Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmitter known to control a wide range of biological functions.[1, 2] 5-HT has in recent years also been implicated in the development of heart valve disease.[1, 3-5] The majority of 5-HT is synthesized in the enterochromaffin cells of the intestine, but evidence also exists of local 5-HT synthesis in the heart.[7-11] Nearly all 5-HT secreted into the blood stream is rapidly taken up by platelets via an active transport mechanism, and then stored in the dense granules of platelets.[6, 12] The cardiovascular system, such as peripheral vascular endothelium, smooth muscles, and heart tissue, readily binds and responds to 5-HT, and increased 5-HT signaling or decreased 5-HT clearance might induce valvular lesions[5, 9, 13, 14]: Rats receiving 5-HT injections over a long period have been shown to develop valvular lesions.[15, 16] Furthermore, 5-HT-producing carcinoid tumors,[4, 17] or intake of serotonergic drugs, have been shown associated with valvulopathy in people.[18-21]
The described 5-HT-induced valvular lesions share many gross and histologic similarities with naturally acquired myxomatous mitral valve disease (MMVD), and 5-HT has recently been suggested to have a role in the development of MMVD in dogs.[10, 14, 22] The disease is characterized by progressive degeneration of the mitral valve.[23-25] The mitral valve leaflets, which normally are thin, translucent and soft, become thickened and elongated with disease progression, leading to mitral regurgitation (MR) and subsequently chronic volume overload with dilatation of the left atrium (LA) and the left ventricle (LV).[25-27] Histopathologic changes in the valvular tissue in MMVD dogs include damage to the endothelial valvular cell-lining. Furthermore, a phenotype transformation occurs of the fibroblast-like valvular interstitial cells, which are responsible for normal valve structure via maintenance of the extracellular valve matrix, into a more active myofibroblast phenotype.[28, 29] 5-Hydroxytryptamine has been suggested to both directly and indirectly (the latter by an up-regulation of transforming growth factor-β [TGF-β1]) stimulate such a phenotype transformation.[10, 13, 14, 30-35] The activated myofibroblasts can induce signaling pathways, resulting in an abnormal structural integrity of the valve with disorganization in the fibrosa layer and marked expansion of the spongiosa layer.[26, 28, 30, 36-38]
Increased circulating 5-HT concentration has been associated with development of heart valve pathology in people.[17, 39, 40] A previous study demonstrated increased serum 5-HT concentrations in dogs with naturally acquired MMVD compared to healthy large-breed control dogs. A potential association between serum 5-HT and MMVD severity has to our knowledge never been investigated. Accordingly, the aim of this study was to investigate association between serum 5-HT concentration and MMVD severity in dogs, and to assess potential associations between serum 5-HT concentrations and dog characteristics, echocardiographic variables, heart rate, systolic blood pressure (SBP), presence of macrothrombocytosis, and plateletcrit (PCT) in the study population.
- Top of page
- Material and Methods
Serum 5-HT concentration was found to decrease with increasing severity of MMVD, and LA/Ao ratio was the variable most strongly associated with 5-HT in both the unilinear and multiple regression analyses. Furthermore, CKCS dogs, which are predisposed to an early onset of MMVD, had higher serum 5-HT concentrations compared with dogs of other breeds, and female dogs had higher serum 5-HT concentrations compared with male dogs.
Associations between 5-HT and MMVD severity was detected by both group-wise comparisons, and unilinear and multiple regression analyses. Group-wise comparisons separated only severe MMVD dogs from healthy dogs and dogs with mild MMVD. The decrease in serum 5-HT concentration with increasing disease severity might suggest that if 5-HT primarily plays a role in progression of early MMVD, it might play a lesser role in dogs with more severe valvular lesions. Although dogs enrolled in the “healthy” group lacked convincing evidence of MMVD on the echocardiogram, very mild, early changes of MMVD might have been undetected in some of these dogs. Such early stages of MMVD could potentially have impacted the results in this study, as a majority of dogs in this “healthy” group were of breeds highly predisposed to an early onset of MMVD.[50-52] Dogs in the healthy group had a median age of 4.5 years. Inclusion of even younger dogs in this group could possibly have led to a statistical separation in serum 5-HT-concentrations between healthy dogs and dogs affected by mild MMVD as the risk of inclusion of dogs with very mild MMVD, classified in the present study as being healthy, would have been substantially reduced. However, people with high circulating 5-HT concentrations have been shown at risk of developing valvulopathy, and dogs highly predisposed to an early onset of MMVD might have high serum 5-HT concentrations already from a comparably young age.
The CKCS breed is predisposed to an early onset of MMVD,[50-52] and the age of onset has been shown to be an inherited trait that is influenced by 2 major loci.[51, 53] These findings strengthen the leading scientific hypothesis that a genetically determined dystrophic process initiates the valve degeneration.[51, 53, 54] Serum 5-HT concentrations were shown to be higher in CKCS dogs compared with other small breed dogs in the present study, according to both unilinear and multiple regression analyses. This finding is similar to the results from a previous study demonstrating higher serum 5-HT concentrations in healthy CKCS compared with other healthy dogs predisposed to MMVD. This might indicate that 5-HT (together with other mechanisms) contributes to the progression of early MMVD in this breed, and that 5-HT potentially could be genetically linked to the etiologic mechanisms of MMVD in CKCS.
CKCS dogs had a high incidence of macrothrombocytosis.[47, 55, 56] 5-HT is normally stored within the dense granules of platelets, and morphologic investigations of macrothrombocytes have revealed normal-appearing dense granules. Still, this does not exclude an excessive content of 5-HT in macrothrombocytes. Nevertheless, significant associations between serum 5-HT concentrations, and macrothrombocytosis and PCT could not be found in the present study. These findings are consistent with findings in a previous study. Furthermore, previous studies have failed to demonstrate an association between presence of macrothrombocytes and MMVD.[58, 59]
Female dogs had higher serum 5-HT concentrations compared with male dogs, according to both unilinear and multiple regression analyses. Furthermore, 5-HT concentration decreased with increasing age in the unlinear regression analysis, which agrees with previously published results. However, an association between 5-HT and age could not be demonstrated in the multiple regression analysis, possibly because of the covariance between age and MMVD severity (indicated by LA/Ao) as dogs with moderate and severe MMVD in the present study were older compared with healthy dogs and dogs with mild MMVD (Table 1). Degenerative changes of the intracardiac valves can be seen in elderly individuals of different species, and although an age influence on serum 5-HT concentrations might exist, the effect of MMVD disease severity on 5-HT concentration appears stronger than age, based on results from the present study.
The present study investigated serum 5-HT concentrations in dogs with different severities of MMVD. However, the role of circulating 5-HT in the regulation of cardiovascular function and its potential contribution to myxomatous changes in the mitral valve interstitium is not yet fully understood. Serum 5-HT concentration results from an equilibrium between 5-HT synthesis, platelet uptake and storage, and metabolism. Hence, increased serum 5-HT concentrations might result from an insufficient 5-HT uptake by the platelets, an increased release of 5-HT from activated platelets, or both, potentially in combination with an excessive 5-HT synthesis, decreased metabolism of 5-HT by the endothelial monoamine oxidase enzymatic system in the liver and lung of the dog, or both.[60, 61] The majority of the 5-HT production occurs in the enterochromaffin cells of the intestines, but findings in studies investigating myoxmatous protein expression patterns in canine mitral valves indicate that an increased autocrine 5-HT production has the potential to mediate myxomatous mitral valve degeneration.[10, 11, 62] Hence, it can be questioned whether altered circulating 5-HT concentrations in dogs with MMVD are caused by alterations in the peripheral production and handling of 5-HT or by alterations in local 5-HT production in the mitral valve (or both). A triggering factor of such a potential alteration in 5-HT production/handling in MMVD dogs remains elusive. Previously published studies have shown that an up-regulation of 5-HT receptors occurs in canine myxomatous degenerative valves tissue,[10, 22] and a 5-HT receptor-dependent regulation of 5-HT uptake could potentially be implicated in the control of circulating 5-HT concentrations.
A limitation with this study is that the samples were stored between 2 and 19 months at −80°C before batch analysis, and degradation of 5-HT might have affected the measured concentrations. However, storage time did not affect 5-HT concentrations in the unilinear and multiple regression analysis. Dogs were recruited during 2 different time periods. From the beginning, we were only aiming for investigating 5-HT concentrations in the dog recruited during 2007–2008. However, we were quite surprised when finding that 5-HT concentration decreased with increasing MMVD severity, and for this reason we decided to verify our findings in an additional group of dogs recruited during 2009–2011. The results from dogs recruited during 2007–2008 were replicated in the group of dogs recruited during 2009–2011. Thus, the groups were combined, and a possible effect from the recruitment period was controlled for in the statistical analyses. Reference values for serum 5-HT concentrations in dogs (both dogs of breeds highly predisposed to MMVD and dogs of other breeds) are unfortunately lacking. Such data would be valuable to allow more comprehensive interpretation of the results from the present study. Concentrations of 5-HT were analyzed in serum in the present study, but further knowledge of 5-HT concentrations in whole blood and platelets in dogs with MMVD of different severities is also desirable. Further limitations with this study were that the PCT and the presence of macrothrombocytosis were only assessed in a subset of the dogs.
In conclusion, serum 5-HT concentration decreased with increasing MMVD severity, which might suggest that if 5-HT plays a role in valvular degeneration it does so primarily in the early stages of the disease. Furthermore, CKCS dogs, which are highly predisposed to MMVD, had higher serum 5-HT concentrations compared with dogs of other breeds. Although abnormal 5-HT signaling is unlikely to be the sole primary cause of MMVD in dogs, alterations in the 5-HT signaling system might be involved in the pathological process of valvular deformation. However, further investigations should be conducted to evaluate the potential role of 5-HT in the pathogenesis of MMVD.