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. 2009 Aug 25;4(8):e6743.
doi: 10.1371/journal.pone.0006743.

Connective tissue growth factor overexpression in cardiomyocytes promotes cardiac hypertrophy and protection against pressure overload

Anna N Panek  1 , Maximilian G Posch, Natalia Alenina, Santhosh K Ghadge, Bettina Erdmann, Elena Popova, Andreas Perrot, Christian Geier, Rainer Dietz, Ingo Morano, Michael Bader, Cemil Ozcelik
Affiliations

Connective tissue growth factor overexpression in cardiomyocytes promotes cardiac hypertrophy and protection against pressure overload

Anna N Panek et al. PLoS One. .

Erratum in

  • PLoS One. 2009;4(9). doi: 10.1371/annotation/818d7cc6-3ec0-4fc5-82e1-8e9b6ceca336. Morano, Rainer Dietz Ingo [corrected to Dietz, Rainer]; Morano, Ingo [added]

Abstract

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca(2+) reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls.Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGF's profibrotic function in the heart.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Construction of cardiomyocyte-specific CTGF-TG mice.
(A) Schematic structure of the transgene. 1 and 2 indicate primers used for genotyping and 3 and 4 show primers used to generate the probe for CTGF ribonuclease protection assay. MLC-2 (myosin light chain-2) promoter; rabbit β-globin exon 2 and 3 for enhanced expression of the transgene; SV40 pA, polyadenylation signal from Simian virus 40. (B) Expression of CTGF mRNA in hearts (He) and other organs (kidney, lung, liver, brain) of transgenic mice shown by ribonuclease protection assay. (C) Western Blot analyses of CTGF protein overexpression in CTGF-TG mouse hearts. GAPDH protein expression was used as loading control. (D) Quantification of the transgenic and endogenous CTGF mRNA expression by TaqMan PCR. (WT n = 4, TG n = 4; *, P<0.05 (E) Histological staining of CTGF in WT and CTGF-TG mice as well as in Wistar-Kyoto rats (WKY) as control for spontaneously hypertensive rat (SHR). Scale bars designate a length of 100 µm.
Figure 2
Figure 2. Characterization of CTGF-TG mice of different ages by echocardiography.
(A, B, C) Measurements of LVEDD, LVESD, calculated ratio of PWD/LVEDD and FS of 3-months-old mice (A, WT n = 9 TG n = 7), 4-months-old mice (B, WT n = 10, TG n = 9) and 7-months-old mice (C, WT n = 7, TG n = 6). * P<0.05; ** P<0.01; *** P<0.001. (D) Examples of M-mode echocardiography in CTGF-TG and WT mice at age of 7 months.
Figure 3
Figure 3. Assessment of cardiac fibrosis at age of 4 and 7 months in CTGF-TG and WT mice.
(A) Masson's trichrome stained cardiac sections of WT and CTGF-TG mice at age of 4 and 7 months. Scale bars designate a length of 100 µm. (B, C) Quantification of the collagen 1α, collagen 3 and fibronectin mRNA expression by TaqMan-PCR performed at age of 4- (B) and 7 months (C) (n = 4 per group). (D) High magnifications of toluidine blue-stained semithin sections, with myofiber hypertrophy apparent in 7 months old CTGF-TG hearts. Scale bars designate a length of 20 µm.
Figure 4
Figure 4. Quantification of expression and activation of cardiac hypertrophy markers.
(A, B) Quantification of ANP and BNP mRNA expression by TaqMan-PCR in 4- (A) and 7 months (B) old mice (n = 4 per group). (C) Quantitative analysis of the phosphorylation status of Akt and JNK at age of 7 months. The band intensities of Akt- or JNK-phosphoproteins are normalized with those of unphosphorylated Akt or unspecific bands respectively.
Figure 5
Figure 5. Characterisation of cardiac function, fibrosis and hypertrophy under pressure overload conditions (3.5 months of age).
(A, B) Echocardiographic measurements of fractional shortening (A) and interventricular septum diameter in diastole (B) in WT (n = 10) and CTGF-TG (n = 11) mice treated with Ang II (*p<0.05; ** p<0.01, *** p<0.001) (C) Masson's trichrome stained cardiac sections of mice treated with Ang II and quantification of collagen 1α mRNA expression by Taqman-PCR. (D) Quantification of ANP and BNP mRNA expression by TaqMan-PCR in mice treated with Ang II (n = 4 per group).
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