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
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.