Spontaneous cardiac calcinosis in BALB/cByJ mice

Abstract

BALB/c mice are predisposed to dystrophic cardiac calcinosis-the mineralization of cardiac tissues, especially the right ventricular epicardium. In previous reports, the disease appeared in aged animals and had an unknown etiology. In the current study, we report a substrain of BALB/c mice (BALB/cByJ) that develops disease early and with high frequency. Here we analyzed hearts grossly to identify the presence and measure the severity of disease and to compare BALB/c substrains. Histologic analysis and fluorescent and immunofluorescent microscopy were used to characterize the calcinotic lesions. BALB/cByJ mice exhibited more frequent and severe calcium deposition than did BALB/c mice of other substrains (90% compared with 3% at 5 wk). At this age, lesions covered an average of 30% of the total ventricular surface area in BALB/cByJ mice, compared with less than 1% in other strains. In bone-marrow-chimeric mice, green fluorescent protein was used as a marker to show that the lesions contain an infiltration of cells of bone marrow origin. Lesion histology showed that calcium deposits were surrounded by fibrosis with interspersed immune cells. Lymphocytes, macrophages, and granulocytes were all present. Internalization of the gap-junction protein connexin 43 was observed in myocytes adjacent to lesions. In conclusion, BALB/cByJ mice exhibit more frequent and severe dystrophic cardiac calcinosis than do other BALB/c substrains. Our findings suggest that immune cells are actively recruited to lesions and that myocyte gap junctions are altered near lesions.

Figure 1.

BALB/cByJ mice developed severe cardiac calcinosis by 5 wk of age. (A) Gross appearance of a mineralized lesion on the epicardial surface of an affected BALB/cByJ subject. (B) Epicardium of an unaffected BALB/cAnTac mouse. (C) Plot comparing the percentages of the ventricular surface occupied by lesion in BALB/cByJ (/cByJ) mice at 3, 5, and 12 wk and in BALB/c mice at 5 and 12 wk. Each symbol represents a single animal; horizontal lines represent the mean. Statistical significance (*, P < 0.01; †, P < 0.001) was determined by using one-way ANOVA followed by the Bonferroni posttest.

Figure 2.

BALB/cByJ lesions contained calcium, collagen, and inflammatory cells. (A) BALB/cByJ heart sectioned transversely and stained with Alizarin red S. Orange–brown areas indicate the presence of calcium (darker area at top right is an artifactual fold in the tissue. (B and C) Heart stained with Masson trichrome, showing abundant collagen fibers within lesions. (D) Note the presence of both mononuclear and polymorphonuclear cells; hematoxylin and eosin stain. The images in panels A and B are composites acquired at a magnification of 100×.

Figure 3.

Mineralized lesions in BALB/cByJ GFP-chimeric mice were GFP-positive. (A) Exteriors of GFP-chimeric hearts viewed under visible light. (B) Chimeric hearts showing GFP fluorescence pseudocolored in red–yellow, such that red areas fluoresce GFP-positive and yellow areas are strongly GFP-positive. (C and D) Fluorescence microscopy of epicardial lesions showing GFP-positive cells in green and DAPI-stained nuclei in blue. Calibration bars are 50 μm.

Figure 4.

Cells within epicardial lesions stained positively for myeloperoxidase, CD3, CD4, and CD11b. (A) Cells shown in green are positive for the neutrophil marker myeloperoxidase. (B) Cells shown in green were positive for the T lymphocyte marker CD3, whereas those in red were positive for CD4. (C and D) Both CD3- and CD11b-staining cells were found within lesions. All sections were counterstained with DAPI (blue)

Figure 5.

Cx43 staining patterns were altered in myocytes adjacent to calcinotic lesions. Z planes are shown in myocytes distant from lesions (A through C) and adjacent to lesions (D through F). Cx43 staining was localized at intercalated disks and within the cytoplasm of myocytes adjacent to lesions.