mRNA and protein expression of SSA/Ro and SSB/La in human fetal cardiac myocytes cultured using a novel application of the Langendorff procedure

Citation
Ce. Tseng et al., mRNA and protein expression of SSA/Ro and SSB/La in human fetal cardiac myocytes cultured using a novel application of the Langendorff procedure, PEDIAT RES, 45(2), 1999, pp. 260-269
Citations number
56
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Pediatrics,"Medical Research General Topics
Journal title
PEDIATRIC RESEARCH
ISSN journal
0031-3998 → ACNP
Volume
45
Issue
2
Year of publication
1999
Pages
260 - 269
Database
ISI
SICI code
0031-3998(199902)45:2<260:MAPEOS>2.0.ZU;2-3
Abstract
Irreversible congenital heart block (CHB) and the transient rash of neonata l lupus are strongly associated with maternal antibodies to SSA/Ro and SSB/ La proteins; however, the precise mechanism by which these antibodies media te organ-specific injury is not yet defined. Culturing of keratinocytes has provided critical insights. Accordingly, successful culturing of human fet al cardiac myocytes at high yield would constitute a powerful tool to direc tly examine conditions that promote expression of the target autoantigens. To accomplish this aim, fetal cardiac myocytes from 18- to 22-wk abortuses were established in culture using a novel technique in which cells were iso lated after perfusion of the aorta with collagenase in a Langendorff appara tus. After preplating to decrease fibroblast contamination, cardiocytes wer e grown in flasks and slide chambers. Staining with monoclonal anti-sarcome ric a-actinin revealed the expected striations typical of cardiac myocytes in 70-90% of the cells after 4 d in culture. Furthermore, the cells were ob served to beat at rates varying between 25-75 beats per minute (bpm) after the addition of 1.8 mM CaCl2. An average yield of 45-60 x 10(6) cells was o btained from a 3- to 5-g heart. Cellular localization of SSA/Ro and SSB/La by indirect immunofluorescence and demonstration of mRNA expression by reve rse transcriptase polymerase chain reaction supports the feasibility of cul tured cardiac myocytes for the study of congenital heart block. In contrast to the increased expression of SSA/Ro reported for keratinocytes, incubati on of cultured human cardiac myocytes with either 17 beta-estradiol or prog esterone did not alter mRNA expression or cellular localization of 48 kD SS B/La, 52 kD SSA/Ro, or 60 kD SSA/Ro. In summary, we describe a novel method to successfully culture human fetal cardiac myocytes that should provide a valuable resource for investigation of the molecular mechanism(s) contribu ting to the development of congenital heart block. Differential constitutiv e and estradiol-induced expression of 52 and 60 kD SSA/Ro in human cardiac myocytes compared with keratinocytes may be a factor contributing to the ma rked discordance of clinically detectable injury in these two target tissue s.