Interrelated lipid alterations and their influence on the proliferation and fusion of cultured myogenic cells

Abstract

We have cultured myogenic cells derived from primary explants and a cell line (L6) in a lipid-depleted medium (LDM) and produced large alterations of the fatty acyl and polar headgroup composition and of the cellular sterol levels. These alterations were produced by altering the composition of the media as follows: removing biotin and providing exogenous fatty acid; removing choline and providing exogenous ethanolamine or choline analogues; and by adding 25-OH cholesterol, an inhibitor of 3-hydroxy-3-methylglutarate (HMG)-CoA reductase. Relatively small, secondary alterations of other lipid classes accompany the large primary alteration. In general, they are not obviously compensatory for the primary alteration by retaining some physical property.We have explored the influence of these lipid alterations on myoblast proliferation and fusion into myotubes. In general, considerable variability appears tolerated, but there also appear to be limits. Long-term cultures grown in media containing a single fatty acid do not proliferate indefinitely, and the fatty acid does not become the sole fatty acyl component of the phospholipids. This phenomenon is also observed for cultures enriched in phosphatidylethanolamine (PE) or phosphatidyldimethylethanolamine (PDME).The influence of the lipid alterations on fusion is particularly interesting. The inclusion of 25-OH cholesterol inhibits fusion. Enrichment of the fatty acyl chains with elaidate or the polar headgroups with PE also inhibits fusion, but in contrast to that by 25-OH cholesterol, a significant fraction of the myoblasts are aligned and interacting with each other. Oleate enrichment enhances the rate of fusion. KEY WORDS membranes lipids fusionMyogenic cells derived from primary explants or available as continuous cell lines can be grown in culture while retaining many of their differentiated membrane properties. When grown under suitable culture conditions, for example, they sort out and fuse into multinucleate myotubes, synthesize acetylcholine receptors, become electrically and chemically excitable, cluster their receptors into "hot spots", and form synapses with appropriate neurons (17-20, 22, 25, 34, 37, 41,43, 50, 56, 57, 59, 61). It is likely that the synthesis and assembly of the relevant molecular 334J. CELL BIOLOOV 9 The Rockefeller University Press 9 0021-9525/78/0501-033451.00 on

Reorganization of membrane cholesterol during membrane fusion in myogenesis in vitro: A study using the filipin-cholesterol complex.

“…Fumagalli et al (8) have shown that membrane fusion does not take place in all areas of the closely apposed plasma membranes of rat myoblast, only in small spots were the two bilayers match and are able to proceed to fusion. By contrast, physicochemical studies of myoblast fusion have been interpreted as indicating that high lipid fluidity is essential for membrane fusion (10,11,12,22). In spite of the publication of these key observations, the mechanisms of membrane fusion are not well understood.…”

Regulation of muscle plasticity and trophism by fatty acids: A short review

“…[7][8][9][10][11] In 1978, Horwitz et al 55 observed that the fatty acids added to the culture medium have a stimulatory effect on the fusion of embryonic myoblasts. The authors observed that the lipid composition of the membrane influences the proliferation and fusion of myoblasts and, consequently, the formation of multinucleated myotubes.…”

Myoblast aminophospholipid asymmetry differs from that of fibroblasts

“…Myoblasts were obtained from the pectoral muscle of 11-day chick embryos as in [13,14]. After digestion for 10 min in 0.1% dispase II (neutral protease from Bacillus polymyxa, Boehringer Mannheim Biochemicals) cells were plated on gelatin-coated 100 mm tissue culture dishes in calcium-free DMEM.…”

“…Fibroblasts were obtained from 11-day chick embryos as in [13]. The cells were maintained for 7-10 days and were subcultured at least twice during that time.…”