Faculty Bibliography

In this study we report, for the first time, on the gene expression of human cardiac SERCA2a, L-type (alpha(1C)) and T-type (alpha(1H)) Ca channels during development, using RNase protection assay, relative quantitative RT-PCR and Western blot. Human hearts during early gestation (8- to 20-wk gestation), neonatal (1- to 4-d-old) and adult (18- to 48-year-old) stages were used. The results show that T-type Ca channel alpha(1H) subunit mRNA decreased and that L-type Ca channel alpha(1C) subunit mRNA increased with development. While the levels of sarcoplasmic reticulum ATPase (SERCA2a) mRNA did not significantly change with development, its protein levels increased with development. In conclusion, SERCA2a, L-type and T-type Ca channel transcripts were detected as early as 8-wk gestation. Defining the profile of Ca handling proteins during development is important to the understanding of excitation-contraction (EC)-coupling of the developing human heart.

Congenital heart block is considered a model of passively acquired autoimmune disease in which the mother generates anti-SSA/Ro and/or anti-SSB/La antibodies that cross the placenta and presumably injure the heart of developing fetus. CHB is accompanied by ECG abnormalities including AV block, sinus bradycardia, and ventricular dysfunction. Our previous data indicate that these abnormalities are caused by maternal autoantibody-mediated disturbance of L-type Ca channels. To investigate the consequence of chronic exposure of L-type Ca channels in newborn pups to maternal autoantibodies during pregnancy, we immunized female rabbits with human 52 kDa-SSA/Ro (Ro52) recombinant protein. ECG revealed that pups from the immunized group had varying degrees of conduction defects. In addition, I(CaL) density and protein were reduced in hearts of pups from the immunized group. Sera and purified IgG from immunized rabbits inhibited I(Ba) recorded from oocytes with expressed alpha(1C) and beta(2a) subunits of L-type Ca channel. Pups born to Ro52 immunized mothers exhibited down-regulation of L-type calcium channels in heart. The data provide new insight into the pathogenesis of congenital heart block.

Congenital heart block (CHB) affects offspring of mothers with autoantibodies (positive IgG) to intracellular SSA/Ro and SSB/La ribonucleoproteins and is associated with high morbidity and mortality. Here, we show that maternal anti-Ro/La antibodies immunoreact with human fetal cardiomyocyte sarcolemma, recognize human L-type Ca channel alpha(1C)-protein and functionally inhibit expressed current in oocytes injected with alpha(1C) cRNA and Purkinje L-type Ca current. Furthermore, cardiac myocytes from pups born to SSA/Ro-immunized mice exhibited reduced L-type Ca current density. All together, the data establish that L-type calcium channel is a target for maternal antibodies and may provide a functional basis for the electrocardiographic abnormalities seen in infants with CHB.

BACKGROUND:Congenital heart block (CHB) is a disease that affects the offspring of mothers with autoimmune diseases. We recently reported that maternal sera containing antibodies against SSA/Ro and SSB/La ribonucleoproteins (positive IgG) inhibited L-type Ca current in isolated cardiac myocytes and induced sinus bradycardia in a murine model of CHB. The direct interaction of positive IgG with L-type Ca channel proteins and the possible inhibition of T-type Ca current that could account for the sinus bradycardia remain unknown. METHODS AND RESULTS/RESULTS:The 2-electrode voltage-clamp technique was used to record currents via L-type (I(Ba)-alpha(1C) or I(Ba)-alpha(1C)+beta(2a)+alpha(2)/delta) and T-type (I(Ba)-alpha(1H)) Ca channels, Na channels (I(Na)-hH1), and K channels (I(Ks)-minK+KvLQT1) expressed in Xenopus oocytes. Positive IgG (350 microgram/mL) inhibited I(Ba)-alpha(1C) by 50.6+/-4.7% (P<0.01) and I(Ba)-alpha(1C)+beta(2a)+alpha(2)/delta by 50.9+/-4.2% (P<0.01); I(Ba)-alpha(1H) was reduced by 18.9+/-1.0% (P<0.01). Immunoblot data show cross-reactivity of positive IgG with alpha(1C) subunit. Pretreatment of oocytes with atropine (1 micromol/L) or acetylcholine (10 micromol/L) did not affect the inhibitory effect of IgG on I(Ba)-alpha(1C) and I(Ba)-alpha(1C)+beta(2a)+alpha(2)/delta (P<0.05). Positive IgG had no effect, however, on either I(Na)-hH1 or I(Ks)-minK+KvLQT1. CONCLUSIONS:Positive IgG inhibited expressed L-type I:(Ba) and cross-reacted with the alpha(1C) subunit in Xenopus oocytes, providing strong evidence that maternal antibodies interact directly with the pore-forming alpha(1)-subunit of Ca channels. In addition, we show for the first time that positive IgG also inhibited T-type I(Ba) but not I(Na)-hH1 or I(Ks)-minK+KvLQT1. This could provide, in part, the ionic basis of sinus bradycardia reported in animal models of CHB and clinically in humans.

OBJECTIVE:T-type Ca2+ currents (I(Ca-T)) are present in neonatal rat myocytes but is not detected in adult ventricular myocytes. The present study was designed to investigate the expression of the T-type Ca2+ channel gene and current in post-infarction remodeled hypertrophied rat left ventricle (LV). METHODS:We compared the expression of T-type Ca2+ channel gene alpha-1G in neonatal rat LV, in adult sham-operated LV and remodeled hypertrophied LV 3 to 4 weeks post-myocardial infarction (MI) using RNase protection assay (RPA). The cDNA fragment of alpha-1G used in RPA was obtained from poorly conserved region of recently published T-type Ca2+ channel coding sequence of rat by RT-PCR. The fragment was verified by restriction enzyme digestion and sequencing. The presence of I(Ca-T) in LV of sham and post-MI rats was examined using patch-clamp techniques. In the presence of K+-free, Na+-free external solution, I(Ca-T) was separated from I(Ca-L) by different holding potentials (HP). I(Ca-T) was also recorded during depolarization to -40 mV from a HP of -80 mV with NaCl in external solution and I(Na) suppressed by 100 microM tetrodotoxin (TTX). RESULTS:The T-type Ca2+ channel gene alpha-1G was expressed in neonatal heart, the expression level decreased by 80%, in adult sham heart and was reexpressed in MI (158% increases compared to sham; P<0.01). I(Ca-T) was recorded in 11 of 31 MI cells in presence of K+-free, Na+-free external solution and in 9 of 14 cells when I(Na) was suppressed by TTX. I(Ca-T) was not detected in any of 21 sham cells. I(Ca-T) density was 1.1+/-0.4 pA/pF. I(Ca-T) was more sensitive to Ni2+ and less sensitive to nisoldipine. CONCLUSIONS:T-type Ca2+ channel gene and current are reexpressed in rat post-MI remodeled LV myocytes. Its functional significance in the post-MI remodeling process remains to be defined.

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.