A novel role for tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in the development of cardiac dysfunction and failure

Abstract

Background: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, is a multifunctional cytokine known to regulate cellular functions in contexts of injury and disease through its receptor, fibroblast growth factor-inducible molecule 14 (Fn14). Although many of the processes and downstream signals regulated by the TWEAK/Fn14 pathway have been implicated in the development of cardiac dysfunction, the role of TWEAK in the cardiovascular system is completely unknown.

Methods and results: Herein, we demonstrate that mouse and human cardiomyocytes express the TWEAK receptor Fn14. Furthermore, we determine that elevated circulating levels of TWEAK, induced via transgenic or adenoviral-mediated gene expression in mice, result in dilated cardiomyopathy with subsequent severe cardiac dysfunction. This phenotype was mediated exclusively by the Fn14 receptor, independent of tumor necrosis factor-alpha, and was associated with cardiomyocyte elongation and cardiac fibrosis but not cardiomyocyte apoptosis. Moreover, we find that circulating TWEAK levels were differentially upregulated in patients with idiopathic dilated cardiomyopathy compared with other forms of heart disease and normal control subjects.

Conclusions: Our data suggest that TWEAK/Fn14 may be important in regulating myocardial structural remodeling and function and may play a role in the pathogenesis of dilated cardiomyopathy.

Figure 1

Expression of Fn14 in the heart and cardiomyocytes. (A). In-situ hybridization for Fn14 in E12.5 mouse hearts, with representative dark field and bright field images (62.5× magnification), demonstrating localization of silver grains (as denoted by arrows) to the ventricles (v), with limited signal within the atria (a). (B) Western blotting analysis showing Fn14 (MW, 12kd) protein expression in isolated adult cardiomyocytes and whole heart tissue and atria. HEK 293 cells serve as negative control. (C) Immunocytochemistry showing the expression of Fn14 (red) in isolated adult cardiomyocytes co-stained with α-actin (green) for myofilament protein and DAPI for nuclei (blue). From the merged picture (bottom row, 63× magnification), Fn14 expression appears most enriched at the membrane surface. Cardiomycoytes stained with control Ig was used as negative control.

Figure 1

Expression of Fn14 in the heart and cardiomyocytes. (A). In-situ hybridization for Fn14 in E12.5 mouse hearts, with representative dark field and bright field images (62.5× magnification), demonstrating localization of silver grains (as denoted by arrows) to the ventricles (v), with limited signal within the atria (a). (B) Western blotting analysis showing Fn14 (MW, 12kd) protein expression in isolated adult cardiomyocytes and whole heart tissue and atria. HEK 293 cells serve as negative control. (C) Immunocytochemistry showing the expression of Fn14 (red) in isolated adult cardiomyocytes co-stained with α-actin (green) for myofilament protein and DAPI for nuclei (blue). From the merged picture (bottom row, 63× magnification), Fn14 expression appears most enriched at the membrane surface. Cardiomycoytes stained with control Ig was used as negative control.

Figure 2

Generation of sTWEAK fl-TWEAK overexpressing mice. (A). Schematic DNA constructs for sTWEAK- and fl-TWEAK–overexpressing mice. (B). Quantitative polymerase chain reaction (PCR) demonstrating TWEAK expression in heart and liver tissue from WT (nontransgenic) and fl-TWEAK transgenic mice. PCR used with primers that detect total TWEAK expression, both endogenous and transgene expression, was performed on cDNA from embryonic stage and adult tissue. ApoE, apolipoprotein E; AAT, alpha antitrypsin. (C). Scatter plot demonstrating circulating TWEAK levels in WT, sTWEAK, and fl-TWEAK mice. (D) Representative hearts from WT and sTWEAK mice showing gross cardiac enlargement. *P<0.01 vs WT; ^†P<0.01 vs fl-TWEAK.

Figure 2

Generation of sTWEAK fl-TWEAK overexpressing mice. (A). Schematic DNA constructs for sTWEAK- and fl-TWEAK–overexpressing mice. (B). Quantitative polymerase chain reaction (PCR) demonstrating TWEAK expression in heart and liver tissue from WT (nontransgenic) and fl-TWEAK transgenic mice. PCR used with primers that detect total TWEAK expression, both endogenous and transgene expression, was performed on cDNA from embryonic stage and adult tissue. ApoE, apolipoprotein E; AAT, alpha antitrypsin. (C). Scatter plot demonstrating circulating TWEAK levels in WT, sTWEAK, and fl-TWEAK mice. (D) Representative hearts from WT and sTWEAK mice showing gross cardiac enlargement. *P<0.01 vs WT; ^†P<0.01 vs fl-TWEAK.

Figure 3

Development of DCM and heart failure in fl-TWEAK mice. Cardiac enlargement in fl-TWEAK mice. (A). Representative whole hearts and trichrome-stained longitudinal sections from WT and fl-TWEAK animals, demonstrating left ventricular (LV), right ventricular (RV), and left atrial (LA) enlargement in transgenic hearts. (B). Heart weight–to–body weight ratios, suggesting the development of cardiac hypertrophy in fl-TWEAK animals. (C). Wet-to-dry lung weights in WT and fl-TWEAK mice. *P<0.01 vs WT. (D). Survival plots for WT and fl-TWEAK animals, showing increased mortality in fl-TWEAK mice. *P<0.01 vs WT. (E). Representative m-mode conscious transthoracic echocardiograms in WT and fl-TWEAK animals. (F). Diastolic and systolic ventricular chamber dimensions and fractional shortening in WT and fl-TWEAK mice, as assessed by echocardiography, showing the development of in vivo idiopathic dilated cardiomyopathy and heart failure with TWEAK overexpression. *P<0.01 vs WT.

Figure 3

Development of DCM and heart failure in fl-TWEAK mice. Cardiac enlargement in fl-TWEAK mice. (A). Representative whole hearts and trichrome-stained longitudinal sections from WT and fl-TWEAK animals, demonstrating left ventricular (LV), right ventricular (RV), and left atrial (LA) enlargement in transgenic hearts. (B). Heart weight–to–body weight ratios, suggesting the development of cardiac hypertrophy in fl-TWEAK animals. (C). Wet-to-dry lung weights in WT and fl-TWEAK mice. *P<0.01 vs WT. (D). Survival plots for WT and fl-TWEAK animals, showing increased mortality in fl-TWEAK mice. *P<0.01 vs WT. (E). Representative m-mode conscious transthoracic echocardiograms in WT and fl-TWEAK animals. (F). Diastolic and systolic ventricular chamber dimensions and fractional shortening in WT and fl-TWEAK mice, as assessed by echocardiography, showing the development of in vivo idiopathic dilated cardiomyopathy and heart failure with TWEAK overexpression. *P<0.01 vs WT.

Figure 3

Development of DCM and heart failure in fl-TWEAK mice. Cardiac enlargement in fl-TWEAK mice. (A). Representative whole hearts and trichrome-stained longitudinal sections from WT and fl-TWEAK animals, demonstrating left ventricular (LV), right ventricular (RV), and left atrial (LA) enlargement in transgenic hearts. (B). Heart weight–to–body weight ratios, suggesting the development of cardiac hypertrophy in fl-TWEAK animals. (C). Wet-to-dry lung weights in WT and fl-TWEAK mice. *P<0.01 vs WT. (D). Survival plots for WT and fl-TWEAK animals, showing increased mortality in fl-TWEAK mice. *P<0.01 vs WT. (E). Representative m-mode conscious transthoracic echocardiograms in WT and fl-TWEAK animals. (F). Diastolic and systolic ventricular chamber dimensions and fractional shortening in WT and fl-TWEAK mice, as assessed by echocardiography, showing the development of in vivo idiopathic dilated cardiomyopathy and heart failure with TWEAK overexpression. *P<0.01 vs WT.

Figure 4

Cellular remodeling and cardiomyocyte dysfunction in fl-TWEAK hearts. (A). Representative cardiomyocyte isolates from wild-type (WT) and fl-TWEAK hearts. Histograms and corresponding average number of (B) cell length and (C) cell width measured in isolated WT and fl-TWEAK cardiomyocytes (N=3 animals each and > 500 cardiomcyocytes counted per heart, *P<0.05 vs WT). (D). Representative cellular tracings for paced WT and fl-TWEAK cardiomyocytes. (E). Percent cell shortening and time to relaxation for WT and fl-TWEAK cardiomyocytes, demonstrating impaired cardiomyocyte contraction and prolonged relaxation with TWEAK overexpression. (N=4 animals each and 20–30 cardiomyocytes function was average per heart, *P<0.05 vs WT)

Figure 4

Cellular remodeling and cardiomyocyte dysfunction in fl-TWEAK hearts. (A). Representative cardiomyocyte isolates from wild-type (WT) and fl-TWEAK hearts. Histograms and corresponding average number of (B) cell length and (C) cell width measured in isolated WT and fl-TWEAK cardiomyocytes (N=3 animals each and > 500 cardiomcyocytes counted per heart, *P<0.05 vs WT). (D). Representative cellular tracings for paced WT and fl-TWEAK cardiomyocytes. (E). Percent cell shortening and time to relaxation for WT and fl-TWEAK cardiomyocytes, demonstrating impaired cardiomyocyte contraction and prolonged relaxation with TWEAK overexpression. (N=4 animals each and 20–30 cardiomyocytes function was average per heart, *P<0.05 vs WT)

Figure 4

Cellular remodeling and cardiomyocyte dysfunction in fl-TWEAK hearts. (A). Representative cardiomyocyte isolates from wild-type (WT) and fl-TWEAK hearts. Histograms and corresponding average number of (B) cell length and (C) cell width measured in isolated WT and fl-TWEAK cardiomyocytes (N=3 animals each and > 500 cardiomcyocytes counted per heart, *P<0.05 vs WT). (D). Representative cellular tracings for paced WT and fl-TWEAK cardiomyocytes. (E). Percent cell shortening and time to relaxation for WT and fl-TWEAK cardiomyocytes, demonstrating impaired cardiomyocyte contraction and prolonged relaxation with TWEAK overexpression. (N=4 animals each and 20–30 cardiomyocytes function was average per heart, *P<0.05 vs WT)

Figure 5

Limited apoptosis in adTWEAK hearts. (A) Percent TUNEL in adTWEAK and adCon hearts at one and three weeks following TWEAK or control viral injection via tail vein (see Methods). Greater than 2000 nuclei were counted from 5–6 random areas from mid-papillary section of each heart. *, p<0.05 vs adTWEAK (N=3 each). (B) Representative TUNEL staining at 3 week post viral injection adTWEAK hearts at 40× magnification and (C) 63× magnification. α-actin stains for cardiac myocytes (red) and DAPI stains for nuclei (blue).

Figure 6

Fn14 mediates the development of TWEAK-induced dilated cardiomyopathy (DCM). (A). Representative whole hearts pictures from WT (wild-type) and Fn14-knockout (Fn14 −/−) treated with control adenovirus (adCon) or adenovirus encoding murine soluble TWEAK (adTWEAK). (B). Heart weight–to–body weight ratios from WT and Fn14 −/− animals after intravenous injection with adCon or adTWEAK, demonstrating attenuated TWEAK-mediated cardiac enlargement in animals that lack the Fn14 receptor. (C). In vivo left ventricular (LV) diastolic chamber dimension and cardiac fractional shortening, as determined by conscious transthoracic echocardiography, in WT and Fn14 −/− animals after intravenously delivery with adTWEAK, showing lack of TWEAK-mediated DCM and contractile failure in mice that lack the Fn14 receptor. N=3–5/group, for (B) *P<0.01 vs WT adCon; ^†P<0.01 vs WT adTWEAK, for (C) *P<0.05 vs WT adTWEAK.

Figure 6

Fn14 mediates the development of TWEAK-induced dilated cardiomyopathy (DCM). (A). Representative whole hearts pictures from WT (wild-type) and Fn14-knockout (Fn14 −/−) treated with control adenovirus (adCon) or adenovirus encoding murine soluble TWEAK (adTWEAK). (B). Heart weight–to–body weight ratios from WT and Fn14 −/− animals after intravenous injection with adCon or adTWEAK, demonstrating attenuated TWEAK-mediated cardiac enlargement in animals that lack the Fn14 receptor. (C). In vivo left ventricular (LV) diastolic chamber dimension and cardiac fractional shortening, as determined by conscious transthoracic echocardiography, in WT and Fn14 −/− animals after intravenously delivery with adTWEAK, showing lack of TWEAK-mediated DCM and contractile failure in mice that lack the Fn14 receptor. N=3–5/group, for (B) *P<0.01 vs WT adCon; ^†P<0.01 vs WT adTWEAK, for (C) *P<0.05 vs WT adTWEAK.

Figure 7

Development of Cardiac Hypertrophy in WT and TNF KO mice following injection with adenovirus encoding murine soluble TWEAK (adTWEAK) as compared to an adenovirus control (AdCon). Cardiac hypertrophy determined by heart weight to body weight ratio (mg/g) in WT and TNF knockout mice following tail vein injection of adTWEAK or control adenovirus (adCon). N=5/group, * indicates P<0.05.