The etiology and pathogenesis of canine myxomatous mitral valve disease (MVD) is poorly understood. Using oligonucleotide microarrays, we have previously reported increased expression of various serotonin (5HT) and transforming growth factor-beta1 (TGF-B1) signaling molecules in myxomatous canine valve tissue. We have also shown that canine interstitial valve cells demonstrate increased downstream mitogen activated protein-kinase (MAP-K) activity after exposure to in vitro 5HT, suggesting that serotonin and TGF-B1 may have the capacity to mediate both the proliferative and degenerative changes seen in MVD. Based on these results, we sought to further characterize the expression of key signaling components of the 5HT and TGF-B1 pathways and extracellular matrix in affected canine mitral valves. Mitral valve leaflets from 7 dogs that were euthanized or died due to severe MVD were excised, snap frozen in liquid nitrogen, and stored at -70C until processing. Mitral valve tissues from 5 healthy dogs that were euthanized for an unrelated study were collected as controls. Total RNA was isolated and analyzed for integrity and quality. Transcription was evaluated by real-time reverse transcriptase quantitative PCR (rt-qPCR) using a commercial system (SybrGreen RT-qPCR, Applied Biosystems). Fifteen different transcripts were analyzed, including various 5HT-related molecules (5HT-receptor subtypes 1A, 1B, 2A, and 2B, and 5HT transporter), TGF-B1-related molecules (TGF-B1, TGF-B1 receptor, SMAD-2, and TAK-1), and extracellular matrix components (MMP-1, 2, and 9, cathepsin K, collagen A, and fibronectin). Results were expressed in units of relative expression. For the 5 5HT-related molecules, transcriptional activity was detected in 33 of 35 MVD samples (94%) vs 9 of 25 control samples (26%) (P<0.0001). Affected valves had significantly increased 5HT-receptor-1B as compared to control (MVD 29.8 vs control 0.0; P<0.05). For TGF-B1 components, no significant difference was found in the presence or absence of expression or relative expression levels between MVD and control. For extracellular matrix components, transcriptional activity was detected in 40 of 42 MVD samples (95%) vs 19 of 30 control samples (63%) (P=0.0005). Affected tissue had significantly increased MMP-9 transcription as compared to control (MVD 98.5 vs control 0.0; P<0.01). Expression of 5HT and extracellular matrix components is more prevalent in affected mitral valves and 5HT signaling may play a role in the pathology of canine MVD.