Vladimir Matchkov

Ca²⁺ entry via L-type voltage-gated Ca²⁺ 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 1^st to 3^rd 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 Ca²⁺ handling processes.