In the mammalian heart, the extracellular matrix plays an important role in regulating cell behavior and adaptation to mechanical stress. In cell culture, a significant number of cells detach in response to mechanical stimulation, limiting the scope of such studies. We describe a method to adhere the synthetic peptides RGD (fibronectin) and YIGSR (laminin) onto silicone for culturing primary cardiac cells and studying responses to mechanical stimulation. We first examined cardiac cells on stationary surfaces and observed the same degree of cellular adhesion to the synthetic peptides as their respective native proteins. However, the number of striated myocytes on the peptide surfaces was significantly reduced. Focal adhesion kinase (FAK) protein was reduced by 50% in cardiac cells cultured on YIGSR peptide compared with laminin, even though beta(1)-integrin was unchanged. Connexin43 phosphorylation increased in cells adhered to RGD and YIGSR peptides. We then subjected the cardiac cells to cyclic strain at 20% maximum strain (1 Hz) for 48 h. After this period, cell attachment on laminin was reduced to approximately 50% compared with the unstretched condition. However, in cells cultured on the synthetic peptides, there was no significant difference in cell adherence after stretch. On YIGSR peptide, myosin protein was decreased by 50% after mechanical stimulation. However, total myosin was unchanged in cells stretched on laminin. These results suggest that RGD and YIGSR peptides promote the same degree of cellular adhesion as their native proteins; however, they are unable to promote the signaling required for normal FAK expression and complete sarcomere formation in cardiac myocytes.
Cardiac myocyte cultures usually require pharmacological intervention to prevent overproliferation of contaminating nonmyocytes. Our aim is to prevent excessive fibroblast cell proliferation without the use of cytostatins. We have produced a silicone surface with 10-microm vertical projections that we term "pegs," to which over 80% of rat neonatal cardiac fibroblasts attach within 48 h after plating. There was a 50% decrease in cell proliferation by 5 days of culture compared with flat membranes (P < 0.001) and a concomitant 60% decrease (P < 0.01) in cyclin D1 protein levels, suggesting a G1/S1 cell cycle arrest due to microtopography. Inhibition of Rho kinase with 5 or 20 microM Y-27632 reduced attachment of fibroblasts to the pegs by over 50% (P < 0.001), suggesting that this signaling pathway plays an important role in the process. Using mobile and immobile 10-microm polystyrene spheres, we show that reactive forces are important for inhibiting fibroblast cell proliferation, because mobile spheres failed to reduce cell proliferation. In primary myocyte cultures, pegs also inhibit fibroblast proliferation in the absence of cytostatins. The ratio of aminopropeptide of collagen protein from fibroblasts to myosin from myocytes was significantly reduced in cultures from pegged surfaces (P < 0.01), suggesting an increase in the proportion of myocytes on the pegged surfaces. Connexin43 protein expression was also increased, suggesting improved myocyte-myocyte interaction in the presence of pegs. We conclude that this microtextured culture system is useful for preventing proliferation of fibroblasts in myocyte cultures and may ultimately be useful for tissue engineering applications in vivo.
Our objective is to alter the surface topography on which cardiac myocytes are grown in culture so that they more closely resemble their in vivo counterparts. Microtextured silicone substrata were made using photolithography and microfabrication techniques and then coated with laminin. Primary cardiac myocytes from newborn rats were plated on microgrooved and nontextured substrata. Myocytes were highly oriented on 5 microm grooves (69.8 +/- 2.0%) and significantly different, p < 0.0001, compared with randomly oriented cells grown on nontextured surfaces (2.9 +/- 0.95%; n = 19). Cells on shallower, 2 microm, grooves were slightly less well oriented (46.9 +/- 4.3%, n = 5, p < 0.001). The lateral spacings of the grooves were altered to examine changes in cell-to-cell contact by confocal immunocytochemistry and quantitative protein analysis. Connexin43 and N-cadherin were distributed around the perimeter of the myocytes plated on 10 x 5 x 5 microgrooved surfaces, similar to the localization found in the neonate. Connexin43 expression in cultures on 5 microm deep grooved substrata was equal to the neonatal heart, whereas it differed in nontextured surfaces. We conclude that it is necessary to combine groove depth (5 microm) and lateral ridge dimensions between grooves (5 microm) in order to recapitulate connexin43 and N-cadherin expression levels and subcellular localization to that of the neonate.
One of the unanswered questions in muscle hypertrophy is how new contractile units are inserted into a stable existing cytoskeletal meshwork. Regulation of actin capping by CapZ may play a role in remodeling processes, therefore, CapZ dynamics are determined during rapid growth of cardiac cells in vitro. Neonatal rat ventricular myocytes were infected with adenovirus expressing green fluorescent protein-CapZ beta1 and responded normally to hypertrophic stimuli. CapZ dynamics were analyzed by fluorescence recovery after photobleaching in cultured myocytes treated with endothelin-1 (100 nM) or phenylephrine (10 muM). Recovery by 30 s was greater with endothelin treatment. Analysis 30 min postbleach showed CapZ-infected cells treated with endothelin recovered more completely than controls (77 +/- 9% vs. 50 +/- 6%, P < 0.001). Similar results were found with phenylephrine (77 +/- 5%, P < 0.05). A potential mechanism for phosphatidylinositol bisphosphate (PIP2) mediation of increased CapZ exchange in endothelin- and phenylephrine-treated cells was tested. PIP2 sequestration with neomycin (500 muM) blocked both endothelin- (43 +/- 6%, P < 0.001) and phenylephrine (36 +/- 4%, P < 0.001)-mediated recovery. The protein kinase C inhibitor chelerythrine chloride (10 muM) also blocked endothelin- (53 +/- 10%, P < 0.001) and phenylephrine (42 +/- 3%, P < 0.001)-mediated recovery. This study demonstrates for the first time that endothelin and phenylephrine alter CapZ dynamics through PIP2- and PKC-dependent pathways, which might destabilize the existing framework and permit sarcomeric remodelling to proceed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.