Role of CaMKII in Ang-II-dependent small artery remodeling

Abstract

Angiotensin-II (Ang-II) is a well-established mediator of vascular remodeling. The multifunctional calcium-calmodulin-dependent kinase II (CaMKII) is activated by Ang-II and regulates Erk1/2 and Akt-dependent signaling in cultured smooth muscle cells in vitro. Its role in Ang-II-dependent vascular remodeling in vivo is far less defined. Using a model of transgenic CaMKII inhibition selectively in smooth muscle cells, we found that CaMKII inhibition exaggerated remodeling after chronic Ang-II treatment and agonist-dependent vasoconstriction in second-order mesenteric arteries. These findings were associated with increased mRNA and protein expression of smooth muscle structural proteins. As a potential mechanism, CaMKII reduced serum response factor-dependent transcriptional activity. In summary, our findings identify CaMKII as an important regulator of smooth muscle function in Ang-II hypertension in vivo.

Keywords: Ang-II; CaMKII; Remodeling; Smooth muscle.

Figure 1. CaMKII is activated in mesenteric arteries by chronic Ang-II infusion

(A) Immunofluorescence for CaMKII (green; smooth muscle actin, red; TO-PRO-III (nuclei), blue). NC, negative control without primary antibody (40X). (B) CaMKII activity assay in mesenteric arteries of WT mice at baseline and on day 14 of Ang-II infusion. Total activity is a measure of total CaMKII protein in the sample. Autonomous activity reflects active CaMKII. (C) Representative immunoblots for active phosphorylated (p-CaMKII) and oxidized (ox-CaMKII) CaMKII and summary data in mesenteric arteries of WT mice at baseline and on day 14 of Ang-II infusion. (D) Representative immunoblots and summary data for active phosphorylated phospholamban Th7-17 (PLB). (E) Expression of CaMKII γ and δ isoforms by qRT-PCR in mesenteric arteries of WT mice at baseline and on day 14 of Ang-II infusion. For B and E, n=3–5 independent experiments; * p<0.05 compared to WT by unpaired, two-tailed t-test.

Figure 2. CaMKII inhibition enhances vasoconstriction in mesenteric arteries after Ang-II infusion

Vasoconstriction of endothelium-denuded second-order mesenteric arteries to (A) Ang-II, (B) phenylephrine (PE), (C) serotonin (5-HT), (D) KCl. (E) Myogenic tone. (F) External diameter under Ca²⁺-free conditions, n=5–8 mice per group. * p<0.05 compared to WT by two-way ANOVA with Sidak’s multiple comparisons tests.

Figure 3. CaMKII inhibition enhances Ang-II-induced inward remodeling in mesenteric arteries

WT and TG SM-CaMKIIN mice were infused with Ang-II for 14 days, and mesenteric arteries isolated. Wall diameters of Ang-II-treated mesenteric arteries were assessed under passive conditions at a pressure of 75 mmHg. (A) External wall diameter, (B) media/lumen ratio, (C) wall thickness, and (D) circumferential stress. n=8–9 mesenteric arteries per group. * p<0.05 compared to WT by unpaired, two-tailed t-test.

Figure 4. CaMKII inhibition decreases ROCK activity and MLCK phosphorylation, consistent with increased MLCP and MLCK activity

(A, B) Representative immunoblots (A) and summary data (B) for p-MYPT and total MYPT (n=4 independent experiments). (C, D) Representative immunoblots (C) and summary data (D) for p-MLCK and total MLCK (n=4 independent experiments). (E, F) Representative immunoblots (E) and summary data (F) for p-MLC20 and total MLC20 (n=5 independent experiments). In all immunoblots, mesenteric arteries of 5–7 Ang-II-treated mice were pooled for one sample. Loading control β-actin. * p<0.05 compared to WT by unpaired, two-tailed t-test.

Figure 5. CaMKII inhibition under Ang-II treatment increases expression of structural proteins

(A) Representative immunoblots for structural proteins myosin heavy chain 11 (MYH11), myosin heavy chain 9 (MYH9), tropomyosin, myosin light chain (MLC), caldesmon, calponin, smooth muscle (SM) α-actin, cyclin E and proliferating cell nuclear antigen (PCNA). β-actin, loading control. Mesenteric arteries of 5–7 Ang-II-treated mice were pooled per lane. (B) Summary data for three independent experiments normalized to β-actin. *p<0.05 compared to WT by unpaired, two-tailed t-test.

Figure 6. CaMKII regulates SRF-dependent promoter activity

(A) qrtPCR for MYH11, MYH9, tropomyosin, myocardin and caldesmon. (B) qrtPCR for AT1A and AT1B receptors. Acidic ribosomal protein-1 (ARP1) as control. n=6. (C) Luciferase promoter assays for SRF-dependent gene transcription in the presence of myocardin and constitutively active (CA) or kinase-dead (KD) CaMKII. The amount of co-transfected CaMKII plasmid is indicated in ng. n=3 * p<0.05 compared to WT. Unpaired, two-tailed t-tests were performed for (A) and (B), Ordinary one-Way ANOVA with Dunnett’s multiple comparisons test for (C).