TY - ABST

T1 - siRNA-induced in vivo downregulation of L-type calcium channels in rat small mesenteric arteries

AU - Møller, Kate

AU - Aalkjær, Christian

AU - Matchkov, Vladimir

N1 - Conference code: 14

PY - 2009

Y1 - 2009

N2 - Previous gene deletion studies have provided insight into the critical role of L-type voltage-gated Ca2+ channels (Cav1.2) in regulation of blood pressure. Homozygous knockout is, however, lethal but this limitation can be overcome by a small RNA interference (siRNA). A specific downregulation of gene expression with siRNA can be a helpful tool in investigations of proteins in the vascular bed. The 1st to 3rd order branches of the mesenteric artery of anestisized Wistar rats were transfected with siRNAs targeting Cav1.2 or with the control non-related siRNAs. The effect of transfection was evaluated after 3 days using qPCR and isometric myography. In comparison to some other genes the expression of Cav1.2 is very sensitive to transfection procedure (see abstract Broegger et al.). The optimization has been made to avoid the unwanted changes in mRNA in the controls transfected with non-related siRNAs. The level of Cav1.2 mRNA correlated with the functional responses, although when Cav1.2 mRNA was above 60% of the control no changes in the contractility were seen. When mRNA was <50% it accompanied with significant reduction in the contractile responses.  We conclude that although in vivo siRNA-induced downregulation in small arteries is possible the optimization of the procedure for the specific gene is necessary. Analyses at both functional and molecular biological levels are beneficial for correct evaluation of the efficiency of downregulation. Due to its the importance for vascular reactivity, Cav1.2 can be suggested as a good candidate for this siRNA optimization procedure.

AB - Previous gene deletion studies have provided insight into the critical role of L-type voltage-gated Ca2+ channels (Cav1.2) in regulation of blood pressure. Homozygous knockout is, however, lethal but this limitation can be overcome by a small RNA interference (siRNA). A specific downregulation of gene expression with siRNA can be a helpful tool in investigations of proteins in the vascular bed. The 1st to 3rd order branches of the mesenteric artery of anestisized Wistar rats were transfected with siRNAs targeting Cav1.2 or with the control non-related siRNAs. The effect of transfection was evaluated after 3 days using qPCR and isometric myography. In comparison to some other genes the expression of Cav1.2 is very sensitive to transfection procedure (see abstract Broegger et al.). The optimization has been made to avoid the unwanted changes in mRNA in the controls transfected with non-related siRNAs. The level of Cav1.2 mRNA correlated with the functional responses, although when Cav1.2 mRNA was above 60% of the control no changes in the contractility were seen. When mRNA was <50% it accompanied with significant reduction in the contractile responses.  We conclude that although in vivo siRNA-induced downregulation in small arteries is possible the optimization of the procedure for the specific gene is necessary. Analyses at both functional and molecular biological levels are beneficial for correct evaluation of the efficiency of downregulation. Due to its the importance for vascular reactivity, Cav1.2 can be suggested as a good candidate for this siRNA optimization procedure.

M3 - Conference abstract in journal

JO - Hypertension

JF - Hypertension

SN - 0194-911X

Y2 - 9 October 2009 through 11 October 2009

ER -