Ca
2+ entry via L-type voltage-gated Ca
2+ channels (Cav1.2) is a key factor in regulation of excitation-contraction coupling in smooth muscle cells (SMCs). Previous gene deletion studies have provided insight into the critical role of the pore-forming α1C subunit in regulation of blood pressure. Homozygous knockout is, however, lethal but this limitation can be overcome by transient downregulation with small 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 different exons of the α1C gene or with control non-related siRNAs. The effect of transfection was analyzed after 3 and 10 days using quantitative PCR and immunohistochemistry. The functional effects of transfection were studied using isometric myography.
Specific transfection downregulates mRNA of Cav1.2 by 93±2% within 3 days but the mRNA level recovered 10 days after transfection (110±13 % of the control level). Immunohistochemistry identified reduced Cav1.2 expression in the arteries transfected with siRNA directed against Cav1.2 α subunit. Surprisingly, normalized internal diameter of Cav1.2 downregulated arteries was significant reduced by 33±10% (n=7) vs. arteries transfected with non-related siRNA. The maximal force development to K+-depolarization was significantly reduced (by 72±10%, n=6). The responses to noradrenaline, vasopressin and caffeine were also suppressed in Cav1.2 downregulated arteries. The reduced force development was accompanied with reduced nifedipine sensitive [Ca2+]i increase. The responses to K+-depolarization and agonist-stimulation in arteries transfected with siRNA directed against α1C were upregulated in comparison with the controls 10 days after transfection. This upregulation was not, however, nifedipine-sensitive.
Using
in vivo transfection of arteries with siRNA we demonstrated the importance of Cav1.2 for vascular structure and reactivity and its tight coupling with other cellular Ca
2+ handling processes.