Fig 1.
TNFR1 and TNFR2 differentially modulated cardiac hypertrophy and inflammation.
WT, TNFR1-/-, and TNFR2-/- mice were subjected to pressure overload for 2 weeks by TAC, and sham-operated mice served as controls (n = 6 per group). (A) Heart size and quantitative data of HW/BW ratio (mg/g). Scale bar, 3 mm. (B) Representative hematoxylin and eosin-stained myocardial sections (200×) and quantitative data of myocyte area. Quantitative RT-PCR analysis of ANP (C), BNP (D), IL-1β (G), IL-6 (H), and IL-10 (I) in myocardial tissues (n = 3 to 5 per group). (E) EF. (F) LV +dP/dt and LV −dP/dt were determined. *P < 0.05, **P < 0.01, ***P < 0.001 versus sham. Individual data are included in S1 Data. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; dP/dtmax, peak instantaneous rate of left ventricular pressure increase; dP/dtmin, peak instantaneous rate of left ventricular pressure increase decline; EF, ejection fraction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HW/BW, heart weight to body weight; IL, interleukin; LV, left ventricle; RT-PCR, real-time PCR; TAC, transverse aortic constriction; TNFR, TNF receptor; WT, wild-type.
Fig 2.
tmTNF-α processing was involved in the opposing effects of TNFR1 and TNFR2 on TAC-induced cardiac hypertrophy.
WT, TNFR1-/-, and TNFR2-/- mice were subjected to overload pressure for 2 weeks by TAC, and sham-operated mice served as controls. (A and C) Representative western blots of TNFR1, TNFR2, and tmTNF-α in myocardial tissues and quantitative data. (B) Quantitative RT-PCR analysis of TNF-α (n = 5 per group). (D and G) Representative images of indirect fluorescence costaining for troponin T and tmTNF-α or TACE on myocardial sections (400×). (E and F) sTNF-α concentrations in heart homogenates and serum detected by ELISA (n = 4 to 5 per group). (H–N) BALB/c mice were injected via the tail vein with rAAV-shTNFR1 or rAAV-shTNFR2 (1 × 1011 virion particles). rAAV-GFP served as a control. After 2 weeks, the mice were subjected to sham operation or TAC for 14 days (n = 6 per group). (H and K) Representative western blots for TNFR1, TNFR2, and tmTNF-α in myocardial tissues and quantitative data for tmTNF-α. (I and J) Quantitative RT-PCR analysis of ANP and BNP (n = 5 per group). (L and M) Concentrations of sTNF-α in heart homogenates and serum determined by ELISA (n = 5 per group). (N) Representative images of fluorescence immunostaining for TACE and troponin T in myocardial sections (400×). *P < 0.05, **P < 0.01, ***P < 0.001 versus sham. See individual data at S1 Data and underlying raw images at S1 Raw Images. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; RT-PCR, real-time PCR; sTNF-α, soluble TNF-α; TAC, transverse aortic constriction; TACE, TNF-α-converting enzyme; tmTNF-α, transmembrane tumor necrosis factor-alpha; TNFR, TNF receptor; WT, wild-type.
Fig 3.
Increased tmTNF-α expression mediated the beneficial effects of TNFR1 KD/KO.
ISO (10 μM) was added to H9C2 cells for 24 h after a 24-h transfection with siRNA targeting TNFR1 or TNFR2 or to primary cardiomyocytes from WT, TNFR1-KO, or TNFR2-KO mice. DMSO served as a vehicle control. (A) Representative images of H9C2 cells stained with Actin-Trakcer Green (200×) and quantitative data of the cell surface area. Scale bar, 50 μm. Quantitative RT-PCR analysis of ANP (B and G), BNP (C and H), and TACE (F and K). (D and I) Representative cytograms and quantitative data for tmTNF-α expression in cardiomyocytes detected by flow cytometry. (E and J) sTNF-α levels in supernatants of H9C2 or primary cardiomyocytes determined by ELISA. All quantitative data represent the means ± SEs of at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle. Find individual data at S1 Data. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ISO, isoproterenol; KO, knockout; KD, knockdown; RT-PCR, real-time PCR; siRNA, small interfering RNA; sTNF-α, soluble TNF-α; TACE, TNF-α-converting enzyme; tmTNF-α, transmembrane TNF-α; TNFR, TNF receptor; WT, wild-type.
Fig 4.
Suppression of TACE alleviated TAC-induced cardiac hypertrophy.
TAPI-1 (8 mg/mL) was administered to WT mice by implantation of osmotic pumps (0.25 μL/h) directly after TAC or sham operation (n = 6 per group). (A) Representative images of tmTNF-α fluorescence immunostaining on myocardial sections (400×) and quantitative data (n = 5 per group). (B) Serum levels of sTNF-α detected by ELISA (n = 3 to 4 per group). (C) Western blot analysis of tmTNF-α, TNFR1, and TNFR2. (D) Heart size and quantitative data of HW/BW ratio (mg/g). Scale bar, 3 mm. (E) Assessment of EF. (F–J), Quantitative RT-PCR analysis of ANP, BNP, IL-1β, IL-6, and IL-10 in myocardial tissues (n = 3 to 4 per group). **P < 0.01, ***P < 0.001 versus corresponding group in sham. Individual data can be found in S1 Data and underlying raw images in S1 Raw Images. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HW/BW, heart weight to body weight; IL, interleukin; RT-PCR, real-time PCR; sTNF-α, soluble TNF-α; TAC, transverse aortic constriction; TACE, TNF-α-converting enzyme; TAPI-1, TNF-α protease inhibitor-1; tmTNF-α, transmembrane TNF-α; TNFR, TNF receptor; WT, wild-type.
Fig 5.
Exogenous tmTNF-α directly protected myocardiocytes from ISO-induced hypertrophy via TNFR2.
(A) The schema of experimental design: Exogenous sTNF-α (20 ng/mL) or tmTNF-α on fixed NIH3T3 cells was added to H9C2 cells transfected with siRNA targeting TNFR1 or TNFR2 or to primary cardiomyocytes from WT, TNFR1-KO, or TNFR2-KO mice at an effector/target ratio of 10:1 and incubated for 24 h in the presence of ISO (10 μM). Vector-transfected NIH3T3 cells served as a control. Ectopic expression of tmTNF-α on the surface of NIH3T3 cells detected by flow cytometry. (B) Representative images of H9C2 cells stained with Actin-Tracker Green (200×) and quantitative data of the cell surface area, as analyzed using Image-Pro Plus 6.0 software. Scale bar, 50 μm. Quantitative RT-PCR analysis of ANP (C), BNP (D and H), IL-1β (E), IL-6 (F and I), and IL-10 (G and J). All quantitative data represent means ± SEs of 5 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus corresponding ISO. Individual data are included in S1 Data. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; ISO, isoproterenol; KO, knockout; KD, knockdown; RT-PCR, real-time PCR; siRNA, small interfering RNA; sTNF-α, soluble TNF-α; tmTNF-α, transmembrane TNF-α; TNFR, TNF receptor; WT, wild-type.
Fig 6.
Down-regulation of TACE by tmTNF-α via TNFR2.
Primary cardiomyocytes from WT, TNFR1-KO, or TNFR2-KO mice were treated with ISO (10 μM) for 24 h in the presence or absence of exogenous sTNF-α (20 ng/mL) and tmTNF-α on fixed NIH3T3 cells (at an effector/target ratio of 10:1). Vector-transfected NIH3T3 cells served as a control. (A and C) Quantitative RT-PCR analysis of TACE. (B and D) Representative cytograms and quantitative data for TACE expression on the cell surface of cardiomyocytes detected by flow cytometry. All quantitative data represent means ± SEs of 5 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus corresponding control. See individual data at S1 Data. FSC, Forward scatter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ISO, isoproterenol; KO, knockout; KD, knockdown; RT-PCR, real-time PCR; sTNF-α, soluble TNF-α; TACE, TNF-α-converting enzyme; tmTNF-α, transmembrane TNF-α; TNFR, TNF receptor; WT, wild-type.
Fig 7.
tmTNF-α promoted pressure overload–induced AKT activation but inhibited the NF-κB pathway via TNFR2.
Representative immunoblots and quantitative data for phosphorylation levels of NF-κB p65 and AKT in LV tissues from WT, TNFR1-KO, and TNFR2-KO mice 2 weeks after TAC operation (A and B) and treatment with TAPI-1 (8 mg/mL) (E and F). (C and D) Representative immunoblots for phosphorylation levels of NF-κB p65 and AKT in H9C2 cells transfected with siRNA for TNFR or in primary cardiomyocytes from TNFR-KO mice, treated with ISO (10 μM) for 24 h. Representative immunoblots and quantitative data for phosphorylation levels of NF-κB p65 and AKT in H9C2 cells transfected with siRNA for TNFR (G–J) or in primary cardiomyocytes from TNFR-KO mice (K–N), treated with ISO (10 μM) and sTNF-α (20 ng/mL) or tmTNF-α on fixed NIH3T3 cells at an effector/target ratio of 10:1. All quantitative data represent means ± SEs of at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus shame (A and B) or vehicle (E and F) or ISO (I–N). See individual data at S1 Data and underlying raw images at S1 Raw Images. ISO, isoproterenol; KO, knockout; LV, left ventricle; NF-κB, nuclear factor kappa B; siRNA, small interfering RNA; sTNF-α, soluble TNF-α; TAC, transverse aortic constriction; tmTNF-α, transmembrane TNF-α; TNFR, TNF receptor; WT, wild-type.
Fig 8.
Schematic summary of the cardioprotective effect of tmTNF-α via TNFR2 on cardiac hypertrophy and inflammation.
In contrast to sTNF-α that exerts prohypertrophy, and pro-inflammation through activating NF-κB pathway and inhibiting AKT pathway via TNFR1 (A), tmTNF-α displays antihypertrophy and anti-inflammation through suppressing NF-κB pathway and activating AKT pathway via TNFR2 in pressure overload–induced cardiac hypertrophy (B). In addition, mechanical stress induces TACE expression followed by enhanced release of sTNF-α that increases TACE expression via TNFR1, which cleaves tmTNF-α to produce more sTNF-α to display detrimental effects (A), whereas inhibition of TACE increases expression of tmTNF-α that down-regulates pressure overload–induced TACE expression via TNFR2, which in turn reduces tmTNF-α processing, and consequence increases tmTNF-α expression to display cardioprotective activities (B). IL, interleukin; NF-κB, nuclear factor-kappa B; sTNF-α, soluble TNF-α; TACE, TNF-α-converting enzyme; tmTNF-α, transmembrane TNF-α; TNFR, TNF receptor.