Fig 1.
TGFBR1 expression is increased in the hearts of HFpEF mice and in “double-damage” H9C2 cells.
(A) Representative western blot images of TGFBR1 and TGFBR2 expression in the left-ventricular samples from the control and HFpEF mice. (B) Quantification of the protein expression of TGFBR1 in (A); n = 6 per group. (D) Relative mRNA levels of TGFBR1 in the left-ventricular samples from the control and HFpEF mice; n = 6 per group. (E) Representative immunofluorescence staining for TGFBR1 (green) in the tissue sections from the left ventricle of the control and HFpEF mice. The nuclei were counterstained with DAPI (blue). Bar = 50 μm. (F) Quantification of fluorescence intensity of TGFBR1 in (E); n = 6 per group. (G) Representative western blot images of TGFBR1 expression in the groups of H9C2 cells; n = 6 per group. (H) Quantification of the protein expression of TGFBR1 in (G). (I) Representative immunofluorescence staining for TGFBR1 (green) in the control and “double-damage” H9c2 cells. The nuclei were counterstained with DAPI (blue). Bar = 50 μm. (J) Quantification of fluorescence intensity of TGFBR1 in (G); n = 6 per group. The normality of data distribution was tested using the Shapiro–Wilk method. Unpaired two-tailed Student’s t tests were applied in (B), (C), (D), (F), (H), and (J). *p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated groups.
Fig 2.
TGFBR1 gene silencing improves cardiac function in HFpEF mice.
(A) Illustrative experimental protocol. (B) Representative western blot images of TGFBR1 expression in the left-ventricular samples from each group of mice. (C) Quantification of the protein expression of TGFBR1 in (B); n = 6 per group. (D) Representative M-mode (upper) and pulse-wave Doppler (bottom) images. (E) Left-ventricular end-diastolic diameter (LVEDD). (F) Left-ventricular end-systolic diameter (LVESD). (G) Interventricular septal thickness in diastole (IVS, d). (H) Left-ventricular ejection fraction (LVEF). (I) LV mass. (J) Ratio between mitral E wave and A wave (E/A). (K) Ratio between mitral E wave and E′ wave (E/E′). (L) Left-ventricular global longitudinal strain (GLS). (M) Heart rate (HR). (N) Diastolic blood pressure (DBP). (O) Systolic blood pressure (SBP). (P) Ratio between heart weight and tibia length (HW/TL). (Q) Ratio between wet and dry lung weight. (R) Running distance during the exercise exhaustion test. (S) Expression levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP) in the left-ventricular samples from each group of mice. (T) Body weight. Mean ± SEM, n = 6 mice per group for panels E–T. The normality of data distribution was tested using the Shapiro–Wilk method. One-way ANOVA was applied in (C) and in (E)–(T). *p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated groups.
Fig 3.
TGFBR1 gene silencing alleviates cardiac remodeling and fibrosis in HFpEF mice.
(A) Representative images showing mouse hearts. Bar = 50 mm. (B) Representative hematoxylin and eosin (H&E) staining images. Bar = 200 μm. (C) Representative Wheat germ agglutinin (WGA) staining images. Bar = 20 μm. (D) Quantification cross-sectional area in (G); n = 100 cells per group. (E) Representative Masson’s trichrome staining images. Bar = 50 μm. (F) Quantification percentage of interstitial fibrosis in (E). (G) and (I) Representative images of myocardial type I collagen (collagen I, Col I) and type III collagen (collagen III, Col III) immunohistochemistry (IHC). Bar = 200 μm. (H) and (J) IHC quantification of collagen I and collagen III. (K) Representative western blot images of Col I, Col III, p-Smad2, and p-Smad3 expression in the left-ventricular samples from each group of mice. (L) Quantification of the protein expression of TGFBR1 in (K). Mean ± SEM, n = 6 mice per group for panels A–L. The normality of data distribution was tested using the Shapiro–Wilk method. One-way ANOVA was applied in (D), (F), (H), (J), and (L). *p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated groups.
Fig 4.
TGFBR1 gene silencing inhibits TAK1 activation and dissociation.
(A) Representative western blot images of p-TAK1 expression in the left-ventricular samples from each group of mice. (B) Quantification of the protein expression of p-TAK1 in (A); n = 6 per group. (C) Representative images of p-TAK1 immunohistochemistry (IHC). Bar = 200 μm; n = 6 per group. (D) Quantification of IHC of p-TAK1. (E) Representative immunoprecipitation images of TGFBR1, TAK1, and RIPK1. (F) Quantification of the binding rate of TAK1 to RIPK1; n = 4 per group. The normality of data distribution was tested using the Shapiro–Wilk method. One-way ANOVA was applied in (B), (D), and (F). *p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated groups.
Fig 5.
TGFBR1 gene silencing alleviates myocardial PANoptosis-like cell death in HFpEF mice.
(A) Representative TUNEL staining images of the left-ventricular samples from each group of mice. Bar = 50 μm. (B) Quantification of TUNEL-positive cells in (A); n = 100 cells per group. (C) Representative western blot images of NLRP3, caspase-1, and GSDMD expression in the left-ventricular samples from each group of mice. (D) Representative western blot images of caspase-3, caspase-7, and caspase-8 expression in the left-ventricular samples from each group of mice. (E) Representative western blot images of p-RIPK1, p-RIPK3, and p-MLKL expression in the left-ventricular samples from each group of mice. (F)–(I) Quantification of the protein expression of NLRP3, cleaved caspase-1, and GSDMD-N in (C). (J)–(L) Quantification of the protein expression of cleaved caspase-3, cleaved caspase-7, and cleaved caspase-8 in (D). (M)–(O) Quantification of the protein expression of p-RIPK1, p-RIPK3, and p-MLKL in (E); n = 6 mice per group for panels A–O. The normality of data distribution was tested using the Shapiro–Wilk method. One-way ANOVA was applied in (B) and in (F)–(O). *p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated groups.
Fig 6.
TGFBR1 gene silencing alleviates myocardial PANoptosis cell death induced by “double-damage.
” (A) Representative western blot images of TGFBR1, NLRP3,caspase-8 and RIPK1 expression in the groups of H9C2 cells; n = 3 per group. (B) Quantification of the protein expression of TGFBR1, NLRP3,caspase-8 and RIPK1. (C) Representative western blot images of caspase-1 expression in the groups of H9C2 cells; n = 6 per group. (D) Quantification of the protein expression of cleaved caspase-1 in (C). (E) Extracellular-fluid IL-1β levels; n = 6 per group. (F) Extracellular-fluid IL-18 levels; n = 6 per group. (G) Representative PI staining images of the groups of H9C2 cells. Red indicates dead cells. Bar = 50 μm. (H) Quantification of PI-positive cells in (G); n = 100 cells per group. The normality of data distribution was tested using the Shapiro–Wilk method. One-way ANOVA was applied in (D)–(F) and in (H). *p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated groups.
Fig 7.
TGFBR1 gene silencing inhibits the assembly of the PANoptosome.
(A) Representative western blot images of ASC, NLRP3, caspase-1, caspase-8, RIPK1, and RIPK3 in the left-ventricular samples from each group of mice. (B) Representative Co-IP images of ASC, NLRP3, caspase-1, caspase-8, RIPK1, and RIPK3 in the left-ventricular samples from each group of mice; n = 3 mice per group for panels A and B.
Fig 8.
TGFBR1 gene silencing inhibits cardiac hypertrophy.
(A) Representative western blot images of TGFBR1, p-MEK1/2, p-ERK1/2, p-p38, and p-JNK1/2 in the left-ventricular samples from each group of mice; n = 4 mice per group. (B) Quantification of the protein expression of TGFBR1, p-MEK1/2, p-ERK1/2, p-p38, and p-JNK1/2 in (A). (C) Representative images of myocardial phalloidin staining. The nuclei were counterstained with DAPI (blue). Bar = 20 μm. (D) Quantification of cell surface area; n = 100 cells per group. (E) Representative western blot images of TGFBR1, p-MEK1/2, p-ERK1/2, p-p38, and p-JNK1/2 expression in the groups of H9C2 cells; n = 4 per group. (F) Quantification of the protein expression of TGFBR1, p-MEK1/2, p-ERK1/2, p-p38, and p-JNK1/2 in (E). The normality of data distribution was tested using the Shapiro–Wilk method. One-way ANOVA was applied in (B), (D), and (F). *p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated groups.
Fig 9.
TGFBR1 gene silencing inhibits TAK1 activation via TRAF6.
(A) Representative western blot images of p-TAK1, p-Smad2, and p-Smad3 in the groups of H9C2 cells. (B) Quantification of the protein expression of p-TAK1, p-Smad2, and p-Smad3 in (A). (C) Representative western blot images of TRAF6 and p-TAK1 in the groups of H9C2 cells. (D) Quantification of the protein expression of TRAF6 and p-TAK1 in (C). (E) Representative western blot images of TRAF6 in the groups of H9C2 cells. (F) Quantification of the protein expression of TRAF6 in (E), and relative TRAF6 mRNA expression levels in the groups of H9C2 cells. (G) Representative immunofluorescence staining of TRAF6 (green) in the groups of H9C2 cells. The nuclei were counterstained with DAPI (blue). Bar = 50 μm. (H) Quantification of fluorescence intensity of TRAF6 in (G); (A)–(H) n = 6 per group. (I) Representative western blot images of TRAF6 in the left-ventricular samples from each group of mice. (J) Quantification of the protein expression of TRAF6 in (I), and relative TRAF6 mRNA expression levels in the left-ventricular samples from each group of mice. (K) Representative images of myocardial TRAF6 immunohistochemistry (IHC). Bar = 200 μm. (L) Quantification of IHC of TRAF6; n = 6 mice per group for panels I–L. The normality of data distribution was tested using the Shapiro–Wilk method. One-way ANOVA was applied in (B), (D), (H), (F), and (L). *p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated groups.
Fig 10.
Potential mechanisms underlying the effects of TGFBR1 gene silencing on the heart of HFpEF mice are as follows.
TGFBR1 gene silencing leads to a reduction in myocardial collagen synthesis through the Smad2/3 signaling pathway and inhibits myocardial hypertrophy in HFpEF mice through the p38/MAPK–JNK1/2 signaling pathway. Furthermore, TGFBR1 gene silencing impedes myocardial remodeling in HFpEF mice by curbing TAK1-mediated PANoptosis, predominantly because TGFBR1 gene silencing hinders the segregation of RIPK1 from TAK1 and reduces TRAF6 expression.