Fig. 7
Phenotype of mice with systemic forced expression of vRANK using the CAG-CreER system (Heart). In CAG-CreER/vRANK mice, systemic induction of vRANK resulted in significant cardiac enlargement and thinning of the left ventricular myocardial wall (yellow double-headed arrows) compared to control WT mice (A, B). At 200× magnification, myocardial tissue from WT mice displayed an orderly arrangement of cardiomyocytes (C), whereas in CAG-CreER/vRANK mice, myocardial cell loss and compensatory fibrosis were observed (D). At higher magnification (400×), cardiomyocyte swelling and myofiber disruption (black arrows), absent in the control (E), were sporadically observed in non-fibrotic regions of the myocardium in CAG-CreER/vRANK mice. Immunohistochemical analysis of TGF-β1 protein localization showed that WT mice had almost no positive cells (G, 400×), whereas in CAG-CreER/vRANK mice, TGF-β1 was localized in spindle-shaped fibroblasts within the fibrotic myocardial regions (H, 400×). In situ hybridization using the RNAscope method was performed to evaluate Opg mRNA expression in myocardial tissue. In both WT (I) and CAG-CreER/vRANK mice (J), Opg mRNA expression was observed as indicated by black arrows. Compared to WT mice, CAG-CreER/vRANK mice showed reduced expression of Opg mRNA (J, 400×). The inset in the lower right shows the expression of ubiquitin C as a positive control for each specimen, with strong signals detected in both groups, indicating that mRNA integrity was well preserved in all samples.
