Extensive induction of important mediators of fibrosis and dystrophic calcification in desmin-deficient cardiomyopathy

Abstract

Mice lacking the intermediate filament protein desmin demonstrate abnormal mitochondria behavior, disruption of muscle architecture, and myocardial degeneration with extensive calcium deposits and fibrosis. These abnormalities are associated with cardiomyocyte hypertrophy, cardiac chamber dilation and eventually with heart failure. In an effort to elucidate the molecular mechanisms leading to the observed pathogenesis, we have analyzed gene expression changes in cardiac tissue using differential display polymerase chain reaction and cDNA atlas array methods. The most substantial changes were found in genes coding the small extracellular matrix proteins osteopontin and decorin that are dramatically induced in the desmin-null myocardium. We further analyzed their expression pattern both at the RNA and protein levels and we compared their spatial expression with the onset of calcification. Extensive osteopontin localization is observed by immunohistochemistry in the desmin-null myocardium in areas with massive myocyte death, as well as in hypercellular regions with variable degrees of calcification and fibrosis. Osteopontin is consistently co-localized with calcified deposits, which progressively are transformed to psammoma bodies surrounded by decorin, especially in the right ventricle. These data together with the observed up-regulation of transforming growth factor-beta1 and angiotensin-converting enzyme, could explain the extensive fibrosis and dystrophic calcification observed in the heart of desmin-null mice, potentially crucial events leading to heart failure.

Figure 1.

Osteopontin expression is dramatically induced in the heart of desmin-null animals. Expression profile of osteopontin mRNA in the heart of wild-type (+/+) and desmin-null (−/−) mice by Northern blot, reveals that osteopontin is induced early in the animal’s life (21 days) and declines as the animal ages. The expression of osteopontin mRNA was undetectable in wild-type animals. D, days; Mo, months. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control (exposure, 12 hours).

Figure 2.

Decorin, ACE, and TGF-β1 mRNAs are up-regulated in the heart of desmin-null mice. mRNA was isolated from the hearts of 2.5-month-old wild-type (+/+) and desmin-null (−/−) animals and analyzed by Northern blot using the corresponding cDNA probes. Shown are representative results for each mRNA. GAPDH was used as a loading control (exposure, 3 days).

Figure 3.

Expression of osteopontin and decorin proteins is elevated in the heart of desmin-null (−/−) animals compared to wild-type (+/+). Heart extracts from 4-month-old animals were electrophoresed in a 9% SDS-PAGE and immunoblotted for osteopontin (A) and decorin (B). Osteopontin could be detected only in extracts from desmin-null heart as a high-molecular weight complex trapped in the stacking gel. Bone extracts were used as a positive control. For the decorin immunodetection, heart extracts were digested with (ABC+) chondroitinase ABC or not (ABC−) before analysis by SDS-PAGE. Note that in the undigested extracts decorin runs as a diffused band centering ∼90 kd and is significantly up-regulated in the hearts of desmin-null animals. After chondroitinase ABC digestion the proteoglycan is converted to an ∼48-kd band clearly overexpressed in the desmin-null animal whereas in the wild-type only a faint band is observed.

Figure 4.

Extensive osteopontin localization is observed in the myocardium of desmin-null animals, in areas with acute inflammatory infiltrate, myocyte degeneration, beginning of calcium precipitation, and coagulative necrosis. A and B (higher magnification of A): Immunohistochemical localization of osteopontin in the right ventricle of a 3-week-old desmin-null animal. Wild-type animals were negative for osteopontin staining, (not shown). D: Von Kossa staining for calcium deposition indicates the beginning of calcium precipitation on the degenerating myocytes. C: H&E (Hem./Eos.) staining, indicating more clearly the infiltrating neutrophils-polymorphs, the degenerating myocytes, and the edema, typical characteristics of acute inflammatory reaction. E: Extended coagulative necrosis is more clearly observed a few days after the initial acute inflammatory infiltration. Degrading myocytes, necrotic cells, debris, and edema fluid are observed by H&E staining of a corresponding area in a desmin-null animal 5 days older than the one described in A–D. F: Negative control for the immunohistochemistry. Unrelated rabbit IgG was used as primary antibody. No staining is observed. A–D and F are from adjacent sections of a 3-week-old heart. For the immunoperoxidase staining (A, B, and F), diaminobenzidine was used as substrate and hematoxylin for nuclei counterstaining. Von Kossa staining for calcium deposition (D) is brown/black and counterstaining for nuclei by Kernechtrot is red.

Figure 5.

Immunohistochemical localization of osteopontin and decorin in advanced stage calcification. Osteopontin is detected in desmin-null animals in necrotic areas with extensive calcification and psammoma body morphology (A and B). These structures are large mineralized deposits with lamellated configurations surrounded by decorin-containing fibrotic tissue (C). A, B, and C are from serial sections in the outer surface of the right ventricle of a 6-month-old animal. Immunohistochemistry staining for osteopontin and decorin (A and C) and von Kossa staining (B) is same as in Figure 4 ▶ .

Figure 6.

Osteopontin co-localizes with calcium in areas with chronic inflammation. Immunohistochemical localization of osteopontin (arrowheads) in a hypercellular area of infiltrate in the right ventricle of desmin-null animal (A) together with mild granular calcium deposits as indicated by a serial section stained by von Kossa (B, arrows). Osteopontin (arrowheads) is also detected in calcified regions of other parts of the myocardium such as the septum (C). A and B are from a 6-month-old animal and C from a 2-month-old animal. Staining procedure is same as in Figure 4 ▶ .

Figure 7.

Abnormal accumulation of decorin and collagen but not laminin in the myocardium of desmin-null animal. Immunofluorescence localization of decorin, collagen, and laminin was performed in cardiac tissue from wild-type (A, C, and E) and desmin-null (B, D, and F) animals. Extensive decorin staining could be seen in fibrotic areas (arrows) of desmin-null-only myocardium (B) but not in wild-type animal (A), where only staining of the endomysium (arrowheads) could be detected. B, D, and F: Staining of serial sections of desmin-null myocardium, for decorin, collagen, and laminin. Note that laminin shows normal pattern and is not a component of the fibrotic areas (compare with the wild-type in E). Decorin (B) has a similar staining pattern as collagen (D) and co-localizes with it in fibrotic areas of desmin-null myocardium. Asterisks in B, D, and F indicate corresponding spots in serial sections.