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Extensive Induction of Important Mediators of Fibrosis and Dystrophic Calcification in Desmin-Deficient Cardiomyopathy

2002; Elsevier BV; Volume: 160; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)64916-4

1525-2191

Manolis Mavroidis, Yassemi Capetanaki,

Nuclear Structure and Function

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-β1 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. 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-β1 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. Ablation of desmin, the muscle-specific intermediate filament protein, by gene targeting in mice leads to transient cardiomyocyte hypertrophy and extensive cardiomyocyte death, followed by cardiac chamber dilation and heart failure.1Milner DJ Weitzer G Tran D Bradley A Capetanaki Y Disruption of muscle architecture and myocardial degeneration in mice lacking desmin.J Cell Biol. 1996; 134: 1255-1270Crossref PubMed Scopus (431) Google Scholar, 2Milner DJ Taffet GE Wang X Pham T Tamura T Hartley C Gerdes AM Capetanaki Y The absence of desmin leads to cardiomyocyte hypertrophy and cardiac dilation with compromised systolic function.J Mol Cell Cardiol. 1999; 31: 2063-2076Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 3Li Z Colucci-Guyon E Pincon-Raymond M Mericskay M Pournin S Paulin D Babinet C Cardiovascular lesions and skeletal myopathy in mice lacking desmin.Dev Biol. 1996; 175: 362-366Crossref PubMed Scopus (286) Google Scholar Similarly, missense mutations of desmin have been identified in humans suffering from idiopathic dilated cardiomyopathy4Li D Tapscoft T Gonzalez O Burch PE Quinones MA Zoghbi WA Hill R Bachinski LL Mann DL Roberts R Desmin mutation responsible for idiopathic dilated cardiomyopathy.Circulation. 1999; 100: 461-464Crossref PubMed Scopus (383) Google Scholar as well as other more generalized myopathies with both skeletal and cardiac dysfunction.5Munoz-Marmol AM Strasser G Isamat M Coulombe PA Yang Y Roca X Vela E Mate JL Coll J Fernandez-Figueras MT Navas-Palacios JJ Ariza A Fuchs E A dysfunctional desmin mutation in a patient with severe generalized myopathy.Proc Natl Acad Sci USA. 1998; 95: 11312-11317Crossref PubMed Scopus (227) Google Scholar, 6Goldfarb LG Park KY Cervenakova L Gorokhova S Lee HS Vasconcelos O Nagle JW Semino-Mora C Sivakumar K Dalakas MC Missense mutations in desmin associated with familial cardiac and skeletal myopathy.Nat Genet. 1998; 19: 402-403Crossref PubMed Scopus (437) Google Scholar, 7Sjoberg G Saavedra-Matiz CA Rosen DR Wijsman EM Borg K Horowitz SH Sejersen T A missense mutation in the desmin rod domain is associated with autosomal dominant distal myopathy, and exerts a dominant negative effect on filament formation.Hum Mol Genet. 1999; 8: 2191-2198Crossref PubMed Scopus (138) Google Scholar, 8Dalakas MC Park KY Semino-Mora C Lee HS Sivakumar K Goldfarb LG Desmin myopathy, a skeletal myopathy with cardiomyopathy caused by mutations in the desmin gene.N Engl J Med. 2000; 342: 770-780Crossref PubMed Scopus (384) Google Scholar, 9Capetanaki Y Desmin cytoskeleton in healthy and failing heart.Heart Failure Rev. 2000; 5: 203-220Crossref PubMed Scopus (20) Google Scholar The cellular and tissue pathology associated with cardiac dysfunction in desmin-null mice has been adequately addressed.1Milner DJ Weitzer G Tran D Bradley A Capetanaki Y Disruption of muscle architecture and myocardial degeneration in mice lacking desmin.J Cell Biol. 1996; 134: 1255-1270Crossref PubMed Scopus (431) Google Scholar, 3Li Z Colucci-Guyon E Pincon-Raymond M Mericskay M Pournin S Paulin D Babinet C Cardiovascular lesions and skeletal myopathy in mice lacking desmin.Dev Biol. 1996; 175: 362-366Crossref PubMed Scopus (286) Google Scholar, 10Thornell L Carlsson L Li Z Mericskay M Paulin D Null mutation in the desmin gene gives rise to a cardiomyopathy.J Mol Cell Cardiol. 1997; 29: 2107-2124Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 11Milner DJ Mavroidis M Weisleder N Capetanaki Y Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function.J Cell Biol. 2000; 150: 1283-1298Crossref PubMed Scopus (294) Google Scholar Briefly, mice lacking desmin demonstrate disruption of muscle architecture with mitochondrial abnormalities, including loss of normal positioning, extensive proliferation and clumping, as well as compromised respiratory function. These abnormalities are followed by myocardial degeneration with extensive fibrosis and dystrophic calcification. The molecular mechanisms underlining the development of these abnormalities are mainly unknown. The inappropriate biomineralization occurring in soft tissues is defined as ectopic calcification. In the absence of a systemic mineral imbalance ectopic calcification is typically called dystrophic calcification and is commonly observed in injury, disease, and aging.12Giachelli CM Ectopic calcification: gathering hard facts about soft tissue mineralization.Am J Pathol. 1999; 154: 671-675Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 13Donley GE Fitzpatrick LA Vascular calcification.Trends Cardiovasc Med. 1998; 8: 199-206Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Although it can occur in all soft tissues, cardiovascular tissues seem particularly prone to dystrophic calcification. In arteries, calcification is correlated with atherosclerotic plaques with the known clinical consequences. Age-related dystrophic calcification in the human cardiovascular system can contribute significantly to cardiac dysfunction and is perhaps more prevalent than ischemic heart disease.12Giachelli CM Ectopic calcification: gathering hard facts about soft tissue mineralization.Am J Pathol. 1999; 154: 671-675Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar Despite the high prevalence and clinical significance, very little mechanistic data exist mainly because of lack of animal models. Dystrophic calcification possesses several features of bone mineralization, including the presence of noncollagenous matrix proteins such as osteopontin, matrix Gla protein, osteocalcin, SPARC (osteonectin), and bone morphogenetic proteins, which all are thought to regulate also pathological calcification.12Giachelli CM Ectopic calcification: gathering hard facts about soft tissue mineralization.Am J Pathol. 1999; 154: 671-675Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 13Donley GE Fitzpatrick LA Vascular calcification.Trends Cardiovasc Med. 1998; 8: 199-206Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Indeed mice lacking matrix Gla protein, by gene targeting inactivation, have extensive calcification of arteries and valves,14Luo G Ducy P McKee MD Pinero GJ Loyer E Behringer RR Karsenty G Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein.Nature. 1997; 386: 78-81Crossref PubMed Scopus (1714) Google Scholar thus supporting the idea that this protein is indeed a natural inhibitor of mineralization. Similar results have been obtained for the osteoprotegerin gene, a member of the transforming growth factor (TGF) receptor superfamily, known to regulate osteoclast differentiation.15Bucay N Sarosi I Dunstan CR Morony S Tarpley J Capparelli C Scully S Tan HL Xu W Lacey DL Boyle WJ Simonet WS Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification.Genes Dev. 1998; 12: 1260-1268Crossref PubMed Scopus (2091) Google Scholar Although dystrophic calcification in all of the above cases is restricted to the vascular system, in desmin-null mice the cardiac muscle is the target tissue and can only be compared to dystrophic cardiac calcinosis. This is an age-related cardiomyopathy that occurs in certain inbred mouse strains that can also lead to congestive heart failure.16Eaton GJ Custer RP Johnson FN Stabenow KT Dystrophic cardiac calcinosis in mice: genetic, hormonal, and dietary influences.Am J Pathol. 1978; 90: 173-186PubMed Google Scholar, 17Rings RW Wagner JE Incidence of cardiac and other soft tissue mineralized lesions in DNA-2 mice.Lab Anim Sci. 1972; 22: 344-352PubMed Google Scholar There are also a few other cases in which mutations in sarcomeric proteins, among other cardiac abnormalities, demonstrated calcification but at minor levels.18Fatkin D Christe ME Aristizabal O McConnell BK Srinivasan S Schoen FJ Seidman CE Turnbull DH Seidman JG Neonatal cardiomyopathy in mice homozygous for the Arg403Gln mutation in the alpha cardiac myosin heavy chain gene.J Clin Invest. 1999; 103: 147-153Crossref PubMed Scopus (92) Google Scholar, 19McConnell BK Jones KA Fatkin D Arroyo LH Lee RT Aristizabal O Turnbull DH Georgakopoulos D Kass D Bond M Niimura H Schoen FJ Conner D Fischman DA Seidman CE Seidman JG Fischman DH Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice.J Clin Invest. 1999; 104: 1235-1244Crossref PubMed Scopus (197) Google Scholar The molecular mechanisms underlying ectopic calcium deposition at sites of inflammation and/or necrosis is a fundamental but poorly understood element of not only dystrophic cardiac calcinosis and desmin-null cardiomyopathy but for any tissue response to injury. Desmin-null mice could serve as a good model to unravel the molecular mechanisms of cardiovascular degeneration, calcification, and the development of heart failure in these animals. Because of the complexity of the observed pathology of desmin-null hearts, it is anticipated that alterations in multiple processes should be responsible for the development of the observed cardiomyopathy. To address this issue we analyzed general gene expression changes in cardiac tissue of the desmin-null mice, using differential display polymerase chain reaction (PCR) and cDNA atlas array methods. The most substantial changes were found for genes coding for extracellular matrix proteins and especially for the small matricellular proteins osteopontin and decorin.13Donley GE Fitzpatrick LA Vascular calcification.Trends Cardiovasc Med. 1998; 8: 199-206Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar We connect their action to the extended inflammatory reaction first observed between the second and third week of the animal's life because of pronounced cardiomyocyte death. These data, together with the observed up-regulation of TGF-β1 and angiotensin I-converting enzyme (ACE) could explain the extensive fibrosis and dystrophic calcification observed in the heart of desmin-null mice. The procedures for the care and treatment of animals were according to institutional guidelines. Mice lacking desmin were generated by gene targeting via homologous recombination as previously described.1Milner DJ Weitzer G Tran D Bradley A Capetanaki Y Disruption of muscle architecture and myocardial degeneration in mice lacking desmin.J Cell Biol. 1996; 134: 1255-1270Crossref PubMed Scopus (431) Google Scholar The mice used for this study were of the C57BL/6–129SV genetic background. Wild-type and desmin-null mice were anesthetized and blood-free hearts were pulverized into powder under liquid nitrogen. RNA was isolated usually from pools of 4 to 5 hearts using the Totally RNA isolation kit (Ambion, Austin, TX). PolyA RNA was isolated using oligo-dT cellulose (Ambion). The differential display PCR method was used essentially as described.20Consalez GG Cabibbo A Corradi A Alli C Sardella M Sitia R Fesce R A computer-driven approach to PCR-based differential screening, alternative to differential display.Bioinformatics. 1999; 15: 93-105Crossref PubMed Scopus (14) Google Scholar The mouse atlas 1.2 k (1185 genes) cDNA array (catalog no. 7853-1) analysis was performed by Clontech (Clontech, San Diego, CA), using pools of three hearts of 4-month-old desmin-null and wild-type animals. Northern blots were performed as previously described2Milner DJ Taffet GE Wang X Pham T Tamura T Hartley C Gerdes AM Capetanaki Y The absence of desmin leads to cardiomyocyte hypertrophy and cardiac dilation with compromised systolic function.J Mol Cell Cardiol. 1999; 31: 2063-2076Abstract Full Text PDF PubMed Scopus (140) Google Scholar using standard techniques. For the ACE probe we have isolated a 0.95-kb fragment of the mouse ACE cDNA (accession no., J04947; area, 2093 to 3044) by RT-PCR amplification and similarly for the TGF-β1 probe we have isolated a 1.54-kb fragment of the mouse cDNA (accession no., AJ009862; area, 414 to 1960). The mouse osteopontin cDNA was kindly provided by Dr. Larry Fisher, National Institute of Dental and Craniofacial Research, Bethesda MD.21Fisher LW Stubbs JT Young MF Antisera and cDNA probes to human and certain animal model bone matrix noncollagenous proteins.Acta Orthop Scand. 1995; 266: S61-S65Google Scholar For the osteopontin Western blot, pulverized tissue (same as in RNA isolation) was extracted either with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (2% SDS, 10% glycerol, 50 mmol/L Tris-HCl, pH 6.8) or with demineralizing buffer that contained all of the above plus 300 mmol/L ethylenediaminetetraacetic acid (EDTA). Bone extracts were prepared from the femur bone of 3-week-old animals. For the decorin Western blot, the pulverized tissue was extracted with guanidine buffer (6 mol/L guanidinine isothiocyanate, 50 mmol/L Tris-HCl, pH 7.3, 5 mmol/L EDTA). For the chondroitinase digestion the guanidine extracts were dialyzed against (50 mmol/L Tris-HCl, 30 mmol/L sodium acetate, pH 7.3, 5 mmol/L EDTA, 3 mmol/L phenylmethyl sulfonyl fluoride) and centrifuged at 13,000 × g for 20 minutes. The supernatant (∼5 μg of total protein) was incubated with 0.04 U of chondroitinase ABC from Sigma (catalog no. C3667; Sigma, St. Louis, MO) in a final volume of 40 μl of 0.5× phosphate-buffered saline (PBS) at 37°C for 3 hours. The samples were analyzed by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed for decorin and osteopontin using the antibodies LF-113 and LF-123, respectively, in a 1:1000 dilution. The antibodies were kindly provided by Dr. Larry Fisher.21Fisher LW Stubbs JT Young MF Antisera and cDNA probes to human and certain animal model bone matrix noncollagenous proteins.Acta Orthop Scand. 1995; 266: S61-S65Google Scholar Blood-free mouse hearts were immersed in OCT compound (Miles Inc., Torrance, CA) and frozen in liquid nitrogen. Frozen tissue sections (7-μm-thick) fixed with 4% paraformaldehyde in PBS were used for immunolabeling as previously described.1Milner DJ Weitzer G Tran D Bradley A Capetanaki Y Disruption of muscle architecture and myocardial degeneration in mice lacking desmin.J Cell Biol. 1996; 134: 1255-1270Crossref PubMed Scopus (431) Google Scholar The anti-decorin (LF-113), anti-osteopontin (LF-123), and anti-collagen-αI (LF-67) antibodies were kindly provided by Dr. Larry Fisher and the anti-laminin antibody was from Sigma (catalog no. L9393). All of the above polyclonal antibodies were used at a 1:300 dilution. The appropriate secondary antibodies (Alexaflour-594 and Alexaflour-488) were from Molecular Probes (Eugene, OR) and used in a 1:800 dilution. Routine histological analysis and hematoxylin-eosin staining was performed as previously described.1Milner DJ Weitzer G Tran D Bradley A Capetanaki Y Disruption of muscle architecture and myocardial degeneration in mice lacking desmin.J Cell Biol. 1996; 134: 1255-1270Crossref PubMed Scopus (431) Google Scholar For immunohistochemical analysis 5-μm-thick paraffin sections, from tissues fixed overnight in 2% paraformaldehyde solution in PBS, were used. The anti-decorin (LF-113) and anti-osteopontin (LF-123) antibodies were used in a 1:800 dilution. Reagents for the immunoperoxidase labeling were from DAKO (Carpinteria, CA). Substitution of primary antibodies by normal rabbit IgG was used as a negative control. Von Kossa staining for calcium detection was performed as described.22Rungby J Kassem M Eriksen EF Danscher G The von Kossa reaction for calcium deposits: silver lactate staining increases sensitivity and reduces background.Histochem J. 1993; 25: 446-451Crossref PubMed Scopus (75) Google Scholar To identify genes that are differentially expressed in the heart of desmin-null mice, a mouse cDNA array was screened with RNA isolated from hearts of 4-month-old wild-type and null animals. Nine percent of the 1185 cDNAs examined displayed at least a twofold difference between wild-type and null animals with 60% of the differentially expressed genes being up-regulated in the null heart. These differentially expressed cDNAs belong to several functional groups (data not shown), but the most substantial changes were found in genes coding for extracellular matrix proteins (Table 1).Table 1Induction of Fibrosis and Calcification Are Linked in the Heart of Desmin-Null MiceDes+Des−Fold XcDNAs14242.0Osteopontin precursor (OP); bone sialoprotein 18465.8Decorin; bone proteoglycan II (PG-S2)11171.5Bone morphogenetic protein 1 (BMP1); biglycan144.0Osteoblast-specific factor 2 (OSF-2)5173.4Fibronectin18553.1Angiotensin-converting enzyme (ACE)General gene expression profile was determined in the heart of desmin-null and wild-type animals, using the mouse 1.2 k (1185 genes) Atlas Array from Clontech Lab. RNA was isolated from 4-month-old, wild-type (Des+) and desmin-null (Des−) animals. Data from extracellular matrix/cell structure proteins are presented. The numbers represent arbitrary units. Open table in a new tab General gene expression profile was determined in the heart of desmin-null and wild-type animals, using the mouse 1.2 k (1185 genes) Atlas Array from Clontech Lab. RNA was isolated from 4-month-old, wild-type (Des+) and desmin-null (Des−) animals. Data from extracellular matrix/cell structure proteins are presented. The numbers represent arbitrary units. Osteopontin RNA, was markedly induced (42-fold) in the heart of desmin-null animals (Table 1). The cDNA array screen results were confirmed by Northern blot analysis (Figure 1), which indeed revealed a dramatic induction of osteopontin RNA in desmin-null hearts. The expression of osteopontin RNA in the heart of wild-type animal is undetectable, even for longer (5 days) exposures. Further analysis of the expression profile of osteopontin RNA at various ages shows maximum induction around 3 weeks of age and then a decline as animals age (compare 6- to 12-month-old animals in Figure 1). In contrast, no detectable expression of osteopontin RNA was observed in skeletal muscle (gastrocnemius) of both desmin-null and wild-type animals (data not shown). The second highest increase (5.8-fold) in the heart of desmin-null animals revealed by the array screen (Table 1) was in the expression of decorin RNA that codes for a proteoglycan, another small extracellular matrix protein.23Iozzo RV Matrix proteoglycans: from molecular design to cellular function.Annu Rev Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1327) Google Scholar The up-regulation of decorin was initially observed by differential display PCR in which a fragment identical to the 1337- to 1763-bp region of the mouse decorin cDNA (accession no. X53928) was isolated. Differential expression of the decorin mRNA in desmin-null heart was also confirmed by Northern blot analysis (Figure 2) using the above fragment as a probe. The induction of the decorin mRNA in the heart of desmin-null animals is approximately threefold compared to the wild-type. This level of decorin induction could be observed as early as 9 days after birth and remains constant at least up to the age of 13 months. Another gene linked to fibrosis, ACE, a key regulator of the renin-angiotensin system24Paradis P Dali-Youcef N Paradis FW Thibault G Nemer M Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling.Proc Natl Acad Sci USA. 2000; 97: 931-936Crossref PubMed Scopus (309) Google Scholar was induced (3.1-fold, Table 1) in desmin-null hearts. ACE induction was also confirmed by Northern blot analysis (Figure 2). The two ACE messages observed, sizes 4.9 and 4.2 kb, are commonly found in Northern blots of somatic tissues.25Bernstein KE Martin BM Edwards AS Bernstein EA Mouse angiotensin-converting enzyme is a protein composed of two homologous domains.J Biol Chem. 1989; 264: 11945-11951Abstract Full Text PDF PubMed Google Scholar Given the established connection between decorin, TGF-β1, and fibrosis26Isaka Y Brees DK Ikegaya K Kaneda Y Imai E Noble NA Border WA Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney.Nat Med. 1996; 2: 418-423Crossref PubMed Scopus (457) Google Scholar, 27Yamaguchi Y Mann DM Ruoslahti E Negative regulation of transforming growth factor-beta by the proteoglycan decorin.Nature. 1990; 346: 281-284Crossref PubMed Scopus (1282) Google Scholar we wanted to see how the expression of TGF-β1 is modulated in the desmin-null heart. Although the cDNA array screen did not reveal any difference, Northern blot analysis did show increase in the expression of TGF-β1 RNA in desmin-null hearts (Figure 2). The increase was ∼2.5-fold in 3-week-old and 4-month-old animals. To further examine whether the observed increase in osteopontin and decorin RNA levels was accompanied by similar changes at the protein level, we performed Western blot analysis. Murine osteopontin has a predicted molecular weight of 35 kd. However, in SDS-PAGE it shows anomalous migration with an apparent molecular weight of 45 to 75 kd,28Rittling SR Feng F Detection of mouse osteopontin by Western blotting.Biochem Biophys Res Commun. 1998; 250: 287-292Crossref PubMed Scopus (42) Google Scholar because of posttranslational modifications, depending on the tissue of origin. Western blot analysis of cardiac extracts from 4-month-old desmin-null animals using anti-osteopontin antibody, showed the presence of high amounts of the protein in a high-molecular weight complex form (Figure 3A). This complex is mainly retained in the stacking gel and has an apparent molecular weight ranging from ∼130 to >200 kd. Even after treatment of the samples with demineralizing buffer for 12 hours, the complex remains the same indicating that osteopontin most possibly is in a stable polymeric form.29Kaartinen MT Pirhonen A Linnala-Kankkunen A Maenpaa PH Transglutaminase-catalyzed cross-linking of osteopontin is inhibited by osteocalcin.J Biol Chem. 1997; 272: 22736-22741Crossref PubMed Scopus (71) Google Scholar Extracts from wild-type hearts were negative for osteopontin protein. Two minor bands of ∼40- to 50-kd molecular weight were also detected in both wild-type and desmin-null cardiac extracts at similar intensity. These could result from either cross-reactivity of the antibody that has been occasionally seen in preparations of different tissues28Rittling SR Feng F Detection of mouse osteopontin by Western blotting.Biochem Biophys Res Commun. 1998; 250: 287-292Crossref PubMed Scopus (42) Google Scholar or could be basic levels of nonmodified or degraded osteopontin (the antibody used recognizes the carboxy half of the molecule). Bone extracts were used as a positive control revealing a major band of ∼65-kd molecular weight and a diffused high-molecular weight band, remained on the top of the gel (Figure 3A) as in the case of desmin-null heart extracts. Western blot analysis revealed that decorin was also significantly increased (approximately threefold) in the heart of desmin-null animal when compared to the wild type (Figure 3B). Heart extracts from 4-week-old, 4-month-old, and 8-month-old animals gave similar results (only data from 4-month-old animals are shown). In SDS-PAGE analysis of cardiac extracts, decorin runs as a diffused band with an apparent molecular weight of 85 to 105 kd. After digestion with chondroitinase ABC, decorin migrates at ∼48 kd (Figure 3B), indicating the existence of a glycosaminoglycan chain as expected. The predicted molecular weight of the core protein is ∼38 kd and usually is modified with one glycosaminoglycan chain and two or three N-linked oligosaccharides.23Iozzo RV Matrix proteoglycans: from molecular design to cellular function.Annu Rev Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1327) Google Scholar Analysis of protein extracts from skeletal muscle (tongue and gastrocnemius) did not show any obvious differences in decorin expression between wild-type and desmin-null animals (data not shown). Immunohistochemical and immunofluorescence analysis was performed to determine the relationship in the spatial and temporal distribution of the different matrix proteins and the pattern of calcium deposits. Extensive osteopontin staining was first observed at the age of 3 weeks in the right ventricle of desmin-null animals (Figure 4, A and B) in areas with pronounced myocyte death, acute inflammatory infiltrate, and calcium precipitation with a gritty appearance (Figure 4, C and D). With the progression of the pathology the extensive myocyte death leads to further tissue remodeling with replacement of cardiomyocytes with fibrosis and accumulation of calcium precipitates in psammoma body structures (Figure 5). These structures of concentric calcium laminations were positive for osteopontin staining, and were observed very frequently. In the right ventricle degeneration and calcification can reach up to 80% of the myocardium thickness. In some cases, after work overload, the damage is so extensive that it leads to rapture of the cardiac wall.9Capetanaki Y Desmin cytoskeleton in healthy and failing heart.Heart Failure Rev. 2000; 5: 203-220Crossref PubMed Scopus (20) Google Scholar, 11Milner DJ Mavroidis M Weisleder N Capetanaki Y Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function.J Cell Biol. 2000; 150: 1283-1298Crossref PubMed Scopus (294) Google Scholar Various degrees of osteopontin staining and calcium precipitation could be also detected in areas with features of chronic inflammation (Figure 6), such as presence of lymphocytes, macrophages, and fibroblast-like cells. These areas could be found spontaneously in different regions of the cardiac tissue, such as the ventricles (Figure 6A), the interventricular septum (Figure 6C), and the papillary muscles. Immunostaining of cardiac tissue sections of wild-type animals for osteopontin were negative in all corresponding cases checked (not shown). In all cases studied, osteopontin seemed to co-localize with calcium deposits (Figure 4, Figure 5, Figure 6), suggesting that this protein plays a crucial role during calcification. Comparison of osteopontin and decorin localization revealed that decorin does not co-localize with osteopontin inside calcified areas, but it is abundant in the immediate surrounding fibrotic region (Figure 5C).Figure 5Immunohistochemical 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 out