Role of domains 3-4 (d3-4) of connective tissue growth factor (CTGF/CCN2) in cardiac remodelling
1.Connective tissue growth factor (CTGF/CCN2) is implicated in several diseases, particularly due to it being a key mediator of tissue fibrosis. We previously reported that overexpression of full length CTGF/CCN2 have minimal implications on fibrosis in mice, and that proteolytic processing of full-length CTGF/CCN2 is necessary for its biological activation. Our data suggests that the CTGF C-terminal domains 3 and 4 are necessary for its signaling activity. To study this, we established a transgenic mouse line (flox) using Cre-lox system with conditional overexpression of the CTGF domains 3-4 (LSLd34CTGF). Using Cre mice with a tissue specific promoter facilitates tissue specific Cre recombinase activity leading to tissue specific overexpression of the transgene.
Here, we investigate the effect of the transgenic overexpression of the active CTGF: d3-4-CTGF in the heart. We will cross our floxed LSLd34CTGF mice (housed at KPM-RH, FOTS 19482) with the well-established Cre model: aMHC-MerCreMer, purchased from Jackson Laboratory. aMHC-MerCreMer transgene contains the mouse cardiac-specific alpha-myosin heavy chain (aMHC) promoter directing expression of a tamoxifen-inducible Cre recombinase (MerCreMer) to cardiac myocytes in young/adult mice. Breeding floxed LSLd34CTGF with aMHC-MerCreMer mice will result in the generation of a hybrid line with Tamoxifen inducible over-expression of d34CTGF in the heart.
Subsequently we will evaluate the direct role of d34CTGF, as a modulator of cardiac fibrosis in mice. Cardiac function and molecular mechanisms in the animals will be evaluated by cardiac echocardiography and by histological and biochemical analyses of cardiac tissue. Understanding of the basic mechanisms regulating the myocardial fibrosis is crucial for the development of intervention strategies to inhibit cardiac fibrosis and promote injury repair in myocardial remodeling.
2.Distress: For induction of transgene, 10-12 week-old hybrid mice will be treated with Tamoxifen by intraperitoneal injection at a dosage of 40 mg/kg (40ug/g body weight) once with endpoint 28 days post Tamoxifen. Cardiac function will be evaluated using echocardiography. None of the two procedures are expected to be distress inflicting procedures.
3. Expected benefit: Myocardial fibrosis commonly occur after myocardial infarction; however, there are various other conditions promoting cardiac fibrosis such as hypertensive heart disease, diabetic hypertrophic cardiomyopathy and idiopathic dilated cardiomyopathy. Therefore, it is necessary to understand the details of the events that lead either to physiological or to pathological cardiac tissue remodeling in order to design new therapeutic targets or strategies. Several scientific reports established that CTGF/CCN2 is a profibrotic molecule. The purpose of this study is to understand the molecular mechanisms of active CTGF/CCN2 (d34CTGF) mediated cardiac fibrosis and enable the design of new, safe and effective treatment strategies.
4.Number of animals: 645 mice
5.How to adhere to 3R: Our previous experience with echocardiographic analysis and physiologic assessment of cardiac function in animal models, facilitate adhering to 3R with lowest number of animals. Well-trained personnel and experienced handling using refined methods will reduce the suffering of the animals.
Here, we investigate the effect of the transgenic overexpression of the active CTGF: d3-4-CTGF in the heart. We will cross our floxed LSLd34CTGF mice (housed at KPM-RH, FOTS 19482) with the well-established Cre model: aMHC-MerCreMer, purchased from Jackson Laboratory. aMHC-MerCreMer transgene contains the mouse cardiac-specific alpha-myosin heavy chain (aMHC) promoter directing expression of a tamoxifen-inducible Cre recombinase (MerCreMer) to cardiac myocytes in young/adult mice. Breeding floxed LSLd34CTGF with aMHC-MerCreMer mice will result in the generation of a hybrid line with Tamoxifen inducible over-expression of d34CTGF in the heart.
Subsequently we will evaluate the direct role of d34CTGF, as a modulator of cardiac fibrosis in mice. Cardiac function and molecular mechanisms in the animals will be evaluated by cardiac echocardiography and by histological and biochemical analyses of cardiac tissue. Understanding of the basic mechanisms regulating the myocardial fibrosis is crucial for the development of intervention strategies to inhibit cardiac fibrosis and promote injury repair in myocardial remodeling.
2.Distress: For induction of transgene, 10-12 week-old hybrid mice will be treated with Tamoxifen by intraperitoneal injection at a dosage of 40 mg/kg (40ug/g body weight) once with endpoint 28 days post Tamoxifen. Cardiac function will be evaluated using echocardiography. None of the two procedures are expected to be distress inflicting procedures.
3. Expected benefit: Myocardial fibrosis commonly occur after myocardial infarction; however, there are various other conditions promoting cardiac fibrosis such as hypertensive heart disease, diabetic hypertrophic cardiomyopathy and idiopathic dilated cardiomyopathy. Therefore, it is necessary to understand the details of the events that lead either to physiological or to pathological cardiac tissue remodeling in order to design new therapeutic targets or strategies. Several scientific reports established that CTGF/CCN2 is a profibrotic molecule. The purpose of this study is to understand the molecular mechanisms of active CTGF/CCN2 (d34CTGF) mediated cardiac fibrosis and enable the design of new, safe and effective treatment strategies.
4.Number of animals: 645 mice
5.How to adhere to 3R: Our previous experience with echocardiographic analysis and physiologic assessment of cardiac function in animal models, facilitate adhering to 3R with lowest number of animals. Well-trained personnel and experienced handling using refined methods will reduce the suffering of the animals.