Study of collagen structure in canine myxomatous mitral valve disease

Abstract

Myxomatous mitral valve disease (MMVD) is the single most common acquired cardiac disease of dogs, and is a disease of significant veterinary importance. It also bears close similarities to mitral valve prolapse in humans and therefore is a disease of emerging comparative interest. Realising the importance of collagen fibres in mitral heart valves and considering the paramount significance of myxomatous mitral valve disease, a better understanding of the pathogenesis of MMVD is essential. Thus, this study was designed to investigate the changes in collagen molecules, including fibril structure, fibril orientation, d-spacing, collagen density, collagen content, thermal stability, and the status of mature and immature crosslinks. A combination of biophysical and biochemical tools such as x-ray diffraction, neutron diffraction, HPLC were utilised in order to fulfil the objectives. Biochemical assay of hydroxyproline revealed a 10% depletion of collagen in mildly affacted (grade I and II) leaflets, while a 20% depletion of fibrillar collagen was revealed by mapping the collagen fibrils onto the anatomy of cardiac leaflets using x-ray data. Differential scanning calorimetry showed that there were no significant differences in the onset temperature of denaturation of collagen between the healthy and affected leaflets. However, in affected areas of leaflets, the enthalpy of denaturation significantly dropped by 20%. In the affected regions, neutron diffraction results showed an increase in the immature reducible cross-links though the low number of the samples can be considered a limiting factor in this regard. However, the HPLC results showed a 25% decrease in the number of mature cross-links. Additionally, the recently introduced imaging technologies to biology and medicine such as differential enhancing imaging (DEI) and coherent anti-Stokes Raman scattering spectroscopy (CARS) were, to the author’s best knowledge, applied for the first time to this disease. In doing so, this thesis furthers our understanding of the pathogenesis of MMVD, especially in relation to the collagen. The thesis provides new findings about MMVD and demonstrates the potential of biophysical tools for studying similar conditions.