Activation of transient receptor potential vanilloid 4 is involved in pressure overload-induced cardiac hypertrophy

  1. Yan Zou
  2. Miaomiao Zhang
  3. Qiongfeng Wu
  4. Ning Zhao
  5. Minwei Chen
  6. Cui Yang
  7. Yimei Du  Is a corresponding author
  8. Bing Han  Is a corresponding author
  1. Department of Cardiology, Xuzhou Central Hospital, China
  2. Xuzhou Institute of Cardiovascular Disease, Xuzhou Central Hospital, China
  3. Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, China
  4. Department of Cardiology, Xiamen Key Laboratory of Cardiac Electrophysiology, Xiamen Institute of Cardiovascular Diseases, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, China
11 figures, 4 tables and 4 additional files

Figures

TRPV4 expression is upregulated in pathological cardiac hypertrophy.

Representative immunoblot image (A) and statistics (B) of TRPV4 protein level in the LV from sham or TAC mice at indicated time points after the operation (n=6 per group). All results represent mean ± SD, an unpaired two-tailed Student’s t-test. (C) Statistical data of Trpv4 mRNA level in the LV from sham or TAC mice at indicated time points after the operation (n=6 per group). All results represent mean ± SD, an unpaired two-tailed Student’s t-test. Representative immunoblot image (D) and statistical data (E) of TRPV4 protein level in human non-failing hearts (n=3) and failing hearts (n=5). All results represent mean ± SD, an unpaired two-tailed Student’s t-test. LV, left ventricle; TAC, transverse aortic constriction.

TRPV4 deficiency attenuates pressure overload-induced cardiac hypertrophy.

(A) Representative images of the heart and statistical results for the ratios of HW/BW and HW/TL of WT and Trpv4 KO mice 1 or 4 weeks after sham or TAC operation (n=7 per group in WT mice, n=5 per group at 1 week in TRPV4−/− mice, n=7 per group at 4 weeks in Trpv4 KO mice) (B) Hematoxylin & eosin staining, wheat germ agglutinin staining, and cross-section area in WT and Trpv4 KO mice 1 or 4 weeks after sham or TAC operation (n=3 per group at 1 week, n=7 per group at 4 weeks). (C) Statistics of hypertrophy-related genes ANP (Nppa) (E) and BNP (Nppb) (F) mRNA levels in mouse hearts from WT or Trpv4 KO 1 or 4 weeks after sham or TAC operation (n=6 per group at 1 week, n=7 per group at 4 weeks). All results represent mean ± SD, a two-way ANOVA followed by the Bonferroni test. BW, body weight; HW, heart weight; TAC, transverse aortic constriction; TL, tibial length; WT, wild-type.

TRPV4 deficiency improves cardiac function and attenuates cardiac fibrosis induced by pressure overload.

Representative images of M-mode echocardiography of WT and Trpv4 KO mice 4 weeks after sham or TAC operation (A). Statistics of EF (B), FS (C), LV mass (D), and LVIDs (E) in mice 4 weeks after sham or TAC operation (n=6 per group). Representative images (F) and statistics (G) of Masson’s trichrome-stained hearts from mice 4 weeks after sham or TAC operation. The statistics were from the panoramic scanning pictures (n=4 per group). Statistics of fibrosis-related genes collagenase-1 (Col1a1) (H), collagenase-3 (Col1a1) (I), and galectin-3 (LGALS3) (J) mRNA levels in mouse hearts 4 weeks after sham or TAC operation (n=7 per group). All results represent mean ± SD, a two-way ANOVA followed by the Bonferroni test. EF, ejection fraction; FS, fractional shortening; LV, left ventricle; TAC, transverse aortic constriction.

TRPV4 deficiency attenuates cardiac fibrosis induced by pressure overload.

Representative immunoblot image (A) and statistics of IL-1β (B), IL-6 (C), and TNF-α (D) protein levels in WT and Trpv4 KO mice 4 weeks after sham or TAC operation (n=6 per group). Statistical data of IL-1β (Il1b) (E), IL-6 (Il6) (F), TNF-α (Tnfa) (G), MCP-1 (Mcp1p) (H), and MIP-2 (Mip2) (I) mRNA levels in mouse hearts 4 weeks after sham or TAC operation (n=7 per group). Representative immunoblot image (J) and statistics of ASC (K), NLRP3 (L), Caspase 1-p20 (M), and p-NFκB p65 (N) protein levels in WT and TRPV4−/− mice at 4 weeks after sham or TAC operation (n=6 per group). All results represent mean ± SD, a two-way ANOVA followed by the Bonferroni test. TAC, transverse aortic constriction; WT, wild-type.

Treatment with TRPV4 antagonist prevents TAC-induced cardiac hypertrophy and dysfunction in mice.

(A) Representative images of heart photo and WGA staining 4 weeks after TAC. Statistical results for HW/BW ratio (n=6 per group) (B), HW/TL ratio (n=6 per group) (C), cross-section area (n=4 per group) (J), ANP (Nppa) (n=6 per group) (E), BNP (Nppb) (n=6 per group) (F), EF (n=6 per group) (G), FS (n=6 per group) (H), LV mass (n=6 per group) (I), and LVIDs (n=6 per group) (J). All results represent mean± SD, an unpaired two-tailed Student’s t-test. Representative images and statistics (K) of Masson’s trichrome-stained hearts 4 weeks after TAC (n=3 per group). All results represent mean ± SD, an unpaired two-tailed Student’s t-test. EF, ejection fraction; FS, fractional shortening; LV, left ventricle; TAC, transverse aortic constriction; WGA, wheat germ agglutinin.

TRPV4 blockade attenuates AngII/PE-induced hypertrophy in NRVMs in vitro.

Representative images (A) and statistics of the cell-surface areas (B) in NRVMs treated with DMSO, AngII, and AngII plus GSK3874 (n=20 cells from three animals). Statistics of ANP (Nppa) (C) and BNP (Nppb) (D) mRNA levels in NRVMs treated with DMSO, AngII, and AngII plus GSK3874 (n=6 per group). Representative images (E) and statistics of the cell-surface areas (F) in NRVMs treated with DMSO, PE, and PE plus GSK3874 (n=20 cells from three animals). Statistics of ANP (Nppa) (G) and BNP (Nppb) (H) mRNA levels in NRVMs treated with DMSO, PE, and PE plus GSK3874 (n=6 per group). All results represent mean ± SD, a one-way ANOVA followed by the Bonferroni test. NRVM, neonatal rat ventricular myocyte.

TRPV4 blockade attenuates AngII/PE-induced Ca2+ overload in NRVMs.

Representative immunoblot image (A) and statistics (B) of TRPV4 protein level in NRVMs treated with DMSO, AngII, and AngII plus GSK3874 (n=6 per group). (C). Statistical data of Trpv4 mRNA level in NRVMs treated with DMSO, AngII, and AngII plus GSK3874 (n=6 per group). Representative recording of changes in intracellular Ca2+ induced by 500 nM GSK 790A and 1 μM A23187 in NRVMs treated with DMSO, AngII, and AngII plus GSK3874 (D). Quantification of [Ca2+]i response induced by GSK790A (E) and A23187 (F) -induced in NRVMs treated with DMSO, AngII, and AngII plus GSK3874 (n=18 per group). Representative recording of changes in intracellular Ca2+ induced by 500 nM GSK 790A and 1 μM A23187 in NRVMs treated with DMSO, PE, and PE plus GSK3874 (G). Quantification of [Ca2+]i response induced by GSK790A (H) and A23187 (I)-induced in NRVMs treated with DMSO, PE, and PE plus GSK3874 (n=12 per group). The arrow indicates the time of addition of GSK1016790A and A21387. F0 represents the average fluorescence intensity before GSK1016790A stimulation. All results represent mean ± SD, a one-way ANOVA followed by the Bonferroni test. NRVM, neonatal rat ventricular myocyte.

TRPV4 activation induces CaMKII phosphorylation.

Representative immunoblot image (A) and statistics (B) of p-CaMKII/CaMKII in NRVMs treated with DMSO, GSK790A, and GSK790A plus GSK3874 (n=4 per group). All results represent mean ± SD, a one-way ANOVA followed by the Bonferroni test. Representative immunoblot image (C) and statistics (D) of p-CaMKII/CaMKII in NRVMs treated with GSK790A in the absence and presence of Ca2+ medium (n=6 per group). All results represent mean ± SD, an unpaired two-tailed student’s t-test. Representative immunoblot image (E) and statistics (F) of p-CaMKII/CaMKII in NRVMs treated with DMSO, AngII, and AngII plus GSK3874 (n=4 per group). All results represent mean ± SD, a one-way ANOVA followed by the Bonferroni test. Representative immunoblot image (G) and statistics (H) of p-CaMKII/CaMKII in the LV from sham or TAC WT mice at indicated time points after the operation (n=6 per group). All results represent mean ± SD, an unpaired two-tailed student’s t-test. Representative immunoblot image (I) and statistics (J) of p-CaMKII/CaMKII in Trpv4 KO mice 1 week or 4 weeks after sham or TAC operation (n=6 per group). All results represent mean ± SD, an unpaired two-tailed student’s t-test. NRVM, neonatal rat ventricular myocyte.

TRPV4 activation induces NFκB phosphorylation via a CaMKII signaling pathway.

Representative immunoblot image (A) and statistics (B) of p-p65 /p65 in NRVMs treated with DMSO, GSK790A, and GSK790A plus GSK3874 (n=6 per group). All results represent mean ± SD, a one-way ANOVA followed by the Bonferroni test. Representative immunoblot image (C) and statistics (D) of p-p65 /p65 in NRVMs treated DMSO, AngII, and AngII plus GSK3874 (n=6 per group). All results represent mean ± SD, a one-way ANOVA followed by the Bonferroni test. Representative immunoblot image (E) and statistics (F) of p-p65 /p65 in NRVMs treated with DMSO, GSK790A, GSK790A plus KN92, and GSK790A plus KN93 (n=6 per group). All results represent mean ± SD, a one-way ANOVA followed by the Bonferroni test.

Schematic illustration of potential mechanisms through which TRPV4 activation promotes pathological cardiac hypertrophy.
Appendix 1—figure 1
Genotyping of TRPV4 wild-type and Trpv4 KO mice and TRPV4 depletion in the heart of Trpv4 KO mice.

A. Representative RT-PCR genotyping gel image of the WT, Trpv4+/-, and Trpv4-/-. B. RT-PCR of total RNA from heart showing Trpv4 mRNA was present in WT mice but absent in Trpv4 KO mice.

Tables

Table 1
Echocardiographic measurements 4 weeks after TAC.
WTTrpv4 KO
ShamTACShamTAC
Heart rate (bpm)457.61±11.77463.11±16.96463.11±18.86443.78±10.38
LVAW,s (mm)1.29±0.141.41±0.181.21±0.21.48±0.17
LVID,d (mm)3.32±0.123.81±0.592.92±0.383.11±0.47
LVPW,d (mm)0.70±0.081.08±0.460.92±0.391.02±0.37
LVPW,s (mm)1.17±0.111.41±0.571.33±0.341.59±0.31
Diameter,s (mm)2.05±0.132.67±0.571.69±0.321.80±0.37##
Diameter,d (mm)3.29±0.123.62±0.482.87±0.423.04±0.45
Volume,s (μl)13.73±2.1728.01±14.43*8.79±4.0010.36±5.41##
Volume,d (μl)43.88±3.8456.39±17.4132.23±11.0237.38±12.30
Stroke volume (μl)30.14±2.4328.39±4.6723.44±7.1927.02±7.52
  1. LVAW,s: systolic left ventricular anterior wall, LVID,s: systolic left ventricular internal diameter, LVPW,d: diastolic left ventricular posterior wall, LVPW,s.: systolic left ventricular posterior wall, Data represent means ± SD, n=6 per group, P<0.05 WT TAC vs WT sham group. ## P<0.01 Trpv4 KO TAC vs WT TAC group

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Chemical compound, drugAng IIMCECat# HY-13948
Chemical compound, drugPEMACKLINCat# I822933
Chemical compound, drugGSK790ASigma-AldrichCat# G0798
Chemical compound, drugGSK3874Sigma-AldrichCat# SML0942
Chemical compound, drugA21387Sigma-AldrichCat# G0798
Chemical compound, drugKN92SelleckCat# S6507
Chemical compound, drugKN93SelleckCat# S6787
Chemical compound, drugPentobarbital sodiumSigma-AldrichCat# 76-74-4
Chemical compound, drugCollagenase IIWorthingtonCat# ls004176
Chemical compound, drugFluo-4/AMAAT BioquestCat# AAT-B20401
Chemical compound, drugPluronic F‐127SolarbioCat# P679
Sequence-based reagentBNP (Nppb)_R
(mice)
This paperPCR primersCAACTTCAGTGCGTTACAGC
Sequence-based reagentCollagenase-1 (Col1a1) _F (mice)This paperPCR primersGAAACCCGAGGTATGCTTGA
Sequence-based reagentCollagenase1 (Col1a1) _R (mice)This paperPCR primersGGGTCCCTCGACTCCTACAT
Sequence-based reagentCollagenase-3 (Col3a1) _F (mice)This paperPCR primersAGCCACCTTGGTCAGTCCTA
Sequence-based reagentCollagenase-3 (Col3a1) _R (mice)This paperPCR primersGTGTAGAAGGCTGTGGGCAT
Sequence-based reagentGalectin-3 (LGALS3)_F (mice)This paperPCR primersCAGGAAAATGGCAGACAGCTT
Sequence-based reagentGalectin-3 (LGALS3)_R(mice)This paperPCR primersCCCATGCACCCGGATATC
Sequence-based reagentIL-1β (Il1b) _F (mice)This paperPCR primersTGCCACCTTTTGACAGTGATG
Sequence-based reagentIL-1β(Il1b)_R (mice)This paperPCR primersTGATGTGCTGCTGCGAGATT
Sequence-based reagentIL-6(Il6)_F (mice)This paperPCR primersGATAAGCTGGAGTCACAGAAGG
Sequence-based reagentIL-6(Il6)_R (mice)This paperPCR primersTTGCCGAGTAGATCTCAAAGT
Sequence-based reagentTNF-α(Tnfa)_F (mice)This paperPCR primersCCCCAAAGGGATGAGAAGTT
Sequence-based reagentTNF-α(Tnfa)_R (mice)This paperPCR primersACTTGGTGGTTTGCTACGA
Sequence-based reagentMIP-2(Mip2)_F (mice)This paperPCR primersCGCCCAGACAGAAGTCATAG
Sequence-based reagentMIP-2(Mip2)_R (mice)This paperPCR primersTCCTCCTTTCCAGGTCAGTTA
Sequence-based reagentMCP-1(Mcp1p)_F (mice)This paperPCR primersTTTTTGTCACCAAGCTCAAGAG
Sequence-based reagentMCP-1(Mcp1p)_R (mice)This paperPCR primersTTCTGATCCTCATTTGGTTCCGA
Sequence-based reagentGapdh_F(mice)This paperPCR primersAAGAAGGTGGTGAAGCAGGCAT
Sequence-based reagentGapdh_F (mice)This paperPCR primersCGGCATCGAAGGTGGAAGAGTG
Sequence-based reagentTrpv4_F (rat)This paperPCR primersCGTCCAAACCTGCGAATGAAGTTC
Sequence-based reagentTrpv4_F (rat)This paperPCR primersCCTCCATCTCTTGTTGTCACTGG
Sequence-based reagentANP (Nppa)_F (rat)This paperPCR primersATCTGATGGATTTCAAGAACC
Sequence-based reagentANP (Nppa)_R
(rat)
This paperPCR primersCTCTGAGACGGGTTGACTTC
Sequence-based reagentBNP (Nppb)_F (rat)This paperPCR primersCAATCCACGATGCAGAAGCT
Sequence-based reagentBNP (Nppb)_R (rat)This paperPCR primersGGGCCTTGGTCCTTTGAGA
Sequence-based reagentGapdh_ F (rat)This paperPCR primersATGGGAAGCTGGTCATCAAC
Sequence-based reagentGapdh_ R (rat)This paperPCR primersGTGGTTCACACCCATCACAA
AntibodyAnti-GAPDH HRP
(mouse monoclonal)
BioworldeCat# MB001H
RRID: AB_2857326
Western blot (1:10,000)
AntibodyAnti-TRPV4
(rabbit polyclonal)
Alomone labsCat# ACC-034
RRID: AB_2040264
Western blot (1:500)
AntibodyAnti-p-CaMKII(Thr287)
(rabbit polyclonal)
Thermo Fisher ScientificCat# PA5-37833
RRID: AB_2554441
Western blot (1:500)
AntibodyAnti-CaMKII
(rabbit monoclonal)
AbcamCat# ab52476
RRID: AB_868641
Western blot (1:1000)
AntibodyAnti-p-P65 (Ser536)
(rabbit polyclonal)
AffinityCat# AF2006
RRID: AB_2834435
Western blot (1:500)
AntibodyAnti-P65
(rabbit polyclonal)
AffinityCat# AF5006
RRID: AB_2834847
Western blot (1:500)
AntibodyAnti-NLRP3
(rabbit monoclonal)
AbcamCat# ab263899
RRID: AB_2889890
Western blot (1:500)
AntibodyAnti-ASC/TMS1
(rabbit monoclonal)
CSTCat# 67824
RRID: AB_2799736
Western blot (1:1000)
AntibodyAnti-Cleaved-Caspase 1, p20
(rabbit polyclonal)
AffinityCat# AF4005
RRID: AB_2845463
Western blot (1:500)
AntibodyAnti-IL 1β
(rabbit monoclonal)
AbcamCat# ab234437Western blot (1:1000)
AntibodyAnti-IL-6
(rabbit monoclonal)
CSTCat# 12912
RRID: AB_2798059
Western blot (1:500)
AntibodyAnti-TNF-α
(rabbit monoclonal)
CSTCat# 11948
RRID: AB_2687962
Western blot (1:1000)
AntibodyGoat Anti-Rabbit IgG HRP
(goat polyclonal)
AffinityCat# S0001
RRID: AB_2839429
Western blot (1:3000)
AntibodyAnti-α-actinin
(rabbit polyclonal)
AbcamCat# Ab137346
RRID: AB_2909405
ICC (1:500)
Commercial assay or kitcDNA reverse transcription kitVazymeCat# R211-01
Commercial assay or kitSYBR Green PCR Master Mix KitCWbioCat# cw3008h
OthersPercollCytivaCat# 17089109A low-viscosity, non-toxic medium suitable for density gradient centrifugation of cells, viruses and subcellular particles
Appendix 1—table 1
Anatomical parameters, early markers of cardiac hypertrophy and fibrosis, and echocardiographic parameters were measured in wild-type (WT) mice after subjected to TAC versus sham-operated controls (n=6–7/group).
2 d1 w2 w4 w
ShamTACShamTACShamTACShamTAC
Anatomical parameter
n77777766
HW/BW4.4±0.14.5±0.15.1±0.27.3±0.7***4.5±0.37.1±1.1***4.5±0.37.1±0.8**
HW/TL6.0±0.26.0±0.16.7±0.68.4±0.6***6.0±0.39.3±1.4***6.3±0.59.7±1.6***
Early markers of cardiac hypertrophy and fibrosis
n66666677
ANP1.0±0.31.1±0.21.0±0.59.1±3.9***1.0±0.428.1±11.5***1.0±0.27.3±1.9***
BNP1.0±0.20.7±0.11.0±0.411.5±2.0***1.0±0.35.2±1.1***1.0±0.42.7±0.4**
Collagenase-11.0±0.41.4±0.71.0±0.41.1±0.31.0±1.31.0±0.71.0±0.26.8±2.5**
Collagenase-31.0±0.21.0±0.51.0±0.41.30±0.251.0±1.12.5±2.11.0±0.33.8±1.2**
Echocardiographic parameters
n77777766
EF (%)66.4±7.265.0±8.971.0±5.968.7±3.575.7±4.460.2±3.9***68.8±3.352.8±10.6**
FS (%)36.0±5.134.2±6.739.8±6.636.1±3.543.4±4.031.3±2.6***37.6±2.726.9±6.5**
  1. HW/BW: heart weight to body weight ratio, HW/TL: heart weight to tibia length ratio, EF: ejection fraction, FS: fractional shortening, Data represent means ± SD, **P<0.01, ***P<0.001, compared between sham- and TAC operated groups.

Appendix 1—table 2
Clinical characteristics of patients with advanced heart failure.
PatientAge (y)GenderEFNYHA
DCM 152Male22.5IV
DCM 247Male12.6III
DCM 346Male15.9IV
DCM 420Male15III
DCM 530Female21IV
Control 135MaleI
Control 227MaleI
Control 340MaleI
  1. DCM: dilated cardiomyopathy, EF: ejection fraction, NYHA: New York Heart Association

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  1. Yan Zou
  2. Miaomiao Zhang
  3. Qiongfeng Wu
  4. Ning Zhao
  5. Minwei Chen
  6. Cui Yang
  7. Yimei Du
  8. Bing Han
(2022)
Activation of transient receptor potential vanilloid 4 is involved in pressure overload-induced cardiac hypertrophy
eLife 11:e74519.
https://doi.org/10.7554/eLife.74519