2. Introduction
Hypertension is a critical risk factor for cardiovascular disease, which is the
leading cause of death and disability worldwide. Angiotensin II (Ang II), as the
major bioactive product of renin-angiotensin system, is crucial for salt and
water homeostasis and vasoconstriction, and thus plays a critical role in the
genesis of hypertension [1]. In vascular smooth muscle cells (VSMCs), activation
of AT1 receptor by Ang II causes phospholipase C (PLC) activation, leading to the
release of the second messengers inositol trisphosphate (IP) and
diacylglycerol (DAG) and results in the rise of intracellular Ca
concentration ([Ca]). Elevated [Ca] initiates a
Ca-calmodulin (CaM) interaction, causing the activation of myosin light
chain kinase (MLCK) and the consequent phosphorylation of myosin light chain
(MLC) [2]. MLC phosphorylation level is the core of VSMCs contraction, which
depends on the balance between MLCK and MLC phosphatase (MLCP) activity. RhoA, a
member of the Rho family of small GTPase binding proteins, is abundantly
expressed in VSMCs. Activation of RhoA and its downstream target Rho-kinase
(Rock) is increasingly being recognized as an important mechanism of
vasoconstriction by Ang II. RhoA activation induces phosphorylation of myosin
phosphatase target subunit 1 (MYPT1), which enhances the dephosphorylation of
MLCP [2]. The reduction of MLCP activity increases the phosphorylation level of
MLC, and thereby maintaining force generation in VSMCs. Deregulation of these
cellular signaling components can lead to an overstimulated state causing
sustained vasoconstriction and the elevation of blood pressure.
Several studies have identified Cl channels expression and characterized
their function in vascular smooth muscle cells (VSMCs), including classic
Ca-activated Cl channel (CaCC), cGMP-dependent CaCC, volume
regulated Cl channel (VRCC) and cystic fibrosis transmembrane conductance
regulator (CFTR) [3]. Evidence from genetic and pharmacological studies point to
a critical role for Cl channels during the development of hypertension [4].
Previous work in our laboratory have demonstrated that VRCC and CaCC were
involved in VSMCs proliferation [5, 6] and cerebrovascular remodeling during
hypertension [7, 8, 9]. CFTR is a member of the ATP-binding cassette transporter
superfamily that functions as a Cl channel across the membrane.
Cftr gene mutations lead to cystic fibrosis (CF) disease, which results
in dysfunction of transepithelial movement of water and electrolyte in exocrine
tissues [10]. Clinical data showed that older female CF mutation carriers
exhibited lower blood pressure (BP), which had been attributed to excessive salt
depletion [11]. Radiotelemetry measurements of physiological blood pressure
demonstrated that average SBP and DBP of 24-hour of Cftr mice
were all significantly higher than those of Cftr mice [12].
These previous studies indicated that CFTR might play an important role in BP
control, however, the regulating effect of CFTR on the genesis of hypertension
remains unclear.
Hypertension is pathologically characterized by augmented vascular
contractility. The role of CFTR function has been described in smooth muscle
cells of airway [13, 14], mesenteric artery [15] and aorta [16]. It was reported
that trachea from CF patients showed greater MLC phosphorylation in response to
IL-8 [17]. Previous studies on blood vessels showed that depolarization induced
aortic tension was significantly increased in Cftr mice
compared to Cftr mice [16]. CFTR corrector compounds (C18
or lumacaftor) normalize pathological alterations in cerebral arterial CFTR
expression, vascular reactivity, and cerebral perfusion [18], suggesting that
CFTR was involved in the regulation of vascular tone. Nevertheless, further
research is needed to investigate the signaling mechanisms underlying the effect
of CFTR on vasoconstriction during hypertension.
In the current study, Ang II infusion induced hypertensive models were
established and the influence of Cftr knockout on BP during this process
was investigated. CFTR protein mRNA/protein expression in the hypertensive
arteries were examined. Besides, the regulating effect of CFTR on Ang II induced
vasoconstriction was assessed in vivo with CFTR pharmacologically
inhibited or genetically knockout. Furthermore, the underlying signaling
mechanisms was investigated centered around the phosphorylation of MLC. This
study reveals the specific involvement of CFTR in the regulation of
vasoconstriction and genesis of hypertension.
3. Materials and methods
3.1 Animals
All animal care and experimental procedures complied with the policies of Ethics
Committee of ZSSOM on Laboratory Animal Care (document No. 2016-080) and
conformed to the “Guide for the Care and Use of Laboratory Animals” of the
National Institute of Health in China. Sprague-Dawley rats were purchased from
the Experimental Animal Center of Sun Yat-sen University. The gut-corrected
Cftr knockout mouse strain STOCK Cftr
Tg(FABPCFTR)1Jaw/J was purchased from the Jackson Laboratory (ME, USA). Females
that are heterozygous for the Cftr knockout allele and homozygous for
the FABP-hCFTR transgene were bred with males that are homozygous for both
Cftr knockout allele and FABP-hCFTR transgene and thus wild-type
(Cftr), heterozygote (Cftr) and Cftr
knockout (Cftr) littermates were obtained. Animals were housed
in standard plastic rodent cages and maintained at a regulated environment (25
C, 12 h light, 12 h dark cycle with lights on at 7:00 and off at 19:00)
with ad libitum access to a normal chow diet with 2.45 g NaCl/1000 g (D12450B, 10
kcal % Fat, Research Diets). The number used followed the principle of minimal
requirement and showed in Figure legends.
Ang II induced hypertension models were established using Cftr,
Cftr mice (5–8 weeks old) or Sprague-Dawley rats (150–200 g).
Ang II was administered via subcutaneously implanted osmotic minipumps (Alzet,
Cupertino, CA, Model 1002 for mice and 2002 for rats) for 2 weeks to establish
Ang II infusion induced hypertension models. The administration rate was 1000
ng/kg/min [19] for mice and 80 ng/min [20] for rats as previously reported and
manufacture’s instruction. BP was measured in conscious mice at 6:00 PM by tail
cuff plethysmography (BP-98A, Softron, Japan) as we previously reported [21].
3.2 Reagents
Antibody to CFTR was from Alomone Labs (Jerusalem, Israel) and antibody to MLCK
was from Sigma Aldrich Corp. (St. Louis, MO, USA). Antibodies to total myosin
light chain (t-MLC), phosphorylated myosin light chain (p-MLC), MYPT1,
phosphorylated MYPT1 (p-MYPT1), -actin and all secondary antibodies were
from Cell Signaling Technology (Boston, MA, USA). CFTR(inh)-172, Y27632, ML-7,
Fura-2AM, RIPA Lysis Buffer (10X), protease inhibitor cocktail, L-nitro-arginine
methyl ester (L-NAME), TritonX-100, EGTA, MnCl, pentobarbital sodium, Ang
II, Hanks’ Balanced Salt Solution (HBSS) and Krebs-Henseleit Modified
Buffer were from were from Sigma Aldrich Corp. (St. Louis, MO, USA).
Polyvinylidene difluoride (PVDF) membrane was from Millipore Corp. (Billerica,
MA, USA). Pierce ECL substrate and sequence-specific primers were from Thermo
Fisher Scientific (Waltham, MA, USA). Trizol reagent was from Invitrogen
(Carlsbad, CA, USA), Transcriptor First Strand cDNA Synthesis Kit (Roche,
Mannheim, Germany) and Fast start universal SYBR Green Master (ROX) were from
Roche Molecular Biochemicals (Mannheim, BW, Germany). RhoA Activation Assay Kit
was from Abcam (Cambridge, UK). Dulbecco’s modified Essential medium/F-12
(DMEM/F12) medium, fetal calf serum were from Gibco (Carlsbad, CA, USA).
3.3 Cell isolation and culture
Basilar artery smooth muscle cells (BASMCs) were isolated from rat and mice
basilar arteries as we previously described [22]. Briefly, 4 weeks old male
Sprague–Dawley rats, 5–8 weeks old Cftr and
Cftr mice were anesthetized with pentobarbital sodium
(50 mg/kg, intraperitoneally). The basilar arteries were collected
immediately and immersed in Krebs solution containing 10 U/L
penicillin and 100 mg/L streptomycin. After the connective tissue
was cleaned, the basilar arteries were cut into small pieces about
0.5 mm long and the vessel segments were placed on the surface of
the dish. The dish was then incubated in DMEM/F12 supplemented with 20% fetal
calf serum at 37 C and 5% CO. Passages 4–6 of
the cultured BASMCs were used for experiments.
3.4 Adenovirus transfection
Ad-Cftr and Ad-Cftr-shRNA were designed and produced by
GENECHEM (Shanghai, CN). The cDNA of Cftr plasmid was a kind gift from
Dr. Jim Hu (University of Toronto, ON, CA). The sequence of Cftr
(NM_031506) shRNA was
5-CCGGGCTGAAAGCAGGTGGGATTCTCAAGAGAAATCCCACCTGCTTTCAGCTTTTTTG-3. Adenovirus was
transfected using the protocol previously described [21]. Briefly, cultured
BASMCs at 50% confluence were infected with adenovirus encoding Cftr or
Cftr shRNA for 6 h in serum-free medium, and then cells were washed and
incubated in fresh complete medium for another 48 h before harvested.
3.5 Western blot analysis
Western blot was performed as previously described [22]. Briefly, cells or
tissues were lysed with RIPA lysis buffer containing protease inhibitor cocktail.
Protein was separated with 8% or 10% SDS-PAGE and transformed to a PVDF
membrane. After blocked with 5% milk for 1 h at room temperature, the membrane
was incubated with primary antibody at 4 C overnight and then with
secondary antibody for 1 h at room temperature. For analysis of CFTR expression
in arteries or in VSMCs, proteins were extracted in lysis buffer and
immunoblotted with anti-CFTR primary antibody (1:500, Alomone Labs) [23], and
detected with HRP-linked secondary antibodies. Immunogen of CFTR antibody:
Peptide (C) KEETEEEVQDTRL, corresponding to amino acid residues 1468–1480 of
human CFTR (Accession P13569). Final band detection was performed with a Pierce
ECL substrate by using a BIO-RAD molecular imager ChemiDoc XRS+ (Bio-Red,
Hercules, CA). The densities of the target bands were determined by ImageJ
software (National Institutes of Health, Bethesda, MD, USA).
3.6 Quantitative real time PCR
Quantitative real time PCR (qRT-PCR) was performed as previously described [24].
Briefly, the total RNA was extracted from basilar arteries using Trizol reagent
according to the manufacturer’s instructions. Purity and concentration of
isolated total RNA were measured using the TECAN infinite M100 PRO Biotek
microplate reader (Tecan Group, Ltd., Männedorf, Switzerland). First-strand
cDNA was generated from 1 g of total RNA using Transcriptor First Strand
cDNA Synthesis Kit and qRT-PCR was performed using the Fast Start Universal SYBR
Green Master (ROX) according to the manufacturer’s instructions. The total
reaction volume was 20 L, including 10 L of 2 SYBR Green
Master, 0.6 L of PCR Forward Primer (10 M), 0.6 L of PCR
Reverse Primer (10 M), 2 L of cDNA (20 ng) and 7 L of
double-distilled water. The qRT-PCR was set at an initial step of 10 min at
95 C, followed by 40 cycles at 95 C for 15 seconds and then
60 C for 60 seconds. qRT-PCR was executed using iQ5 real-time PCR
detection system (Bio-Rad Laboratories, Inc, CA, USA). All experiments were done
in triplicate, and all samples were normalized to GAPDH. The expression levels
were calculated using 2 methods. Melting curve
analysis was performed in the range of 60 C to 95 C, 0.5
C per 5 seconds increments. The sequence-specific primers were used as
follows: CFTR, 5-GGATGCTGAGGAAGCAACTC-3 (forward) and
5-CCAGCCTGGAACTCTCTTTG-3 (reverse); GAPDH, 5-AGGTCGGTGTGAACGGATTTG-3
(forward) and 5-TGTAGACCATG TAGTTGAGGTCA-3 (reverse).
3.7 Isometric tension measurement in arteries
Vascular rings of thoracic aorta and basilar artery were prepared and isometric
tension measurement experiments were performed as previously described [25].
Briefly, mice were euthanized and then thoracic aortas or basilar arteries were
quickly removed and placed into ice-cold Krebs-Henseleit Modified Buffer
containing 2.5 mM Ca (Krebs buffer), and detached from fatty and
connective tissues. Aortic ring segments (3–5 mm) and basilar artery ring
segments (2 mm) were fixed on the Wire Myograph System (Danish Myo Technology,
Denmark) and immersed in Krebs buffer aerated with 95% O/5% CO and
maintained at 37 C. The rings were stretched to an optimal resting
tension (3 mN) and allowed to stabilize for 1 h. 60 mM of KCl was used to
stimulate vessel contraction for 3 times, the difference of isometric force
should be less than 5% between each time. At the end of each stimulation, the
original equilibrium tension of vessels were recovered after washed by Krebs
buffer for 3 times. The vessels were then balanced for 30 min, followed by
subsequent experiments. During the measurement process, endothelium of the
arteries was chemically denudated by L-NAME (10 M). Isometric force
were recorded using PowerLab data acquisition system (AD Instruments Inc, US).
3.8 Intracellular Ca concentration measurement
[Ca] was measured as we previously described [26]. Briefly, VSMCs
were digested and incubated with DMEM/F12 medium containing 2 M of
Fura-2/AM for 45 min at 37 C, then the cells were washed with HBSS
buffer containing 2 mM Ca 3 times and resuspended. The number of cells was
adjusted to 10 cells per mL. Fluorescence emission was monitored at 500 nm
using RF-5301 fluorescence Spectrophotometer (Shimadzu, Tokyo, Japan) with an
excitation at 340 and 380 nm. [Ca] was determined from the formula:
[Ca] = Kd Sf380/b380(R–Rmin)/(Rmax–R), where Kd is 224
nm at 37 C; Sf380/b380 is the ratio of the intensities of the free and
bound dye forms at 380 nm; R is the fluorescence ratio (340 nm/380 nm) of the
intracellular Fura-2/AM; Rmax and Rmin are the maximal and minimal fluorescence
ratios obtained by addition of Triton X-100 (0.09%) and EGTA (3 mM)
respectively. Ang II (100 nM) was added to the cell suspension when the
fluorescence intensity was stable.
3.9 Measurements of Mn quenching of fura-2 fluorescence
As we described previously [27], VSMCs were digested and incubated with 2
M of Fura-2/AM for 45 min at 37 C, then the cells were washed
with HBSS buffer containing 1.3 mM Ca 3 times and resuspended with
Ca-free HBSS buffer. The number of cells was adjusted to 10 cells
per ml. After baseline fluorescence got to steady state, Mn was injected
into the cuvette with the final concentration of 500 M. Fluorescence
intensity was detected by RF-5301 Fluorescence Spectrophotometer (Shimadzu,
Tokyo, Japan) with magnetic stirring at 37 C. The excitation wavelength
was 360 nm and the absorption wavelength was 510 nm. After the fluorescence value
was relatively stable, Ang II were added to stimulate the flow of divalent
cations.
3.10 RhoA activation assay
RhoA-GTP and total RhoA were detected using a RhoA Activation Assay Kit
according to manufacturer’s instruction. Briefly, the anti-active RhoA (RhoA-GTP)
mouse monoclonal antibody was incubated with cell lysates. The bound RhoA-GTP was
then pulled down by protein A/G agarose beads at 4 C for 1 h. The beads
were centrifuged and washed, and then boiled in SDS-PAGE sample buffer. Samples
were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes and
incubated with anti-RhoA Rabbit polyclonal antibody. Total RhoA was detected by
western blot of the cell lysates using anti-RhoA rabbit polyclonal antibody
provided in the kit.
3.11 Statistics
All data are expressed as mean SEM with n representing the number of
independent experiments on different batches of cells or different mice. All
statistical analyses were performed using Prism 9 (GraphPad software, San Diego,
CA). Student’s t test (2-tailed) was used to detect significant
differences between two groups while one-way ANOVA followed by Bonferroni
multiple comparison test was used to compare differences between three groups or
more. Values were considered statistically significant when p 0.05.
4. Results
4.1 CFTR participates in the progress of Ang II induced
hypertension
Ang II of various infusion rates (125, 250, 500 and 1000 ng/kg/min) was
administrated to Cftr mice using osmotic pumps for 14 days. In
comparison to sham group, mice administrated with 500 and 1000 ng/kg/min Ang II
exhibited significant higher systolic blood pressure (SBP), which were 139.33
2.58 and 159.67 4.57 mmHg (Fig. 1A). Meanwhile, Cftr
mRNA level in basilar arteries was significantly reduced in these two groups
(Fig. 1B) and negative correlation between Cftr mRNA and SBP was
observed (Fig. 1C). Moreover, CFTR protein expression was decreased in basilar
arteries from Ang II induced hypertensive rats (Fig. 1D). These in vivo
data suggested that arterial CFTR was downregulated only if SBP was elevated.
Fig. 1.
Expression of CFTR is downregulated in hypertensive arteries and
Cftr knockout enhanced Ang II induced blood pressure elevation. (A) Ang
II of 0, 125, 250, 500 and 1000 ng/kg/min was administrated for 14 days to obtain
Ang II induced hypertensive mice model. Systolic blood pressure (SBP) was
significantly elevated in 500 and 1000 ng/kg/min groups. (B) Cftr mRNA
level in basilar arteries was also significantly reduced in 500 and 1000
ng/kg/min groups (n = 6, *p 0.05 and ***p 0.001 vs. 0
ng/kg/min group). (C) Correlation analysis of basilar artery Cftr mRNA
expression and SBP in Ang II induced hypertensive mice. (D) CFTR protein
expression in basilar arteries was reduced in Ang II induced hypertensive rats (n
= 6, *p 0.05 vs. Sham group). (E,F) No significant difference was
observed in baseline SBP and DBP levels between Cftr,
Cftr and Cftr mice (n = 6). (G,H)
Cftr and Cftr mice were used to establish Ang
II induced hypertensive model, SBP and DBP were measured 0, 3, 6, 9, 12, 14 days
after 1000 ng/kg/min of Ang II infusion operation. In comparison to
Cftr mice, SBP and DBP measured in Cftr mice were
increased (n = 7–8, *p 0.05 and **p 0.01 vs.
Cftr group).
In previous study, average SBP and DBP of 24-hour, daytime and night time of
Cftr mice were all significantly higher than those of
Cftr mice, however, Cftr knockout did not alter SBP or
DBP recorded from 4:00 PM to 8:00 PM [12]. To investigate whether CFTR
play a part in Ang II induced hypertension, Cftr and
Cftr mice were used to establish Ang II infusion induced
hypertension model, SBP and DBP was measured at 6:00 PM 0, 3, 6, 9, 12 and 14
days after the pumps were implanted. We found that SBP of Cftr
mice was significantly higher than that of Cftr mice since day 6
(Fig. 1G). DBP was also additionally increased in Cftr mice on
day 3, 12, and 14 (Fig. 1H). Baseline SBP and DBP of Cftr,
Cftr and Cftr mice were also measured at
6:00 PM, as shown in Supplementary Fig. 1, Cftr knockout did not
affect BP in physiological state. These in vivo data indicated that
Cftr knockout aggravated Ang II induced hypertension.
4.2 Cftr knockout or pharmacological inhibition enhanced
vasoconstriction in response to Ang II
In both sham and Ang II induced hypertensive mice, Ang II induced tension of
thoracic aorta was significantly higher in Cftr mice vs.
Cftr mice (Fig. 2A,B). Furthermore, preincubation of CFTR
specific inhibitor CFTR(inh)-172 (5 M) for 5 min significantly
increased Ang II induced vasoconstriction in thoracic aorta and basilar artery,
which were isolated from Cftr mice and SD rats, respectively
(Fig. 2C,D). Additionally, KCl induced arterial
tension was also enhanced by CFTR(inh)-172 (Fig. 2E), while no significant effect
could be observed when thromboxane analogue U46619, 5-HT, or phenylephrine were
used as stimulators (Fig. 2F–H). These data demonstrated that short-term
pharmacological or genetic inhibition of CFTR increased vasoconstriction in
response to Ang II in both normotensive and hypertensive arteries.
Fig. 2.
Effect of Cftr knockout or pharmacological inhibition
on vasoconstriction in response to different stimulus. (A–B)
Cftr and Cftr mice were used to establish Ang
II infusion induced hypertensive model and thoracic aortas were isolated.
Endothelium of the vessel was chemically denudated by L-NAME (10
M). Representative tracings of isometric tension in response to Ang
II (1 M) in thoracic aorta were shown. Maximum tension was adopted
for analysis (n = 5–6, *p 0.05). (C,D) Thoracic aortas and basilar
arteries were isolated from C57BL/6J mice and SD rats, respectively, and
conducted endothelial denudation. CFTR was pharmacologically inhibited by 5 min
of preincubation with CFTR(inh)-172 (5 M), while DMSO was used as
solvent control. Arterial rings were incubated with Ang II (1 M)
for 5 min and representative tracings of isometric tension were shown. Ang II
induced tension was expressed in the percentage of KCl (60 mM) induced tension in
the absence of CFTR(inh)-172 (n = 6, *p 0.05). (E) Arteries were
pretreated with CFTR(inh)-172 (5 M) for 5 min and KCl (60 mM) induced
tension were measured (n = 8, *p 0.05). (F–H)
Concentration-response curves of U46619, 5-HT and phenylephrine (Phe) were drawn
in the presence or absence of CFTR(inh)-172, no significant difference in tension
could be observed (n = 5).
4.3 Effect of CFTR on the phosphorylation of MLC in VSMCs in vivo
and in vitro
The reversible phosphorylation of regulatory myosin light chain (MLC) is the
core of VSMCs contraction. To investigate the effect of CFTR on MLC
phosphorylation in vivo, Cftr and Cftr
mice were used to establish Ang II infusion induced hypertension model, and the
phosphorylated MLC (p-MLC) and total MLC (t-MLC) were detected. As shown in Fig. 3A, p-MLC in thoracic aortas was increased by Ang II infusion, and significantly
higher in Cftr mice in comparison to Cftr ones
in the hypertension group. No significant alteration was observed in t-MLC level
(Fig. 3B). In vitro, CFTR overexpression adenovirus (Ad-Cftr),
CFTR shRNA silencing adenovirus (Ad-Cftr-shRNA) and CFTR specific
inhibitor CFTR(inh)-172 were used to alter the expression or activity of CFTR. As
shown in Fig. 3C,D, p-MLC in primarily isolated basilar artery smooth muscle
cells (BASMCs) was decreased by CFTR overexpression and increased by CFTR
silencing or CFTR(inh)-172 incubation for 48 h, while no significant alteration
was observed in t-MLC level (Supplementary Fig. 1). These data
demonstrated that both CFTR expression and long-term activity inhibition could
affect MLC phosphorylation level in resting VSMCs.
Fig. 3.
Downregulation/inhibition of CFTR increased the phosphorylation
level of myosin light chain in vivo and in vitro. (A,B)
Cftr and Cftr mice were used to establish Ang
II infusion induced hypertensive model. Thoracic aortas were isolated from
hypertensive group and sham group, phosphorylated myosin light chain (p-MLC) and
total myosin light chain (t-MLC) were detected by western blot (n = 6 mice,
*p 0.05). Cftr knockout increased p-MLC in Ang II induced
hypertensive arteries. (C,D) p-MLC level in basilar artery smooth muscle cells
(BASMCs) were measured with CFTR overexpression, silencing or pharmacological
inhibition. Ad-Cftr, Ad-Cftr-shRNA or CFTR(inh)-172 (5
M, 48 h) were used, respectively. BASMCs were treated with
Ad-Cftr or Ad-Cftr-shRNA for 6 h followed by cultivation with
DMEM/F12 medium containing 10% FBS for another 48 h. Ad-lacZ and Ad-Scr
were used as negative control, respectively. p-MLC (C,D) and t-MLC (see in
Supplementary Fig. 1) in cell lysates were detected by western blot (n =
6, *p 0.05).
4.4 Effect of CFTR on Ang II induced Ca influx in VSMCs
Cytosolic Ca concentration elevation is a trigger for vascular
contraction. A defect in the regulation of Ca signaling plays a role in
hypertension associated vascular dysfunction. For smooth muscle cell contraction
to occur, cytosolic Ca binds to calmodulin, and Ca/calmodulin
complex interacts with MLC, leading to the activation of MLCK and phosphorylation
of MLC. To investigate whether CFTR works through Ca-dependent pathway,
effect of CFTR on Ang II induced increase in [Ca] was detected.
BASMCs were cultured from Cftr or Cftr mice,
and CFTR overexpression in Cftr BASMCs was conducted using
Ad-Cftr. 100 nM of Ang II was added to stimulate [Ca]
increase with 2 mM of extracellular Ca. As shown in Fig. 4A,C, CFTR did
not affect basal [Ca] in resting BASMCs, however, BASMCs from
Cftr mice exhibited higher [Ca] in response to Ang
II compared to that from Cftr mice (Fig. 4B), while CFTR
overexpression caused a decrease in Ang II induced [Ca] rise (Fig. 4D). Then we went further for the Mn quenching rate measurement and found
that Cftr knockout also enhanced Ang II induced Mn influx (Fig. 4E). These results for the first time demonstrated that CFTR was involved in Ang
II induced Ca influx, and thus initiated MLCK activation and the
consequent MLC phosphorylation.
Fig. 4.
Effect of CFTR on Ang II induced Ca influx in cultured
BASMCs. (A) BASMCs were isolated from Cftr and
Cftr mice and cultured in DMEM/F12 medium with 20% FBS.
Intracellular calcium concentration ([Ca]) was monitored and
recorded every 10 seconds with 2 mM of extracellular Ca. Ang II (100 nM)
was added after the fluorescence was stable. (B) Maximum of [Ca] in
response to Ang II were compared between BASMCs from Cftr and
Cftr mice (n = 6, **p 0.01). (C) BASMCs were
treated with Ad-lacZ (100 MOI) or Ad-Cftr (100 MOI) for 6 h
followed by cultivation with DMEM/F12 medium containing 10% FBS for another 48
h. [Ca] was monitored and recorded every 10 seconds with 2 mM of
extracellular calcium concentration. Ang II (100 nM) was added after the
fluorescence was stable. (D) Maximum of Ang II induced [Ca] rise
were compared between Ad-lacZ and Ad-Cftr group. (n = 6,
**p 0.01). (E) Measurement of Mn quenching rate. BASMCs were
cultured from Cftr and Cftr mice, digested and
incubated with 2 M Fura-2AM for 45 min and then washed and resuspended
with Ca free HBSS. Fluorescence in response to Ang II was recorded every
second. Quenching rate was expressed as percentage change of the maximum rate.
4.5 Regulating effect of CFTR on the phosphorylation of MYPT1 via
RhoA/Rock pathway
As shown in Fig. 3C,D,4A,C, in resting VSMCs, CFTR could regulate the
phosphorylation of MLC without affecting resting [Ca]. In addition,
CFTR overexpression did not alter MLCK protein expression level (Fig. 5A), and
with MLCK activity inhibited by ML-7, CFTR overexpression could still decrease
p-MLC (Fig. 5B), suggesting that CFTR might regulate resting tension of VSMCs
through Ca-independent pathway.
Fig. 5.
Effect of CFTR on phosphorylation of MYPT1 via adjusting RhoA
activity. (A) BASMCs were treated with Ad-Cftr or Ad-lacZ for
6 h and cultured with 10% FBS DMEM/F12 medium for another 48 h. MLCK expression
was detected by western blot and no significant difference was observed (n = 6).
(B) ML-7 (inhibitor of MLCK, 10 M) was applied during the whole
process of CFTR overexpression treatment in BASMCs. Cell lysates were collected
and p-MLC was detected by western blot (n = 6, *p 0.05).
(C–F) BASMCs were treated with Ad-Cftr, Ad-Cftr-shRNA or
negative control vector for 6 h and cultured with DMEM/F12 medium with 10% FBS
for another 48 h. Cell lysates were collected and MYPT1 phosphorylation at
Ser507, Thr696 and Thr855 and total MYPT1 expression were detected using western
blot (see in Supplementary Fig. 2), -actin was used as loading
control. (n = 6, **p 0.01). (G) BASMCs were treated with
CFTR(inh)-172 (5 M, 48 h) and/or Y-27632 (10 M, 48 h) and
terminated by liquid nitrogen. Cell lysates were collected and p-MYPT1
was detected by western blot (n = 6, *p 0.05, ***p
0.001). (H) BASMCs were treated with Ad-Cftr or Ad-lacZ, Ang II
(100 nM) was added for 5 min to induce cell contraction and then liquid nitrogen
was used to terminate the process immediately. Cell lysates were collected and
RhoA-GTP and total RhoA were detected using RhoA Activation Assay Kit (n = 6,
*p 0.05, **p 0.01).
While MLCK-mediated constriction is strictly dependent on [Ca], MLC
phosphorylation and the subsequent constriction can be maintained even after
[Ca] returns to basal levels via inhibition MLCP. MLCP holoenzyme
consists of three subunits: catalytic subunit of protein phosphatase 1 (PP1),
myosin phosphatase target subunit 1 (MYPT1) and a subunit of unknown function
[28]. The binding of MYPT1 to PP1 unit increases MLCP catalytic activity, while
phosphorylated MYPT1 (p-MYPT1) represses the catalytic activity of MLCP and thus
enhances MLC phosphorylation and VSMCs contraction. p-MYPT1 is modulated by
RhoA/ROCK pathway, which can be activated by various stimuli, including Ang II.
As shown in Fig. 5C–F, CFTR affected p-MYPT1 at site Thr855, but not at Ser507
or Thr696 in BASMCs. Incubation with selective Rock inhibitor Y-27632 decreased
p-MYPT1 in resting BASMCs and abolished the enhancement of p-MYPT1 induced by
long-term CFTR activity inhibition (48 h) (Fig. 5G), while CFTR overexpression
decreased RhoA activation in resting and Ang II stimulated BASMCs (Fig. 5H).
These data indicated that RhoA/Rock mediated MYPT1 phosphorylation might be
another signaling pathway involved in the regulating effect of CFTR on
vasoconstriction and thus hypertension. Our finding is in agreement with a
previous report demonstrating that continuous inhibition CFTR-Cl
conductance for 3–5 days resulted in a threefold increase in RhoA expression
[29].
5. Discussion
Both human and murine studies suggest the Cftr mutation is associated
with blood pressure and vascular disfunction, however, the role of CFTR in the
genesis of hypertension remains quite unillustrated. In the present study, we
provided the first piece of evidence that Cftr participated in the
progression of Ang II induced hypertension. Our findings are as follows: (1)
Cftr knockout enhanced BP elevation in Ang II infusion induced
hypertensive mice, while CFTR mRNA/protein expression were decreased in
hypertensive arteries. (2) Both genetic and pharmacological inhibition of CFTR
increased Ang II induced vasoconstriction and the phosphorylation of MLC in
VSMCs. (3) [Ca] rise induced by Ang II in VSMCs was increased by
Cftr knockout and decreased by CFTR overexpression, while basal
[Ca] was not affected by CFTR in resting VSMCs. (4) CFTR regulated
the phosphorylation of MYPT1 at site Thr855 via RhoA/Rock pathway in both resting
and Ang II stimulated VSMCs.
CFTR has been studied in cardiovascular system, including left ventricular
remodeling [30], elevated myocardial contractility [31], cardio-protection
against acidosis/ischemia [32] and cardiac development during early embryogenesis
[33]. CFTR is a cAMP-dependent Cl channel best known for its role in
Cl and HCO transport in epithelial tissues [34]. Older female
CF mutation (not gene deletion) carriers showed lower BP than matched control
subjects [11] and F508del heterozygous mice had lower arterial pressures than
wild-type mice [35], which had been attributed to excessive salt depletion.
Ya-Ping Zhang et al. [12] demonstrated that 24-hour average
physiological SBP and DBP of Cftr mice were all significantly
higher than those of Cftr mice.
It is noteworthy that previous studies only mentioned the relationship between
physiological BP and CFTR, however, the effect of CFTR on pathophysiological BP
level in the process of hypertension remains unillustrated. In the present study,
we established the Ang II infusion induced hypertension model and found that CFTR
expression in hypertensive arteries were decreased and negatively related with BP
level. Moreover, Cftr knockout enhanced Ang II induced BP elevation
during the development of hypertension, without altering BP in normotensive mice.
Previous study indicated that CFTR might play a little role in circadian
regulation of BP [12]. Both Cftr and Cftr mice
displayed a bimodal circadian pattern over 24-hour period but BP peaks of
Cftr mice were higher and later when compared to
Cftr mice. As a result of this regulatory effect, Cftr
knockout did not alter SBP or DBP measured from 4:00 PM to 8:00 PM. Because the
aim of our study was to investigate the role of CFTR in the development of
hypertension, all BP values recorded in our study was measured at 6:00 PM of the
day, avoiding the influence of the Cftr knockout on physiological BP.
The effect of Cftr mutation on blood pressure in previous studies had
been attributed to excessive salt depletion or absorption [11, 35, 36], however,
hypertension is pathologically characterized by augmented vascular contractility
and chloride channels in VSMCs have been identified as important regulators of
vascular tone. As a small outwardly rectifying chloride channel, the effect of
CFTR on vasoconstriction should get more attention. In previous studies, aortic
diameter measured at valsalva sinuses was significantly higher in patients with
CF mutations [37]. CFTR inhibition enhanced pressure-induced myogenic
vasoconstriction [15], and aortic rings from Cftr mice showed a
much stronger depolarization induced vasoconstriction than those from wild-type
mice [16]. In addition, the loss of CFTR function (F508del) enhanced posterior
cerebral arteries myogenic vasoconstriction in response to phenylephrine, while
CFTR corrector compounds normalize pathological alterations in cerebral artery
CFTR expression, vascular reactivity, and cerebral perfusion [18]. Conversely,
aortas from piglets with F508del mutation showed decreased vascular tone in
response to 60 mM of KCl, while no difference was observed in CFTR null piglets
[38]. Regulating effect of CFTR on vasoconstriction remains controversial. In
this study, we demonstrated that Cftr knockout enhanced Ang II induced
constriction of thoracic aortas in both Sham and Ang II induced hypertensive
mice. In addition, short-term inhibition of CFTR channel activity by
CFTR(inh)-172 increased vascular tension in response to Ang II in both thoracic
aortas and basilar arteries. These results were consistent with the data of blood
pressure we observed in Ang II induced hypertensive Cftr mice.
Notably, phenylephrine, 5-HT and U46619 can cause adenylate cyclase (AC)
activation and the production of cAMP [39], which can activate CFTR channel.
Therefore, it is conceivable that phenylephrine, 5-HT or U46619 induced arterial
tension was not altered by short-term CFTR activity inhibition, which might be
due to the counterbalance of cAMP and CFTR(inh)-172.
Growing evidence indicates that Ca signaling and CFTR are interdependent.
Binding of CFTR to isolated calmodulin activated CFTR independent of protein
kinase A (PKA) [40], and CFTR was necessary for forskolin induced
[Ca] response in human airway epithelia [41]. Endoplasmic reticulum
(ER) localized F508del-CFTR affected store-operated calcium channel (SOCC) Orai1
function and strongly enhanced calcium signaling in nasal epithelial cells [42, 43]. Our data further revealed that CFTR modulated Ang II induced Ca
influx in VSMCs. However, it has not been clarified in this work that how CFTR
modulated the influx of Ca in Ang II-stimulated VSMCs. SOCC,
voltage-dependent Ca channels, CaM-CFTR complex and all other pathways
mediating the rise of [Ca] in response to Ang II might be involved
in this process, further investigation is required in the future.
In resting VSMCs, CFTR did not alter basal [Ca] but regulated MLC
phosphorylation even with MLCK activity inhibited, suggesting that
Ca-dependent MLC phosphorylation through MLCK cannot explain all
modalities of the effect of CFTR in arterial contraction. It was reported that
CFTR disruption induced an increase in baseline myocardial contractility
in vivo, in the absence of changes in the magnitude of Ca
transients [31]. Thus, we speculate that there might be different mechanisms
underlying the regulating effect of CFTR on VSMCs contraction in resting and
contractile states. As mentioned above, phosphorylation of MLC is controlled by a
balance in the activity of MLCK and myosin MLCP and RhoA/Rock pathway regulates
MLCP via phosphorylating its regulatory subunit MYPT-1 [44]. We found that
selective Rock inhibitor Y-27632 decreased MYPT1 phosphorylation, and CFTR
silencing increased RhoA activation, in both resting and Ang II stimulated
BASMCs. Our results are in agreement with a previous report demonstrating that
continuous inhibition CFTR-Cl conductance for 3–5 days resulted in a
threefold increase in RhoA expression [29], the effect of long-term inhibition of
CFTR channel activity might be due to the augmented RhoA activation caused by the
increased intracellular chloride concentration [Cl] [45]. Based on
these findings, we considered two possible mechanisms accounting for the
regulation of CFTR on vasoconstriction of VSMCs: in resting VSMCs, CFTR altered
MLC phosphorylation through RhoA/Rock pathway, while in Ang II stimulated VSMCs,
the modulation was conducted via both Ca influx and RhoA/Rock mediated
pathway.
In general, as the graphical summary shown in Fig. 6, both chloride channel
activity and protein expression of CFTR are involved in vasoconstriction and Ang
II induced hypertension. Pharmacological inhibition of CFTR activity (short-term,
pre-incubation for 5 min) enhanced Ang II induced artery constriction, but not
resting arterial tension, while long-term inhibition (48 h) increased MLC and
MYPT1 phosphorylation in VSMCs. CFTR protein expression level affected Ang II
induced Ca influx, RhoA activity, MYPT1, MLC phosphorylation and Ang II
induced VSMC contraction and hypertension. Cl equilibrium potential is
normally above the resting membrane potential in VSMCs [46]. Opening of Cl
channels in the plasma membrane of VSMCs causes a Cl efflux, membrane
depolarization, and increased contractile force. For instance, TMEM16A chloride
channel promotes cell contraction and myogenic tone in different vascular beds
[47, 48, 49, 50]. Conversely, in our study, suppression of CFTR expression or activity
enhanced Ang II stimulated vasoconstriction, indicating loss of active CFTR may
have other consequences in addition to impairing Cl transport. For example,
as a membrane protein, CFTR might directly interact with the upstream signaling
molecule of Ca or RhoA during the vasoconstriction process.
Fig. 6.
Summary of the mechanisms underlying the regulating effect of
CFTR on vasoconstriction. In VSMCs, Ang II binding to ATR regulates
contraction through G Ca-sensitive MLCK activation and
G Rho/Rho kinase-mediated inhibition of MLC phosphatase (MLCP).
Production of arachidonic acid derived hydroxyeicosatetraenoic acids (HETEs) and
formation of NADPH oxidase-derived reactive oxygen species (ROS) were also
involved in Ang II/AT1R mediated vasoconstriction. Activation of AT1 receptor by
Ang II in VSMCs results in PLC activation leading to the release of the second
messengers IP and DAG. Activation of IPR stimulates intracellular
Ca release from sarcoplasmic reticulum and DAG causes PKC activation.
Different Ca entry channels, such as voltage-dependent, receptor-operated,
and store-operated Ca channels are involved in the elevation of
intracellular Ca concentration. The elevation in [Ca]
initiates contractile activity by a Ca-CaM interaction, causing MLCK
activation and consequent MLC phosphorylation, which in turn enables the
interaction between myosin and actin and initiates VSMCs contraction. In the
absence of significant increase in [Ca], MLC phosphorylation can
still be induced by pathway mediated by RhoA, which switches between an inactive
GDP-bound state and an active GTP-bound state in response to various stimuli,
including Ang II. RhoA activation induces phosphorylation of MYPT1, which
inhibits the dephosphorylation of MLCP and thereby maintains force generation. In
Ang II stimulated VSMCs, CFTR down-regulation and short-term inhibition increased
Ca influx and the subsequent MLC phosphorylation, besides, RhoA/Rock
mediated MLCP inactivation could also be involved. In resting VSMCs, CFTR
down-regulation or long-term inhibition increased MLC phosphorylation by
affecting RhoA mediated MYPT1 phosphorylation.
6. Conclusions
The present study demonstrated the critical role of CFTR in the genesis of Ang
II induced hypertension. We showed for the first time that genetic ablation of
Cftr aggravated elevation of BP in Ang II induced hypertensive mice.
Both pharmacological and genetic inhibition of CFTR enhanced Ang II induced
vasoconstriction, which was associated with Ca-dependent signaling pathway
and RhoA/Rock mediated phosphorylation of MYPT1. Our findings indicated that
modification of CFTR might be a novel therapeutic strategy for hypertension.
7. Author contributions
LZ and GW designed the experiments; LZ, FY, NP, YY, HY, YL and RW conducted the
experiments; LZ and FY analyzed the data; GW conceived and supervised the
project; LZ drafted the manuscript; BZ and GW participated in data analysis and
finalized the manuscript writing.
8. Ethics approval and consent to participate
All animal care and experimental procedures complied with the policies of Ethics
Committee of ZSSOM on Laboratory Animal Care (approval number: 2016-080) and
conformed to the “Guide for the Care and Use of Laboratory Animals” of the
National Institute of Health in China.
9. Acknowledgement
Thanks to all the peer reviewers for their opinions and suggestions.
10. Funding
This study was supported by National Natural Science Foundation of China
(81903687, 62172452, 82073848 and 81773722), Natural Science Foundation of
Guangdong Province (2020A1515010045, China), Guangdong Provincial Department of
Science and Technology (2017A020215104, China), the Science and Technology
Program of Guangzhou City (201803010092, China).
11. Conflict of interest
The authors declare no conflict of interest.
Abbreviations
Ang II, angiotensin II; VSMCs, vascular smooth muscle cells; CaCC,
Ca-activated Cl channel; VRCC, volume regulated Cl channel;
CFTR, cystic fibrosis transmembrane conductance regulator; CF, cystic fibrosis;
SBP, systolic blood pressure; DBP, diastolic blood pressure; MLC, myosin light
chain; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase;
PLC, phospholipase C; IP, inositol trisphosphate; DAG, diacylglycerol; SR,
sarcoplasmic reticulum; PKC, protein kinase C; [Ca], intracellular
Ca concentration; CaM, calmodulin; SHR, spontaneously hypertensive rat;
2k2c, 2-kidney 2-clip; t-MLC, total myosin light chain; p-MLC, phosphorylated
myosin light chain; MYPT1, myosin phosphatase target subunit 1; p-MYPT1,
phosphorylated MYPT1; L-NAME, L-nitro-arginine methyl ester; HBSS, Hanks’
Balanced Salt Solution; PVDF, polyvinylidene difluoride; DMEM/F12, Dulbecco’s
modified Essential medium/F-12; BASMCs, basilar artery smooth muscle cells; PKA,
protein kinase A; SOCC, store-operated calcium channel.