TRPA1 Promotes Cardiac Myofibroblast Transdifferentiation after Myocardial Infarction Injury via the Calcineurin-NFAT-DYRK1A Signaling Pathway

Figure 1. Effect of TRPA1 on the course of cardiac fibrosis after MI injury. (a) Survival curves of Trpa1+/+ (WT), Trpa1−/− (KO), and CA-pretreated WT (CA) littermates after sham or permanent coronary ligation surgery (MI). n = 20 per group. (b and c) Western blot analysis and quantification of the TRPA1 protein level in the ventricles of MI-injured mice. Molecular weights in kDa are shown to the right of the blots. n ≥ 3 per group. (d) Representative M-mode echocardiography images of mice. (e) Measurement of the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and left ventricular end-diastolic dimension (LVEDD). n ≥ 10 per group. (f) Representative images of Masson's trichrome staining. Red, viable myocardium; blue, fibrosis due to infarction damage (scale bar = 1 mm). (g) Quantitative analysis of the fibrotic area (blue). n ≥ 5 per group. (h) Picrosirius red staining for collagens in sections of myocardium as observed via optical microscopy (scale bar = 1 mm). (i) Under polarized light, the orange and red colors indicate type I collagen deposition and the green and yellow colors indicate type III collagen deposition in the myocardial infarct…

Figure 2. TRPA1-mediated CMF activation in the fibrotic response to MI injury. (a and b) Western blot analysis and quantification of TRPA1 protein expression from adult CFs isolated from the ventricles of sham or MI-injured mice. Molecular weights in kDa are shown to the right of the blots. n ≥ 3 per group. (c and d) Immunofluorescence staining and quantification of CMF numbers (white arrows) in the cardiac infarction border zone 7 days after injury. Myofibroblasts are positive for α-SMA (red) and negative for CD-31 (green) (scale bar = 25 μm). Five sections were measured per heart, n ≥ 3 per group. (e and f) Western blot analysis and quantification of Postn protein expression in adult CFs isolated from the ventricles of sham or MI-injured mice. Molecular weights in kDa are shown to the right of the blots. n ≥ 3 per group. The error bars represent the means ± s.e.m.∗ P < 0.05 and ∗∗ P < 0.01. The data are representative of the results of three or more independent experiments.

Figure 3. Deletion of TRPA1 in resident CFs abrogates TGF-β-mediated CMF transdifferentiation. (a and b) Immunofluorescence staining (a) and quantification (b) of α-SMA-positive (red) stress fibers and DAPI (blue) in neonatal Trpa1+/+ and Trpa1−/− mouse primary CFs with and without TGF-β stimulation (scale bars = 25 μm). (c and d) Images (c) and quantification (d) of the contraction of floating collagen gel matrices seeded with Trpa1+/+ or Trpa1 −/− CFs at 24 hr after TGF-β stimulation. The white arrows show the direction of contraction. n ≥ 3 per group. (e and f) Western blot analysis and quantification of Postn protein expression in primary Trpa1+/+ and Trpa1−/− CFs, with and without TGF-β stimulation. Molecular weights in kDa are shown to the right of the blots. n ≥ 3 per group. The error bars represent the means ± s.e.m.∗ P < 0.05 and ∗∗ P < 0.01. The data are representative of the results of three or more independent experiments.

Figure 4. Overexpression of TRPA1 in primary CFs enhances CMF transdifferentiation. (a) Western blot analysis and quantification of TRPA1 protein expression in neonatal WT mouse primary CFs with and without TGF-β stimulation. Molecular weights in kDa are shown to the right of the blots. n ≥ 3 per group. (b and c) Immunofluorescence staining (b) and quantification (c) of α-SMA-positive (red) stress fibers and DAPI (blue) in CFs treated with TGF-β transfected with negative control (NC) adenoviral vector (not shown), Ad-TRPA1 (Ad), negative control (NC) siRNA (not shown), or si-TRPA1 (si) and treated 48 hr later with TGF-β. (scale bars = 25 μm). n ≥ 3 per group. (d) α-SMA-luciferase promoter activity in CFs treated as described above. n ≥ 3 per group. (e) Western blot analysis and quantification of Postn protein expression in CFs treated as described above. Molecular weights in kDa are shown to the right of the blots. n ≥ 3 per group. (f and g) Images (f) and quantification (g) of the contraction of floating collagen gel matrices seeded with CFs treated as described above. n ≥ 3 per group. The error bars represent the means ± s.e.m.∗ P < 0.05 and ∗∗ P < 0.01. The data are representative of…

Figure 5. TRPA1 overexpression stimulates Ca2+ influx events in CFs. (a and b) Immunofluorescence staining (a) and quantification (b) of Fluo-4 in neonatal WT mouse primary CFs treated with or without TGF-β or transfected with Ad-TRPA1 (Ad) or si-TRPA1 (si) and treated 48 hr later with TGF-β (scale bars = 25 μm). n ≥ 3 per group. (c and d) Representative traces (c) and frequencies (d) of Ca2+ oscillation (n = 50 cells) in single-cell Ca2+ imaging experiments. Ca2+ signals are presented as F/F 0 values, where F is the Fluo-4 fluorescence value after treatment and F 0 is the value before treatment. n ≥ 3 per group. The error bars represent the means ± s.e.m.∗ P < 0.05 and ∗∗ P < 0.01. The data are representative of the results of three or more independent experiments.

Figure 6. TRPA1 mediates CMF transdifferentiation via the Ca2+-calcineurin- (CaN-) NFAT pathway. (a) Quantification of CaN activity in cultured neonatal WT mouse primary CFs treated with TGF-β or transfected with negative control (NC) adenoviral vector (not shown), Ad-TRPA1 (Ad), negative control (NC) siRNA (not shown), or si-TRPA1 (si) and treated 48 hr later with TGF-β. n ≥ 3 per group. (b and c) Immunofluorescence staining (b) and quantification (c) of α-SMA-positive (red) stress fibers and DAPI (blue) in CFs treated with TGF-β or transfected with Ad-TRPA1 and stimulated 48 hr later with TGF-β, with and without FK506 treatment (scale bars = 25 μm). n ≥ 3 per group. (d) α-SMA-luciferase promoter activity in CFs treated as described above. (e and f) Images (e) and quantification (f) of the contraction of floating collagen gel matrices seeded with CFs treated as described above. n ≥ 3 per group. (g and h) Western blot analysis and quantification of NFATc3 protein expression in the cytosolic and nuclear fractions of CFs treated as described above. Molecular weights in kDa are shown to the right of the blots. n ≥ 3 per group. The error bars represent the means ± s.e.m.∗ P < 0.05 and ∗∗ P <…

Figure 7. DYRK1A inhibits CaN-mediated NFATc3 nuclear localization in CMF transdifferentiation. (a) Western blot analysis and quantification of DYRK1A protein expression in neonatal WT mouse cultured primary CFs treated with TGF-β or transfected with negative control (NC) adenoviral vector (not shown), Ad-TRPA1 (Ad), negative control (NC) siRNA (not shown), or si-TRPA1 (si) and treated 48 hr later with TGF-β. Molecular weights in kDa are shown to the right of the blots. n ≥ 3 per group. (b and c) Immunofluorescence staining (b) and quantification (c) of α-SMA-positive (red) stress fibers and DAPI (blue) in CFs treated with TGF-β or transfected with Ad-TRPA1 and stimulated 48 hr later with TGF-β, with and without harmin (Harm) treatment. (d) α-SMA-luciferase promoter activity in CFs treated as described above (scale bars = 25 μm). n ≥ 3 per group. (e and f) Western blot analysis and quantification of NFATc3 protein expression in the cytosolic and nuclear fractions of CFs treated as described above. Molecular weights in kDa are shown to the right of the blots. n ≥ 3 per group. The error bars represent the means ± s.e.m.∗ P < 0.05 and ∗∗ P < 0.01. The data are representative of the results of…

Figure 8. Schematic of the proposed mechanism underlying TRPA1-mediated CMF transdifferentiation and cardiac fibrosis after MI injury. Signaling model for CMF transdifferentiation whereby MI injury upregulates TRPA1 expression, enhancing Ca2+ entry and leading to CaN activation, resulting in the translocation of NFAT to the nucleus to participate in CMF phenotypic conversion. DYRK1A, as the downstream signaling effector of TRPA1, mediates NFAT transcription factors as likely regulators of CMF transdifferentiation-related gene expression.