Characterization of myosin heavy chains in cultured aorta smooth muscle cells. A comparative study.

S Kawamoto,R S Adelstein

doi:10.1016/s0021-9258(18)48234-8

Abstract

Myosin heavy chains (MHCs) from rat aorta smooth muscle cells were analyzed prior to and after these cells were placed into cell culture using sodium dodecyl sulfate-5% polyacrylamide gels, immunoblots, and two-dimensional peptide maps of tryptic digests. Rat aorta smooth muscle cells prior to culture were found to contain two MHCs (mass = 204 and 200 kDa) which cross-reacted with antibodies raised to smooth muscle myosin, but not with antibodies raised to platelet myosin. Tryptic peptide maps of these two MHCs showed no major differences when compared to each other and to maps of vas deferens and uterus smooth muscle MHCs. When rat aorta smooth muscle cells were placed into culture, the MHCs isolated from the cell extracts differed, depending on whether the cells were rapidly growing or postconfluent. Extracts from log-phase cultures contained predominantly MHCs that migrated more rapidly than smooth muscle myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mass = 196 kDa) and cross-reacted with antibodies raised to platelet myosin, but not to smooth muscle myosin. Tryptic peptide maps of this MHC were very similar to those obtained with MHCs from non-muscle sources such as platelets and fibroblasts. In contrast, extracts from postconfluent rat aorta cell cultures contained three MHCs (mass = 204, 200, and 196 kDa). Using immunoblots and peptide maps, the fastest migrating MHC was found to be identical to the 196-kDa non-muscle MHC, while the two slower migrating MHCs had the same properties as aorta smooth muscle MHCs prior to culture. These results suggest that smooth muscle cells grown in primary culture contain predominantly (greater than 80%) non-muscle myosin while actively growing, but at a postconfluent stage, contain more equivalent amounts of smooth muscle and non-muscle myosins.

Highlights

From the Laboratory of Molecular Cardiolo” gy.,National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
The proteins 204- and 200-kDa Myosin heavy chains (MHCs), we find that these ratiosdiffer
In comparing the peptide map obtained from the 204-kDa The purpose of the present study was to characterize the MHC fromprimary culture (Fig. 5B) with that obtained from MHCs found in adult aorta smooth muscle cells before and

Summary

Introduction

From the Laboratory of Molecular Cardiolo” gy.,National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892. Myosin heavy chains (MHCs) from rat aorta smooth the amino-terminal region of each heavy chain assumes a muscle cells were analyzed prior to and after these cells were placed into cell culture using sodium dodecyl sulfate-5% polyacrylamide gels, immunoblots, and two-dimensional peptide maps of tryptic digests. Rat aorta smooth muscle cells prior to culture werefound to contain twoMHCs (mass = 204 and 200 kDa) which cross-reacted with antibodies raisedto smooth muscle myosin, but not withantibodiesraisedtoplatelet myosin. It is generally accepted that quiescent differentiated smooth muscle cells can be triggered to dedifferentiate and proliferate in certain pathological conditions such as atherosclerosis [11, 12] and in cell culture [13] This feature of smooth muscle cells is unique compared to striated muscle muscle MHCs prior to culture. These cells undergo marked changes in phenotype, loss of contractility, and alteration in

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