Faculty Bibliography

Congenital heart block (CHB), detected at or before birth in a structurally normal heart, is strongly associated with autoantibodies reactive with the intracellular soluble ribonucleoproteins 48kD SSB/La, 52kD SSA/Ro, and 60kD SSA/Ro. CHB is presumed to be due to the transplacental passage of autoantibodies from the mother into the fetal circulation. Varying degrees of heart block have been reported. Although second degree block has, on rare occasion, reverted to normal sinus rhythm, complete atrio-ventricular (AV) block is irreversible. CHB carries substantial mortality and morbidity, with > 60% of affected children requiring lifelong pacemakers. The recurrence rate exceeds, by at least twofold, that of the first birth and is likely to influence the decision to have more children. Curiously, the mother's heart is almost never affected (with complete heart block) despite exposure to identical circulating autoantibodies. As part of our continuing effort to understand the complex factors contributing to the pathogenesis of CHB, we have established an animal model of CHB by immunizing female mice with recombinant proteins/antigens, reproduced the human complete AV block in an isolated Langendorff perfused fetal heart, and correlated these findings with L-type Ca channel inhibition by maternal antibodies from mothers of children with CHB. In addition, we established a passive animal model by directly injecting maternal antibodies into pregnant mice and reported significant sinus bradycardia, indicating that the spectrum of conduction abnormalities may extend beyond the AV node. All together, the data provided strong evidence supporting an etiologic role of antibody/Ca channel involvement in the pathogenesis of CHB. However, other yet unknown factors seem necessary to explain the full expression of CHB.

OBJECTIVE:In immature animal hearts, lower activity of sarcoplasmic reticulum and lower densities of Ca2+ channels highlight the potentially vital role of the Na+/Ca2+ exchanger (NCX) to excitation-contraction coupling. To date, studies on NCX expression have been restricted to late developmental stages. The distribution and gene expression of NCX during early ontogeny is not known, especially in humans. In the present report, we systematically characterized changes in NCX gene expression in human heart during development, with particular emphasis in early ontogeny. METHODS:Human hearts during early gestation (9- to 20-week gestation), neonatal (1 to 2 days after birth) and adulthood (18-40 years old) were used. NCX mRNA levels were studied using RNase Protection Assay (RPA) and NCX protein levels were assessed by Western blot. Wet weight was also used as the tissue base. Immunolocalization studies using confocal microscopy were performed in isolated fetal cardiac myocytes. RESULTS:Normalization of NCX mRNA derived from ventricles against an early gestational age (10-week gestation) shows that NCX mRNA levels nominally increased from 1 to 1.13 at 19-week gestation then decreased to 0.74 (P < 0.05) at neonate and further decreased to 0.23 (P < 0.05) at adult stages. NCX protein levels increased from 1 at 9-week gestation to 3 (P < 0.05) at 20-week gestation and then decreased to 1.8 (P < 0.05) at neonate and to 1.87 (P < 0.05) at adult stages. Confocal imaging of fetal cardiac myocytes revealed intense homogeneous membrane staining and abundance of NCX protein at this stage. CONCLUSIONS:The data demonstrate changes in NCX transcript and NCX protein levels as well as total RNA and proteins during human heart development. Per wet weight, NCX mRNA was 4.5 times greater at early fetal than adult stages and NCX protein was 2 times greater at adult than the early fetal stage indicating considerable post-transcriptional regulation. These findings provide new insights into the understanding of temporal changes in NCX in the developing heart at the gene level. The functional significance remains to be determined.

Three weeks after myocardial infarction (MI) in the rat, remodeled hypertrophy of noninfarcted myocardium is at its maximum and the heart is in a compensated stage with no evidence of heart failure. Our hemodynamic measurements at this stage showed a slight but insignificant decrease of +dP/dt but a significantly higher left ventricular end-diastolic pressure. To investigate the basis of the diastolic dysfunction, we explored possible defects in the beta-adrenergic receptor-G(s/i) protein-adenylyl cyclase-cAMP-protein kinase A-phosphatase pathway, as well as molecular or functional alterations of sarcoplasmic reticulum Ca(2+)-ATPase and phospholamban (PLB). We found no significant difference in both mRNA and protein levels of sarcoplasmic reticulum Ca(2+)-ATPase and PLB in post-MI left ventricle compared with control. However, the basal levels of both the protein kinase A-phosphorylated site (Ser16) of PLB (p16-PLB) and the calcium/calmodulin-dependent protein kinase-phosphorylated site (Thr17) of PLB (p17-PLB) were decreased by 76% and 51% in post-MI myocytes (P<0.05), respectively. No change was found in the beta-adrenoceptor density, G(salpha) protein level, or adenylyl cyclase activity. Inhibition of phosphodiesterase and G(i) protein by Ro-20-1724 and pertussis toxin, respectively, did not correct the decreased p16-PLB or p17-PLB levels. Stimulation of beta-adrenoceptor or adenylyl cyclase increased both p16-PLB and p17-PLB in post-MI myocytes to the same levels as in sham myocytes, suggesting that decreased p16-PLB and p17-PLB in post-MI myocytes is not due to a decrease in the generation of p16-PLB or p17-PLB. We found that type 1 phosphatase activity was increased by 32% (P<0.05) with no change in phosphatase 2A activity. Okadaic acid, a protein phosphatase inhibitor, significantly increased p16-PLB and p17-PLB levels in post-MI myocytes and partially corrected the prolonged relaxation of the [Ca(2+)](i) transient. In summary, prolonged relaxation of post-MI remodeled myocardium could be explained, in part, by altered basal levels of p16-PLB and p17-PLB caused by increased protein phosphatase 1 activity.

INTRODUCTION/BACKGROUND:L-type calcium channels were studied in cell-attached patches from ventricular cell membranes of human fetal heart. METHODS AND RESULTS/RESULTS:Experiments were performed in the presence of 70 mM Ba2+ as the charge carrier at 22 degrees C to 24 degrees C. Unitary current sweeps were evoked by 300-msec depolarizing pulses to 0 mV from a holding potential of -50 mV at 0.5 Hz. Recorded currents were blocked by nisoldipine (1 microM) and stimulated by (-)Bay K 8644 (1 microM). During control, channel activity was seen in 13.9%+/-4.2% of the total 200 sweeps. Ensemble average current amplitude was 0.03+/-0.01 pA (n = 6) and average conductance was 20.4+/-0.2 pS (n = 5). Analysis of single channel kinetics showed open time and closed time histograms were best fit by one and two exponentials, respectively. Mean open time was tau(o) = 0.99+/-0.05 msec (n = 6). Mean closed time fast (tau(cf)) and slow (tau(cs)) component values were tau(cf) = 0.85+/-0.09 msec and tau(cs) = 8.0+/-0.94 msec (n = 6), respectively. With intrapipette (-)Bay K 8644 (1 microM), mean open time was best fit by two exponentials, tau(of) = 0.9+/-0.2 msec (n = 10) and tau(os) = 13.4+/-2.6 msec (n = 10); mean close time values were tau(cf) = 0.6+/-0.1 msec (n = 10) and tau(cs) = 9.8+/-1.9 msec (n = 10), respectively. With (-)Bay K 8644, channel activity was 66.5%+/-7.4%, the ensemble average current was 0.52+/-0.04 pA (n = 10) and the conductance 20.7+/-0.5 pS (n = 5). CONCLUSION/CONCLUSIONS:(1) the data establishes the characteristics of L-type Ca channels of human fetal hearts and their modulation by dihydropyridines; (2) the open time kinetics differ from those of avian embryonic and rat fetal hearts; and (3) the findings provide new and relevant information for understanding the physiologic behavior of unitary Ca2+ channels in the developing human heart and the baseline comparison for diseases that implicate Ca2+ channels in their etiology, such as autoimmune-associated congenital heart block.

BACKGROUND:It is a widely held view that congenital heart block (CHB) is caused by the transplacental transfer of maternal autoantibodies (anti-SSA/Ro and/or anti-SSB/La) into the fetal circulation. To test this hypothesis and to reproduce human CHB, an experimental mouse model (BALB/c) was developed by passive transfer of human autoantibodies into pregnant mice. METHODS AND RESULTS/RESULTS:Timed pregnant mice (n=54) were injected with a single intravenous bolus of purified IgG containing human anti-SSA/Ro and anti-SSB/La antibodies from mothers of children with CHB. To parallel the "window period" of susceptibility to CHB in humans, 3 groups of mice were used: 8, 11, and 16 days of gestation. Within each group, we tested 10, 25, 50, and 100 microg of IgG. At delivery, ECGs were recorded and analyzed for conduction abnormalities. Bradycardia and PR interval were significantly increased in 8-, 11-, and 16-day gestational groups when compared with controls (P<0.05). QRS duration was not significantly different between all groups. Antibody levels measured by ELISA in both mothers and their offspring confirmed the transplacental transfer of the human antibodies to the pups. CONCLUSIONS:The passive transfer model demonstrated bradycardia, first-degree but not complete atrioventricular block in pups. The greater percentage and degree of bradycardia and PR prolongation in the 11-day mouse group correlates with the "window period" of susceptibility observed in humans. The high incidence of bradycardia suggests possible sinoatrial node involvement. All together, these data provide relevant insights into the pathogenesis of CHB.

Irreversible congenital heart block (CHB) and the transient rash of neonatal lupus are strongly associated with maternal antibodies to SSA/Ro and SSB/La proteins; however, the precise mechanism by which these antibodies mediate organ-specific injury is not yet defined. Culturing of keratinocytes has provided critical insights. Accordingly, successful culturing of human fetal cardiac myocytes at high yield would constitute a powerful tool to directly 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 isolated after perfusion of the aorta with collagenase in a Langendorff apparatus. After preplating to decrease fibroblast contamination, cardiocytes were grown in flasks and slide chambers. Staining with monoclonal anti-sarcomeric alpha-actinin revealed the expected striations typical of cardiac myocytes in 70-90% of the cells after 4 d in culture. Furthermore, the cells were observed 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 obtained from a 3- to 5-g heart. Cellular localization of SSA/Ro and SSB/La by indirect immunofluorescence and demonstration of mRNA expression by reverse transcriptase polymerase chain reaction supports the feasibility of cultured cardiac myocytes for the study of congenital heart block. In contrast to the increased expression of SSA/Ro reported for keratinocytes, incubation of cultured human cardiac myocytes with either 17beta-estradiol or progesterone did not alter mRNA expression or cellular localization of 48 kD SSB/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) contributing to the development of congenital heart block. Differential constitutive 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 marked discordance of clinically detectable injury in these two target tissues

To correlate the arrhythmogenic effects of maternal autoantibodies with the genesis of congenital heart block, female BALB/c mice were immunized with human recombinant 48-kDa SSB/La, 60-kDa SSA/Ro, 52-kDa SSA/Ro (52alpha), and 52beta (amino acids 169-245 deleted) as well as with murine recombinant 52-kDa SSA/Ro. Control animals received beta-galactosidase or a polypeptide encoded by pET-28 alone. Following primary immunization and two boosters, high titer responses to the respective Ags were established by ELISA, immunoblotting, and immunoprecipitation. Sera from mice immunized with either human 52alpha or 52beta immunoprecipitated murine 52Ro. mRNA and protein expression of 52Ro was demonstrated in the newborn murine heart. A spectrum of atrioventricular nodal conduction abnormalities was identified by electrocardiogram. First-degree block was detected in 7% of 27 pups born to mothers immunized with 48La, 20% of 54 pups born to 60Ro-immunized mothers, 6% of 56 pups born to 52alpha-immunized mothers, 7% of 86 pups born to 52beta-immunized mothers, and 9% of 22 pups born to mothers immunized with murine 52Ro. Advanced conduction abnormalities were only identified in offspring of 52alpha- or 52beta-immunized mice. In the 52alpha group, one pup had complete block and another had second-degree block (Wenckebach type); in the 52beta group, five pups had complete block. Maternal Abs to the primary immunogens were detected in the pups. No control had any conduction abnormalities. This Ab-specific animal model provides strong evidence for a pathogenic role of anti-SSA/Ro-SSB/La Abs, particularly 52Ro, in the development of congenital heart block. The range and frequency of conduction defects suggest that additional factors promote disease expression

Although a strong clinical association exists between congenital heart block (CHB) and an immune response to SSA/Ro and SSB/La proteins, a causative role of these antibodies in the pathogenesis is just emerging. In a preliminary report, we have demonstrated that IgG fractions isolated from the sera of mothers whose children have CHB are arrhythmogenic in the human fetal heart. To more precisely define the arrhythmogenic effect of anti-SSA/Ro-SSB/La antibodies, we used the readily available rat heart model to record: 1) ECGs from Langendorff beating hearts; 2) action potentials from atrioventricular (AV) nodal preparations; 3) L-type Ca currents, I(Ca) at the whole-cell and single channel levels; and 4) other currents such as the transient outward K+ current, I(to), the inward rectifier K+ current, I(K1), and the Na+ current, I(Na). Perfusion of hearts with purified IgG (800 microg/mL), isolated from the serum of a mother with SSA/Ro and SSB/La antibodies whose child had CHB, resulted in bradycardia associated with 2:1 AV block. Simultaneous action potentials were recorded from dissected atrial and AV nodal areas of the rat heart. Superfusion of these preparations with the same mother's IgG fraction resulted in 2:1 AV block followed by complete inhibition of AV nodal action potential. Because AV nodal electrogenesis is largely dependent on I(Ca), the effect of these antibodies on I(Ca) was subsequently determined. Superfusion of myocytes with whole serum or purified IgG (80 microg/mL) from the same mother consistently inhibited whole cell I(Ca), ensemble average Ba2+ currents (I(Ba)) and open state probability, p(o), without affecting the channel conductance. IgG had no significant effect on I(to), I(K1), or I(Na). Whole sera and IgG fractions from a healthy mother with no detectable anti-SSA/Ro or SSB/La antibodies did not inhibit I(Ca) or I(Ba). These results demonstrate that IgG containing anti-SSA/Ro and -SSB/La antibodies induces complete AV block in beating hearts and in multicellular preparations, thus implicating a preferential interaction of these autoantibodies with Ca channels and/or associated regulatory proteins. This is consistent with the observed inhibition of Ca channels that may be a critical factor contributing to the pathogenesis of CHB