The long-term goal is to elucidate the mechanisms underlying autoimmune-associated sinus bradycardia. Significant sinus bradycardia was observed in infants from mothers with anti-Ro/La autoantibodies, and in pups from Ro/La immunized mice. Recent findings that alphalD L-type Ca channel activates at as low as -65mV, and the fact that alphalD knock out mice exhibited unexpected significant sinus bradycardia, suggest that alpha1D plays important role in pacemaking activity of SA node. I hypothesize that L-type Ca channel alphalD subunit could be the preferential target for anti-Ro/La antibodies, and may contribute to the development of autoimmune-associated sinus bradycardia. Information to be gained from this proposal will provide novel diagnostic and therapeutic insights into the autoimmune-associated sinus bradycardia. ? The specific aims are: 1. To test the effect of anti-Ro/La antibodies on L-type Ca channel alphalD expressed in Xenopus oocytes, because of the unavailable pharmacological agent to separate L-type alphalD from alphalC current in native cells. 2. To test the effect of anti-Ro/La antibodies on L-type Ca current activity of SA node cells obtained from alpha1D+/+ mice and alpha1 D-I- mice. Whole-cell L-type Ca current density before and after application of anti-Ro/La antibodies will be recorded and compared using patch-clamp technique on SA node cells obtained from alphalD+/+ and from alphalD-/- transgenic mice. 3. To identify the expression of Ca channel alphalD subunit in human fetal heart SA node cells. Single cell RT-PCR and indirect immunostaining will be used since alphalD is also expressed in neuronal and vascular tissue. ? ?
