L-type Ca2+ channel blockers promote vascular remodeling through activation of STIM proteins

Abstract

Voltage-gated L-type Ca²⁺ channel (Ca_v1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca²⁺ signaling machinery, including down-regulation of Ca_v1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca²⁺ sensor proteins and the plasma membrane ORAI Ca²⁺ channels. STIM/ORAI proteins mediate store-operated Ca²⁺ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca²⁺, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca²⁺ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Ca_v1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca²⁺ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.

Keywords: Cav1.2; STIM1; calcium signaling; hypertension; vascular remodeling.

Fig. 1.

LCCBs increase intracellular Ca²⁺ and induce VSMC remodeling. (A) Synthetic VSMCs immune-stained with α-SMA antibody and Hoechst. (Scale bar: 100 μm.) (B) Cytosolic Ca²⁺ measurements in VSMCs (Fura2) stimulated with 20 μM amlodipine or vehicle. (C) Quantification of maximal Ca²⁺ entry from B. (D) Cytosolic Ca²⁺ in VSMCs stimulated with 20 μM diltiazem or vehicle. (E) Quantification of maximal Ca²⁺ entry from D. (F) Cytosolic Ca²⁺ in VSMCs (Fluo-4) stimulated with 0.5 to 20 μM amlodipine. (G) Quantification of maximal Ca²⁺ entry from F. (H and I) Quantification of migration in VSMC stimulated with vehicle, amlodipine (0.5 μM), PDGF (0.5 ng/mL), amlodipine (0.5 μM) + PDGF (0.5 ng/mL), or PDGF (10 ng/mL) at 12 to 24 h (H and I). (J) Bright field images of VSMC migration. (Scale bar: 500 μm.) (K) VSMC proliferation over 72 h with vehicle, amlodipine (0.5 μM), PDGF (0.5 ng/mL), amlodipine (0.5 μM) + PDGF (0.5 ng/mL), or PDGF (10 ng/mL). Ca²⁺ imaging: ****P < 0.0001. VSMC migration and proliferation: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 when compared to vehicle. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared with 0.5 ng/mL PDGF. Unpaired Student’s t test for two comparisons and ANOVA with Dunnett’s test for multiple comparisons.

Fig. 2.

LCCBs activate I_CRAC which require STIM and ORAI. (A) Cytosolic Ca²⁺ measurements in HEK293 cells stimulated with amlodipine or vehicle. (B) Quantification of maximal Ca²⁺ entry from A. (C) Whole-cell patch-clamp recording of native I_CRAC in HEK293 cells stimulated first with vehicle (dimethyl sulfoxide [DMSO]) and then 20 μM amlodipine. (D) Ca²⁺ current/voltage (I/V) curves from C, taken where indicated by “1” and “2”. (E) Whole-cell patch-clamp recording from HEK293 cells expressing eYFP-STIM1 and mCherry-ORAI1 stimulated first with DMSO and then amlodipine. (F) Ca²⁺ I/V curves of E, taken where indicated by “3” and “4”. (G) Quantification of peak current density activated by amlodipine. (H) Quantification of peak current density activated by amlodipine in HEK293 cells coexpressing eYFP-STIM1 and mCherry-ORAI1. (I) Cytosolic Ca²⁺ measurement in ORAI1/2/3-TKO cells, STIM1/2-DKO cells, and HEK293 cells stimulated with amlodipine. (J) Quantification of maximal Ca²⁺ entry from I. (K) STIM1/2-DKO cells expressing 1 μg of either an empty plasmid, or CFP-ORAI1, or coexpressing STIM1-eYFP (2 μg) and CFP-ORAI1 (1 μg) and stimulated with amlodipine. (L) Quantification of maximal Ca²⁺ entry from K along with that of similar experiments performed on STIM1/2-DKO cells transfected with CFP-ORAI2 (1 μg), or CFP-ORAI3 (1 μg) either alone or cotransfected with STIM1-eYFP (2 μg) and stimulated with amlodipine. Also quantified experiments using cells coexpressing STIM2-eYFP (instead of STIM1-eYFP) and CFP-ORAI1 and stimulated with amlodipine (traces in SI Appendix, Fig. S2). (M–R) Cytosolic Ca²⁺ traces and quantifications of maximal Ca²⁺ entry from STIM1/2-DKO cells with STIM/ORAI expression combinations in K and L, except now cells are stimulated with other LCCBs. Cells are stimulated with either nifedipine (M and N), verapamil (O and P), or diltiazem (Q and R). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Unpaired Student’s t test for two comparisons and ANOVA with Dunnett’s test for multiple comparisons.

Fig. 3.

LCCBs cause STIM1/ORAI1 interaction and puncta formation. (A) Confocal images of localization of CFP-ORAI1 (1 μg) and STIM1-eYFP (2 μg) and the merged distribution in STIM1/2-DKO cells. Images of cells before (0) and after treatment (8 min) with vehicle. (B and C) Cells before (0) and after (8 min) stimulation with either amlodipine or diltiazem. (Scale bar: 10 μm.) (D) Sensitized emission Förster resonance energy transfer (seFRET) between STIM1-eYFP (2 μg) and CFP-ORAI1 (1 μg) expressed in STIM1/2-DKO cells and stimulated with diltiazem or vehicle. (E) Quantification of maximum seFRET. (F) YFP/CFP ratios from E. (G) seFRET between STIM1-eYFP (2 μg) and CFP-ORAI1 (1 μg) expressed in STIM1/2-DKO cells and stimulated with either thapsigargin or vehicle. (H) Quantification of maximum seFRET from G. (I) YFP/CFP ratios from G. ****P < 0.0001; ns, not significant (unpaired Student’s t test).

Fig. 4.

LCCBs do not deplete ER Ca²⁺ stores. (A) Cytosolic Ca²⁺ measurements and (B) quantification of maximal cytosolic Ca²⁺ in HEK293 cells stimulated with either amlodipine or vehicle. Amlodipine was first added to cells in nominally Ca²⁺-free, and when 2 mM Ca²⁺ was restored to the bath. (C) ER Ca²⁺ measurements in HEK293 cells transfected with the ER Ca²⁺ sensor GCaMP6-150 after addition of either amlodipine or vehicle. At the end of each recording, 1 μM ionomycin was added to fully empty Ca²⁺ stores. (D) Similar to C, diltiazem or vehicle was added. (E) At 14 min, normalized GCaMP6-150 fluorescence between vehicle, amlodipine, and diltiazem was quantified. (F) ER Ca²⁺ measurements in HEK293 cells after stimulation with either CPA or (G) carbachol. (H) Quantification of GCaMP6-150 fluorescence after carbachol or CPA stimulation at 14 min. (I) Images of HEK293 cells expressing GCaMP6-150. (Scale bar: 10 μm.) ****P < 0.0001, ns, not significant (ANOVA with Dunnett’s multiple comparison test).

Fig. 5.

The target of LCCBs is STIM. (A) Confocal images of distribution of CFP-ORAI1 (1 μg) expressed in STIM1/2-DKO cells before and after treatment for 8 min with either vehicle, diltiazem, or amlodipine. (Scale bar: 10 μm.) (B) Confocal images show distribution of STIM1-eYFP (1 μg) in ORAI1/2/3-TKO cells before and after treatment for 8 min with either vehicle, diltiazem, or amlodipine. (Scale bar: 10 μm.) (A and B) Zoom-in Insets. (Scale bar: 2.5 μm.) (C) Quantification of ORAI1 and STIM1 puncta upon stimulation with vehicle, diltiazem, and amlodipine. (D) Truncated STIM1 constructs (STIM1_1–310-CFP) and YFP-tagged SOAR1 (YFP-SOAR1) with the denoted domains: signal peptide (SP), EF hand, sterile α motif (SAM), transmembrane (TM), and coiled-coil 1 (CC1). (E) Epifluorescence images of STIM1/2-DKO cells coexpressing STIM1_1–310-CFP (1 μg), YFP-SOAR1 (1 μg), colocalization, and seFRET. (Scale bar: 10 μm.) (F) seFRET between STIM1_1–310-CFP and YFP-SOAR1-SOAR1 expressed in STIM1/2-DKO cells stimulated with either diltiazem or vehicle. At the end, cells were stimulated with 1 μM ionomycin (Iono) for maximal dissociation of SOAR1 from STIM1_1–310. (G) Quantification of seFRET at 8 min. (H) Quantification of YFP/CFP ratios from F. (I) Western blots of HeLa cell membranes expressing STIM1 (A230C). Prior to isolating membranes, cells were incubated with vehicle, amlodipine, or diltiazem. Isolated membranes are then incubated with ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (0 µM Ca²⁺) or Ca²⁺ at final concentrations ranging from 0.3 to 2,000 μM during iodine-induced cross-linking. (J) The percentage of STIM1 dimer formation at each indicated Ca²⁺ concentration for cells treated with vehicle, amlodipine, or diltiazem. *P < 0.05, ****P < 0.0001, ns, not significant (unpaired Student’s t test for FRET experiments and ANOVA with Dunnett’s test for puncta experiments).

Fig. 6.

LCCBs effect requires a small N-terminal region of STIM1. (A) Confocal images of Xenopus STIM1-YFP (xSTIM1, 4 μg) and Xenopus ORAI1-CFP (xORAI1, 2 μg) in STIM1/2-DKO cells before and after treatment for 8 min with thapsigargin (TG). (B) Similar images showing localization of xSTIM1-YFP and xORAI1-CFP before and after treatment with amlodipine. (C) Quantification of xSTIM1or hSTIM1 puncta after stimulation with amlodipine. (D) SOCE triggered by thapsigargin in STIM1/2-DKO cells cotransfected with either xSTIM1-YFP and xORAI1-CFP or an empty plasmid. (E) Quantification of maximal Ca²⁺ entry from D. (F) Cytosolic Ca²⁺ measurements on STIM1/2-DKO cells cotransfected with either xSTIM1 + xORAI1 or with hSTIM1 + hORAI1. Cells were stimulated with amlodipine. (G) Quantification of maximal Ca²⁺ signals from F. (H) SOCE was induced with thapsigargin in STIM1/2-DKO cells expressing ORAI1-CFP (1 µg) with either STIM1 (2 µg), or STIM1-Δ29–46, or STIM1-Δ31–40. (I) Quantification of maximal Ca²⁺ entry from H. (J) Confocal images of STIM1, STIM1-Δ29–46, and STIM1-Δ31–40 expressed in STIM1/2-DKO cells before and after thapsigargin. (K) Amlodipine was used to stimulate Ca²⁺ entry in STIM1/2-DKO cells expressing ORAI1-CFP (1 µg) with 2 µg plasmid of either full-length STIM1, or STIM1-Δ29–46, or STIM1-Δ31–40. (L) Quantification of maximal Ca²⁺ entry from K. (M) Western blots of HeLa cell membranes expressing STIM1 (A230C). For “Intact Cells,” cells were incubated with either amlodipine or vehicle and then lysed, and membranes were isolated. For the “Membranes” condition, cells were lysed first, and the isolated membranes were incubated with either amlodipine or vehicle. For both conditions, the isolated membranes were then incubated with EGTA (0 µM Ca²⁺) or Ca²⁺ at final concentrations ranging from 0.3 to 2,000 μM, cross-linked and analyzed by nonreducing SDS/PAGE. (N) Quantification of the percentage of STIM1 dimer after treatment with either amlodipine or vehicle at each Ca²⁺ concentration. (A, B, and J) Scale bars: 10 μm and 2.5 μm for Zoom in Insets. **P < 0.01, ****P < 0.0001 (unpaired Student’s t test for two comparisons and ANOVA with Dunnett’s test for multiple comparisons).

Fig. 7.

LCCBs promote vascular remodeling through STIM1. (A) STIM1 Western blots of VSMCs transfected with Scrambled shRNA (shScr.) and two STIM1 shRNA (shSTIM1#1 [shS1 #1] and shSTIM1#2 [shS1 #2]). (B) STIM1 protein quantification from A using densitometry normalized to tubulin. (C) SOCE triggered by thapsigargin in VSMC stably expressing either shScramble, or shSTIM1#1, or shSTIM1#2. (D) Quantification of maximal Ca²⁺ entry from C. (E) Quantification of VSMC migration at 24 h in ShScramble and shSTIM1-expressing VSMCs treated with either 0.5 ng/mL PDGF or 0.5 μM amlodipine + 0.5 ng/mL PDGF. (F) Quantification in normalized relative fluorescence units (RFU) of proliferation in ShScramble- and shSTIM1-expressing VSMCs treated with either 0.5 ng/mL PDGF or 0.5 μM amlodipine + 0.5 ng/mL PDGF. (G) Bright field images of VSMC migration from E. (Scale bar: 500 μm.) (H) STIM1 and STIM2 Western blots in VSMCs acutely isolated from endothelial-denuded aortic rings of SHR and WKY rats. (I and J) Quantification of STIM1 (I) and STIM2 (J) in WKY and SHR normalized to tubulin. (K) SOCE measurements from SHR and WKY VSMCs. (L) Quantification of maximal Ca²⁺ entry from K. (M and N) Whole-cell I_CRAC induced by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) in VSMCs from WKY (M) and SHR (N). Divalent free (DVF) bath solution was used to potentiate a Na⁺ current. (O) Ca²⁺ I_CRAC and (P) Na⁺ I_CRAC I/V relationships obtained from M and N where indicated by numbers (1 to 4). (Q) Peak Ca²⁺ I_CRAC and (R) peak Na⁺ I_CRAC densities from SHR and WKY VSMCs. (S) Data from Penn State University Hospital with reported cases of heart failure when patients are treated with LCCBs or other antihypertensive drugs. Exposed control did not develop heart failure to date. In italics is the percentage of patients in that exposed group. The last row reports pooled patients exposed to antihypertensive drugs that are not LCCBs. (T) Odds ratio of tabulated results in S. Error bars are 95% CIs (*P < 0.05, ***P < 0.001; unpaired Student’s t test). ANOVA and Dunnett’s multiple comparison test were used for B and D, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E and G) #P < 0.05, ##P < 0.01, ###P < 0.001 when compared to 0.5 ng/mL PDGF.