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(Circulation. 1995;92:705-709.)
© 1995 American Heart Association, Inc.

Articles

Osteopontin Expression Is Increased in the Heritable Cardiomyopathy of Syrian Hamsters

Errol B. Williams, MD; Igor Halpert, MD; Samuel Wickline, MD; Glenn Davison, MD; William C. Parks, PhD; Jeffrey N. Rottman, MD

From the Department of Internal Medicine, Divisions of Cardiology and Dermatology (W.C.P.), Washington University School of Medicine, St Louis, Mo.

Correspondence to Jeff Rottman, MD, Cardiology Division, Jewish Hospital at Washington University Medical Center, 216 S Kingshighway, St Louis, MO 63110.

	Abstract

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Background Osteopontin, a noncollagenous matrix protein, is transiently expressed in the heart after experimental cardiac injury, but its expression in states of continuing cardiac remodeling is unknown. We evaluated osteopontin expression in the heritable cardiomyopathy of the Syrian hamster.

Methods and Results Hamster hearts were obtained for RNA isolation and analysis and in situ hybridization from two groups: normal control animals (n=4) and untreated cardiomyopathic hamsters (n=5). Osteopontin mRNA was 12-fold greater in cardiomyopathic hearts compared with normal controls (1.76±0.31 versus 0.14±0.04 arbitrary units normalized to GAPDH, mean±SEM, P<.05). In situ hybridization was used to define the origin of osteopontin in the heart. Osteopontin mRNA above background levels was not detected in sections from noncardiomyopathic hamster hearts but was readily detected in sections from cardiomyopathic hamsters, in which it originated in cells morphologically consistent with tissue macrophages.

Conclusions In the hamster, osteopontin is expressed in heritably cardiomyopathic hearts under conditions of chronic injury and repair, and the source of osteopontin message appears to be tissue macrophage-like cells in foci of inflammation. This model could be used to evaluate the biological role of osteopontin in myocardial inflammation and remodeling.

Key Words: osteopontin • proteins • cardiomyopathy • RNA

	Introduction

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The inbred Syrian hamster, Mesocricetus auratus auratus, develops a hereditary cardiomyopathy marked by diffuse degenerative changes consisting predominantly of myofibrillar calcinosis and early myocytolysis.¹ This condition, transmitted by an autosomal recessive gene, ultimately results in death through congestive heart failure and, as such, has served as a model for physiological and pharmacological studies of cardiomyopathy.² Previous studies have demonstrated that the process of cardiac necrosis in this hamster model is associated with elevated levels of heart calcium that can be mitigated by administration of taurine (2-aminoethane sulfonic acid). This effect may occur at the level of the sarcolemmal membrane by prevention of excessive calcium accumulation.^{3 4 5} Verapamil, cinnarizine, and certain other pharmacological agents that function as calcium antagonists have been shown to have a similar influence.¹

Calcium-mediated necrosis of vascular tissue has been well documented in atherosclerosis. Osteopontin (OPN),a noncollagenous matrix protein, is highly expressed in atherosclerotic lesions.⁶ This finding has prompted investigation of the cardiovascular role of this protein, which is known to be central to the process of physiological and possibly pathological biomineralization in other tissues.⁷ OPN is a highly acidic secreted phosphoglycoprotein. Although it was first described in bone, subsequent studies have implicated it in the processes of cell adhesion, cell migration, and signal transduction in nonosseous tissues, including kidney,⁸ epithelial surfaces,⁹ and vessel wall.^{6 10 11} Many of the biological effects of OPN appear to be mediated by binding of a tripeptide arginine-glycine aspartate (RGD) sequence to cell-surface integrin receptors.^{11 12 13}

Recently, OPN expression was examined after experimental cardiac injury in rats and in human myocardial infarction.¹⁴ OPN is synthesized by macrophages as part of a generalized reaction to tissue injury; however, levels of OPN in myocardial lesions decreased dramatically over a period of weeks as healing progressed.¹⁴ The expression of OPN in conditions of chronic cardiac injury has not been studied previously. We show that OPN expression is increased in Syrian hamster cardiomyopathy, which involves a continuing process of injury and repair. In situ hybridization localizes the sites of expression to foci of acute inflammatory infiltrates. These data suggest that the Syrian hamster cardiomyopathy may serve as a valuable model system in which to study the regulation and consequences of OPN expression in cardiac remodeling and repair.

	Methods

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Animal Preparations
Nine 1-month-old male Syrian hamsters (cardiomyopathic and normal) were divided into two groups: five untreated cardiomyopathic hamsters (TO-2 strain, Biobreeders, Inc) and four normal control hamsters (F1-B strain, Biobreeders, Inc). Animals were housed three to a cage in HEPA-filtered "stay-clean" units with a 12-hour light/dark cycle and had free access to laboratory rodent chow (Purina). All animals received fresh drinking water daily. No animals were lost to morbidity or mortality. At 16 weeks, hamsters were anesthetized with methoxyflurane and then euthanatized with sodium pentobarbital 120 mg/kg IP (Abbot Laboratories). Hearts were quickly excised, the apexes were snap-frozen in liquid nitrogen for subsequent RNA preparation, and the remaining portions were placed in 10% buffered formalin for in situ hybridization. The study protocol was approved by the Animal Care Committee of the Jewish Hospital at Washington University Medical Center.

RNA Isolation and Analysis
Total RNA was prepared from frozen tissue by the guanidinium–acid phenol method (RNAzol B, Test-Test, Inc). Total RNA (10 µg) was resolved on a 1% formaldehyde-agarose gel and then transferred and cross-linked to MagnaGraph membranes (Micro Separations Inc) according to the manufacturer's instructions. The OPN probe, corresponding to the 1.1-kb BamHI fragment from the plasmid pGEM4-2arcDNA (gift of Dr Marian F. Young, Bone Research Branch, NIH, Bethesda, Md), was labeled with [³²P]dCTP, random hexamers, and the Klenow fragment of DNA polymerase I. This cDNA fragment contains most of the coding sequence of the murine OPN gene. The exact sequence of the hamster OPN gene is unknown, but comparison of OPN cDNA nucleotide sequences from a number of other species (including human, rat, mouse, cow, and chicken) demonstrated high conservation within this fragment.¹⁵ Blots were prehybridized and hybridized overnight at 42°C in 50% formamide, 5x Denhardt's solution, 4x SSPE, 2.5 µg/mL herring testis DNA, and 1% SDS. The highest-stringency wash was at 50°C in 0.1x SSPE/1% SDS, followed by autoradiography at -80°C overnight with Kodak XAR 5 film. The relative intensities of the resultant bands were quantified in their linear range by computer densitometry. To assess potential variations in gel loading and transfer, the blots were then reprobed for GAPDH mRNA under the same conditions.

Once the quality of the RNA and the specificity of the probe were confirmed on Northern blotting, a Minifold II slot-blot system (Schleicher & Schuell) was used to create multiple blots for purposes of quantification. Yeast tRNA was mixed with total sample RNA to produce a 4x range of sample concentrations. After the sample was applied to the slot-blot apparatus, hybridization, wash, and analysis were performed as described above. Data were compiled and analyzed by the Statistical Analysis System procedures (SAS Institute). ANOVA was used to test for significant differences among treatments, and means were separated with Tukey's studentized range test (P<.05).

In Situ Hybridization
The mouse OPN probe was a full-length antisense transcript prepared by linearizing pGEM4-2arcDNA (as above) at the Bgl II site. A 500-nucleotide sense RNA probe transcribed from a bovine tropoelastin cDNA was used as a control for nonspecific hybridization. The validity of this probe as a negative control has been confirmed previously.^{16 17} The riboprobes were labeled with ³²S-UTP (>1200 Ci/mmol; NEN) by use of Promega reagents. The manufacturer's instructions were modified as follows to increase the specific activity of the probes¹⁷ : 150 µCi of -³⁵S-UTP was dried under vacuum with gentle centrifugation to increase the effective concentration. Transcription reagents, 5 U of the appropriate RNA polymerase, and 1 µg of linearized DNA template were added to a reaction volume of 20 µL and incubated at 37°C for 1 hour. An additional 5 U of polymerase was then added, and the incubation was continued for another 3 hours. These modifications typically resulted in a 5- to 10-fold increase in the specific activity of the RNA probes. Specificity of the mouse riboprobe was demonstrated with Northern blotting under conditions similar to those used for in situ hybridization. Heart samples were fixed in formalin and embedded in paraffin, and normal bone was processed as a positive control. Five-micrometer sections were treated with 1 µg/mL nuclease-free proteinase K (Sigma Chemical Co), washed in freshly prepared 0.1 mol/L triethanolamine buffer containing 0.25% acetic anhydride, and then covered with hybridization buffer (50% formamide, 300 mmol/L NaCl, 30 mmol/L sodium citrate, pH 7.0, 1x Denhardt's solution, 1 mmol/L EDTA, 10% dextran sulfate, 100 mmol/L DTT, 0.5 mg/mL yeast tRNA) containing 2.5x10⁴ cpm/µL of ³⁵S-labeled riboprobe. After hybridization for 18 hours at 55°C, nonspecific background was reduced by incubation with 20 µg/mL RNase A (Sigma) in 0.5 mol/L NaCl, 10 mmol/L Tris, pH 8.0, and 1 mmol/L EDTA, followed by washes at increasing stringency up to 0.1x SSC at 60°C. Slides were then dipped in Kodak NTB-2 emulsion and processed for autoradiography. After a 21-day exposure, the emulsion was developed, and the slides were stained with hematoxylin-eosin. Areas of calcification were demonstrated with von Kossa or alizarin staining.¹⁸ To quantify calcification, photomicrographs were digitized with a Javelin CCD camera (Javelin Electronics) and a NuVista Framebuffer (Truevision, Inc) and analyzed with NIH Image 1.47 as previously described.¹⁹

	Results

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The heart walls of cardiomyopathic hamsters were thicker than those of the F1-B controls (2.6±0.14 mm, n=5, versus 1.6±0.22 mm, n=4, mean±SEM, P<.05). Body weights of the cardiomyopathic hamsters were less than those of the controls, but the ratio of heart weight to body weight was increased (0.36±0.01 versus 0.32±0.01, P<.05). Diffuse areas of infiltrative fibrous scar were macroscopically visible on sections of the ventricles of cardiomyopathic hamsters. These findings are consistent with our previous observations.¹⁹

A single band of 1.8 kb, the approximate size of the OPN mRNA in human, rat, mouse, and cow,¹⁵ was consistently observed in the RNA samples from cardiomyopathic hearts (Fig 1a, lane 2). A band of the same size was observed with hamster RNA from kidney and bone, two tissues known to express OPN, but not in RNA from spleen or liver, two nonexpressing tissues (data not shown). A corresponding band in RNA prepared from a nonmyopathic hamster heart was only faintly visible at the same exposures (Fig 1a, lane 1). Slot blotting was used to assess differences in OPN mRNA levels more quantitatively. Cardiomyopathic hamster hearts consistently exhibited higher levels of OPN mRNA than did control hamster hearts: when normalized to GAPDH, there was 12-fold more OPN mRNA in cardiomyopathic hamster hearts than in the normal control hearts (Fig 1b, 1.76±0.31 versus 0.14±0.04 normalized units, P<.05).

View larger version (18K): [in this window] [in a new window]   Figure 1. a, Northern blots of normal (NM) and cardiomyopathic (CM) hamster hearts, probed with osteopontin (OPN) (upper) and GAPDH (lower). The OPN mRNA is 1.8 kb in size. b, Quantification of OPN mRNA relative to GAPDH mRNA; error bars represent SEM. The difference between the levels of OPN mRNA in normal and cardiomyopathic hearts was significant at the P=.05 level.

We performed in situ hybridization to determine the distribution of OPN mRNA in the heart. Signal for OPN mRNA was not detected (0 of 3) in the hearts of noncardiomyopathic hamsters (Fig 2a and 2b). In contrast, OPN mRNA was readily detected in all (4 of 4) sections from cardiomyopathic hamsters (Fig 2c and 2d). Grains were localized exclusively to regions of inflammatory infiltrate within zones of fibrous scarring (Fig 3A and 3C). Not all areas of inflammatory infiltrate demonstrated OPN expression, but the areas that did were frequently more hypercellular than those that did not (see Fig 3A). Almost all areas of infiltrate included or abutted patches of calcification, and more exuberant calcification was not reliably associated with either the presence or absence of OPN mRNA signal (see Fig 3A and 3B). Regression analysis disclosed no relation between the level of OPN mRNA and the cross-sectional extent of calcification (data not shown). The morphology of the positive cells (Fig 3D and 3E) was consistent with that of tissue macrophages.

View larger version (0K): [in this window] [in a new window]   Figure 2. In situ hybridization of sections of normal (a and b) and cardiomyopathic (c and d) hamster heart, with an antisense osteopontin (OPN) probe, on hematoxylin-eosin staining (a and c) and dark-field microscopy (b and d). Intense OPN signal is observed in the area of inflammatory infiltrate. Arrows indicate representative cells, morphologically consistent with macrophages, expressing high levels of OPN. a through d, bar=2.5 µm.

View larger version (0K): [in this window] [in a new window]   Figure 3. Overview of histology, osteopontin (OPN) mRNA in situ signal, and calcification in a section from a cardiomyopathic hamster heart. Discrete areas of inflammatory infiltrate, identified by arrows, are apparent on hematoxylin-eosin staining (A; bar=25 µm). These areas are generally associated with calcification, which is red on alizarin stain (B, same scale as A). In this section only one inflammatory focus, indicated by the heavy arrows, demonstrates substantial OPN mRNA signal; this and an adjacent negative focus are shown enlarged in the dark-field image in C (bar=10 µm). OPN mRNA signal localized to large cells with pale cytoplasm and pale nuclei, morphologically consistent with macrophages (D, hematoxylin-eosin; E, dark-field microscopy; bar=2.5 µm).

	Discussion

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OPN is increasingly recognized as a prominent extracellular matrix molecule in the cardiovascular system. An earlier report by Murry et al¹⁴ identified transient expression of OPN by tissue macrophages after acute injury to the heart. OPN expression was localized to tissue macrophages. Despite the persistence of macrophages throughout the healing response, production of OPN diminished dramatically during the later process of scar formation. Our data, however, address a different biological process, characteristic of many cardiomyopathies, in which tissue injury and remodeling occur on a continuing basis. In the Syrian hamster cardiomyopathy model, OPN is expressed without antecedent discrete traumatic injury to the heart. This may more closely model the process of injury and repair that occurs in many human cardiomyopathies.

As in the recent report by Murry et al,¹⁴ the source of OPN mRNA appears to be tissue macrophages in foci of inflammation. Considerable variability in the levels of OPN expression among individual cells was observed in these inflammatory foci, with expression most apparent in the especially hypercellular regions. These regions may represent areas of more recent injury, with expression of OPN constituting an early response, and OPN mRNA decreasing as the scar matures. OPN expression could also result in a chemotactic stimulus that generates the observed hypercellularity. The chemotactic properties of OPN have been demonstrated in other tissues.^{8 11 20 21}

The heart comprises a complex and heterogeneous mixture of cell types. Thus, to evaluate the significance of localized OPN expression, it will be necessary to define the distribution of OPN receptors, including the _vß₃ integrin,^{11 22 23} in different cell populations in the heart. The existence of validated, high-resolution ultrasonic techniques to study the material properties of the matrix in the Syrian hamster cardiomyopathy model¹⁹ may prove particularly useful in evaluating the microscopic structural consequences of OPN expression and signaling. In ischemic cardiomyopathies, OPN could also be expressed in cells within coronary atherosclerotic lesions.^{6 7 10} It is not known whether OPN from this source could also affect myocardial remodeling.

Although scar calcification was a prominent and nearly ubiquitous histological finding in the cardiomyopathic hamsters, we could not deduce a clear association between the extent of local calcification and the level of osteopontin expression. Because many areas with the histological appearance of acute inflammation also exhibited adjacent calcification, presumably representative of a more chronic response, repeated waves of acute injury and inflammation may occur at a single location. Tissue calcification may represent a temporally integrated response, whereas OPN expression may or may not occur when sampled at a single time point. The OPN protein may persist in the extracellular matrix in regions in which expression at the RNA level, as detected by in situ hybridization, is only transient. Further experiments may help in defining the temporal course of OPN expression and protein in specific cardiomyopathic lesions.

Received May 10, 1995; revision received June 21, 1995; accepted June 21, 1995.

	References

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Williams, E. B. Articles by Rottman, J. N. Search for Related Content PubMed PubMed Citation Articles by Williams, E. B. Articles by Rottman, J. N. Pubmed/NCBI databases Substance via MeSH Medline Plus Health Information Cardiomyopathy