Isometric myography is a feasible method to identify and quantify endothelial dysfunction in dogs with myxomatous mitral valve disease.

OBJECTIVE: To assess the feasibility of isometric myography in pet dogs with myxomatous mitral valve disease (MMVD) to determine its use in quantifying endothelial dysfunction. ANIMALS: 9 dogs euthanized for medical reasons. METHODS: Femoral, renal, and mesenteric arteries were collected postmortem and stored in physiological saline solution at 4 °C for myography. Mitral valves were scored for myxomatous degeneration (grades 1 to 4). Sections of arteries were mounted in wells, immersed in physiological saline solution perfused with 95% O2 and 5% CO2 at 37 °C, and stretched to an internal circumference (IC) that generated the maximal difference between active and passive wall tension (IC1). Normalization factors were calculated by dividing the IC1 by the IC at which the passive wall tension was 100 mm Hg (IC100). Vasoconstriction to phenylephrine and vasodilation to acetylcholine (endothelial dependent) and sodium nitroprusside (endothelial independent) were assessed by cumulative dose-response curves. RESULTS: Median MMVD grade was 3. Mean values of normalization factors were 1.00 ± 0.14 (renal, n = 15), 1.00 ± 0.10 (femoral, 8), and 1.05 ± 0.12 (mesenteric, 6). Responses to phenylephrine were similar between dogs (P = .14). Reduced responses to acetylcholine compared with sodium nitroprusside were identified in 15 arteries, suggesting endothelial dysfunction. CLINICAL RELEVANCE: Isometric myography of arteries from pet dogs is feasible and can identify loss of endothelial-dependent relaxation in dogs with MMVD postmortem. Its use in further research can lead to a better understanding of the pathophysiology mechanisms of this disease.

Survival of activated myofibroblasts in canine myxomatous mitral valve disease and the role of apoptosis.

Myxomatous mitral valve disease (MMVD) is the single most important acquired cardiovascular disease of the dog. Much is known about the cellular changes and the contribution of activated myofibroblasts (valve interstitial cells (aVICs) to the valve extra-cellular matrix remodelling characteristic of the disease. However, little is known on how aVIC survival might contribute to disease pathogenesis. This study examined the temporal (disease severity-dependent) and spatial distribution of aVICs in MMVD valves, the expression of a range of apoptosis-related genes in cultured VICs from both normal (quiescent VIC (qVIC) and diseased (aVIC) valves, and the differential effects of doxorubicin treatment, as a trigger of apoptosis, on expression of the same genes. Activated myofibroblasts were identified in normal valves at the valve base only (the area closest to the annulus), and then became more numerous and apparent along the valve length as the disease progressed, with evidence of cell survival at the valve base. There were no significant differences in basal gene expression comparing qVICs and aVICs for CASP3, FAS, BID, BAX, BCL2, CASP8, DDIAS, XIAP and BIRC5. After doxorubicin treatment (2 mM) for 8 h there was significant difference (P < .05) in the expression of BID, BCL2, DDIAS, and CASP8, but when assessed for interactions using a mixed model ANOVA only CASP8 was significantly different because of treatment (P < .05). These data suggest aVIC survival in MMVD valves may be a consequence of heightened resistance of aVICs to apoptosis, but would require confirmation examining expression of the relevant proteins.

Evaluation of innervation of the mitral valves and the effects of myxomatous degeneration in dogs.

OBJECTIVE: To map aspects of the innervation of the mitral valve complex and determine any association with the development or progression of myxomatous mitral valve disease (MMVD) in dogs. SAMPLE POPULATION: Septal mitral valve leaflets from 11 dogs aged 6 months to > 10 years. PROCEDURES: Expression of protein gene product 9.5 (general neuronal marker), tyrosine hydroxylase (adrenergic innervation marker), vasoactive intestinal peptide (parasympathetic innervation marker), and calcitonin gene-related peptide (sensory innervation marker) was assessed by use of a standard immunohistochemical technique. Innervation was assessed qualitatively and semiquantitatively. Differences between valvular zones and between groups were analyzed statistically. RESULTS: MMVD was present in leaflets of all dogs > or = 5 years of age. Innervation was confirmed in all leaflets but was markedly reduced in leaflets of dogs > 10 years of age. Innervation was most dense at the base of valves and mainly associated with the epimysial, perimysial, and endomysial layers of the muscle and blood vessels within the valve. Innervation was reduced within the middle zone of the valve and lacking at the free edge. Innervation was not identified at the tip of the leaflet, the free edge, or the chordae. Nerve fibers were mostly sympathetic, with the remainder being parasympathetic or sensory. Existence of MMVD did not alter the pattern or density of innervation. CONCLUSIONS AND CLINICAL RELEVANCE: Mitral valve leaflets in the study dogs were innervated, with most of the nerve fibers associated with the myocardium in the valve base. Development of MMVD appeared to precede the reduction of innervation associated with advancing age.

Distribution of myofibroblasts, smooth muscle-like cells, macrophages, and mast cells in mitral valve leaflets of dogs with myxomatous mitral valve disease.

OBJECTIVE: To map the cellular distribution and phenotypic alteration of the predominant stromal cell population throughout the entire valve length of dogs with myxomatous mitral valve disease (MMVD). SAMPLE POPULATION: 31 mitral valve complexes (ie, mitral valve leaflets) collected from 4 clinically normal dogs and 27 dogs with MMVD of varying severity. PROCEDURES: A combination of standard histologic and immunohistochemical techniques was used to identify pathologic changes, the presence of mast cells, and the density and distribution of cells expressing vimentin, desmin, alpha-smooth muscle actin (alpha-SMA), smooth muscle myosin, and the macrophage marker MAC387. RESULTS: Vimentin-positive cells predominated in the mitral valve leaflets from clinically normal dogs and were located throughout the leaflet, but cell density was appreciably decreased with disease progression, and minimal cell numbers were found in distinct myxomatous areas. Cells that were positive for alpha-SMA were uncommon in the mitral valve leaflets from clinically normal dogs and only seen in appreciable numbers in mitral valves of dogs with severe late-stage disease, in which cells were typically located close to the ventricularis valve surface. A slight increase in mast cell numbers was observed in the distal zone of affected leaflets. CONCLUSIONS AND CLINICAL RELEVANCE: Activated-myofibroblasts (alpha-SMA-positive cells) were increased and inactive-myofibroblasts (vimentin-positive cells) were reduced in mitral valve leaflets of dogs with MMVD, compared with that of clinically normal dogs. Impact on Human Medicine-This is the first description of spatial and temporal alterations in mitral valve cells of any species with MMVD and has clinical importance in the understanding of disease development in dogs and humans.