siRNA-mediated knockdown of h-caldesmon in vascular smooth muscle.
Smooth muscle contraction involves phosphorylation of the regulatory myosin light chain. However, this thick-filament system of regulation cannot account for all aspects of a smooth muscle contraction. An alternate site of contractile regulation may be in the thin-filament-associated proteins, in particular caldesmon. Caldesmon has been proposed to be an inhibitory protein that acts either as a brake to stop any increase in resting or basal tone, or as a modulatory protein during contraction. The goal of this study was to use short interfering RNA technology to decrease the levels of the smooth muscle-specific isoform of caldesmon in intact vascular smooth muscle tissue to determine more carefully what role(s) caldesmon has in smooth muscle regulation. Intact strips of vascular tissue depleted of caldesmon produced significant levels of shortening velocity, indicative of cross-bridge cycling, in the unstimulated tissue and exhibited lower levels of contractile force to histamine. Our results also suggest that caldesmon does not play a role in the cooperative activation of unphosphorylated cross bridges by phosphorylated cross bridges. The velocity of shortening of the constitutively active tissue and the high basal values of myosin light chain phosphorylation suggest that h-caldesmon in vivo acts as a brake against contractions due to basally phosphorylated myosin. It is also possible that phosphorylation of h-caldesmon alone in the resting state may be a mechanism to produce increases in force without stimulation and increases in calcium. Disinhibition of h-caldesmon by phosphorylation would then allow force to be developed by activated myosin in the resting state.
Smolock
Elaine M
EM
Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th St., MS #488, Philadelphia, PA 19102, USA.
Trappanese
Danielle M
DM
Chang
Shaohua
S
Wang
Tanchun
T
Titchenell
Paul
P
Moreland
Robert S
RS
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002339:Carotid Arteries / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D055785:Gene Knockdown Techniques; D006632:Histamine / Q000494:pharmacology; D007551:Isotonic Contraction; D053580:Muscle Strength; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D009924:Organ Culture Techniques; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D013552:Swine; D013997:Time Factors; D014661:Vasoconstriction / Q000187:drug effects / Q000235:genetics; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D002148:Calmodulin-Binding Proteins; D018994:Myosin Light Chains; D034741:RNA, Small Interfering; D014662:Vasoconstrictor Agents; D006632:Histamine; D002118:Calcium