Transcription Factor 7-like 2 Mediates Canonical Wnt/β-Catenin Signaling and c-Myc Upregulation in Heart Failure

Abstract

Background: How canonical Wnt/β-catenin signals in adult hearts, especially in different diseased states, remains unclear. The proto-oncogene, c-Myc, is a Wnt target and an early response gene during cardiac stress. It is not clear whether c-Myc is activated or how it is regulated during heart failure.

Methods and results: We investigated canonical Wnt/β-catenin signaling and how it regulated c-Myc expression in failing hearts of human ischemic heart disease, idiopathic dilated cardiomyopathy, and murine desmin-related cardiomyopathy. Our data demonstrated that canonical Wnt/β-catenin signaling was activated through nuclear accumulation of β-catenin in idiopathic dilated cardiomyopathy, ischemic heart disease, and murine desmin-related cardiomyopathy when compared with nonfailing controls and transcription factor 7-like 2 (TCF7L2) was the main β-catenin partner of the T-cell factor (TCF) family in adult hearts. We further revealed that c-Myc mRNA and protein levels were significantly elevated in failing hearts by real-time reverse transcription polymerase chain reaction, Western blotting, and immunohistochemical staining. Immunoprecipitation and confocal microscopy further showed that β-catenin interacted and colocalized with TCF7L2. More importantly, chromatin immunoprecipitation confirmed that β-catenin and TCF7L2 were recruited to the regulatory elements of c-Myc. This recruitment was associated with increased histone H3 acetylation and transcriptional upregulation of c-Myc. With lentiviral infection, TCF7L2 overexpression increased c-Myc expression and cardiomyocyte size, whereas shRNA-mediated knockdown of TCF7L2 suppressed c-Myc expression and cardiomyocyte growth in cultured neonatal rat cardiomyocytes.

Conclusions: This study indicates that TCF7L2 mediates canonic Wnt/β-catenin signaling and c-Myc upregulation during abnormal cardiac remodeling in heart failure and suppression of Wnt/β-catenin to c-Myc axis can be explored for preventing and treating heart failure.

Keywords: acetylation; cardiomyopathy; cell signaling/signal transduction; desmin; heart failure.

Figure 1

Accumulation of β-catenin in the nucleus of CMs from human failing hearts. A: Representative Western blots of total (β-catenin) and non-phosphorylated active β-catenin (ABC) in the cytoplasmic (25 μg), nuclear (25 μg), and whole (12.5 μg) lysates from idiopathic dilated cardiomyopathy (IDC), ischemic heart disease (IHD), and non-failing heart (NFH). B: Fold increases in β-catenin mRNA levels by real-time RT-PCR and in protein levels of total β-catenin and ABC by relative densitometric values of Western blots (N=10 in each group).C: Immunohistochemical staining (top row) revealed that there was no significant difference in β-catenin at intercalated disks (IDs) of CMs from IDC, IHD, and NFH; but increased nuclear β-catenin was observed in CMs of IDC and IHD compared to that of NFH. Representative confocal microscopy of triple stains with desmin, β-catenin, and 4′,6-diamidino-2-phenylindole (DAPI, bottom row). Desmin increased in CMs from IDC and IHD relative to that of NFH. Again, all CMs in IDC, IHD, and NFH showed β-catenin positivity at IDs. Nuclear β-catenin was strong in CMs of IDC and IHD, but not readily detected in CMs of NFH. Representative images from 5 sets of staining were shown. Data were mean ± SEM, P<0.05 indicated statistically significant differences compared with NFH. Scale bar, 50μm.

Figure 2

Accumulation of β-catenin in the nucleus of CMs from 14- to 16-week-old mice with D7-Des mutation (DES). A: Histology demonstrated cardiac hypertrophy with nuclear enlargement in DES hearts similar to human failing hearts compared with wild type (WT) mice. B: Heart weight/body weight ratio and left ventricle weight/body weight ratio were increased in DES hearts. C: Representative Western blots of total (β-catenin) and non-phosphorylated active β-catenin (ABC) in the cytoplasmic (25 μg), nuclear (25 μg), and whole (12.5 μg) lysates from DES and WT hearts. D: Fold increases in β-catenin mRNA levels by real-time RT-PCR and in protein levels of total β-catenin and ABC by relative densitometric values of Western blots from DES over WT hearts. N=5 in each group. E: Representative images of immunohistochemical staining from 5 mice in each group revealed similar β-catenin intensity at intercalated disks (IDs) between DES and WT CMs; but obvious nuclear β-catenin was present in CMs from DES, but not detected that from WT. Data were mean ± SEM, P<0.05 indicated statistically significant differences between DES and WT. Scale bar, 50μm.

Figure 3

Upregulation of TCF7L2 and c-Myc in human failing and mouse DES hearts. A and B: Representative Western blots (A) of whole lysate and fold increases (B) in relative densitometric values of c-Myc and total long (78 kDa) and short (55 kDa) isoforms of TCF7L2 in idiopathic dilated cardiomyopathy (IDC) and ischemic heart disease (IHD) over non-failing heart (NFH). C and D: Semi-quantitative RT-PCR (C) and fold increases by real-time (D) RT-PCR of TCF7L2 with primers common to both long and short isoforms and c-Myc in IDC, IHD, and NFH. E and F: Representative Western blots (E) and fold increases (F) in relative densitometric values of c-Myc and total long (78 kDa) and short (55 kDa) isoforms of TCF7L2 in mouse DES and WT hearts. G and H: Semi-quantitative RT-PCR (G) and fold increases by real-time (H) RT-PCR of TCF7L2 with primers common to both long and short isoforms and c-Myc in mouse DES and WT hearts. Ten hearts were included in IDC, IHD and NFH while 5 hearts were used in DES and WT groups for every analysis.I and J: Representative images of immunohistochemical staining of TCF7L2 and c-Myc in 5 sets of human and mouse hearts in each group. Data were mean ± SEM, P<0.05 indicated statistically significant differences compared to control groups. Scale bar, 50μm.

Figure 4

Colocalization and co-recruitment of β-catenin with TCF7L2. A: Colocalization of TCF7L2 (red) with β-catenin (green) by confocal microscopy in idiopathic dilated cardiomyopathy (IDC, top row), ischemic heart disease (IHD, middle row), and non-failing heart (NFH, bottom row); nuclear counterstaining of DAPI (blue) and bar=10μm. Representative images are from 5 sets of staining in each group. B: Immunoprecipitation and Western blotting of TCF7L2 and β-catenin in IDC, IHD and NFH. Ten human samples were used in each group. C and D: Semi-quantitative PCR of c-Myc promoter after chromatin immunoprecipitation with β-catenin, TCF7L2, and acetylated histone H3 (acetylH3) in human (C) and mouse (D) hearts. Data are representative of PCR and the densitometric analysis from 10 human and 5 mouse hearts in each group. DES indicates D7-Des mutation; and WT, wild-type.

Figure 5

TCF7L2 levels control c-Myc expression in cultured neonatal rat cardiomyocytes (NRCMs). A to C: Morphological changes of NRCMs by TCF7L2 transfection. Cells transferred with Tcf7l2 shRNA were narrow and thin spindle-like shape (A, left lower) with smaller size (B) and perimeter (C) compared with non-infected control group. NRCMs enlarged and varied from round-spindle to polygonal by transfection with short form full-length Tcf7l2 (Lenti WT) (A, right lower), while the size and perimeter of cells dramatically increased (B and C). D and E, Nppa and Myh7 mRNA levels were downregulated by Tcf7l2 shRNA, but upregulated by infection with full-length Tcf7l2. F to H: Representative TCF7L2 and c-Myc Western blots (F) as well as fold changes of TCF7L2 (G) and c-Myc (H) in NRCMs transfected with no virus, control virus (pGPIZ), Tcf7l2 shRNA, and full-length short form Tcf7l2. I to K: Semi-quantitative RT-PCR (I) and fold increases by real-time RT-PCR of Tcf7l2 (J) and c-Myc (K) in NRCMs transfected with no virus, control virus (pGPIZ), Tcf7l2 shRNA, and full-length short form Tcf7l2. Data are representative of five independent repeats. P<0.05 indicated statistically significant differences compared to control groups. Scale bar, 100μm.