Canine myxomatous mitral valve disease (MMVD) is the most common cardiac
disease in dogs affecting all breeds, and it shares many similarities with the
equivalent human disease. From the only transcriptomic report for canine MMVD
published in 2006, serotonin signalling was identified as a contributing factor and has
been widely studied since. Two transcriptomic profiling studies in human MMVD
have also identified oxidative stress response and bone morphogenic protein
signalling contributing to disease pathology. All studies at the transcriptional level
have identified a variety of biological functions in MMVD suggesting dynamic
extracellular matrix (ECM) remodelling processes are on-going. Moreover, cellular
changes found in MMVD are somewhat reminiscent of the events seen in early heart
valve, suggesting possible re-activation of signalling pathways of which those
driving development and endothelial-to-mesenchymal transition (EndoMT) are
particularly interesting. EndoMT, in which endothelial cells change their identity to
mesenchymal phenotype and migrate into the cardiac jelly underneath the
endothelium, is a crucial mechanism in valvulogenesis. Whether or not gene
regulation of EndoMT and valve development also plays a role in MMVD is
unknown.
In this study, the MMVD cellular changes in the Cavalier King Charles Spaniel
(CKCS), a breed with the highest prevalence, earliest onset, and rapid progression of
the disease, was investigated. Secondly, transcriptional profiling was conducted
using the latest canine microarray chips, a single affected breed (CKCSs), stringent
sample quality control and statistical thresholds, with quantitative polymerase chain
reaction (Q-PCR) for data validation. After transcriptional mapping, multi-platform
in silico analysis was conducted to identify relationship between differentially
expressed genes and their relevant biological functions. Next, a comparison study
using immunohistochemistry was performed on different severities of myxomatous
valves to localize the proteins of interest. Finally, to model the transcriptional factors
and their downstream targets, mitral valve endothelial cell (MVEC) clones were
derived from the canine normal mitral valves for future in vitro studies.
Cellular changes of MMVD between CKCS and non-CKCS populations showed no
difference in their distribution, number and phenotypic markers. Global genomic
expression analysis identified similar (inflammation, up-regulation of serotonin
receptor and bone morphogenic protein) and novel biological functions (epithelial-to-mesenchymal
transition) compared to the previous study in 2006. Key transcriptional
factors and genes associated with EndoMT including SNAI1, TAGLN, ACTA2,
ACTG2, HAS2, and CTNNB1 were found up-regulated, and NID1, LAMA2, CDH5
were down-regulated in the MMVD group. In myxomatous mitral valves, increased
expression of HAS2 in myofibroblasts, SNAI1 expression in endothelial cells, and
co-expression of CDH5 and α-smooth muscle actin (α-SMA) also suggested the
presence of EndoMT compared to normal valves. Nevertheless, there is also
evidence of EndoMT in normal valves (α-SMA positive endothelial cells) which
might suggest contribution to life-long valve re-modelling. In addition, there was a
decreased expression of microRNAs associated with modulation of extracellular
matrix transcripts, including miR-23, miR-29, and miR-218, indicating epigenetic
regulation in MMVD.
Based on the cellular changes, MMVD in CKCS appears to be representative of
MMVD in all breeds and the early-onset of MMVD in that breed does not lead to
different end-stage pathology. Novel biological functions such as EndoMT, were
identified by transcriptional profiling, and by using powerful bioinformatic tools
providing insight into understanding gene regulation in MMVD. Furthermore, a
relationship between developmental biology processes and MMVD pathogenesis was
established, with a likely important role for epigenetics in disease pathogenesis