The echocardiographic data from seven WT compared with eight MMP-2 TG mice at an average age of 4 months (range 3.5–4.5 months) are summarized in . All studies were done in conscious animals. There was no evidence for mitral valve thickening or prolapse in mice at this age and most cardiac functional parameters were the same in both the WT and MMP-2 TG mice. There was a modest, but significant, decrease in left ventricular fractional shortening in the TG mice at this time.
Echocardiographic assessment of function in control and MMP-2 transgenic mice
The echocardiographic data from seven WT mice compared with five MMP-2 TG mice at 14±0.5 months are summarized in . There was no difference in heart rate between the two groups. As reported [11
], the MMP-2 TG mice exhibited marked impairment of LV function with significant declines in LV ejection fraction and fractional LV shortening. In the transgenics, LVend-systolic volumes were more than twice the WT values. LV mass was also significantly elevated, as were long axis dimensions in both diastole and systole. Of note, aortic root dilatation was also present in the transgenics ( and ). The MMP-2 TG mice consistently displayed echocardiographic evidence of mitral valve thickening and prolapse ().
Fig. 3 Transgenic MMP-2 induces aortic root dilatation. (Panel A) Masson trichrome stain through the midsection of the aortic root from WT mice with normal sinuses of Valsalva. (Panel B) The aortic root diameter at the level of the sinuses of Valsalva is increased (more ...)
Fig. 1 (I) Representative echocardiograms on 14.5-month-old conscious WT (Panel A) and MMP-2 TG (Panel B) mice. Studies were performed as detailed in Materials and Methods. The WT mice demonstrate normal mitral structure and motion. The anterior leaflet of the (more ...)
(Panel A) shows a normal mitral valve apparatus stained with PSR for interstitial collagen from a 14-month-old WT mouse. The anterior and posterior valve leaflets are of normal dimensions and contain a well-defined, brightly stained lamina fibrosa composed of organized collagen. Immediately adjacent there is a small amount of ground substance (glycosoaminoglycans) which characterizes the normal lamina spongiosa. In contrast, the mitral valve apparatus from the MMP-2 TG hearts is grossly distorted. Both the anterior and posterior leaflets were elongated and redundant with focal areas of massive spongiosa expansion. We measured the lengths of the mitral valve posterior leaflets from the WT and MMP-2 TG mice. The posterior leaflets from the WT mice averaged 135±25 μm in length, while the posterior leaflets from the MMP-2 transgenics averaged 210±13 μm in length (n=4 for each group; P<.05). Valve thickness was not determined as there was extensive variation along the length of any individual leaflet due to zones of either rarefaction or massive acidic glycosoaminoglycan accumulation in the zona spongiosa that precluded accurate quantitation.
Collagen volume fractions of the posterior mitral valve leaflets were determined on PSR-stained sections. The total amounts of interstitial collagen in the WT and MMP-2 TG posterior mitral valve leaflets were not significantly different (WT: 29.7±4.8%; TG: 31.5±4.2%, P>.05, n=4 for each group). There was a major loss of collagen bundle organization within the lamina fibrosa in the MMP-2 TG mice (, cf. Panels A and B). The collagen bundles comprising the lamina fibrosa are tightly organized in the WT mitral valves, with clear delineation from the surrounding ground substance. In sharp contrast, the collagen bundles in the TG valves are loosely associated and expanded within areas of intervening ground substance. As shown in the inset in (Panel B), there are nodules of highly disorganized collagen in the TG valves.
Fig. 2 MMP-2 TG valves and chordae tendineae exhibit myxomatous degeneration and chondrocyte transformation. Isolated mitral valve/papillary muscle preparations were sequentially stained with PAS and Alcian blue to define neutral (PAS) and acidic (Alcian blue) (more ...)
Similar collagen organizational changes, although less dramatic, were seen in the aortic valve cusps of the MMP-2 transgenics (, cf. Panels A and B). The aortic valve lamina fibrosa was disorganized and there was moderate expansion of the spongiosa. Valvular myxomatous degeneration was limited to the left side of the heart, as the tricuspid and pulmonary valves in the MMP-2 transgenics were structurally intact (data not shown).
Combined PAS and Alcian blue staining was performed on isolated mitral valves to determine whether neutral (PAS) or acidic (Alcian blue) glycosoaminoglycans accumulated in the lamina spongiosa of the MMP-2 transgenics. Representative sections of WT (Panels A and B) and MMP-2 transgenics (Panels C and D) are shown in . The normal mitral valves contain modest amounts of neutral (pink) and acidic (blue) glycosoaminoglycans within the lamina spongiosa. In contrast, there is massive accumulation of acidic glycosoaminoglycans (chondroitin-6-sulfate, hyaluronate) within the lamina spongiosa of the MMP-2 transgenics. Expansion of the spongiosa in the MMP-2 transgenics was not uniform and was distributed in a nodular or irregular pattern. Expansion of the spongiosa in the MMP-2 transgenics was accompanied by a modest, but significant, increase in the number of interstitial valvular cells (WT: 23.5±3.8 cells/625 μm2; TG: 34.7±3.9/625 μm2; P=.02). The valvular interstitial cells did acquire an elongated morphology characteristic of a myofibroblast phenotype (, Panel D insert). Similar to findings in human disease, the chordae tendineae from the MMP-2 transgenics demonstrated extensive myxomatous degeneration, with focal accumulation of acidic glycosoaminoglycans (, Panel E). There was also evidence for fibrin deposition and hemorrhage within the chordae tendineae. Cells with the morphologic features of hypertrophic chondrocytes were detected within the mitral valve and the tips of the papillary muscles in the MMP-2 transgenics (, Panel F).
Analysis of serial sections of the proximal aortas confirmed the finding of aortic root dilatation in the MMP-2 transgenics obtained by ultrasound studies. Representative sections are shown in . The aortic valves were irregularly thickened along the length of the cusps primarily due to substantial expansion of the spongiosa. In addition, the TG valves demonstrated the hypercellularity of the interstitial cells along with occasional foci of inflammatory cells (, Panel B, blue arrow).
Aortic dilatation in the MMP-2 transgenics was confined to the level of the coronary sinus and in the example shown there was a greater than 1.5-fold increase in diameter at this level. Aortic diameters more distally were not enlarged in the MMP-2 transgenics and we did not detect histologic evidence for medial cystic degeneration or dissection. The limited extent of the aortic dilation may be the consequence of limited diffusion of the TG MMP-2 protein from the surrounding cardiomyocytes.
A key question relates to how cardiomyocyte expression of active MMP-2 under the control of the α-MHC promoter contributes to valvular myxomatous degeneration. Possible explanations include the effects of systolic dysfunction and ventricular remodeling induced by MMP-2 on mitral and aortic valve functional dynamics, or possible diffusion of active MMP-2 from the cardiomyocytes into the valvular structures, per se. Alternatively, it was possible that MMP-2 trangene expression was induced in the setting of progressive ventricular failure due to activation of the α-myosin heavy chain promoter. To gain insight into this issue, we performed immunohistochemical staining of isolated mitral valves from 14.5-month-old mice for the c-myc epitope encoded at the C-terminal of the TG MMP-2 expression cassette. In our initial efforts, we detected little c-myc epitope staining in the mitral valves of the MMP-2 transgenics when the sections were conventionally processed with a methyl green counterstain and examined under standard microscopic conditions. However, examination of noncounterstained sections using Nomarksi interference contrast microscopy revealed detectable Vector Purple reaction product within the mitral valve leaflets of the MMP-2 transgenics with a pattern consistent with valvular interstitial cell localization (). No c-myc epitope immunoreactive product was detected in isolated mitral valves from 4- to 6-month-old TG mice, a time which precedes the development of myxomatous degeneration (data not shown). These results are consistent with delayed expression of the MMP-2 transgene by valvular interstitial cells, presumably due to recruitment of α-myosin heavy chain promoter activity.
Fig. 4 Expression of c-myc-tagged MMP-2 transgene in posterior leaflet mitral valve at 14.5 months. (Panel A) Wild-type control leaflet stained for the c-myc epitope tag without counterstaining and photographed using Nomarski optics. Minimal background staining (more ...)
The cellular morphology of the myxodematous valves, coupled with the massive expansion of acidic glycosoaminoglycans, is consistent with differentiation of the interstitial valve cells to a chondrocytic or chondroblastic phenotype. To substantiate this impression, we performed RT-PCR on excised mitral valve tissues and assessed the expression of three discrete genes. SOX9 is a transcription factor expressed during osteoblast and chondroblast differentiation and during gonadal differentiation [12
]. Matrilin-1 is a cartilage-specific noncollagenous protein expressed during cartilage development [13
]. Cartilage oligomeric matrix protein is an acidic oligomeric protein detected only in differentiated cartilage [14
]. RT-PCR of extracted mitral valve RNA from 8-month-old mice did not reveal any detectable SOX9 or matrilin-1 transcripts in either the WT or TG mitral valves (data not shown). In contrast, low levels of COMP transcript were detected in the wild types and there was a significant, 2.7-fold increase in COMP transcript abundance in the TG mitral valves (). These results are consistent with a partial differentiation of the MMP-2 TG interstitial valve cells to a chondrocytic phenotype and confirm the impressions obtained with cellular morphology and expression of acidic glycosoaminoglycans.
Fig. 5 RT-PCR analysis of COMP expression. RT-PCR was performed on RNA templates isolated from 8-month-old WT and MMP-2 TG mitral valves. Relative transcript abundance of GAPDH-normalized PCR products is given, in which the control, WT abundance of COMP is assigned (more ...)