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THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 261, No. 31, Issue of November 5 , pp. 14740-14745,1986 Printed in U.S.A.

0 1986 by The American Society of Biological Chemists, Inc.

Expression of Smooth Muscle and Nonmuscle Myosin Heavy Chains in Cultured Vascular SmoothMuscle Cells* (Received for publication, May 5, 1986)

Arthur S. Rovner, RichardA. Murphy, and GaryK. Owens$ From the Department of Physiology, School of Medicine, University of Virginia, Charlottesville, Virginia22908

We explored the hypothesis that discrepancies in the muscle MHC form in these smooth muscle cells indiare required toinduce the fully literature concerning the natureof myosin expression cates that other factors in culturedsmooth muscle cells are due to the appear- differentiated state while in culture. ance of a new form of myosin heavy chain (MHC) in vitro. Previously, we used a very porous sodium dodecyl sulfate gel electrophoresis system to detect two Chamley et al. (1977) developed a technique for smooth MHCs in intactsmooth muscles (SM1and SM2) which differ byless than 2%in molecular weight (Rovner,A. muscle cell (SMC‘) culture based on an enzymatic dissociation S., Thompson, M.M., and Murphy, R. A. (1986) Am. of intact tissues. These investigators found that vascular J. Physiol. 250, (3861-C870). Myosin-containing ho- smooth muscle cells underwent a process of “phenotypic modmogenates of rat aorta cells in primary culture were ulation”inculture (Chamley-Campbell et al., 1979) which electrophoresed on thisgel system, and Western blots involved their apparentconversion to a cell type characterized were performed using smooth muscle-specific and non- by proliferation, synthesis, andsecretion rather thancontracmuscle-specific myosin antibodies. Subconfluent, rap- tion. During this process, the cells acquired large amounts of idly proliferating cultures contained a form of heavy rough endoplasmic reticulum and Golgi apparatus, whereas chain not found in rat aorta cells i n vivo (NM) with there was a loss of cytoplasmic thick filaments and the proelectrophoretic mobility and antigenicity identical to gressive diminution of myosin immunofluorescent staining by the single unique heavy chain seen in nonmuscle cells. a smooth muscle-specific antibody produced against chicken Moreover, these cultures expressed almost none of the gizzard myosin (Chamley and Campbell, 1974; Chamley et al., smooth muscle heavy chains.In contrast,postconfluent 1977; Groschel-Stewart et al., 1975). Sometimes, primary culgrowth-arrested cultures expressed increased levels of tures which had been plated at an initial high density (>5 X the twosmooth muscle heavy chains, along with large lo4 cells/ml) showed a return of smooth muscle myosin imamounts of NM. Analysis of cultures pulsed with [35S] munofluorescence after the attainmentof confluency (Chammethionine indicatedthat subconfluent cells were syn-ley-Campbell and Campbell, 1981), although in passaged culthesizing almost exclusivelyNM, whereas postconflu- tures, there was a persistence of the “synthetic state” and a ent cells synthesized SM1 and SM2 as well as larger continued lack of smooth muscle myosin antigenicity (Chamamounts of NM. Similar patternsof MHC content and ley and Campbell, 1974; Chamley et al., 1977). These types of synthesis were found in subconfluent and postconflu- observations led Chamley-Campbell et al. (1979) to suggest ent passaged cells. These results show that cultured that cell culture caused a permanent loss of many of the vascular smoothmusclecellsundergo differential characteristics typical of contractile smooth muscle cells, inexpression of smooth muscle- and nonmuscle-specific cluding the possession of myosin. MHC forms with changes in their growth state, which Several recent studies suggested that the concept of “synappear to parallel changes in expression of the smooth thetic” uersw “contractile” phenotype is overly simplistic in muscle and nonmuscle forms of actin (Owens, G. K., describing the state of differentiation of SMCs in culture. Loeb, A., Gordon, D., and Thompson, M. M. (1986)J. Vascular SMCs display contractionsafter several days in Cell Biol. 102, 343-352). The reappearance of the culture in response to the application of agonists such as smooth muscle MHCs in postconfluent cells suggests angiotensin I1 (Gunther et al., 1982). Established smooth that density-related growth arrest promotes cytodif- muscle cultures continue to express high affinity receptors to ferentiation, but the continued expressionof the non- contractile agonists (Gunther et al., 1982; Colucciet al., 1984). After growth arrest by heparin, changes in ionic conductance * This research was supported by a National Institutes of Health in response to theapplication of vasoactive amines are evident Predoctoral Training Grant 5 T32 HL07284-09 (to A. S. R.) and (Martin and Gordon, 1983). Myosin light chain phosphorylNational Institutes of Health Grants 2 PO1 HL19242-09 and 5 PO1 HL19242-10. Portions of this work have previously been presented ation is seen in postconfluent cultures of rat mesenteric artery at the 1985 Federation of American Societies for Experimental Biol- SMCs in response to thebinding of angiotensin I1 (Anderson et al., 1981).It has also been shown that theamount of smooth ogy Summer Conference on Smooth Muscle Physiology, June 24-28, 1985, Saxton’s River, VT, and at the 1986 Federation of American muscle-specific a-actin,asa proportion of totalactin,is Societies for Experimental Biology meeting, St. Louis, MO, April 13- greatly decreased during the proliferative phase of cultured 18,1986. The costs of publication of this article were defrayed in part cell growth (Fatigati and Murphy, 1984). However, smooth be hereby by the payment of page charges. This article must therefore marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 muscle actin increases dramaticaIIy upon growth arrest by the withdrawal of mitogens or by contact inhibition at high cell solely to indicate this fact. $ To whom reprint requests should be addressed Dept. of Physiology, School of Medicine, Box 449, University of Virginia, Charlottesville, VA 22908.

‘The abbreviations used are: SMC, smooth musclecell; sodium dodecyl sulfate.

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SDS,

Myosin Heavy Chain Expression Cultured in

Smooth Muscle Cells

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, 1 mM EDTA. The majority of this homogenate densities (Owens etal., 1986; Strauch and Rubenstein, 1984). oride (200 p ~ )and was mixed with an equal volume of twice-concentrated SDS sample These observations illustrate that SMCs may exhibit a conbuffer and boiled. The remainder wasassayed for DNA content tinuum of phenotypes with varying relative degrees of con- (Labarca and Paigen,1980)or protein (Lowry et al., 1951). This tractile and synthetic characteristics while in culture. Howmethod allowed us to normalize the volume of sample loaded from ever, the issue of whether myosin expression is altered by thecells at different stages of culture to a constant amount of DNA or protein. conditions of culture remains unclarified. Denaturing gel samples from intact cardiac and smooth muscle Larson et al. (1984b) showed that both subconfluent and were prepared by mixing pyrophosphate extracts from these tissues postconfluentcultures of rat mesentericartery cellswere with an equal volume of twice-concentrated denaturing sample buffer hu- and boiling as above. These extracts were prepared as described by immunofluorescently stained by polyclonal antibodies to man uterine (ASMM) and platelet (APM) myosins. These Hoh et al. (1978) except that the extracting bufferwas 80 mM in investigators also showed by quantitative SDS-polyacrylam- tetrasodium pyrophosphate and contained the proteolytic enzyme ide gel densitometry that the total amountof myosin present inhibitor mixture described above. Discontinuous SDS-Polyacrylamide Gels-The denaturing SDS gel at these stages of culture remained fairly constant. A small intact myosin diminution in ASMM-specificstainingwasnoted a t con- system of Laemmli (1970) was used for all analyses of acrylamide heavy chains. A veryporous separating gelof3-4% fluency, whereasAPM staining remained constant (Larsonet gradient with low myosin loadings (