L-type Ca2+ channel blockers promote vascularremodeling through activation of STIM proteinsMartin T. Johnsona, Aparna Gudlurb, Xuexin Zhanga

, Ping Xina, Scott M. Emricha, Ryan E. Yoasta,Raphael Courjaretc, Robert M. Nwokonkoa, Wei Lid,e,f, Nadine Hempele,g, Khaled Machacac, Donald L. Gilla,Patrick G. Hoganb

, and Mohamed Trebak ( قبيرطدمحم )a,e,1

aDepartment of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033; bDivision of Signaling andGene Expression, La Jolla Institute for Immunology, La Jolla, CA 92037 cDepartment of Physiology and Biophysics, Weill Cornell Medicine Qatar, EducationCity, Qatar Foundation, Doha, Qatar; dDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey,PA 17033; ePenn State Cancer Institute, The Pennsylvania State University College of Medicine, Hershey, PA 17033; fDepartment of Pediatrics, ThePennsylvania State University College of Medicine, Hershey, PA 17033; and gDepartment of Pharmacology, The Pennsylvania State University College ofMedicine, Hershey, PA 17033

Edited by Mark T. Nelson, University of Vermont, Burlington, VT, and approved June 2, 2020 (received for review April 26, 2020)

Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) aremajor drugs for treating hypertension, the preeminent risk factorfor heart failure. Vascular smooth muscle cell (VSMC) remodeling isa pathological hallmark of chronic hypertension. VSMC remodel-ing is characterized by molecular rewiring of the cellular Ca2+ sig-naling machinery, including down-regulation of Cav1.2 channelsand up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasmamembrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene pro-grams during cardiovascular remodeling. SOCE is activated by ago-nists that induce depletion of ER Ca2+, causing STIM to activateORAI. Here, we show that the three major classes of LCCBs activateSTIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM Nterminus to cause STIM relocalization to junctions and subsequentORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodel-ing requires STIM1, which is up-regulated in VSMCs from hyperten-sive rats. Epidemiology showed that LCCBs are more associatedwith heart failure than other antihypertensive drugs in patients.Our findings unravel a mechanism of LCCBs action on Ca2+ signalingand demonstrate that LCCBs promote vascular remodeling throughSTIM-mediated activation of ORAI. Our data indicate cautionagainst the use of LCCBs in elderly patients or patients with ad-vanced hypertension and/or onset of cardiovascular remodeling,where levels of STIM and ORAI are elevated.

Cav1.2 | STIM1 | calcium signaling | vascular remodeling | hypertension

Globally, one in four adults is diagnosed with systemic arterialhypertension, which remains the highest risk factor for

cardiovascular disease and mortality (1). Clinical complicationsinclude retinopathy, chronic kidney disease, ischemic stroke,peripheral artery remodeling, ischemic heart disease, and heartfailure. Clinical treatment of hypertension uses a spectrum of anti-hypertensive drugs, including α-blockers, β-blockers, angiotensinreceptor blockers (ARBs), diuretics, angiotensin-converting enzyme(ACE) inhibitors, and Cav1.2 voltage-gated L-type Ca2+ channelblockers (LCCBs). However, treated patients exhibit multiplecomplications, suggesting that the molecular mechanisms and cel-lular side effects of antihypertensive drugs are still elusive (2).In healthy vessels, medial vascular smooth muscle cells (VSMCs)

are quiescent and rarely proliferate and migrate. Their mainphysiological role is to contract and relax to regulate vascular tone(3). In addition to enhanced arterial tone, a significant pathologicalhallmark of hypertension is the structural remodeling of arteries,which contributes to increased peripheral resistance. Vascularremodeling is the thickening of the medial layer of arteries fromenhanced VSMC proliferation and migration, ultimately decreasingthe luminal area of the vessel and enhancing arterial resistance (4).Emerging evidence reveals that Ca2+ signaling is a significant

contributor to VSMC remodeling (5–8). VSMC remodeling ischaracterized by reprogramming of the Ca2+ signaling machin-ery (6–8). The Ca2+ channels essential for excitation–contractioncoupling, ryanodine receptors (RyRs), and Cav1.2 L-type voltage-gated Ca2+ channels are down-regulated (6, 9–12). Reciprocally,receptor-activated Ca2+ channels such as stromal-interactingmolecule (STIM)/ORAI channels, which mediate store-operatedCa2+ entry (SOCE), are up-regulated (6, 13–17).Mammals express two STIM homologs, STIM1 and STIM2,

which span the endoplasmic reticulum (ER) membrane (18, 19).In VSMCs, stimulation by agonists to phospholipase C (PLC)-coupled receptors triggers the formation of inositol-1,4,5-tri-sphosphate (IP3) (20). Diffusible IP3 induces ER Ca2+ releasethrough IP3 receptors, which depletes ER Ca2+ and causes Ca2+

dissociation from the low-affinity luminal EF-hand domains ofSTIM proteins (21–24). This causes STIM to undergo a confor-mational switch, migrate toward ER–plasma membrane (PM)junctions, and expose their C-terminal STIM-ORAI activatingregion (SOAR) (25, 26). SOAR is able to physically trap andactivate the Ca2+-selective family of PM ORAI channels. Thethree mammalian ORAI homologs, ORAI1, ORAI2, and ORAI3,mediate SOCE from the extracellular milieu into the cytosol (22).

Significance

L-type Ca2+ channel (Cav1.2) blockers (LCCBs) represent a largefamily of drugs widely used in the clinic for over 70 y to treathypertension, angina, and cardiac arrhythmias. Using geneti-cally modified cells, animal models, and human studies, wedemonstrate that all the three major classes of LCCBs activateSTIM proteins by acting on a 10-amino acid N-terminal regionlocated in the endoplasmic reticulum lumen. The activation ofSTIM triggers store-operated Ca2+ entry and promotes vascularremodeling. These results provide unique mechanistic insightsinto how widely used drugs activate a Ca2+ signaling pathwayand suggest that the use of LCCBs in patients with chronichypertension, where levels of STIM proteins and vascularremodeling are already enhanced, should be avoided.

Author contributions: M.T.J., K.M., P.G.H., and M.T. designed research; M.T.J., A.G., X.Z.,P.X., S.M.E., R.E.Y., R.C., and R.M.N. performed research; W.L., N.H., and D.L.G. contrib-uted new reagents/analytic tools; M.T.J., A.G., X.Z., R.C., K.M., P.G.H., and M.T. analyzeddata; and M.T.J. and M.T. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

See online for related content such as Commentaries.1To whom correspondence may be addressed. Email: mtrebak@psu.edu.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2007598117/-/DCSupplemental.

First published July 8, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.2007598117 PNAS | July 21, 2020 | vol. 117 | no. 29 | 17369–17380

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STIM/ORAI-mediated SOCE drives transcriptional gene pro-grams, including nuclear factor for activated T cells (NFAT)transcription factors, that promote vascular remodeling in vitroand in animal models of restenosis and hypertension (7, 8, 14, 16,22, 27–31).The three major classes of LCCB drugs used to treat hyper-

tension are as follows: dihydropyridines (e.g., amlodipine), phe-nylakylamines (e.g., verapamil), and benzothiazepines (e.g.,diltiazem); all block Cav1.2 channels to cause vasorelaxation (32).However, during chronic hypertension, VSMCs dedifferentiatefrom a contractile quiescent phenotype into a proliferative andmigratory phenotype (termed synthetic). Synthetic VSMCs havereduced contractile responses due to the decrease in expression ofcontractile proteins, including the expression of Cav1.2 channels.In contrast, synthetic VSMCs up-regulate proproliferative andpromigratory signaling pathways, including STIM/ORAI channelsactivated by receptors to growth and vasoactive agonists. Here, wereveal that all of the three major classes of LCCBs, well-describedas specific Cav1.2 channel blockers (33), unexpectedly also targetthe N terminus of STIM proteins to induce their relocalization tojunctions and subsequent activation of ORAI channels. We showthat this LCCB-mediated activation of STIM/ORAI-dependentCa2+ entry occurs independently of store depletion and Cav1.2.We further reveal that clinically relevant concentrations of LCCBsfound in the plasma of hypertensive patients treated with thesedrugs (34) stimulate VSMC proliferation and migration in a STIM1-dependent manner. We further show that STIM1 is up-regulatedand SOCE is enhanced in VSMC isolated from chronically hyper-tensive rats but not in VSMC from normotensive rats. Intriguingly,epidemiological evidence suggests that LCCBs are associated withheart failure, a clinical consequence of prolonged vascular remod-eling, more than other antihypertensive drugs. Our results revealthat LCCBs promote vascular remodeling through STIM-mediatedactivation of ORAI and indicate caution in the use of LCCBs inpatients with advanced hypertension or vascular remodeling.

ResultsLCCBs Increase Cytosolic Ca2+ and Induce VSMC Migration andProliferation. When contractile VSMCs are isolated fromhealthy vessels and cultured in vitro, they dedifferentiate into a“synthetic” phenotype, which recapitulates many of the charac-teristics of dedifferentiated VSMCs found in remodeled vesselsduring restenosis, atherosclerosis, and chronic hypertension (7,15, 35). These synthetic VSMCs represent an excellent in vitromodel for remodeled VSMCs (36). We isolated VSMC fromaortas of normotensive rats using enzymatic dispersion as de-scribed earlier (13). To ensure VSMC purity, we used immu-nofluorescence of the smooth muscle marker α-smooth muscleactin (α-SMA) (Fig. 1A). We recently showed that 20 μM of thedihydropyridine LCCB amlodipine activated a Ca2+ entry routein glioblastoma cells that bears the pharmacological features ofSOCE (37). We and others have noted that LCCBs interferewith the signal of the Ca2+ dye Fura2 and strongly blunt itsfluorescence, thus underestimating the magnitude of LCCBs-activated Ca2+ signals (37, 38). Therefore, we used Fura2 (be-ing ratiometric) with 10 mM extracellular Ca2+ to enhance thedriving force and amplify Ca2+ signals. We show that 20 μMamlodipine stimulates a robust and sustained cytosolic Ca2+

signal in synthetic VSMCs (Fig. 1 B and C). Similar results wereobtained with 20 μM of the benzothiazepine LCCB, diltiazem(Fig. 1 D and E). To ensure that low concentrations of amlodi-pine (0.5 μM), which are reminiscent of physiological concen-trations found in the plasma of hypertensive patients treated byLCCBs (34), can activate a Ca2+ signal in VSMCs, we performeda dose–response curve using the nonratiometric Ca2+ dye Fluo4and show that 0.5 μM amlodipine activated a small but statisti-cally significant Ca2+ entry in VSMCs (Fig. 1 F and G). Unlessstated otherwise, in subsequent experiments, cells were loaded

with Fura2 and stimulated with 20 μM of LCCBs in 2 mMextracellular Ca2+.Increased cytosolic Ca2+ in synthetic VSMCs has been shown

to induce VSMC proliferation and migration (6). Since the ex-pression of Ca2+ channels crucial for contraction (Cav1.2 andRyR) is decreased while that of proproliferative promigratoryCa2+ channels (STIM/ORAI) is augmented in “synthetic”VSMCs (6, 7, 13, 27), we determined whether concentrations ofLCCBs that are reminiscent of those found in the plasma ofhypertensive patients treated by LCCBs (0.5 μM) (34) can in-crease VSMC proliferation and migration. The potent mitogen,platelet-derived growth factor (PDGF), which is secreted inpathological vascular remodeling and induces smooth musclecells to proliferate and migrate (39), was used as a controlthroughout. VSMC migration was measured using the gap clo-sure assay. VSMCs are treated in low-serum media (0.4% fetalbovine serum [FBS]) with either 1) vehicle, 2) submaximal con-centration (0.5 μM) of amlodipine, 3) submaximal concentration(0.5 ng/mL) of PDGF, 4) 0.5 μM amlodipine + 0.5 ng/mLPDGF, or 5) maximal concentration (10 ng/mL) of PDGF. Torule out contributions from VSMC proliferation, 0.4% FBSculture media was supplemented with 10 μg/mL mitomycin C.VSMCs migrated minimally in vehicle and only slightly morewhen either 0.5 μM amlodipine or 0.5 ng/mL PDGF were added.However, VSMCs migrated significantly more when treated with10 ng/mL PDGF (Fig. 1 H–J). Interestingly, VSMCs treated withthe combination of 0.5 μM amlodipine + 0.5 ng/mL PDGFshowed a synergetic effect and migrated significantly more than0.5 μM amlodipine or 0.5 ng/mL PDGF alone (Fig. 1 H–J). Thisincreased migration was similar to that induced by maximalconcentrations of PDGF (10 ng/mL). Similarly, VSMC pro-liferation was measured over 72 h with the dye CyQUANT. Thecombination of 0.5 μM amlodipine + 0.5 ng/mL PDGF causedsignificantly higher proliferation than 0.5 μM amlodipine or0.5 ng/mL PDGF alone (Fig. 1K). These data suggest that lowconcentrations of amlodipine, reminiscent of circulating con-centrations in hypertensive patients, synergize with submaximalconcentrations of PDGF to further enhance VSMC proliferationand migration and promote vascular remodeling.

LCCBs Activate a Pathway Bearing the Biophysical Signature of SOCE.To determine the molecular identity of the Ca2+ signal activatedby LCCBs, we used HEK293 cells in which we can relativelyeasily perform gene knockout; 20 μM amlodipine induced a ro-bust cytosolic Ca2+ signal in HEK293 cells, exhibiting similarkinetics and amplitude to the Ca2+ signal induced by amlodipinein VSMCs (Fig. 2 A and B). To rule out nonspecific effects onmembrane potential, we used whole-cell patch clamp to measurethe current elicited by LCCBs. When Ca2+ in the pipette wasbuffered to 150 nM, 20 μM amlodipine in the bath elicited asmall (∼0.5 pA/pF at −100 mV) Ca2+ current in HEK293 cells(Fig. 2 C, D, and G), reminiscent of the current mediated bySOCE, the Ca2+ release-activated Ca2+ current (ICRAC) that isencoded by STIM/ORAI and triggered by ER Ca2+ store de-pletion (40). When we coexpressed STIM1 and ORAI1 inHEK293 cells, 20 μM amlodipine activated a large (∼40 pA/pFat −100 mV) inwardly rectifying Ca2+-selective current withpositive reversal potential (∼ +60 mV) (Fig. 2 E, F, and H).

STIM and ORAI Are Required for LCCB-Activated Ca2+ Signaling.Mammals express two STIM and three ORAI proteins, withSTIM1 and ORAI1 contributing the dominant proportion ofSOCE in most cells (21, 24). To determine if STIM and ORAImediate the increase in cytosolic Ca2+ in response to amlodipine, wegenerated STIM1 and STIM2 double knockout (STIM1/2-DKO)and ORAI1/2/3 triple knockout (ORAI1/2/3-TKO) HEK293 cellsusing CRISPR/Cas9 technology and confirmed STIM1/2 andORAI1 knockout by Western blots (41–43) (SI Appendix, Fig.

17370 | www.pnas.org/cgi/doi/10.1073/pnas.2007598117 Johnson et al.

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S1 A–E). Since there are currently no reliable ORAI2/3 anti-bodies, we utilized two guide RNAs (gRNAs) flanking the targetgene to completely excise the whole gene. Hence, mRNA mea-surements are a reliable means to document knockout. ORAI1/2/3-TKO cells have no detectable ORAI2 and ORAI3 mRNAcompared to parental HEK293 cells (SI Appendix, Fig. S1 F andG). Genomic sequencing further confirmed knockout. Thapsi-gargin (2 μM), a pharmacological inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) added to cells innominally Ca2+-free solution, causes passive ER Ca2+ depletion.When 2 mM Ca2+ was restored to the bath, robust SOCE wasmeasurable only in wild-type HEK293 cells (SI Appendix, Fig.S1 H and I), providing functional evidence that STIM/ORAIproteins have been knocked out.The significant increase in cytosolic Ca2+ with 20 μM amlo-

dipine was absent in ORAI1/2/3-TKO and STIM1/2-DKO cells(Fig. 2 I and J). To provide evidence that these results are notdue to off-target effects from CRISPR/Cas9, we overexpressedORAI1 in the STIM1/2-DKO cells either with or without res-cuing STIM1 expression. As a control, STIM1/2-DKO cells weretransfected with an empty GFPN-1 construct (Fig. 2 K, blacktrace and L, black data), which did not support Ca2+ entry inresponse to 20 μM amlodipine (Fig. 2 K and L). Similarly, 20 μMamlodipine did not stimulate Ca2+ entry in STIM1/2-DKO cellsexpressing ORAI1 alone (Fig. 2 K, light blue trace and L, lightblue data). However, expression of both STIM1 and ORAI1caused 20 μM amlodipine to stimulate Ca2+ entry (Fig. 2 K, darkblue trace and L, dark blue data). Similar results were obtainedwhen either ORAI2 or ORAI3 was coexpressed with STIM1 in

STIM1/2-DKO cells (traces in SI Appendix, Fig. S2 A and E;statistics in Fig. 2L). Expression of ORAI2 or ORAI3 alone inSTIM1/2-DKO cells did not rescue Ca2+ entry in response to20 μM amlodipine (traces in SI Appendix, Fig. S2 A and E; sta-tistics in Fig. 2L). Further, coexpression of STIM2 (instead ofSTIM1) with ORAI1 in STIM1/2-DKO cells was sufficient tosupport amlodipine-mediated Ca2+ entry (traces in SI Appendix,Fig. S2I; statistics in Fig. 2L) although this Ca2+ entry was lessrobust than that supported by STIM1, consistent with the knownweaker activity of STIM2 relative to STIM1. These results sug-gest that the combination of one STIM and one ORAI isoform isnecessary for amlodipine to activate a cytosolic Ca2+ signal.We next sought to determine whether the activation of STIM/

ORAI-dependent Ca2+ signal can be triggered by other LCCBsbelonging to the two other major families, namely phenylalkyl-amines and benzothiazepines. We showed that 20 μM of anotherdihydropyridine, nifedipine, as well as the benzothiazepine, dil-tiazem, and the phenylalkylamine, verapamil, were equally ca-pable of stimulating an increase in cytosolic Ca2+ when eitherORAI1, ORAI2, or ORAI3 was coexpressed with either STIM1or STIM2 in STIM1/2-DKO cells (Fig. 2 M–R and SI Appendix,Fig. S2 B–L). Expression of the empty GFPN-1 vector or ex-pression of either ORAI1, ORAI2, or ORAI3 alone (withoutSTIM) did not support LCCB-mediated increase in cytosolicCa2+. The T-type voltage-gated Ca2+ channel blocker Mibefradilalso blocks L-type Ca2+ channels (44). Mibefradil stimulated aCa2+ signal in STIM1/2-DKO cells coexpressing STIM1 andORAI1 (SI Appendix, Fig. S3 A and B). Furthermore, stimulationwith 20 μM of the (+) enantiomer of Bay K 8644, which is a

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blocker of Cav1.2 (45), stimulated an increase in cytosolic Ca2+

in STIM1/2-DKO cells coexpressing STIM1 and ORAI1 (SIAppendix, Fig. S3 C and D) while the (−) enantiomer of Bay K8644, which activates Cav1.2, was less efficient (SI Appendix, Fig.S3 C and D).We also tested drugs inhibiting other voltage-gated Ca2+

channels. We tested whether ω-conotoxin, a neurotoxic peptidethat blocks N-type voltage-gated Ca2+ channels (46), can acti-vate a STIM/ORAI-dependent cytosolic Ca2+ signal; 20 μMω-conotoxin did not activate a Ca2+ signal in STIM1/2-DKOcells coexpressing STIM1 and ORAI1 (SI Appendix, Fig. S3 Eand F). We also explored the possibility that voltage-gated Na+

channel blockers, such as local anesthetics, tetracaine, and ben-zocaine (47), can activate STIM/ORAI-dependent cytosolicCa2+ signal. Stimulation with 20 to 200 μM tetracaine or 20 μMto 2 mM benzocaine did not activate Ca2+ entry in STIM1/2-DKO cells coexpressing STIM1 and ORAI1 (SI Appendix, Fig.S3 G–J). These data suggest that the broad chemical class ofLCCBs, which have diverse structures and binding mechanismsto Cav1.2, increase cytosolic Ca2+ by activating STIM andORAI proteins.

LCCBs Cause STIM and ORAI Puncta and STIM–ORAI Interaction. Theactivation of SOCE requires STIM proteins to migrate to theER–PM junctions where they form puncta. At rest, the SOAR

domain of STIM which activates ORAI (25, 26) is engaged in anintramolecular clamp with the adjacent coiled coil-1 (CC1) do-main. On store depletion, STIM gains an extended conforma-tion, opening the CC1-SOAR clamp and exposing SOAR, whichbinds the C terminus of PM ORAI channels to cause their acti-vation (48). We used STIM1/2-DKO cells coexpressing C-terminalfluorescently tagged STIM1 (STIM1-YFP) and the N-terminalfluorescently tagged ORAI1 (CFP-ORAI1). Using a confocal mi-croscope, we show that, at rest, STIM1 proteins have the typicalreticular organization while ORAI1 proteins are diffusely localizedin the PM with little colocalization (Fig. 3 A–C). Upon stimulationwith either amlodipine or diltiazem, both STIM1 and ORAI1formed puncta and showed enhanced colocalization at 8 min(Fig. 3 A–C). To document STIM1–ORAI1 close interactions, wemeasured Förster resonance energy transfer (FRET) betweenSTIM1-YFP and CFP-ORAI1. Diltiazem induced an increase inSTIM1-ORAI1 FRET (Fig. 3 D–F). The maximal FRET signalinduced by diltiazem was of similar magnitude to that induced bythapsigargin (Fig. 3 G–I). Because amlodipine emits some fluo-rescence within the range of emission of CFP (49), this precludedreliable FRET measurements with amlodipine.

LCCBs Activate STIM without Depleting ER Ca2+ Stores. The move-ment of STIM proteins, their interaction with ORAI, and acti-vation of SOCE are triggered physiologically through depletion

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Fig. 2. LCCBs activate ICRAC 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 ICRAC in HEK293 cells stimulated first with vehicle (dimethylsulfoxide [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-clamprecording from HEK293 cells expressing eYFP-STIM1 and mCherry-ORAI1 stimulated first with DMSO and then amlodipine. (F) Ca2+ I/V curves of E, takenwhere indicated by “3” and “4”. (G) Quantification of peak current density activated by amlodipine. (H) Quantification of peak current density activated byamlodipine in HEK293 cells coexpressing eYFP-STIM1 and mCherry-ORAI1. (I) Cytosolic Ca2+ measurement in ORAI1/2/3-TKO cells, STIM1/2-DKO cells, andHEK293 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, orCFP-ORAI1, or coexpressing STIM1-eYFP (2 μg) and CFP-ORAI1 (1 μg) and stimulated with amlodipine. (L) Quantification of maximal Ca2+ entry from K alongwith 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 withSTIM1-eYFP (2 μg) and stimulated with amlodipine. Also quantified experiments using cells coexpressing STIM2-eYFP (instead of STIM1-eYFP) and CFP-ORAI1and stimulated with amlodipine (traces in SI Appendix, Fig. S2). (M–R) Cytosolic Ca2+ traces and quantifications of maximal Ca2+ entry from STIM1/2-DKO cellswith 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 ANOVAwith Dunnett’s test for multiple comparisons.

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of ER Ca2+ stores (21, 24). Therefore, we reasoned that LCCBs-activated Ca2+ entry through STIM-ORAI was likely the resultof LCCBs causing Ca2+ store depletion. We first used Fura2 toperform cytosolic Ca2+ measurements in HEK293 cells stimu-lated with 20 μM amlodipine in a nominally Ca2+-free extra-cellular solution. Under these conditions, amlodipine failed tocause an increase in cytosolic Ca2+ (Fig. 4 A and B). However,when extracellular Ca2+ was restored to 2 mM, now amlodipinecaused an increase in cytosolic Ca2+ (Fig. 4 A and B), suggestingthat the action of amlodipine on STIM-ORAI is independent ofstore depletion. To provide stronger evidence that the ER Ca2+

content is not affected by amlodipine, we performed direct ERCa2+ measurements in HEK293 cells using the ER-targetedCa2+ sensor, GCaMP6-150 (50). We first documented thatstimulation of cells with either the agonist carbachol (Cch) or thereversible SERCA inhibitor cyclopiazonic acid (CPA) caused adecrease in ER Ca2+ (Fig. 4 F–I). When CPA was washed out,we could readily observe the refilling of ER Ca2+ stores. How-ever, the addition of either amlodipine or diltiazem did not causeany detectable decrease in ER Ca2+ (Fig. 4 C–E and I). Yet, thesubsequent addition of 1 μM ionomycin to the same cells causedcomplete depletion of ER Ca2+ stores identical to that ofvehicle-treated cells, arguing that activation of STIM-ORAIproteins by LCCBs is store-independent.So far, we showed that LCCBs can activate Ca2+ entry only

when one STIM and one ORAI isoform are present in cells, but

not when an ORAI isoform is expressed alone (Fig. 2 A–D),suggesting that LCCBs cannot directly activate ORAI channelsin a STIM-independent manner. Although unlikely, it is howeverpossible that LCCBs could first act on ORAI causing their ag-gregation, which would then trap STIM leading to ORAI gatingand activation. To rule out this “outside-in” mode of LCCBsaction, we used STIM1/2-DKO cells expressing CFP-ORAI1(without STIM) to determine whether amlodipine or diltiazemcan cause ORAI1 puncta formation. In these cells, amlodipineand diltiazem did not cause ORAI1 puncta (Fig. 5 A and C).However, both drugs caused significant STIM1 puncta formationin ORAI1/2/3-TKO cells expressing STIM1-YFP (Fig. 5 B andC), strongly arguing that the target of LCCBs is STIM.Additional evidence that that the target of LCCBs is STIM

was gleaned from experiments utilizing a two-component FRETsystem of STIM1 (51), which consists of 1) a construct encodingthe N terminus, the transmembrane domain, and CC1 domain ofSTIM1 (STIM11–310) tagged with CFP on the C terminus; and 2)a construct encoding the SOAR domain of STIM1 (SOAR1)tagged with YFP on the N terminus (Fig. 5D). These two-taggedconstructs were coexpressed in STIM1/2-DKO cells where theywere shown to associate through CC1-SOAR1 interactions torecapitulate full-length STIM1 in the ER (51). Indeed, at rest,STIM11–310 and SOAR1 were highly colocalized at the ER anddisplayed high FRET values due to CC1 tethering to SOAR1(Fig. 5 E and F). Diltiazem induced SOAR1 to dissociate from

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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 themerged 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 ratiosfrom 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).

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STIM11–310 manifested by a dramatic decrease in FRET (Fig. 5 Eand F). The subsequent addition of 1 μM ionomycin caused only amarginal additional decrease in FRET (red trace, Fig. 5F). How-ever, the addition of ionomycin to cells expressing STIM11–310 andSOAR1 and treated first with vehicle (no diltiazem) caused a robustdecrease in FRET (black trace, Fig. 5F).Further evidence that STIM proteins are the target of LCCBs

were obtained from in vitro disulfide cross-linking assays thatprobe STIM1 activation in isolated ER membranes (52). HeLacells expressing an engineered STIM1 (A230C) protein (SI Ap-pendix, Fig. S4A) were treated with 20 μM amlodipine, or 20 μMdiltiazem, or with vehicle. Cells were lysed, and isolatedmembranes—still in the presence of the compounds—weresubjected to cross-linking at concentrations of Ca2+ ranging from0 to 2 mM. The principle of the assay is that low concentrationsof Ca2+, mimicking store depletion, favor a STIM1 conforma-tional change that brings the transmembrane helices together

and allows disulfide cross-linking via the engineered cysteineresidues. STIM1 cross-linking was analyzed by nonreducing so-dium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and Western blotting with STIM1-specific antibody. Asexpected, decreasing the concentration of Ca2+ increasedSTIM1-STIM1 cross-linking in all samples (Fig. 5 I and J). Im-portantly, pretreatment of cells with amlodipine or diltiazemresulted in significantly higher proportions of STIM1 dimers atCa2+ concentrations in the range 300 μM to 1 mM, compared tocells exposed only to vehicle (Fig. 5 I and J). Neither STIM1from cells treated with LCCBs nor STIM1 from cells treated with1 μM thapsigargin exhibited appreciable cross-linking when thereaction was conducted in vitro in the presence of 2 mM Ca2+

(Fig. 5 I and J and SI Appendix, Fig. S4B), indicating that STIM1in isolated ER membranes does not retain a conformationalmemory of its activation and oligomerization in vivo prior toisolation. These results establish that LCCBs render STIM1

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Fig. 4. LCCBs do not deplete ER Ca2+ stores. (A) Cytosolic Ca2+ measurements and (B) quantification of maximal cytosolic Ca2+ in HEK293 cells stimulated witheither 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+ mea-surements 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 μMionomycin was added to fully empty Ca2+ stores. (D) Similar to C, diltiazem or vehicle was added. (E) At 14 min, normalized GCaMP6-150 fluorescence be-tween 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).

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susceptible to activation even at resting ER Ca2+ concentrations,thus accounting for STIM-ORAI pathway activation by LCCBsindependent of ER Ca2+ store depletion.

A STIM1 N-Terminal Region Is Required for SOCE Activation by LCCBs.We explored the possibility that LCCBs directly bind to STIM1to induce a conformational change and subsequent activation.To identify the potential domain of STIM1 required for activa-tion by LCCBs, we used a variety of truncated STIM1-YFPconstructs expressed in HEK293 cells (SI Appendix, Fig. S5A).Cells were then treated with diltiazem, followed by the SOCEmodifier 2-aminodiphenylborate (2-APB), which has been shownto unfold the CC1-SOAR clamp of the C-terminal region ofSTIM proteins and activate SOCE independently of store de-pletion (42, 53). We generated four different C-terminally YFP-tagged STIM1 truncated constructs: 1) STIM1235–685, lacking boththe N-terminal and the transmembrane domain; 2) STIM1215–685,lacking the N-terminal domain; 3) STIM1120–685; and 4)STIM175–685 (SI Appendix, Fig. S5A).We then coexpressed theses STIM1 constructs with CFP-ORAI1

in STIM1/2-DKO cells. When the YFP C-terminally taggedSTIM1235–685 construct, which consists of STIM1 C-terminal

domain (STIM1-Ct) containing the CC1-SOAR1 clamp, isexpressed in HEK293 cells, it mostly fails to support significantSOCE activity (25) (however, see ref. 54). Indeed, we show thatthe STIM1235–685 construct diffusely localizes in the cytosol andfails to support significant SOCE upon thapsigargin stimulation(SI Appendix, Fig. S5 B and C). However, subsequent additionof 50 μM 2-APB induced a robust and transient Ca2+ entry,consistent with previous studies showing that 2-APB can tran-siently cause STIM1-Ct to activate ORAI1 (53). Diltiazem failedto induce significant STIM-Ct–mediated Ca2+ entry (SI Appendix,Fig. S5 D and E). However, 2-APB caused a significant cytosolicCa2+ signal in the same cells (SI Appendix, Fig. S5D). These dataargue that STIM1-Ct alone is incapable of supporting Ca2+ entryin response to LCCBs. The other three STIM1 constructs (SIAppendix, Fig. S5A) showed ER localization (SI Appendix, Fig.S5 B, F, H, and J) but failed to support Ca2+ entry in response to20 to 50 μM diltiazem (SI Appendix, Fig. S5 D, E, G, I, and K).These results suggest that the LCCBs action on STIM is distinctfrom the direct mechanism of action of 2-APB on STIM1-Ct, andthat LCCBs action requires the full-length STIM protein.To gain insights into the STIM domain mediating LCCBs

action, we tested whether LCCBs activation of STIM was conserved

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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 for8 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 cellsbefore 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 (STIM11–310-CFP) andYFP-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 STIM11–310-CFP (1 μg), YFP-SOAR1 (1 μg), colocalization, and seFRET. (Scale bar: 10 μm.) (F)seFRET between STIM11–310-CFP and YFP-SOAR1-SOAR1 expressed in STIM1/2-DKO cells stimulated with either diltiazem or vehicle. At the end, cells werestimulated with 1 μM ionomycin (Iono) for maximal dissociation of SOAR1 from STIM11–310. (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 in-dicated Ca2+ concentration for cells treated with vehicle, amlodipine, or diltiazem. *P < 0.05, ****P < 0.0001, ns, not significant (unpaired Student’s t test forFRET experiments and ANOVA with Dunnett’s test for puncta experiments).

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in other vertebrates. Current measurements demonstrated thatLCCBs do not activate native SOCE in Xenopus oocytes (SI Ap-pendix, Fig. S6). We then coexpressed Xenopus STIM1 andORAI1 (xSTIM1-YFP and xORAI1-CFP) in STIM1/2-DKOcells. Confocal microscopy confirmed that, under resting condi-tions, the tagged xSTIM1 and xORAI1 are located in the ER andPM, respectively (Fig. 6A). The addition of thapsigargin (+TG)for 8 min induced xSTIM1 and xORAI1 to form puncta andcolocalize (Fig. 6A). However, xSTIM1 and xORAI1 failed toform significant puncta and to colocalize in response to 20 μMamlodipine (Fig. 6B). Side-by-side comparisons between xSTIM1and human STIM1 (hSTIM1) showed significant hSTIM1 punctaformation upon amlodipine stimulation (Fig. 6C). Furthermore,SOCE induced by thapsigargin was rescued in STIM1/2-DKO bycoexpression of xSTIM1 and xORAI1 (Fig. 6 D and E). Inter-estingly, amlodipine failed to cause significant Ca2+ entry inSTIM1/2-DKO cells coexpressing xSTIM1 and xORAI1, whileinducing a significant Ca2+ signal in cells coexpressing hSTIM1and hORAI1 (Fig. 6 F and G). These results suggest that LCCBs-mediated activation of SOCE is specific to human STIM and isnot supported by xSTIM1 and xORAI1.

We performed sequence alignments of hSTIM1 and xSTIM1and identified 10 to 18 amino acids within the N terminus ofhSTIM1 that are either absent or not conserved in xSTIM1 (SIAppendix, Fig. S6H). We then created two truncations within thisN-terminal region of hSTIM1, namely STIM1Δ29–46 andSTIM1Δ31–40 (SI Appendix, Fig. S6H). Importantly, thesetruncated hSTIM1 constructs were fully functional and mediatedSOCE identical to that of full-length STIM1 (Fig. 6 H and I).Furthermore, STIM1Δ29–46 and STIM1Δ31–40 distributed intopuncta in response to stimulation with thapsigargin in a mannersimilar to full-length STIM1 (Fig. 6J). However, these twotruncated constructs failed to support Ca2+ entry in response toamlodipine (Fig. 6 K and L), suggesting that this N-terminalregion of hSTIM1 is necessary for LCCB-mediated activationof SOCE.

An Intermediate Pathway Is Required for LCCBs to Activate STIM. Todetermine whether LCCBs directly activate STIM1, we used thecross-linking described earlier. Instead of treating cells withLCCBs before lysis, we first lysed the cells expressing the engi-neered STIM1-A230C (SI Appendix, Fig. S4A), isolated themembranes, and incubated these membranes with LCCBs in the

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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 bythapsigargin in STIM1/2-DKO cells cotransfected with either xSTIM1-YFP and xORAI1-CFP or an empty plasmid. (E) Quantification of maximal Ca2+ entry fromD. (F) Cytosolic Ca2+ measurements on STIM1/2-DKO cells cotransfected with either xSTIM1 + xORAI1 or with hSTIM1 + hORAI1. Cells were stimulated withamlodipine. (G) Quantification of maximal Ca2+ signals from F. (H) SOCE was induced with thapsigargin in STIM1/2-DKO cells expressing ORAI1-CFP (1 μg) witheither 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, andSTIM1-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 expressingORAI1-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 orvehicle. For both conditions, the isolated membranes were then incubated with EGTA (0 μM Ca2+) or Ca2+ at final concentrations ranging from 0.3 to 2,000μM, cross-linked and analyzed by nonreducing SDS/PAGE. (N) Quantification of the percentage of STIM1 dimer after treatment with either amlodipine orvehicle 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 twocomparisons and ANOVA with Dunnett’s test for multiple comparisons).

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presence of varying concentrations of Ca2+. The original pro-tocol used in Fig. 5 where intact cells are treated with LCCBswas performed side-by-side. Unlike intact cells treated withLCCBs, when we treated isolated membranes with amlodipine,we failed to see increases in the proportion of STIM1 dimers athigher concentrations of Ca2+ (Fig. 6 M and N). These findingssuggest that STIM1 activation by LCCBs requires intact cells,presumably because a cellular protein or factor required for mod-ifying or binding to STIM1 is missing from isolated membranepreparations.We considered the possibility that the protein required for

LCCBs action on STIM is Cav1.2. While the Cav1.2 channel isonly functional in excitable cells, the α1C subunit of Cav1.2 is stillexpressed HEK293 cells (33). Previous studies showed thatCav1.2 is negatively regulated by STIM1 through direct bindingof SOAR1 to the C terminus of the α1C subunit of Cav1.2,causing α1C internalization (55, 56). We reasoned that perhapsLCCBs disrupt resting Cav1.2-STIM1 interactions, unlocking thispool of STIM1 in an extended conformation to activate ORAI.To generate functional Cav1.2 channels in HEK293 cells, we

coexpressed the YFP-tagged Cav1.2 α1C along with its auxiliarysubunits β1A and α2δ1 (57), which allowed trafficking of thechannel to the membrane (SI Appendix, Fig. S7A). An antibodyspecific to α1C detected the native Cav1.2 α1C and the YFP-tagged Cav1.2 α1C (SI Appendix, Fig. S7B). Depolarization with134 mM KCl caused a robust Ca2+ entry only in cells expressingCav1.2 subunits (SI Appendix, Fig. S7 C and D). As expected, thisCav1.2-mediated Ca2+ signal was blocked by 0.5 μM amlodipine(SI Appendix, Fig. S7 C and D). Interestingly, both native SOCEinduced by thapsigargin (SI Appendix, Fig. S7 E and F) and Ca2+

entry induced by amlodipine (SI Appendix, Fig. S7 G and H)were not enhanced as would be expected if Cav1.2 is mediatingthe effects of LCCBs on STIM proteins. Quite the opposite,SOCE and amlodipine-activated Ca2+ entry were slightly re-duced when Cav1.2 was expressed in HEK293 cells, suggestingthat Cav1.2 is likely sequestering native STIM proteins and re-ducing their accessibility to ORAI.Next, we knocked down the α1C subunit of Cav1.2 in HEK293

cells using short hairpin RNA (shRNA). Western blots con-firmed ∼50% knockdown (SI Appendix, Fig. S7 I and J). NativeSOCE activated by thapsigargin (SI Appendix, Fig. S7 K and L)and Ca2+ entry induced by amlodipine (SI Appendix, Fig. S7 Mand N) were slightly augmented in shCav1.2-expressing cells, inagreement with previous studies showing that Cav1.2 knockdownliberates a STIM1 pool that can now activate ORAI channels(55, 56). These results suggest that LCCBs action on theSTIM1 N terminus is Cav1.2-independent and mediated by anintermediary mechanism.

LCCBs Activate VSMC Remodeling through STIM1. To determinewhether STIM1 is required for low doses (0.5 μM) of amlodipineto mediate enhanced VSMC proliferation and migration, wegenerated VSMCs with stable STIM1 knockdown using two dif-ferent shRNAs. The shSTIM1#1- and shSTIM1#2-transfectedVSMC showed reduced STIM1 protein (Fig. 7 A and B) and re-duced SOCE (Fig. 7 C and D) compared to the shScramble-transfected VSMCs. Importantly, the synergistic effect of 0.5 μMamlodipine +0.5 ng/mL PDGF on VSMC migration (Fig. 7 E andG and SI Appendix, Fig. S8 A and B) and proliferation (Fig. 7F andSI Appendix, Fig. S8C) was significantly reduced in shSTIM1#1-and shSTIM1#2-transfected VSMCs, suggesting that the effectsof amlodipine on VSMC proliferation and migration is STIM1-dependent.

VSMCs from Chronically Hypertensive Rats Have Up-Regulated STIM1and Enhanced SOCE. An excellent model for chronic hypertensionassociated with VSMC remodeling is the spontaneously hyper-tensive rats (SHRs) and respective control Wistar-Kyoto rats

(WKYs). SHRs have systolic blood pressures over 150 mm Hgafter 1 mo of age (58). After 12-wk, the aortas of SHRs undergoconsiderable remodeling, with VSMC hyperplasia and vesselthickening (59). Using 12-wk-old male rats, endothelial denudedaortic rings were dissected, and VSMCs were isolated from SHRand WKY rats. VSMCs acutely isolated from SHR showed en-hanced STIM1 protein expression (Fig. 7 H and I) but only amarginal increase in STIM2 protein expression (Fig. 7 H and J).SOCE activated by thapsigargin and ICRAC were significantlyincreased in SHR VSMCs compared to WKY VSMCs (Fig. 7 K–N). Both peak current densities at −100 mV of Ca2+ currents(Fig. 7 O and Q) and Na+ ICRAC recorded in divalent free bathsolutions (Fig. 7 P and R) were increased in SHR VSMCs. Thus,in vivo VSMC remodeling in chronic hypertension is associatedwith enhanced STIM1 expression and increased SOCE and ICRACactivities. It is established that isolating VSMC and culturing themcauses their dedifferentiation into a synthetic phenotype (7, 15,35). We show that, whether VSMCs are isolated from SHR orWKY, once they are placed into cell culture for 3 d, both SHRand WKY VSMCs show equally enhanced SOCE in response tothapsigargin (SI Appendix, Fig. S8 D and E), strongly arguing that,regardless of the origin of VSMCs, once in culture, they equallydedifferentiate into a synthetic phenotype.

LCCBs Are Associated with Increased Incidence of Heart Failure inPatients. The pathological VSMC remodeling in chronic hyper-tension is accompanied with decreased luminal diameter, in-creased systemic peripheral resistance (4), and heart failure (60).Therefore, we considered the possibility that amlodipine andother LCCBs could be associated with enhanced incidence ofheart failure in hypertensive patients treated with those drugs.Using the Penn State clinical database known as i2b2, we com-pared the incidence of heart failure in hypertensive patientstreated exclusively with LCCBs (exposed cases) to those treatedwith none-LCCBs antihypertensive medications (unexposedcases). Our analysis showed that hypertensive patients treatedwith LCCBs had a significantly higher incidence of heart failure(23.632%) compared to patients treated with other antihyper-tensive drugs (18.549%) (Fig. 7S). Statistically, patients treatedwith LCCBs were more prone to heart failure when compared topooled patients treated with other antihypertensive drugs(Fig. 7T) (95% CI for odds ratio = [1.222,1.252]). The de-mographics including sex, race, and age among the cohorts weresimilar (SI Appendix, Fig. S9 A–C). After stratification based onsex, LCCBs were still more associated with heart failure thanother antihypertensive drugs (SI Appendix, Fig. S9D) (95% CIfor odds ratio = [1.100, 1.138] and [1.369, 1.417] for males andfemales, respectively), with the exception of diuretics andβ-blockers in male patients.We also examined other smooth muscle proliferative diseases

and certain types of cancers that are associated with enhancedSTIM expression and increased SOCE activity (61). The smoothmuscle neoplasms, leiomyomas of the uterus (fibroids; 95% CIfor odds ratio = [0.900, 1.001]), leiomyomas (1.000, 1.201), orleiomyosarcomas (0.777, 1.081) were no more and no less asso-ciated with LCCBs than other antihypertensive drugs (SI Ap-pendix, Fig. S10 A–F). However, LCCBS were more associatedwith prostate (1.245, 1.381), breast (1.057, 1.176), and bladdercancer (1.156, 1.240) (SI Appendix, Fig. S11 A–F).

DiscussionHere, we unravel a mechanism whereby LCCBs activate STIM totrigger SOCE and smooth muscle proliferation and migration.These findings were epidemiologically associated with increasedheart failure in hypertensive patients on LCCBs. In previousstudies, we showed that STIM1-ORAI1 proteins, SOCE andICRAC are increased in neointimal VSMCs from rat and micemodels of vascular remodeling (14, 28). Knockdown or knockout

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of STIM1 or ORAI1 in rat and mice inhibited neointima for-mation and hypertension (14, 29). In the present study, we showthat STIM1 proteins, SOCE and ICRAC are up-regulated inVSMCs isolated from vessels of 12-wk-old spontaneously hy-pertensive rats, suggesting that enhanced SOCE is a commontheme in VSMC remodeling. It is established that sub-μM con-centrations of LCCBs inhibit Cav1.2 channels and induce vaso-relaxation (62). Thus, LCCBs have been widely prescribed fortreatments of hypertension for over 70 y. However, duringchronic hypertension, Cav1.2 channels are down-regulated inremodeled VSMCs, and many Ca2+ signaling proteins likeSTIM-ORAI are up-regulated (6, 7). Since Cav1.2, which rep-resents the target of LCCBs, is decreased during vascularremodeling, the efficacy of LCCBs during long-term chronichypertension is uncertain.Our unbiased screen of ∼1,700 FDA-approved drugs in glio-

blastoma discovered that LCCBs activated a Ca2+ entry pathwaythat was inhibited by pharmacological blockers of SOCE (37).Here, we determined the molecular target of LCCBs and theirimpact on VSMC function. LCCBs activate SOCE and ICRACmediated by STIM-ORAI. Using gene knockout in HEK293cells, we determined that one STIM and one ORAI isoform are

necessary to support LCCB-activated Ca2+ entry. The target forLCCBs in VSMCs is STIM1, and STIM1 activation by LCCBsrequires an N-terminal portion that is absent from XenopusSTIM1. LCCBs activation of STIM1 occurs independently of ERCa2+ depletion. Interestingly, all major classes of LCCBs withdifferent chemical structures, including dihydropyridines, ben-zothiazepines, and phenylalkylamines, activate STIM-ORAI.Each of these three classes of LCCBs binds to distinct sites onCav1.2 (63, 64). Since STIM1/2 were activated by all classes ofLCCBs, this offers further support that the mechanism of STIMactivation by LCCBs is indirect. This and the failure of LCCBs toactivate STIM1 dimer formation in isolated membranes arecompatible with the requirement of an intermediary factor orprocess. Because Cav1.2 has been shown to interact with STIM1(55, 56), and is a target common to all LCCBs, we excludedCav1.2 as this intermediary protein. Although the identificationof this intermediary mechanism requires further investigations,this mechanism might involve a soluble factor, a protein–proteininteraction, or a posttranslational modification. This includesmodification by phosphorylation or reactive oxygen species,which have been shown to activate STIM1 independently ofstore depletion (65–67).

STIM1

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Cases ControlsN % N %

LCCBs 10,670 23.632% 45,150 76.368%Diuretics 8,600 21.603% 39,810 78.397%β-Blockers 8,450 20.575% 41,070 79.425%ACE Inhibitors 5,970 15.355% 38,880 84.645%ARBs 2,140 13.342% 16,040 86.658%⍺-Blockers 2,180 18.809% 11,590 81.191%

Pooled non-LCCBs 27,340 18.549% 147,390 81.451%

1.0 1.5 2.0

Pooled non-LCCBs

ACE Inhibitors

Diuretics

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α-Blockers

Association of LCCBs withheart failure by comparison toother antihypertensive drugs

Odds Ratio

H I J K L

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A

Fig. 7. LCCBs promote vascular remodeling through STIM1. (A) STIM1 Western blots of VSMCs transfected with Scrambled shRNA (shScr.) and two STIM1shRNA (shSTIM1#1 [shS1 #1] and shSTIM1#2 [shS1 #2]). (B) STIM1 protein quantification from A using densitometry normalized to tubulin. (C) SOCE triggeredby thapsigargin in VSMC stably expressing either shScramble, or shSTIM1#1, or shSTIM1#2. (D) Quantification of maximal Ca2+ entry from C. (E) Quantificationof 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/mLPDGF 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 inVSMCs 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 SHRnormalized to tubulin. (K) SOCE measurements from SHR and WKY VSMCs. (L) Quantification of maximal Ca2+ entry from K. (M and N) Whole-cell ICRACinduced 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 wasused to potentiate a Na+ current. (O) Ca2+ ICRAC and (P) Na+ ICRAC I/V relationships obtained from M and N where indicated by numbers (1 to 4). (Q) Peak Ca2+

ICRAC and (R) peak Na+ ICRAC densities from SHR and WKY VSMCs. (S) Data from Penn State University Hospital with reported cases of heart failure whenpatients are treated with LCCBs or other antihypertensive drugs. Exposed control did not develop heart failure to date. In italics is the percentage of patientsin 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.

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STIM1-mediated SOCE enhances VSMC proliferation andmigration (14, 28, 68). During vascular remodeling, local PDGFsecreted from endothelial and immune cells induces VSMCproliferation and migration (39). STIM1 is required for PDGF-induced smooth muscle migration and proliferation (27). Here,we show that low concentrations of amlodipine (reminiscent ofthose circulating in patients treated with those drugs) (34) syn-ergize with submaximal concentrations of PDGF to enhanceSTIM1-dependent VSMC proliferation and migration. In-terestingly, this effect seems unique to LCCBs since other anti-hypertensive medications like ACE inhibitors inhibit vascularremodeling (69, 70). SHR rats have exacerbated vascularremodeling during the chronic phase of hypertension. We showthat acutely isolated VSMCs from 12-wk-old SHRs have in-creased STIM1 protein, and enhanced SOCE and ICRAC, con-sistent with previous studies (71). We previously showed thatacutely isolated VSMCs have little ORAI1 protein, marginalSOCE activity, and undetectable ICRAC (13, 14, 27). We alsodemonstrated that smooth muscle-specific STIM1 knockoutmice were protected against hypertension induced by angiotensinII infusion (29). These findings and the established role ofSTIM1 in driving VSMC neointimal hyperplasia during balloonangioplasty (14) strongly argue that STIM1 and SOCE play anactive role in exacerbating hypertension.Heart failure is a clinical consequence of chronic hypertension

and vascular remodeling (4, 60). Epidemiological evidence sug-gests that hypertensive patients on LCCBs are more likely todevelop heart failure compared to hypertensive patients on otherantihypertensive medications. This may be due to the mecha-nisms described above and summarized herein. Although largerepidemiological studies and drug interventional studies in ani-mals are warranted, our data suggest that the management ofchronic hypertension with LCCBs should be reevaluated in el-derly patients or in patients with chronic hypertension or a his-tory of cardiovascular remodeling. Female hypertensive patientstreated with amlodipine for 2 wk show a greater decrease inblood pressure than males (72); and acutely isolated VSMCsfrom normotensive female mice have larger Cav1.2 channelclusters, activity, and myogenic tone than male VSMCs (73),suggesting that Cav1.2 function is more prominent in femalesthan males. However, our study showed female patients onLCCBs have stronger association with heart failure than males.This is likely because VSMCs from the chronically hypertensivepatients in our study are remodeled with down-regulated Cav1.2and enhanced STIM1 expression.

STIM1 protein has been reported to be enhanced in certainneoplasms and necessary for their development (61). We foundthat LCCBs administration is no more associated with smoothmuscle neoplasms than other antihypertensive medications. Thismight be because STIM1 does not provide the oncogenic triggerto transform the relatively oncogenic-resistant smooth musclecells (74). However, prostate, breast, and bladder cancers aremore associated with LCCBs use than other antihypertensivemedications. Although STIM1 has not been studied in bladdercancer, it was shown to drive breast (75) and prostate (76) cancergrowth. Interestingly, studies have also shown a similar associa-tion of LCCBs with breast cancer (77). Our results identifyLCCBs as activators of STIM proteins. This causes enhancementof SOCE and stimulation of VSMC remodeling. Treatment withLCCBs is clinically associated with elevated incidence of heartfailure, which prompts a careful examination of the use ofLCCBs during chronic hypertension where vascular remodelingis evident.

MethodsDetails of VSMC isolation from rat aortas and maintenance, complementaryDNA construct cloning, generation of CRISPR/Cas9 knockout cell lines for STIMand ORAI isoforms, VSMC and HEK293 cell culture, stable transfections,patch-clamp recordings of CRAC currents, ER and cytosolic Ca2+ measure-ments with chemical and genetically encoded indicators, colocalization ofSTIM/ORAI by confocal imaging, STIM1 cross-linking assays, FRET analysis,Western blot analysis, animal and human patient studies, and statistics areprovided in SI Appendix.

Data Availability. All materials and experimental protocols; all raw data andraw unprocessed gels and Western blots; and the source and referencenumber of reagents, recombinant DNA, and animals necessary for replicationare included in SI Appendix. The exact P values for all statistical comparisonsare also included in SI Appendix.

ACKNOWLEDGMENTS. We thank the following for kindly providing con-structs: Dr. Kurt Beam (University of Colorado) for CMV-YFP-Cav1.2-⍺1c,CMV-Cav1.2-β1A, and CMV-Cav1.2-⍺2△; and Dr. Yubin Zhou (Texas A&MUniversity) for CMV-STIM1(1-310)-CFP. This work was supported by NIH/NationalHeart, Lung, and Blood Institute Grant R35-HL150778 (to M.T.), Grant F30-HL147489-01A1 (to M.T.J.), NIH/National Institute of General Medical SciencesGrant 1R35 GM131916 (to D.L.G.), NIH/National Institute of Allergy andInfectious Diseases Grant R01-AI084167 (to P.G.H.) and Grant R01-AI040127(to Anjana Rao and P.G.H.), NIH Grant TL1TR002016 (to M.T.J.), Depart-ment of Defense Grant CDMRP W81XWH1810209 (to W.L.), Qatar NationalResearch Fund Grants NPRP7-542-3-145 and NPRP8-110-3-021 (to M.T. andK.M.), and funds from Weill Cornell Medicine-Qatar of the QatarFoundation.

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