Affinity-purified maternal CHB serum antibodies co-localize with α_1G.

<p>(<b>A</b>) Cardiomyocytes from ventricle of human fetal hearts, gestational week 20.6, were dissociated, immunofluorescence stained, and visualized with confocal microscopy. Cardiomyocytes are stained for Cav3.1 (α_1G) (Red), for the cardiomyocyte marker anti-Troponin-T (Green), and nuclei visualized with DAPI (Blue). Secondary antibodies alone gave no stain. Inset image demonstrates expression on the surface in a cross section image. Images shown are representative of various cardiomyocyte samples; similar staining patterns were obtained in four replicate experiments. Secondary antibodies, anti-rabbit-IgG-Cy3 (Red) and anti-mouse-IgG-Alexa488 (green) alone give no stain (data not shown). Note that DAPI stains the nuclei of cardiomyocytes and of other cells present in the samples, such as fibroblasts. Scale bar represents 12 µm. (<b>B</b>) Dissociated and non-permeabilized cardiomyocytes from ventricle of 20.6 week human fetal hearts, immunofluoresence stained for α_1G affinity-purified serum (red), and α_1G (green). Co-localization of staining (yellow) was assessed with confocal microscopy. Scale bar represents 12 µm.</p

Maternal sera antibody profile screening of α_1G peptides.

<p>Maternal sera from one CHB⁺ pregnancy (<b>A, C, E</b>) and one CHB⁻ pregnancy (<b>B, D, F</b>) were tested in ELISA against 15aa long overlapping peptides derived from extracellular regions of α_1G: T-type aa130–380 (<b>A, B</b>); aa774–963 (<b>C, D</b>); and, aa1308–1536 (<b>E, F</b>). Reactivity above threshold (HC: Average+3×St. Dev; set at 0.59, 0.64, 0.48 respectively for A/B, C/D, and E/F) was observed in the CHB⁺ but not CHB⁻ serum to α_1G peptides p305 (aa305–319, light grey bar) and p315 (aa315–323, dark grey bar) was observed in the CHB⁺ but not the CHB⁻ serum. Above threshold reactivity was also observed among peptides in the region spanning aa1308–1536.</p

T-type calcium channel α_1G is expressed in the AV node of human fetal hearts.

<p>(<b>A</b>) Masson's trichrome staining of 21-week human fetal heart demonstrates morphology of the AVJ region with light pink staining of the spindle-like cells in the AV node (AVN) and the AV bundle (AVB), green staining corresponding to collagen fibres. (<b>B</b>) α_1G staining (Red) is present in AVJ and AV bundle regions which are distinguishable by positive NF-160 staining (Green). Scale bars represent 100 µm (A) and 150 µm (B).</p

Expression analysis, and CHB maternal sera immune reactivity towards α_1G.

<p>(<b>A</b>) Although both α_1G and α_1H are expressed in human fetal hearts, real time PCR demonstrates that transcripts from <i>CACNA1G</i> (α_1G) are 2.2- to 4-fold higher in the AVJ than in the apex tissue (between 18–22.6 weeks gestation), whereas <i>CACNA1H</i> expression levels are between 0.7- and 1.1-fold in the AVJ compared to the apex. (<b>B</b>) Combining the data from 3 hearts (18.0 weeks-22.6 weeks), shows that <i>CACNA1G</i> expression in the AVJ is significantly higher than <i>CACNA1H</i> in the AVJ (p<0.05). (<b>C</b>) Western blot with human fetal heart lysate (20.4 weeks) demonstrates α_1G expression by a Cav3.1 commercial antibody (lane 1), that is blocked by the peptide immunogen of this antibody (aa1–22 of rat α_1G) (lane 2). CHB sera (anti-Ro and anti-La titer >100 IU) also binds to α_1G (lane 3); this reactivity is blocked by the α_1G p305 peptide (lane 4). Sera from mothers with anti-Ro/La antibodies (anti-Ro = 60 IU and anti-La titer >100 IU) giving birth to normal babies do not have immune reactivity to the α_1G protein (lane 5), and a commercial α_1H antibody confirms that the band seen is not α_1H. (<b>D</b>) α_1G isolated from human fetal heart (AV junction, ventricle, and apex) was immunoprecipitated with a Cav3.1 antibody, and the immunoblot was probed with human sera. Immunoblot was subsequently stripped and re-probed with a second Cav3.1 antibody towards a different epitope, demonstrating presence and specificity of the IP towards α_1G. Note that CHB sera is defined as anti-Ro^/La positive sera from pregnancies affected by CHB. Normal sera are from pregnancies with a healthy outcome and not affected by CHB.</p

Ion channel blocker effects on Wenckebach cycle length in atrial paced newborn and adult rabbit hearts.

<p>Wenckebach cycle length (WBCL) was recorded during perfusion with calcium channel and <i>I</i>_f channel blockers in newborn and adult rabbit hearts (WBCL in milliseconds). Equivalent effects were observed in adult and newborn hearts treated with L-type calcium channel blockers (<b>A</b>) Diltiazem and (<b>B</b>) Verapamil and with the <i>I</i>_f blocker ZD7288 (<b>C</b>) (p = <i>ns</i> repeated measures ANOVA). The T-type calcium channel blocker Mibefradil (<b>D</b>) demonstrated significant preferential AV block in the newborn hearts as compared to the adult hearts (p<0.05). Values shown are mean ± SEM, newborn (n = 7) and adult (n = 8). Diltiazem showed a trend toward a difference in sensitivity of adult and newborn hearts, however there was great variability in results between experiments. (<b>E</b>) Effects of the α_1H T-type calcium channel blocker NiCl₂ were also investigated on WBCL in newborn Langendorff rabbit hearts. WBCL measurements do not differ from each other significantly with mean ± SEM (repeated measures ANOVA p = <i>ns</i>). One heart out of 6 that appeared to be an outlier showed some variability in response to NiCl₂, and was excluded from statistical analysis.</p

Effect of maternal sera on calcium current in mouse sinoatrial node (SAN) cells.

<p>(<b>A</b>) CHB maternal serum antibodies, but not healthy control serum, show reactivity to mouse α_1G in Western blot. Commercial α_1G (Cav3.1) antibody demonstrates the α_1G positive band and calnexin (CNX) antibody probing shows equal loading of mouse heart lysate. (<b>B</b>) Calcium current density-voltage curve with CHB serum, (<b>C</b>) Calcium current density-voltage curve with normal serum, (<b>D</b>) representative Calcium current traces recording at −50 mV from holding at −80 mV with CHB serum, (<b>E</b>) representative Calcium current traces recording at −50 mV from holding at −80 mV with normal serum. CHB serum is defined as an anti-Ro^/La positive serum from a pregnancy affected by third-degree AVB or CHB. Normal serum was obtained from a mother with a pregnancy unaffected by CHB, resulting in a healthy outcome.</p

Reactivity in CHB pregnancies is specific for the p305 peptide of α_1G and can be blocked.

<p>Four overlapping 15aa peptides, and one 20aa peptide were selected to further characterize maternal sera reactivity to α_1G in mothers with CHB pregnancies (CHB⁺) compared to mothers with unaffected pregnancies (CHB⁻). Reactivity to (<b>A</b>) peptide p300 (aa300–314), (<b>B</b>) p305 (aa305–319), (<b>C</b>) p310 (aa310–324), (<b>D</b>) p315 (aa315–329) demonstrate that sera from mothers with pregnancies affected by CHB (CHB⁺), have significantly higher p305 antibody levels compared to unaffected (CHB⁻) pregnancies (p<0.05). Although p305 (aa305–319) had the highest reactivity, a longer peptide combining p305 and p310 designated p305/310 (aa305–24) demonstrated a significant difference between CHB⁺ and CHB⁻ maternal serum (<b>E</b>). Pre-incubation of one CHB⁺ maternal sera, and one healthy control (HC) sera with increasing concentration (0, 20, 40 µg/ml) of peptide p305 (aa305–319) demonstrates that the reactivity of sera is specific for this peptide and can be blocked (<b>F</b>). Error bars indicate mean ± SE.</p

Sequence identity and maternal sera reactivity for α_1G and α_1H T-type calcium channel peptides.

<p>(<b>A</b>) Alignment of peptides derived from human α_1G and α_1H S5–S6 extracellular loop I sequences. The ‘core sequence’ of interest with 10 amino acids in common among the α_1G peptides is underlined, and the seven residues that are identical in the α_1G and α_1H sequences are indicated in bold type. Note that this group is present in the sequence of each immunoreactive peptide, whereas the non-reactive peptides lack two or more of these amino acid residues. Sequences shown correspond to human CAC1G (α_1G) and human CAC1H (α_1H) (UniProt accession numbers O43497 and 095180, respectively). (<b>B</b>) Peptides derived from the aligned loop regions of α_1G and α_1H exhibit a similar pattern of CHB⁺/CHB⁻ reactivity. CHB maternal sera ELISA reactivity (OD 405 nm) was plotted with interleaved bars for comparison. Error bars represent Mean ± SE. (<b>C</b>) Reactivity of CHB⁺ maternal sera was observed to be significantly higher than HC sera, where p<0.05 for both α_1G p305 (aa305–319) and α_1H p330 (aa330–343), but did not reach significance for comparison with CHB⁻ sera (p = 0.075 and p = 0.184 for α_1G and α_1H, respectively).</p