Ultrastructural Adaptation of the Cardiomyocyte to Chronic Mitral Regurgitation

Figures

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  Figure 1. Representative images of the whole heart and mid-left ventricular cross-sectional morphology from H&E staining in the sham, MR 2-week, and MR 10-week groups. Left, a two- or four-chamber longitudinal view of the heart in the experimental groups. Right, a cross-sectional view of the midventricular level. Wall thickness in the sham group appears to be less than the MR 2- and MR 10-week groups.

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  Figure 2. (A) Representative cross-sectional view of the midventricular level of the entire rat heart on gomori elastin trichrome staining. (B) Representative ×20 image of longitudinally aligned fibers from the from the sham group. (C) MR 2-week group; and (D) MR 10-week groups. Intersitial collagen between the fibers are shown in blue in each group, and the density of the fibers appears to be increased in the MR 2- and MR 10-week groups compared with sham.

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  Figure 3. Cross-sectional view of the cardiomyocytes on H&E staining in the three samples from the (A) sham, (B) MR 2-week, and (C) MR 10-week groups. (D) Average cardiomyocyte cross-sectional area (CSA) quantified from the H&E staining in each group using ImageJ. Data are represented as mean ± 1 standard deviation, and p-values <0.05 were considered statistically significant. Both the MR 2- and 10-week group had a significantly higher cardiomyocyte CSA compared with sham (p < 0.0001).

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  Figure 4. Immunohistochemistry of the intracellular cytoskeleton protein, desmin, in sham (A) and MR hearts (B,C) at ×63 magnification. Staining of desmin alone is shown in the left column for each group; DAPI staining is shown in the middle column; merged staining is shown in the right column. In the sham group, desmin is regularly distributed throughout the myocytes and concentrated at the Z-discs. In the MR 2- and 10-week group, desmin staining is irregular and focally concentrated at the intercalated disc region and not the Z-discs.

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  Figure 5. Transmission electron microscopy images detailing the sarcomere structure, intracellular organelles, and their spatial organization. (A1–A3) Sarcomere structure from three different rats, depicting an organized sarcomeric network, with clearly visible and condensed Z-line, I-band, and A-band. A linear registry of mitochondria that are interspersed parallel to the myofibrils and adjacent to the sarcomeres are evident. (B1–B3) After 2 weeks of mitral regurgitation, ultrastructural changes are clearly evident. The I-band was not evident, and the density of the Z-band was reduced. The mitochondria interspersed between the sarcomeres were fragmented and disorganized. (C1–C3) At 20 weeks of mitral regurgitation, damage to the sarcomeres was evident.

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  Figure 6. (A) Sarcomere length measured from TEM images of the sham, MR 2-week, and MR 20-week groups. (B) Cardiomyocyte length measured from immunohistochemistry images from the sham, MR 2-week, and MR 20-week groups. Data are represented as mean ± standard deviation. p < 0.05 represent statistical significance.

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  Figure 7. Transmission electron microscopy images detailing the mitochondrial organization in each rat left ventricle from the sham, MR 2-week, and MR 20-week groups. Representative images from three individual samples from different rats are shown in the sham group (A1–A3), MR 2-week (B1–B3), and MR 20-week group (C1–C3). The nucleus (N) and mitochondria (M) is labeled in each image.

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  Figure 8. Transmission electron microscopy images detailing the presence and ultrastructure of vacuoles in each rat left ventricle from the sham (A), MR 2-week (B), and MR 20-week groups (C). The vacuoles are depicted by yellow arrows in images. Sham rats had fewer vacuoles present within the myocardium, and when visualized, vacuoles were small in size and were located near the Z-disc. In the MR samples at 2 and 20 weeks, there was a higher presence of vacuoles of increased size which were filled with electron-dense material. Vacuoles were present near mitochondria, Z-discs, and the intercalated disc region. (D) Quantitative analysis of relative vacuole size measured using ImageJ. Data are represented as median with interquartile range (IQR: 25–75%). The non-parametric data were compared using the Kruskal-Wallis test. p < 0.05 was considered statistically significant.

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  Figure 9. Transmission electron microscopy images detailing focal areas of myocytolysis and fragmentation (A) and glycogen accumulation (B) in two MR 2-week samples. Myocytoloysis and fragmentation of the sarcomeres were observed in some areas of the sample, which are depicted in the yellow rectangle. Some areas also showed significant accumulation of glycogen granules between the sarcomeres and within the mitochondria, which are depicted by the red asterisk.