Figure 8. Loss of SMC differentiation and arterial contractility in i8-YT-KO mice at 8 weeks.

(A) Proteins with a role in SMC contraction that were significantly reduced in the proteomic experiment at 8 weeks after tamoxifen. Several of these, including integrin α8 (ITGA8), calponin-1 (CNN1), transgelin (SM22α), membrane primary amine oxidase (SSAO), and myosin light chain kinase (MLCK), are targets of the transcription factor myocardin (Myocd), the master regulator of SMC differentiation. To examine contractility, we mounted caudal arteries in Mulvany myographs and applied a basal tension of 5 mN. FC, fold change. (B) Shows that the internal circumference was greater in i8-YT-KO mice compared with controls (Ctrl) at 5 mN (n ≥ 18 mice and 38 arteries). (C–E) Following equilibration, arteries were stimulated with 60 mM K⁺ (C, n ≥ 18 mice and 38 arteries), the α1-adrenergic receptor agonist cirazoline (D, n ≥ 10 mice and 20 arteries), and vasopressin (E, n ≥ 10 mice and 20 arteries). Preparations were washed and maintained in a relaxed state for 25 minutes between stimuli. Transcriptomic data indicated reduced expression of Myocd at 8 weeks after tamoxifen. (F) This reduced expression was confirmed using RT-qPCR in time-course studies of the aorta (n ≥ 4). (G and H) Parallel reduction of Acta2 (G, n ≥ 4) and Mylk (H, n ≥ 4) was observed. (I) Western blot for MLCK using 8-week aortae along with quantification of the bands at 210 and 130 kDa (n = 3). (J) MLCK in caudal arteries at 8 weeks (n ≥ 6). (K) Reduction in smooth muscle myosin heavy chain (MYH11) was also confirmed by Western blotting (n = 3). (L) Results from the Godet test (n ≥ 6). This test measures time taken (in seconds) for skin to rebound from pitting and is considered an indication of edema. *P < 0.05; **P < 0.01; ***P < 0.001 by Mann-Whitney test (B and L), 2-tailed Student’s t test (C and F–K), or 2-way ANOVA with Bonferroni’s post hoc test (D and E).