ABSTRACT
Background and Purpose
Substitution of Cys674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) causes SERCA2 dysfunction which leads to activated inositol requiring enzyme 1 alpha (IRE1α) and spliced X-box binding protein 1 (XBP1s) pathway accelerating cell proliferation of pulmonary artery smooth muscle cells (PASMCs) followed by significant pulmonary vascular remodeling resembling human pulmonary hypertension. Based on this knowledge, we intend to investigate other potential mechanisms involved in SERCA2 dysfunction-induced pulmonary vascular remodeling.
Experimental Approach
Heterozygous SERCA2 C674S knock-in (SKI) mice of which half of cysteine in 674 was substituted by serine to mimic the partial irreversible oxidation of C674 were used. The lungs of SKI mice and their littermate wild-type mice were collected for PASMC culture, protein expression, and pulmonary vascular remodeling analysis.
Results
SERCA2 dysfunction increased intracellular Ca2+ levels, which activated Ca2+-dependent calcineurin (CaN) and promoted the nuclear translocation and protein expression of the nuclear factor of activated T-lymphocytes 4 (NFAT4) in an IRE1α/XBP1s pathway-independent manner. In SKI PASMCs, the scavenge of intracellular Ca2+ by BAPTA-AM or inhibition of CaN by cyclosporin A can prevent PASMC phenotypic transition. CDN1163, a SERCA2 agonist, suppressed the activation of CaN/NFAT4 and IRE1α/XBP1s pathways, reversed the protein expression of PASMC phenotypic transition markers and cell cycle-related proteins, and inhibited cell proliferation and migration when given to SKI PASMCs. Furthermore, CDN1163 ameliorated pulmonary vascular remodeling in SKI mice.
Conclusions and Implications
SERCA2 dysfunction promotes PASMC phenotypic transition and pulmonary vascular remodeling by multiple mechanisms, which could be improved by SERCA2 agonist CDN1163.
SUMMARY
‘What is already known’
l The dysfunction of SERCA2 promotes PASMC hyperproliferation and pulmonary vascular remodeling through activation of the IRE1α/XBP1s pathway.
‘What this study adds’
l The dysfunction of SERCA2 activates the Ca2+-dependent CaN-mediated NFAT4 pathway to promote the PASMC phenotypic transition.
l Revitalization of SERCA2 suppresses PASMC phenotypic transition and pulmonary vascular remodeling caused by SERCA2 dysfunction.
‘Clinical significance’
l SERCA2 dysfunction-induced pulmonary vascular remodeling involves more than one mechanism, implicating that more drugable targets are to be discovered.
l SERCA2 is a potential therapeutic target for preventing pulmonary vascular remodeling.
Introduction
Pulmonary hypertension (PH) is a life-threatening, intractable disease characterized by progressive remodeling of the distal precapillary pulmonary arterioles and the muscularization of peripheral arteries causing increased pulmonary vascular resistance, right ventricular hypertrophy, and failure (Citation1). PH is clinically classified into five groups, among which pulmonary artery hypertension (PAH) is a severe subtype characterized by slow clinical onset and progressive organ deterioration (Citation2–4). The hypertrophy and/or hyperplasia of endothelial cells, smooth muscle cells, fibroblasts, and the increased deposition of extracellular matrix (ECM) components all contribute to pulmonary vascular remodeling manifested by the thickening of all vascular layers. In particular, pulmonary artery smooth muscle cells (PASMCs) undergo a phenotypic transition from a contractile phenotype to a synthetic phenotype gaining the function of proliferation, migration, and production of an alternative number of ECM proteins contributing to the pulmonary vascular remodeling (Citation5).
Increased intracellular Ca2+ level is a well-known contributor to the proliferation of PASMCs. Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) is key to maintaining low Ca2+ concentration in the cytoplasm by transporting Ca2+ from the cytoplasm to the sarcoplasmic reticulum and endoplasmic reticulum. SERCA2 mainly includes SERCA2a and SERCA2b and is the main subtype of SERCA in the vasculature. The S-glutathiolation of the amino acid residue Cys674 (C674) is key for the enzymatic activity of SERCA2 under physiological conditions, however, prevented by irreversible oxidation under pathological situations with excessive ROS (Citation6). We have found that C674 is irreversibly oxidized in hypoxic PH (Citation7). Using heterozygous SERCA2 C674S knock-in (SKI) mice, where one copy of C674 was substituted by serine (S674) mimicking partial C674 oxidative inactivation (Citation8), we have reported for the first time that the substitution of C674 causes the dysfunction of SERCA2 in PASMCs, which promotes PASMC proliferation and leads to pulmonary vascular remodeling resembling human PH by the activation of inositol requiring enzyme 1 alpha (IRE1α) and spliced X-box binding protein 1 (XBP1s) pathway (Citation7). However, SERCA2 itself is an important node of the network in the complicated Ca2+ signaling pathways, which makes it reasonable to assume that how SERCA2 dysfunction causes pulmonary vascular remodeling has not been fully explored. It has been reported that the activated calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) pathway contributes to the pathogenesis of monocrotaline-induced pulmonary arterial hypertension rats (Citation9). In this study, we discovered that the dysfunction of SERCA2 activates the Ca2+-dependent CaN-mediated NFAT4 pathway which directly contributes to PASMC phenotypic transition, while revitalization of SERCA2 by CDN1163 could prevent PASMC phenotypic transition and pulmonary vascular remodeling in SKI mice.
Materials and methods
Animals
All animal care and study protocols complied with the guidelines for the ethical use of animals and were approved by the Laboratory Animal Welfare and Ethics Committee of Chongqing University (IACUC No.: CQU-IACUC-RE-202112-005, Chongqing, China). We minimized animal suffering and reduced the number of animals used.
Mice were bred and housed in the animal room at Chongqing University under specific pathogen-free conditions. Mice were kept in open polypropylene cages with clean chip bedding. The animal room was maintained at a controlled temperature (22 ± 3°C) and a 12 h cycle of light and dark. Five mice in each cage were free to drink water and were fed on a regular diet (0.3–0.8% sodium, 1.0–1.8% calcium, 0.6–1.2% phosphorus, Beijing Keao Xieli Feed Corporation, China). To obtain tissues, all mice were sacrificed by intraperitoneal injection instantly with avertin (2,2,2-tribromoethanol, Sigma-Aldrich, Cat# T48402) at a dose of 300 mg·kg−1. We chose avertin over the isoflurane or ketamine combination because it does not need to consider the anesthetic effect and is suitable for the final execution of animals.
SERCA2 C674S knock-in (SKI) mouse construct
All mice used in this study were of C57BL/6J background (The Jackson Laboratory, Bar Harbor, Maine, USA). The construct of the SKI mouse was generated as previously described (Citation8). Briefly, the genetic variation was TGT at C674 to TCC (S674) in exon 14 of SERCA2 governed by the unaltered upstream native SERCA2 promoter. The presence of the C674S mutation was verified by sequencing both genomic DNA and cDNA from the heart. Due to embryo lethality of homozygous SKI mice, only heterozygous SKI mice with 50% of C674 and 50% of S674 expressed were used in this study, and their littermate wild-type (WT) mice without S674 were used as controls. From our preliminary data, SKI male mice have relatively more severe lesions than female mice of the same age. To control variate gender, all experiments used healthy male mice, whose number in each study is indicated in the corresponding figure legend.
In vivo treatment
CDN1163 is a well-known SERCA agonist, and the selected dose of CDN1163 is based on the literature (Citation10) and our previous experiments. The 4-week-old male WT and SKI mice were randomly divided into two groups and given CDN1163 (50 mg·kg−1·day−1, 100 μL, MCE, Cat# HY-101455, Shanghai, China) or solvent control by intraperitoneal injection once a day for 4 weeks. CDN1163 dissolved in a solvent containing 5% DMSO, 40% PEG400, 10% Tween 80, and 40% saline.
Measurement of right ventricular parameters
To obtain a stable anesthetic effect, we used urethane instead of isoflurane or ketamine combinations. Mice were anaesthetized once with 15% urethane (Chengdu Huaxia Chemical Reagent Co., Ltd.) at a dose of 10 mL/kg body weight. One end of a polyethylene catheter was inserted into the right ventricle via the right external jugular vein, and the other end of the catheter was connected to a transducer. The right ventricular parameters, including right ventricular systolic pressure (RVSP) and right ventricle end-diastolic pressure (RVEDP), were simultaneously recorded by a physiological recorder (PowerLab system, AD Instruments, Castle Hill, NSW, Australia), and analyzed by the chart program supplied by the system. After all measurements were complete, the right ventricle (RV) free wall, interventricular septum (S), and the left ventricle (LV) to measure their weight were removed. The Fulton Index [RV/(LV + S) weight] assessed right ventricular hypertrophy.
Histology, immunohistochemistry, and immunofluorescence in lung sections (Citation7)
The middle lobes of the right lung were fixed in 4% paraformaldehyde for 24 h, and then in 30% sucrose solution for 24 h. After that, embedded lungs in optimum cutting temperature compound (the middle biggest lobe) or paraffin (the middle second-biggest lobe) to prepare 7 μm serial sections. The leftover lungs were snap-freezed in liquid nitrogen and stored at −80°C for protein analysis. Serial sections of the lung for each mouse were stained individually with Verhoeff-Van Gieson (VVG) and Masson’s trichrome for morphological analysis. Masson’s trichrome staining showed that the fibrosis was blue. For immunofluorescence analysis of lung cryosections, incubated sections at 4°C for 12 h with specific antibodies against the following proteins: α-smooth muscle actin (α-SMA, Invitrogen, Cat# MA5–11547), von Willebrand factor (vWF, Proteintech, Cat# 11778–1-A), Ki67 (Abcam, Cat# ab15580), followed by Alexa Fluor 488 goat anti-rabbit IgG (H + L) (Jackson immunoresearch, Cat# 111-545-144) or Cy3-conjugated Affinipure goat anti-mouse IgG (H + L) (Proteintech, Cat# SA00009–1) for 2 h. 4,’6-diamidino-2-phenylindole (DAPI, Solarbio, Cat# C0065) stained nuclei for 10 min. Fluorescence images were taken by the microscope (Leica DM6, Germany) and analyzed by Las X software (Leica, Germany). For the distal pulmonary arterioles (<50-μm-diameter), the levels of muscularization were evaluated by α-SMA and vWF co-staining. vWF staining showed the endothelial cells (ECs) of arterioles, and α-SMA staining showed the existence of smooth muscle cells (SMCs) outside the endothelial layer. Full muscularization showed that α-SMA staining formed a complete circle, non-muscularization showed no α-SMA staining and partial muscularization was between them. The total number of arterioles to calculate their relative percentages was counted. The pulmonary artery (PA) tunica media was indicated by α-SMA staining, and the percentage of Ki67-positive proliferating cells in the total number of nuclei showed by DAPI staining was calculated. The calculation by different observers in at least three random fields was compared and represented by the average value from different observers.
Morphology analysis of pulmonary vascular lesions (Citation7)
Tunica media thickness, identified by α-SMA staining, was used to evaluate tunica media hypertrophy in PAs (outer diameter of 50‒100 μm), calculated as [(outer diameter ‒ inner diameter)/outer diameter] × 100%. Cellular neointima, identified by vWF staining, consists of ECs in small PAs (around 50 μm diameter) characterized by remarkable cellular filling inside of lumen, whose incidence was calculated by the ratio of the number of PAs with cellular neointima to the total number of PAs. We scored overall PA lesions based on the standard of Heath-Edward classification that was later revised by Wagenvoort et al. (Citation11), and the plexiform lesions were divided into four grades (I‒IV). Heath-Edward score 0, normal PAs; score 1 (grade I), tunica media hypertrophy of PAs, muscularization of distal pulmonary arterioles, without intimal alteration; score 2 (grade 2), tunica media hypertrophy, cellular neointima in the smaller PAs; score 3 (grade 3), extensive vascular occlusion with intimal lamellar changes; score 4 (grade 4), plexiform lesions, thrombosis. Venous lesions were characterized by the thickening of pulmonary veins and dense cell clusters derived from SMCs inside and outside pulmonary veins. The severity of venous lesions was evaluated according to the ratio of the venous wall area to lumen area: score 0, ratio < 0.25 (its ratio in normal vein < 0.25); score 1, 0.25 < ratio < 0.50; score 2, 0.50 ≤ ratio < 0.75; score 3, 0.75 ≤ ratio < 1.00; score 4, ratio > 1.00. The neointimal lesions in PAs were evaluated by VVG staining or α-SMA staining and vein lesions by VVG staining.
Isolation and culture of PASMCs
PASMCs were isolated from 8-week-old male WT and SKI mice using an explant method (Citation7). Briefly, rinse the PA branches below grade 3 with sterile saline to wash out blood, then digest with 0.2% collagenase II (Worthington Biochemical, Cat# LS004177) for 5 min. Cut the artery longitudinally, and scrape intima and adventitia to remove ECs and fibroblasts. The left tissue was placed to the bottom of a 35 mm dish and was cultured in DMEM supplemented with 20% FBS (ExCell Bio, Cat# FSP500) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37°C in a humidified atmosphere containing 5% CO2. Once the cells grew from the explants to a radius of around 0.5 cm, digested with 0.2% trypsin and cultured in DMEM containing 10% FBS and antibiotics. PASMCs from passages 3 to 8 were used.
Western blot
Homogenize and lyse the lungs or PASMCs in a RIPA buffer (Enogene, Cat# E1WP106) and separate the proteins by SDS-PAGE electrophoresis using standard methods, then transfer them to PVDF membrane. Immunoblot PVDF membrane at 4°C overnight with specific antibodies against the following proteins: SERCA2 (C498 antibody was a custom polyclonal antibody from Bethyl Laboratories, Inc., 110 kDa, dilution 1:1000), phosphorylated IRE1α (p-IRE1α, Genetex, GTX132808, 133 kDa, dilution 1:1000), XBP1s (Biolegend, Cat# 658802, 55 kDa, dilution 1:1000), CaN (Proteintech, Cat# 13422–1-AP, 59 kDa, dilution 1:1000), NFAT4 (Abcam, Cat# Ab93628, 150 kDa, dilution 1:1000), cyclin A1 (abcepta, Cat# AP51052, 55 kDa, dilution 1:1000), cyclin A2 (abcepta, Cat# AP7292a, 50 kDa, dilution 1:1000), cyclin B1 (abcepta, Cat# AP20411b, 60 kDa, dilution 1:1000), cyclin-dependent kinase 1 (CDK1, abcepta, Cat# AP16160b, 35 kDa, dilution 1:1000), α-SMA (Proteintech, Cat# 14395–1-AP, 43 kDa, dilution 1:1000), SM22α (Proteintech, Cat# 10493–1-AP, 23 kDa, dilution 1:2000), OPN (Proteintech, Cat# 22952–1-AP, 44 kDa, dilution 1:1000), MMP2 (Proteintech, Cat# 10373–2-AP, 64 kDa, dilution 1:2000), collagen type I (Col I, Engene, Cat# E110154C, 139 kDa, dilution 1:1000), collagen type III (Col III, Abcam, Cat# Ab7778, 138 kDa, RRID:AB_306066, dilution 1:2000), β-Actin (EnoGene, Cat# E12-051-3, 42 kDa, dilution 1:2000), followed by incubation with HRP-conjugated goat-anti-rabbit secondary antibody (Sino Biological Inc., Cat# SSA003) 1 h at room temperature. Proteins were visualized with an X-ray film system (Fujifilm, Japan) or ChemiDoc TM Touch System (Bio-Rad, USA). Band density was quantified by NIH Image J software and was normalized to β-Actin and expressed as a ratio to WT or solvent (vector) control.
Intracellular calcium measurements
Seed WT and SKI PASMCs on glass coverslips overnight in a 24-well plate. According to the manufacturer’s procedure, the cells were incubated with 4 μM Fluo-4 AM (Solarbio, Cat# F8500) in HBSS (Hyclone, Cat#SH30030.02) containing 0.02% pluronic F12 in the dark at 37°C for 20 min, retained for 40 min after adding a twofold volume of HBSS containing 10% FBS. Wash cells five times with HEPES buffer and incubate them in the dark at 37°C for 10 min. Record the fluorescence signals with excitation λ494 nm and emission λ516 nm by fluorescence microscopy (Leica DM6, Germany) and analyze the average fluorescence signals by Las X software (Leica, Germany).
Cell proliferation assay
The cell proliferation rate was determined by counting the cell number or using a tetrazolium-based non-radioactive proliferation assay kit (Quick Cell Proliferation Assay Kit II, BioVision, Cat# K301–500). To count cell numbers, PASMCs were seeded in a 12-well plate at a density of 5 × 104 cells per well in DMEM supplemented with 0.2% FBS for 24 h, and then switched to DMEM containing 10% FBS for 48 h. Harvest cells by mild trypsinization and count them with a hemocytometer. For the tetrazolium-based assay, PASMCs were seeded in a 96-well plate at a density of 5 × 103 cells per well in DMEM supplemented with 0.2% FBS overnight. Cell proliferation was stimulated by a medium supplemented with 10% FBS, and 0.2% FBS medium was used as a control. Change culture medium with high or low serum daily. The cell number in each well was determined 72 h later by the proliferation assay kit according to the manufacturer’s protocol.
Wounded monolayer migration assay
Briefly, 106 cells/well in 12-well plates were seeded in 0.2% FBS DMEM overnight to reach confluence, then scratch wounds were applied to PASMC monolayer with a pipette tip. Immediately after scratching, the cells were treated with 10% FBS DMEM to stimulate cell migration. In some experiments, SKI PASMCs were pre-treated with BAPTA-AM (1 μM, MCE, Cat# HY-100545, Shanghai, China), cyclosporin A (CsA, 1 μM, MCE, Cat# HY-B0579, Shanghai, China), 4μ8C (10 μM, MCE, Cat# HY-19707, Shanghai, China), or CDN1163 (10 μM, MCE, Cat# HY-101455, Shanghai, China) for 48 h before migration assay. DMSO acts as solvent control. Photographs were taken at 0 h and 6 h at three fixed locations along the scratch with a light microscope and analyzed using NIH Image J software.
Flow cytometry analysis of cell cycle
PASMCs (2 × 105 cells) were cultured in a 60 mm dish with 0.2% FBS DMEM for 24 h and then switched to DMEM containing 10% FBS for 24 h. Harvest cells by mild trypsinization and centrifugation. Wash the cells twice with pre-chilled PBS and then use pre-chilled 70% ethanol to fix the cells at 4°C overnight. The next day, the cells were collected by centrifugation and washed with 1 mL of PBS, then added 500 μL PBS containing 50 μg/mL propidium iodine (PI), 100 μg/mL RNase A, 0.2% Triton X-100. Incubate at 4°C for 30 min in the dark. Cell cycle assay was determined by flow cytometry (CytoFLEX A00-1-1102; Beckman Coulter, Brea, CA, USA), and data were analyzed using CytExpert software (Beckman Coulter). Use ModFit LT 3.2 software (Verity Software House, Topsham, ME, USA) to measure the percentage of cells in each phase of the cell cycle.
Cell treatments
For in vitro treatments of BAPTA-AM, CsA, CDN1163, or 4μ8C, culture SKI PASMCs in 0.2% FBS DMEM for 24 h, and then switch to media containing 10% FBS and BAPTA-AM (1 μM), CsA (1 μM), CDN1163 (10 μM), or 4μ8C (10 μM) for 24 h for analysis of protein expression, immunofluorescence staining, and cell cycle, or for 48 h for proliferation assay. The DMSO served as solvent control, and its final concentration was less than 0.1%.
Immunofluorescence staining in PASMCs
PASMCs were incubated with antibodies namely XBP1s (Biolegend, Cat# 658802), Ki67 (Abcam, Cat# ab15580), NFAT4 (Abcam, Cat# Ab93628) for 12 h, followed by Alexa Fluor 488 goat anti-rabbit IgG (H + L) (Jackson immunoresearch, Cat# 111-545-144) or Cy3-conjugated Affinipure goat anti-mouse IgG (H + L) (Proteintech, Cat# SA00009–1) for 2 h. Stain nuclei with DAPI or Hoechst 33 258 for 10 min. Monitor the fluorescence signals by microscopy (Leica DM6, Germany) and analyze them by Las X software (Leica, Germany).
Transfection of PASMCs
Adenovirus SERCA2b S674, or empty adenovirus were transfected to WT PASMCs. Meanwhile, adenovirus SERCA2b C674 or empty adenovirus was transfected to SKI PASMCs. Both transfections were conducted at 50 MOI/cell in DMEM without serum and antibiotics for 6 h before switching to DMEM containing 5% FBS for 48 h. Collect cells for western blot.
Data and statistical analysis
All studies were randomized into groups of the same size. For data analysis, the outcome assessors were blinded to animal code. That is, the animal code was assigned by one person, while data analysis was done by others without knowing the animal code, and then data were regrouped later based on the animal code. Group size is the number of independent repeats indicated in the figure legend, and statistical analysis was undertaken using only these independent repeats with n ≥ 5. Sample sizes in each group subjected to statistical analysis were determined based on our previous studies, preliminary results, and power analysis. In some experiments, we used ratio to control to avoid larger variations among different experiments. We normalized the mean values of the control group to 1. In the figures, the Y-axis shows the ratio of the values in the experimental group to that in the control group. Results presented as mean ± SEM. Statistical analysis was performed with GraphPad Prism 9.00 (http://www.graphpad.com/). Comparisons between experimental groups were performed with an unpaired t test, one-way ANOVA with Tukey’s multiple comparisons test or two-way ANOVA with Bonferroni’s multiple comparisons test. ROUT (Q = 1%) was used to find any number of outliers. To determine whether groups differed, the level of probability was set at P < .05 for statistical significance.
Materials
CsA (Cat# HY-B0579), BAPTA-AM (Cat# HY-100545), CDN1163 (Cat# HY-101455), and 4μ8C (Cat# HY-19707) were supplied by MCE (Shanghai, China). Avertin (2,2,2-tribromoethanol, Cat# T48402) was supplied by Sigma-Aldrich (Shanghai, China).
Results
The replacement of C674 by S674 promotes PASMC phenotypic transition
PH is characterized by a PASMC phenotypic transition. We have reported that the dysfunction of SERCA2 in PASMCs accelerates cell cycle and cell proliferation (Citation7), suggesting that SERCA2 dysfunction might promote PASMC phenotypic transition from a contractile phenotype to a synthetic phenotype, thus enhancing PASMCs proliferation, migration, and expression of fibrotic and inflammatory proteins. ECM remodeling of the pulmonary arteries plays a central role in PH, characterized by the increased collagen deposition, cross-linkage of collagen, and breakdown of elastic laminae (Citation12). PASMCs can synthesize and secrete ECM. Col I, Col III, and MMP2 play crucial roles in ECM remodeling, which promotes the proliferation of pulmonary vascular cells via various signaling pathways (Citation12). Matrix cytokine osteopontin (OPN) is a pleiotropic cytokine involved in the proliferation and migration of PASMCs and pulmonary vascular remodeling, whose expression is upregulated in the lungs of PAH patients and correlated to hemodynamic severity (Citation13,Citation14).
Due to the embryo’s lethality, only heterozygous SKI mice that had half C674 and half S674 were used. We have reported that the replacement of C674 by S674 causes SERCA2 dysfunction, leading to increased intracellular Ca2+ levels in PASMCs (Citation7). As shown in , in SKI PASMCs, the expression levels of PASMC synthetic phenotypic markers (Col I, Col III, MMP2, and OPN) were higher than those of WT PASMCs, while the expression levels of PASMC contractile phenotypic markers (α-SMA and SM22α) were lower than those of WT PASMCs. Similar to previous findings (Citation7), SKI PASMCs proliferated faster than WT PASMCs (). In addition, SKI PASMCs migrated faster than WT PASMCs (). All these indicate that the dysfunction of SERCA2 causes PASMC phenotypic transition.
Figure 1. The replacement of C674 by S674 promotes PASMC phenotypic transition. (a) Representative western blots of phenotype-related proteins in PASMCs and quantification of band intensities in the graph. (b) cell proliferation and migration. Data shown are means ± SEM. n = 5–7. *P < .05, SKI vs. WT; unpaired t test.
The replacement of C674 by S674 activates CaN/NFAT4 pathway
Ca2+ is a universal intracellular messenger whose change has a significant impact on identifying pathogenic mechanisms in PH (Citation15). Increased intracellular Ca2+ was detected by Fluo-4 in SKI PASMCs compared to WT PASMCs (). CaN is a Ca2+/calmodulin-dependent serine/threonine phosphatase that integrates alterations in intracellular Ca2+ levels into downstream signaling pathways, such as NFAT (Citation16). The Ca2+-activated phosphatase CaN is necessary for the nuclear import of NFATs. NFAT proteins remain phosphorylated in the cytoplasm in a quiescent state. Following a sustained increase in intracellular Ca2+, activated CaN directly dephosphorylates NFATs, resulting in the exposure of their nuclear localization sequence to lead to nuclear importation. Once located in the nucleus, NFATs bind to their target promoter elements either alone or in combination with nuclear partners to initiate the transcription of target genes (Citation17). NFAT4 is the predominant isoform of NFAT in smooth muscle, and its activation requires a global increase in intracellular Ca2+ (Citation18). As shown in , the expression levels of CaN and NFAT4 in SKI PASMCs were significantly higher than that of WT PASMCs. The majority of NFAT4 is located in the cytoplasm of WT PASMCs, while about half of NFAT4 in SKI PASMCs were in the cytoplasm and nucleus (), indicating that the dysfunction of SERCA2 increased intracellular Ca2+ levels in PASMCs and may activate NFAT4 via altering the Ca2+ level.
Figure 2. The replacement of C674 by S674 activates CaN/NFAT4 pathway in PASMCs. (a) the intracellular Ca2+ level was detected by Fluo-4 (green). (b) Representative western blots of CaN and NFAT4 and quantification of band intensities in the graph. (c) Representative images of NFAT4 immunofluorescence staining (green) and quantitative the proportion of cells with NFAT4 in the nucleus in the graph. Nuclei are indicated by DAPI (blue). Data shown are means ± SEM. (a)–(b), unpaired t test; (c), two-way ANOVA with Bonferroni’s multiple comparisons test; *P < .05, SKI vs. WT; n = 6. Scale bar: 50 μm.
Scavenging intracellular Ca2+ by BAPTA-AM inhibits CaN/NFAT4 pathway and PASMC phenotypic transition in SKI PASMCs
To validate our hypothesis that the increased intracellular Ca2+ which activates CaN/NFAT4 pathway and causes PASMC phenotypic transition is due to SERCA2 dysfunction, we used a highly selective intracellular Ca2+ chelating agent BAPTA-AM to scavenge Ca2+ in SKI PASMCs. BAPTA-AM significantly inhibited the protein expression of CaN and NFAT4 (). The immunofluorescence results of NFAT4 showed that after treatment with BAPTA-AM, the proportion of cells, with NFAT4 entering the nucleus decreased (). Furthermore, the administration of BAPTA-AM in SKI PASMCs downregulated the protein expression of PASMC synthetic phenotypic markers (Col I, Col III, MMP2, and OPN), while not affecting PASMC contractile phenotypic markers α-SMA and SM22α (), inhibited cell proliferation and migration (). Previously, we reported that the dysfunction of SERCA2 promotes PASMC proliferation by increasing the protein expression levels of p-IRE1α, an active form of IRE1α, and its downstream target XBP1s (Citation7). However, in SKI PASMCs, BAPTA-AM did not affect the protein expression of p-IRE1α and XBP1s (). These results proved that SERCA2 dysfunction leads to the activation of the CaN/NFAT4 pathway and the promotion of PASMC phenotypic transition via increased intracellular Ca2+ level.
Figure 3. Scavenging intracellular Ca2+ by BAPTA-AM inhibits CaN/NFAT4 pathway and PASMC phenotypic transition in SKI PASMCs. (a) Representative western blots of SKI PASMCs treated with BAPTA-AM, and quantification of band intensities in graph. (b) Representative images of NFAT4 (green) immunofluorescence staining in SKI PASMCs treated with BAPTA-AM, and quantitative the proportion of cells with NFAT4 in the nucleus in the graph. Nuclei are indicated by DAPI (blue). (c) cell proliferation and migration. Data shown are means ± SEM. (a) and (c), unpaired t test; (b), two-way ANOVA with Bonferroni’s multiple comparisons test; *P < .05, BAPTA-AM vs. solvent control (Ctrl); n = 5–8. Scale bar: 100 μm.
CaN inhibitor CsA suppresses the activation of NFAT4 and inhibits PASMC phenotypic transition in SKI PASMCs
To further test the contribution of the CaN/NFAT pathway to the PASMC phenotypic transition in SKI PASMCs, we used CsA, a CaN inhibitor, to inhibit the activity of CaN. The administration of CsA downregulated the protein expression of NFAT4, PASMC synthetic phenotypic markers (Col I, Col III, MMP2, OPN), and cell cycle-related proteins (cyclin A2, cyclin B1, CDK1), while upregulated the protein expression of PASMC contractile phenotypic marker α-SMA (). Accordingly, CsA decreased the percentage of NFAT4 in the nucleus and Ki67-positive cells (), prevented cells of the G0/G1 phase from entering the S and G2/M phases (), and inhibited cell proliferation and migration (). In SKI PASMCs, CsA did not affect the protein expression of CaN, p-IRE1α, and XBP1s. These results proved that SERCA2 downstream CaN/NFAT4 pathway mediates the PASMC phenotypic transition.
Figure 4. CaN inhibitor CsA suppresses the activation of NFAT4 and inhibits PASMC phenotypic transition in SKI PASMCs. (a) Representative western blots of SKI PASMCs treated with CsA, and quantification of band intensities in the graph. (b) Representative images of NFAT4 (green) and Ki67 (green) immunofluorescence staining in SKI PASMCs treated with CsA, and quantitative the proportion of cells with NFAT4 in the nucleus or percentage of Ki67-positive (green) cells. Nuclei are indicated by DAPI (blue). (c) Representative cell cycle analyzed by flow cytometry (left), and the relative percentage of cells in each phase (right). (d) cell proliferation and migration. Data shown are means ± SEM. NFAT4 immunofluorescence staining, two-way ANOVA with Bonferroni’s multiple comparisons test; others, unpaired t test; *P < .05, CsA vs. solvent control (Ctrl); n = 5–7. (c), n = 3. Scale bar: 100 μm.
The activation of CaN/NFAT4 is independent from IRE1α/XBP1s pathway in SKI mice
We have previously reported that 4μ8C, a substrate-specific inhibitor of IRE1α endoribonuclease, inhibited cell proliferation in SKI PASMCs (Citation7). Here, we showed that 4μ8C also inhibited cell migration in SKI PASMCs (). However, in SKI PASMCs, 4μ8C did not affect the proportion of cells with NFAT4 in the nucleus (), nor the protein expression of CaN and NFAT4 (). We tested the lung samples from a previous study (Citation7), and 4μ8C treatment still did not affect the protein expression of CaN and NFAT4 (). Together with the results above, we conclude that the activation of CaN/NFAT4 is independent of IRE1α/XBP1s pathway in the SKI background.
Figure 5. The activation of CaN/NFAT4 is independent of IRE1α/XBP1s pathway in SKI. (a) migration. n = 7–8. (b) Representative images of NFAT4 (green) immunofluorescence staining in SKI PASMCs treated with 4μ8C, and quantitative the proportion of cells with NFAT4 in the nucleus in the graph. Nuclei are indicated by DAPI (blue), n = 6. Scale bar: 50 μm. (c) Representative western blots of SKI PASMCs treated with 4μ8C, and quantification of band intensities in the graph, n = 5. (d) Representative western blots of SKI lung tissues treated with 4μ8C, and quantification of band intensities in the graph, n = 8. Data shown are means ± SEM. NFAT4 immunofluorescence staining, two-way ANOVA with Bonferroni’s multiple comparisons test; others, unpaired t test; *P < .05, 4μ8C vs. solvent control (Ctrl).
The redox status of C674 in the SERCA2b regulates CaN/NFAT4 pathway
SERCA2b is the predominant isoform of SERCA2 in cultured PASMCs. The overexpression of SERCA2b S674 activates IRE1α/XBP1s pathway and promotes cell proliferation in WT PASMCs, while the overexpression of SERCA2b C674 inhibits IRE1α/XBP1s pathway and cell proliferation in SKI PASMCs (Citation7). As shown in , overexpression of SERCA2b S674 upregulated the expression of CaN and NFAT4 in WT PASMCs (), while overexpression of SERCA2b C674 downregulated the expression of CaN and NFAT4 in SKI PASMCs (), proving that the redox status of C674 in the SERCA2b regulates the function of CaN/NFAT4 pathway.
Figure 6. The redox status of C674 in the SERCA2b regulates the CaN/NFAT4 pathway. (a) overexpression of adenovirus SERCA2b S674 or empty adenovirus control (Ctrl) on protein expression of CaN and NFAT4 in WT PASMCs. (b) overexpression of adenovirus SERCA2b C674 or empty adenovirus control (Ctrl) on protein expression of CaN and NFAT4 in SKI PASMCs. Data shown are means ± SEM. n = 5–7. *P < .05, significantly different as indicated; unpaired t test.
Activation of SERCA2 by CDN1163 inhibits SKI PASMC phenotypic transition
CDN1163 is a well-known SERCA agonist (Citation10), and its activation of the SERCA2 function is independent of the redox status of C674. In SKI PASMCs, the administration of CDN1163 did not affect the protein expression of SERCA2, but downregulated the protein expression of p-IRE1α, XBP1s, CaN, and NFAT4 (), decreased the expression of intracellular XBP1s detected by immunofluorescence staining () and the percentage of NFAT4 in the nucleus (). CDN1163 also reduced the protein expression of cell cycle related-proteins cyclin A1, cyclin A2, cyclin B1, and CDK1 (), decreased the percentage of Ki67-positive cells, and prevented cells in the G0/G1 phase from entering the S and G2/M phases in SKI PASMCs (). In addition, CDN1163 inhibited the protein expression of PASMC synthetic phenotypic markers (Col I, Col III, OPN) and promoted the protein expression of PASMC contractile phenotypic marker SM22α, without affecting the expression of MMP2 and α-SMA (). Overall, CDN1163 inhibited SKI PASMCs proliferation and migration (). These results indicate that the activation of SERCA2 inhibits the SKI PASMC phenotypic transition by suppressing both CaN/NFAT4 and IRE1α/XBP1s pathways.
Figure 7. Activation of SERCA2 by CDN1163 inhibits SKI PASMC phenotypic transition. (a) Representative western blots of SKI PASMCs treated with CDN1163, and quantification of band intensities in the graph. (b) Representative images of XBP1s (red) immunofluorescence staining in SKI PASMCs treated with CDN1163 and quantitative analysis of fluorescence density in the graph. Nuclei are indicated by DAPI (blue). (c) Representative images of NFAT4 (green) immunofluorescence staining in SKI PASMCs treated with CDN1163, and quantitative the proportion of cells with NFAT4 in the nucleus in the graph. Nuclei are indicated by DAPI (blue). (d) Representative percentage of Ki67-positive (green) cells and cell cycle analyzed by flow cytometry in SKI PASMCs treated with CDN1163, and the relative percentage of cells in each phase. Data shown are means ± SEM. NFAT4 immunofluorescence staining, two-way ANOVA with Bonferroni’s multiple comparisons test; others, unpaired t test; *P < .05, CDN1163 vs. solvent control (Ctrl); cell cycle (n = 3), others (n = 5–9). Scale bar: 100 μm.
Revitalization of SERCA2 by CDN1163 ameliorates pulmonary vascular remodeling in SKI mice
We previously found that SKI mice had various types of pulmonary vascular remodeling, including pulmonary venous hypertrophy and PA remodeling (tunica media hypertrophy, neointimal lesions, plexiform lesions, fibrosis, thrombosis), especially at an older age (Citation7). Next, we gave the 4-week-old WT and SKI mice CDN1163 or solvent control (Ctrl) for 4 weeks to test if improving the function of SERCA2 could prevent pulmonary vascular remodeling. The pulmonary vascular remodeling in SKI mice at this age endpoint is reversible based on the Heath-Edward score, while their right ventricular pressure and Fulton Index are not affected (Citation7). The major types of pulmonary vascular remodeling in 2-month-old SKI mice include venous hypertrophy, tunica media thickness of the resistance PAs (50- to 100-μm diameter), cellular neointima in small PAs (around 50 μm diameter), and the muscularization of distal pulmonary arterioles. In addition, the percentage of Ki67-positive cells in PA tunica media in SKI mice is increased compared to WT mice (Citation7).
In the first round of in vivo studies, between WT/CDN1163 and WT/Ctrl, there was no difference in body weight, right ventricular pressure, and Fulton Index (Supplemental ). CDN1163 treatment did not affect pulmonary vascular remodeling indexes, including venous lesion score (Supplemental ), PA tunica media thickness (Supplemental ), the muscularization of distal pulmonary arterioles (Supplemental ), the percentage of cellular neointima, and the overall Heath-Edward score (Supplemental ), nor did affect the percentage of Ki67-positive cells in PA tunica media (Supplemental ). Thus, we did not apply CDN1163 further in WT mice, and only compared three groups (WT/Ctrl, SKI/Ctrl, and SKI/CDN1163).
Among these three groups (WT/Ctrl, SKI/Ctrl, and SKI/CDN1163), there were no differences in body weight, right ventricular pressure, and Fulton Index (Supplemental ). The pulmonary vascular remodeling indexes (venous lesion score, PA tunica media thickness, percentage of cellular neointima, Heath-Edward score, percentage of partial muscularization of distal pulmonary arterioles, and percentage of Ki67-positive cells in PA tunica media) in SKI mice were improved by CDN1163 to the levels comparable to WT mice (). In lung samples of SKI mice, the increased protein expression of p-IRE1α, XBP1s, CaN, NFAT4, and cell cycle related-proteins was reversed by CDN1163 to the levels comparable to WT mice as well (), suggesting that the activation of SERCA2 by CDN1163 could ameliorate pulmonary vascular remodeling in SKI mice by suppressing IRE1α/XBP1s and CaN/NFAT4 pathways.
Figure 8. Revitalization of SERCA2 by CDN1163 ameliorates pulmonary vascular remodeling in SKI mice. (a) Representative pulmonary vein remodeling is indicated by Verhoeff-van Gieson staining (top) and pulmonary artery remodeling is indicated by H&E staining (bottom). (b) summary of the cellular neointima and Heath-Edward score in pulmonary arteries. (c) muscularization of distal pulmonary arterioles indicated by a-smooth muscle actin (a-SMA) staining (red) and von Willebrand factor (vWF) staining (green). (d) the percentage of Ki67 (green) positive staining in pulmonary artery tunica media indicated by a-SMA (red). Nuclei are indicated by DAPI (blue). (e) the protein expression of p-IRE1α, XBP1s, CaN, NFAT4 and cell cycle related-proteins in lungs. Data shown are means ± SEM. *P < .05, SKI treated with solvent control (SKI/ctrl) vs. WT treated with solvent control (WT/ctrl), #P < .05, SKI treated with CDN1163 (SKI/CDN) vs. SKI/Ctrl, one-way ANOVA with Tukey’s multiple comparisons test, n = 7. Scale bar: 25 μm.
Discussion and conclusion
The redox status of C674 in the SERCA2 has been proven to be associated with cardiovascular health and disease (Citation19–21). These findings lead us to explore its role in other diseases. We first observe that the irreversible oxidation of SERCA2 C674 is increased in PASMCs and remodeled PAs in the hypoxia-induced PH mouse model. Then we utilize heterozygous SKI mice to mimic partial C674 oxidation and find that the substitution of C674 causes SERCA2 dysfunction and promotes PASMC proliferation and pulmonary vascular remodeling by activating IRE1α/XBP1s pathway (Citation7). In this study, we further show that suppressing IRE1α/XBP1s pathway by 4μ8C inhibits cell migration in SKI PASMCs, supporting that the activation of IRE1α/XBP1s pathway accounts for the PASMC phenotypic transition induced by the SERCA2 dysfunction. The association of SERCA2 dysfunction and PASMC phenotypic transition interests us. In this study, we find that the dysfunction of SERCA2 promotes the PASMC phenotypic transition by activating the cytoplasmic calcium-dependent CaN/NFAT4 pathway, which is independent of the activation of IRE1α/XBP1s pathway, and suppressing CaN/NFAT4 pathway by CsA inhibits SKI PASMC phenotypic transition. Revitalizing the function of SERCA2 by CDN1163 or overexpression of SERCA2b suppresses both CaN/NFAT4 pathway and IRE1α/XBP1s pathway. Furthermore, CDN1163 can prevent PASMC phenotypic transition and pulmonary vascular remodeling caused by SERCA2 dysfunction. summarizes the mechanisms of how SERCA2 dysfunction promotes PASMC phenotypic transition and pulmonary vascular remodeling.
Figure 9. Schematic diagram summarizes the proposed mechanisms underlying how the irreversible oxidation of SERCA2 C674 thiol promotes PASMC phenotypic transition. Increases in ROS production cause the irreversible oxidation of SERCA2 C674 thiol, which further 1) activates the p-IRE1α/XBP1s pathway (Citation7), 2) activates the calcium-dependent CaN/NFAT4 pathway (the current study), and promotes the PASMC phenotypic transition and eventually causes pulmonary vascular remodeling. CaN/NFAT4 and p-IRE1α/XBP1s pathways are independent of each other in promoting PASMC phenotypic transition. p-IRE1α/XBP1s inhibitor 4μ8C, CaN/NFAT4 inhibitor cyclosporin a (CsA), SERCA2 agonist CDN1163, or overexpression of SERCA2b could reverse PASMC phenotypic transition caused by C674 inactivation.
SERCA activity and calcium homeostasis are key to maintaining vascular homeostasis (Citation19). SERCA2 activity can be altered under hypoxia, which promotes transglutaminases 2-mediated SERCA2 serotonylation, further increases the calcium influx through the TRPC6 channel, and leads to pulmonary vein remodeling (Citation22). A decrease in SERCA2a expression is found in pulmonary vessels of PAH patients and monocrotaline-induced PH rats, while overexpression of SERCA2a improves monocrotaline-induced rat pulmonary vascular remodeling and right ventricular hypertrophy (Citation23). There is no report on whether the dysfunction of SERCA2a could activate the CaN/NFAT pathway or not in PASMCs. However, in human PASMCs, overexpression of SERCA2a suppresses the protein expression of CaN and inhibits NFAT transcriptional activity, as well as cell proliferation and migration (Citation23). As data is not shown, the overexpression of SERCA2a S674 in WT PASMCs upregulated the expression of CaN and NFAT4, proving the capacity of SERCA2a dysfunction on the regulation of CaN/NFAT activation. We find that the mRNA level of SERCA2a is hardly detectable in cultured PASMCs and SERCA2b is the predominant isoform of SERCA2 in cultured PASMCs (Citation7). The overexpression of SERCA2b S674 in WT PASMCs activates IRE1α/XBP1s pathway (Citation7) and CaN/NFAT pathway, while the overexpression of SERCA2b C674 in SKI PASMCs inhibits IRE1α/XBP1s pathway (Citation7) and CaN/NFAT pathway. All these indicate that the redox status of C674 in the SERCA2b is key to controlling the PASMC phenotypic transition by regulating IRE1α/XBP1s and CaN/NFAT pathways. In SKI PASMCs, the expression of p-IRE1α and XBP1s are not affected by intracellular Ca2+ chelating agent BAPTA-AM or CaN inhibitor CsA, while the expression of CaN and NFAT4 or its nuclear localization are not affected by p-IRE1α/XBP1s pathway inhibitor 4μ8C, indicating that the activation of calcium-dependent CaN/NFAT pathway and IRE1α/XBP1s pathway caused by SERCA2 dysfunction work independently.
Currently, there is no report of SERCA agonist treatment of pulmonary vascular remodeling and PH. CDN1163 is a small molecule allosteric SERCA2 activator that directly binds and interferes with the structure of SERCA2, leading to its activation (Citation24). Selective screening tests were conducted on CDN1163 to evaluate its impact on various ion pumps and channels and found that it only has activity on SERCA (Citation25). Considering the lipophilic nature of CDN1163, it was proposed that the binding site(s) for CDN1163 is localized in the SERCA transmembrane region (Citation24). C674 is located on the cytoplasmic side, thus its redox state might have little effect on the binding efficiency of CDN1163 to SERCA2. We find that CDN1163 inhibits the expression of p-IRE1α, XBP1s, CaN, NFAT4, cell cycle related-proteins, and PASMC synthetic phenotypic markers, slows down cell cycle, and inhibits cell proliferation and migration in SKI PASMCs. The pulmonary vascular remodeling indexes and the increased protein expression of p-IRE1α, XBP1s, CaN, and NFAT4 in SKI lungs were reversed by CDN1163 to the levels comparable to WT mice, indicating that SERCA2 agonist CDN1163 could compensate for SERCA2 dysfunction to ameliorate PASMC phenotypic transition and pulmonary vascular remodeling by suppressing IRE1α/XBP1s and CaN/NFAT pathways. All these indicate that CDN1163 improves SERCA2 function independent of C674 redox state.
The fundamental function of SERCA2 is to maintain calcium homeostasis. We have previously reported that SERCA2 dysfunction increased intracellular Ca2+ level in PASMCs (Citation7). Intracellular Ca2+ is essential for cellular physiology and cell cycle progression. Exacerbated intracellular Ca2+ signaling promotes PASMC hyperproliferation and exacerbates migration. Since the increased intracellular Ca2+ level is a well-known contributor to the proliferation of PASMCs, we infer that SERCA2 dysfunction could accelerate PASMC proliferation by activating a calcium-dependent pathway. Based on our experience in aortic aneurysms, we found that the dysfunction of SERCA2 could exacerbate aortic aneurysms by multiple independent mechanisms, including the activation of calcium-dependent CaN/NFAT4 pathway (Citation20). The CaN/NFAT pathway has been reported to contribute to the pathogenesis of monocrotaline-induced pulmonary arterial hypertension rats (Citation9). Therefore, in this study, we explored the contribution of the calcium-dependent CaN/NFAT pathway in the SERCA2 dysfunction-induced pulmonary artery remodeling.
CaN is a calcium-dependent serine/threonine phosphatase, whose activation requires sustained increase in cytoplasmic Ca2+ and consequently phosphorylates its downstream targets, including NFAT. The classical CaN inhibitor CsA inhibits hypoxia-induced rat PH and monocrotaline-induced rat PAH (Citation9,Citation26). The most studied substrates of CaN are NFAT transcription factors, whose family consists of five isoforms, NFAT1‒5. NFAT1–4 is regulated by calcium signaling. NFAT1–4 contains the DNA binding domain Rel-homology region (RHR) and regulatory domain NFAT homology region (NHR). NFAT5 does not contain NHR. NHR is highly phosphorylated in resting cells to restrict NFAT to the cytoplasm, and its dephosphorylation by CaN results in the exposure of NFAT nuclear localization sequence and leads to NFAT nuclear translocation being active (Citation27). NFAT1 and NFAT4 are the main isotypes of NFAT involved in the pathogenic process of PH (Citation17). NFAT1 is activated in human PH PASMCs and rat monocrotaline-induced PAH (Citation28). The activation of NFAT1 mediates platelet-derived growth factor-induced PASMC proliferation and migration (Citation29), and serotonin-induced cyclin A expression, CDK2 activation, and DNA synthesis (Citation30). The activation of NFAT4 is responsible for hypoxia-induced PA wall thickness and right ventricular hypertrophy (Citation31). In SOD1 knockout mice, the elevated superoxide/hydrogen peroxide ratio activates NFAT4 but not NFAT1 in PAs and PASMCs, which induces pulmonary vascular remodeling and PH (Citation32). As data show, we observed increased superoxide production and downregulation of SOD1 in SKI PASMCs. NFAT4 is broadly activated in SKI aortic SMCs (Citation20), cardiac fibroblasts (Citation33), and bronchial SMCs (unpublished data). Although we have not particularly discriminated whether NFAT1 or other isoforms of NFAT are involved, we infer that the activation of NFAT4 plays an important role in regulating SKI PASMC phenotypic transition. In addition, the regulation of different isoforms of NFAT on cell cycle-related proteins is controversial, which might be due to the various cell types and distinct signaling pathways activated by different NFAT isoforms (Citation34). Here, we provide evidence that the activation of the CaN/NFAT4 pathway accelerates the cell cycle by upregulation of cell cycle-related proteins cyclin A2, cyclin B1, and CDK1.
PH is characterized by progressive distal PA obstruction, leading to right ventricular hypertrophy and failure. ECM remodeling occurs before the increase of the initial and medial thickness, and promotes the proliferation of pulmonary vascular cells by multiple mechanisms, while inhibition of ECM remodeling prevents experimental PAH (Citation12). Col I, Col III, and MMP2 play crucial roles in ECM remodeling. OPN promotes PASMC phenotypic transition and pulmonary vascular remodeling, and its expression is significantly correlated to PAH severity (Citation13,Citation14). We have reported that OPN as a marker protein for SMC differentiation could upregulate SMC synthetic phenotypic markers (Col I, Col III, and MMP2) and downregulate SMC contractile phenotypic marker SM22α in aortic SMCs and further promote cell proliferation and migration (Citation35,Citation36). There is no report about the direct regulation of OPN on Col I, Col III, and MMP2 in PASMCs. We infer that there might be a similar regulatory effect of OPN on PASMC phenotypic markers, and OPN might play a key role in regulating PASMC phenotype. Similar to our findings in SKI aortic SMCs (Citation20), inhibition of CaN activity by CsA also suppresses the expression of OPN, Col I, Col III, and MMP2 in SKI PASMCs. In rat PASMCs, sphingosine-1-phosphate could upregulate OPN expression by activation of the CaN/NFAT4 pathway and stimulate PASMC proliferation (Citation37). Our results support the idea that the activation of the CaN/NFAT4 pathway is responsible for the upregulation of OPN, Col I, Col III, and MMP2 caused by SERCA2 dysfunction, thus promoting PASMC phenotypic transition.
We have reported that the inactivation of C674 causes SERCA2 dysfunction, by activating IRE1α/XBP1s pathway to promote PASMC hyperproliferation and pulmonary vascular remodeling (Citation7). Here we show that the dysfunction of SERCA2, especially SERCA2b, could also activate the cytoplasmic calcium-dependent CaN/NFAT4 pathway to promote PASMC phenotypic transition. Our ongoing study suggests that mitochondrial dysfunction also contributes to the PASMC phenotypic transition and pulmonary vascular remodeling of SKI mice, indicating that the dysfunction of SERCA2 promotes PASMC phenotypic transition and pulmonary vascular remodeling by multifaceted mechanisms. SERCA2 agonists have therapeutic potential in clinics for the prevention and treatment of pulmonary vascular remodeling and PH.
Author contributions
Y.W., Z.Q., P. H., and X.T. participated in the research design and performed the data analysis and interpretation; Y.W., L.S., and X.T. wrote the manuscript; Y.W., Z.Q., Y.Q., C.H., X.H., X.L., and X.G. conducted the experiments. All authors reviewed and revised the final version of this manuscript and approved its submission.
Significance
We first report that the dysfunction of SERCA2 causes pulmonary vascular remodeling by promoting PASMC phenotypic transition via activation of CaN/NFAT4 pathway, which could be reversed by SERCA2 agonist CDN1163.
Abbreviations
C674, Cys674; CaN, calcineurin; CDK, cyclin-dependent kinase; Col I, collagen type I; Col III, collagen type III; CsA, cyclosporin A; DAPI, 4,’6-diamidino-2-phenylindole; ECM, extracellular matrix; ECs, endothelial cells; IRE1α, inositol requiring enzyme 1 alpha; LV, left ventricle; NFAT, nuclear factor of activated T-lymphocytes; OPN, osteopontin; PA, pulmonary artery; PAH, pulmonary artery hypertension; PASMCs, pulmonary artery smooth muscle cells; PH, pulmonary hypertension; p-IRE1α, phosphorylated IRE1α; RV; right ventricle; RVEDP, right ventricular end diastolic pressure; RVSP, right ventricular systolic pressure; S, interventricular septum; S674, Ser674; SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase; SKI, SERCA2 C674S knock-in; SMA, smooth muscle actin; SMCs, smooth muscle cells; VVG, Verhoeff-Van Gieson; vWF, von Willebrand factor; WT, wild-type; XBP1s, spliced X-box binding protein 1
Supplemental Material
Download Zip (248.9 KB)Acknowledgments
This work was supported by the National Natural Science Foundation of China (31571172 and 81870343, X.T., and 81700237, P.H.), Chongqing Natural Science Foundation (cstc2021jcyj-msxmX0043, X.T.), Special Research Assistant of the Chinese Academy of Sciences (W.Y.), Postdoctoral Research Program of Chongqing Human Resources and Social Security Bureau (W.Y.).
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/10641963.2023.2272062
Additional information
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