Essential Role of Smooth Muscle STIM1 in Hypertension and Cardiovascular Dysfunction

Abstract

Objectives: Chronic hypertension is the most critical risk factor for cardiovascular disease, heart failure, and stroke.

Approach and results: Here we show that wild-type mice infused with angiotensin II develop hypertension, cardiac hypertrophy, perivascular fibrosis, and endothelial dysfunction with enhanced stromal interaction molecule 1 (STIM1) expression in heart and vessels. All these pathologies were significantly blunted in mice lacking STIM1 specifically in smooth muscle (Stim1(SMC-/-)). Mechanistically, STIM1 upregulation during angiotensin II-induced hypertension was associated with enhanced endoplasmic reticulum stress, and smooth muscle STIM1 was required for endoplasmic reticulum stress-induced vascular dysfunction through transforming growth factor-β and nicotinamide adenine dinucleotide phosphate oxidase-dependent pathways. Accordingly, knockout mice for the endoplasmic reticulum stress proapoptotic transcriptional factor, CCAAT-enhancer-binding protein homologous protein (CHOP(-/-)), were resistant to hypertension-induced cardiovascular pathologies. Wild-type mice infused with angiotensin II, but not Stim1(SMC-/-) or CHOP(-/-) mice showed elevated vascular nicotinamide adenine dinucleotide phosphate oxidase activity and reduced phosphorylated endothelial nitric oxide synthase, cGMP, and nitrite levels.

Conclusions: Thus, smooth muscle STIM1 plays a crucial role in the development of hypertension and associated cardiovascular pathologies and represents a promising target for cardiovascular therapy.

Keywords: ER stress; cardiac hypertrophy; endothelial nitric oxide synthase; hypertension; nicotinamide adenine dinucleotide phosphate; stromal interaction molecule 1; vascular reactivity.

Figure 1. STIM1 and CHOP mediate hypertension-induced cardiac damage and fibrosis

Body weight (A, n=6) and Systolic blood pressure measured by tail cuff machine (B, n=6) in WT, heterozygous (Stim1SMC−/+) and homozygous Stim1 knockout specifically in SMC (Stim1SMC−/−), and CHOP knockout (CHOP−/−) mice infused with or without Ang II. Cardiac hypertrophy determined by evaluating heart weight/tibia length ratios (C, n=6), Cardiac fibrosis stained with the collagen-specific Sirius-red (D, n=3), Western blot and quantification showing phosphorylated and total SMAD 2/3, STIM1 (E, n=3) and ER stress markers (ATF6, BIP CHOP) (F, n=3), mRNA levels of ER stress marker (ATF6 CHOP and BIP) (G, H, I, n=3) and STIM1 (J, n=3) and, Immunohistochemistry showing STIM1 (K, n=3) and CHOP (L, n=3) in heart from WT, Stim1SMC−/− and CHOP−/− mice infused with saline or Ang II. Two-way repeated measured ANOVA followed by Tukey's Post-Hoc test were applied for figures (A, B). One-way ANOVA followed by Bonferroni Post-Hoc test were applied for figures (G, H, I, J, E, F). @p<0.05 between STIMSMC−/−, STIMSMC−/− + AngII VS WT, WT + Ang II, STIMSMC−/+, STIMSMC−/+ + Ang II, CHOP−/−, CHOP−/− + Ang II. $p<0.05 between STIMSMC−/− + AngII, CHOP−/− + Ang II vs. STIMSMC−/+ + Ang II, WT + Ang II. ^p<0.05 between WT, STIMSMC−/−, CHOP−/− vs WT + Ang II, STIMSMC−/− + AngII, CHOP−/− + Ang II. *p<0.05 between WT + Ang II vs WT, STIMSMC−/−, STIMSMC−/− + AngII, CHOP−/−, CHOP−/− + Ang II. #p<0.05 between STIMSMC−/− vs STIMSMC−/− + AngII. &p<0.05 between CHOP−/− vs CHOP−/− + Ang II.

Figure 2. STIM1 and CHOP regulate hypertension-induced vascular damage

Wire Myograph vascular reactivity showing vessel contraction response to phenylephrine (A, D, n=5–6) and ThromboxaneA2 analogue U-46619 (B, E, n=5), and endothelial-dependent relaxation in response to acetylcholine (C, F, n=5) in mesenteric resistance arteries (MRA) from WT, heterozygous (Stim1SMC−/+) and homozygous Stim1 knockout specifically in SMC (Stim1SMC/−), and CHOP knockout (CHOP−/−) mice infused with saline or Ang II. Western blot showing phosphorylated and total eNOS (G, n=3), Elisa showing cGMP levels using a sandwich enzymelinked immunosorbent assay (H, n=3) and nitrites/nitrate levels using the Griess reaction (I, n=3), protein levels and quantification of ER stress markers (ATF6, BIP and CHOP) (J, n=3), NADPH oxidase activity using lucigenin chemiluminescence (K, n=5), Western blot showing STIM1 (L, n=3), mRNA of ER stress markers (ATF6, BIP and CHOP) (M, N, O, n=3) and STIM1 (P, n=3) in mesenteric resistance arteries from WT, Stim1SMC−/− and CHOP−/− mice infused with or without Ang II. Two-way repeated measured ANOVA followed by Tukey's Post-Hoc test were applied for figures (A, B, C, D, E, F). One-way ANOVA followed by Bonferroni Post-Hoc test were applied for figures (K, L, H, I, J, M, N, O, P). *p<0.05 between STIMSMC−/− vs. WT, STIMSMC−/+, and CHOP−/−. &p<0.05 between STIMSMC−/− + AngII vs WT + Ang II, STIMSMC−/+ + Ang II, CHOP−/− + Ang II. ?p<0.05 between STIMSMC−/− + AngII, CHOP−/− + Ang II VS WT + Ang II, STIMSMC−/+ + Ang II. ^p<0.05 between WT + Ang II, STIMSMC−/+ + Ang II vs. STIMSMC−/− + AngII. #p<0.05 between WT + Ang II, STIMSMC−/+ + Ang II Vs. STIMSMC−/− + AngII vs CHOP−/− + Ang II. @p<0.05 between WT + Ang II vs WT, STIMSMC−/−, STIMSMC−/− + AngII, CHOP−/−, CHOP−/− + Ang II. $p<0.05 between STIMSMC−/− vs STIMSMC−/− + AngII. %p<0.05 between CHOP−/− vs. CHOP−/− + Ang II.

Figure 3. Effect of inhibition of NADPH oxidase and TGF-β signaling on Vascular reactivity in STIM1 and CHOP knockout mice

Wire Myograph vascular reactivity showing endothelial-dependent relaxation in response to acetylcholine before and after incubation with TGF-β inhibitors (SB431542) and NADPH oxidase inhibitor (gp91 ds-tat) in mesenteric resistance arteries from: - WT mice infused with saline or Ang II (A, B, n=5) - Heterozygous Stim1 knockout specifically in SMC (Stim1SMC−/+) mice infused with saline or Ang II (C, D, n=5) - Homozygous Stim1 knockout specifically in SMC (Stim1SMC−/−) mice infused with saline or Ang II (E, F, n=5) - CHOP knockout (CHOP−/−) mice infused with saline or Ang II (G, H, n=5) Two-way repeated measured ANOVA followed by Tukey's Post-Hoc test were applied for figures (A, B, C, D, E, F, G H). *p<0.05 between WT + Ang II + gp91 ds-tat vs WT + Ang II, WT + Ang II + SB. ^p<0.05 between WT, STIMSMC−/+ + Ang II + gp91 ds-tat vs STIMSMC−/+ + Ang II, STIMSMC−/+ + Ang II + SB. $p<0.05 between WT vs. STIMSMC−/− + Ang II + gp91 ds-tat, STIMSMC−/− + Ang II, STIMSMC−/− + Ang II + SB.

Figure 4. Effect of tunicamycin-induced ER stress on vascular reactivity in STIM1 knockout mice

Body weight (A, n=5) and systolic blood pressure measured by tail cuff machine (B, n=5), Wire Myograph vascular reactivity showing endothelial-dependent relaxation in response to acetylcholine (C, n=5) and eNOS levels determined by an ELISA Kit (D, n=3) in MRA from WT, heterozygous (Stim1SMC−/+) and homozygous Stim1 knockout specifically in SMC (Stim1SMC−/−) mice treated with saline or Tunicamycin. Two-way repeated measured ANOVA followed by Tukey's Post-Hoc test were applied for figures (A, B, C). One-way ANOVA followed by Bonferroni Post-Hoc test were applied for figures (D). $p<0.05 between STIMSMC−/+ + Tunica vs. WT, STIMSMC−/−, STIMSMC−/+. *p<0.05 between STIMSMC−/+ + Tunica, WT + Tunica vs. WT, STIMSMC−/−, STIMSMC−/+. &p<0.05 between STIMSMC−/− vs. STIMSMC−/− + Tunica. ^p<0.05 between STIMSMC−/−, STIMSMC−/− + Tunica vs. STIMSMC−/+ + Tunica, WT + Tunica. %p<0.05 between STIMSMC−/− + Tunica vs. STIMSMC−/+ + Tunica and WT + Tunica.

Figure 5. Effect of inhibition of NADPH oxidase and TGF-β signaling on Vascular reactivity in tunicamycin-treated STIM1 knockout mice

Wire Myograph vascular reactivity showing endothelial-dependent relaxation in response to acetylcholine before and after incubation with TGF-β inhibitor (SB431542) and NADPH oxidase inhibitor (gp91 ds-tat) in mesenteric resistance arteries from: - WT mice treated with saline or Tunicamycin (A, n=5) - Heterozygous Stim1 knockout specifically in SMC (Stim1SMC−/+) mice treated with saline or Tunicamycin (B, n=5) - Homozygous Stim1 knockout specifically in SMC (Stim1SMC−/−) mice treated with saline or Tunicamycin (C, n=5) Two-way repeated measured ANOVA followed by Tukey's Post-Hoc test were applied for figures (A, B, C). *p<0.05 between WT, WT + Tunica + gp91 ds-tat vs Sham + Tunica, WT + Tunica + SB. $p<0.05 between STIMSMC−/+ vs STIMSMC−/+ + Tunica + gp91 ds-tat. @p<0.05 between STIMSMC−/+ + Tunica + gp91 ds-tat vs STIMSMC−/+ + Tunica, STIMSMC−/+ + Tunica + SB. $p<0.05 between STIMSMC−/− vs STIMSMC−/− + Tunica, STIMSMC−/− + Tunica + gp91 ds-tat, STIMSMC−/− + Ang II, STIMSMC−/− + Tunica + SB.