Fig 1.
Increased echogenic foci in CVB3 infected mice.
(A) Two-dimensional parasternal long axis echocardiograms in infected and uninfected mice. In uninfected mice (top panels), the left ventricular myocardium has a relatively homogenous echodensity. However, in infected DysWT mice (lower left panel) there are foci of increased echogenicity scattered throughout the ventricular myocardium. In infected mice in which the dystrophin-gene is mutated to prevent cleavage by CVB3 protease 2A, (lower right panel) the echogenic areas were less than in the infected WT-mice. (S1–S4 Videos are from the same echocardiograms.) (B) Using semi-quantitative grading of the echogenic areas (Echogenicity scored from 0–4) there was a significant increase in the infected hearts when compared to the uninfected hearts. Furthermore, the extent of echogenicity was lower in infected DysKI mice than the infected DysWT mice (N = 43 total mice, data is mean+/- s.e. ** p<0.01 ***p<0.001).
Table 1.
Echocardiographic functional analysis of the left ventricle.
Data shown from mice 8 days post-infection. Values in both infected (DysWT n = 12, DysKI n = 14) and uninfected (DysWT n = 9, DysKI n = 9) mice are considered within a normal functional range. There were no statistically significant differences between groups. Data shown as mean ± SD.
Fig 2.
Correlation of extent of echogenicity with histologic markers of myocarditis.
(A) von Kossa (left panels), hematoxylin and eosin (center panels), and picrosirius red (right panels) stains are shown from a representative uninfected and infected wild type mouse heart. In each case, abnormal staining was only present in infected mouse hearts. (B) The extent of von Kossa staining was quantitated as a percent of the total myocardial area that was von Kossa positive. The inflammation score was determined using a semi-quantitative scoring system (0–4) as previously described (12). Extent of fibrosis, as determined by picrosirius red staining, is expressed as a percent of total myocardial area. As shown in panel B, there was a significant correlation between extent of myocardial echogenicity and area of von Kossa staining, inflammation score, and fibrosis.
Fig 3.
Increased radiodensity by microCT in CVB3 infected mouse hearts.
(A) MicroCT images obtained from uninfected DysWT and DysKI are shown in the upper panels. MicroCT images from infected DysWT and DysKI are in the lower panels. The white areas are the areas of increased radiodensity. (B) Tomographic images of hearts from infected DysWT and DysKI and an uninfected DysWT mice were visualized with a background stain that is visible on microCT (white) to identify the cardiac structures. The areas of radiodensity are superimposed in blue in these images. (C) MicroCT images localize the radiodense areas in the heart of DysWT (top) and DysKI (bottom) infected hearts. Background staining alone is shown in the left images. The radiodense foci are shown in the middle images (blue), and the merged images are shown on the right. (D) Zoomed image of the three-dimensional reconstruction of the microCT of the infected DysKI heart from the middle of the lower panel in C demonstrates that areas of radiodensity can be detected when as small as the 100 x 40μm as represented by the red bar (arrow). 1 mm scale bars are shown in the bottom right of each image.
Table 2.
Volume measurements of radiodense foci.
Table demonstrates the volume of the heart tissue compared to the volume of radiodense foci and the percent of the volume of the heart tissue that contains radiodense foci in the infected and uninfected DysWT and DysKI hearts shown in the figures. Each measurement is from a single heart.