest novel influences of fibroblasts on cardiomyocyte phenotype and plasticity as well as on the formation of myofibroblasts.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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), medium obtained after conditioning on cardiac fibroblast plates (
), or cardiac medium diluted with PBS but with horse serum concentrations equivalent to standard medium (x). Duplicate plates were fixed and stained for myosin heavy chain (MyHC), and positive cells were counted every 24-h increment to obtain the curves depicted. The results were compiled as means ± SD of the cell counts. There were no statistically significant differences in cell numbers regardless of treatment at any of the time points. Similar results were obtained for 2 experiments when initial seedings were at a density of 5 x 104 cells/100-mm plate. B: immunocytochemical analysis of pooled cardiomyocytes. Cardiomyocytes were pooled, purified, and seeded at 7 x 104 cells across 18 100-mm plates per time point for comparison of control and conditioned media regarding MyHC, connexin 43, and 
