The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has recently been suggested to play a role in the development of naturally acquired myxomatous mitral valve disease (MMVD) in dogs.
The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has recently been suggested to play a role in the development of naturally acquired myxomatous mitral valve disease (MMVD) in dogs.
To investigate the 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, presence of macrothrombocytosis, and plateletcrit.
A total of 120 client-owned dogs.
Dogs were prospectively recruited and were classified by standard echocardiography into healthy (dogs of breeds predisposed to MMVD, but without echocardiographic evidence of the disease), mild, moderate, or severe MMVD groups. Serum 5-HT concentrations were analyzed using an ELISA.
Dogs with severe MMVD had lower serum 5-HT concentrations than healthy dogs (P = .0025) and dogs with mild MMVD (P = .0011). Unilinear and multiple regression analyses showed that serum 5-HT concentrations decreased with increasing left atrial to aortic root ratio (LA/Ao), were higher in Cavalier King Charles Spaniel (CKCS) dogs compared to dogs of other breeds, and were higher in female dogs than in male dogs. The LA/Ao was the variable most strongly associated with serum 5-HT concentration.
The finding of higher serum 5-HT concentrations in dogs of breeds predisposed to the early onset of MMVD (CKCS) and dogs with mild MMVD suggests that alterations in 5-HT signaling might play a role in progression of early stages of MMVD.
Cavalier King Charles Spaniel
% fractional shortening
end-diastolic interventricular septum dimension
end-systolic interventricular septum dimension
left atrial to aortic root ratio
end-diastolic left ventricular internal dimension
percentage increase in end-diastolic left ventricular internal dimension
end-systolic left ventricular internal dimension
percentage increase in end-systolic left ventricular internal dimension
end-diastolic left ventricular posterior wall dimension
end-systolic left ventricular posterior wall dimension
myxomatous mitral valve disease
systolic blood pressure
transforming growth factor-β
valvular interstitial cell
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.
The study was approved by the Local Ethical Committee in Uppsala, Sweden. Informed owner consent was obtained. Client-owned dogs were prospectively recruited at the cardiology unit of the Faculty of Veterinary Medicine and Animal Sciences in Uppsala between May 2007 and October 2008 and December 2009 and August 2010, respectively. Each dog was included only once in the study. Dogs were included if they had either (1) evidence of MMVD or (2) a breed predisposition to MMVD and an absence of physical or echocardiographic evidence of MMVD. Dogs were excluded if they had congenital heart disease, other acquired cardiovascular disorders or significant organ-related or systemic diseases. Dogs with MMVD in need of heart failure therapy were allowed into the study. Only dogs with a body weight <15 kg were included.
All examinations were performed without sedation and during a single visit in a quiet examination room. An owner interview was conducted to collect data concerning age, sex, and medical history. Dogs underwent a general physical examination. Blood pressure was indirectly measured using an automated oscillometric device (1on dogs recruited 2007–2008 and 2on dogs recruited 2009–2010). Dogs were minimally restrained in a standing position, and an appropriate neonatal cuff, with a width of approximately 40% of the tail circumference, was applied to the base of the tail with the artery marker placed ventrally. Once reliable consecutive readings were obtained, the mean of 5 consecutive blood pressure measurements was calculated.
Echocardiographic examinations, which included M-mode, 2D, and color flow Doppler, were performed by use of either an ultrasonographic unit3 equipped with a 5 MHz transducer (dogs recruited 2007–2008) or an ultrasonographic unit4 equipped with a 5-1 MHz transducer (dogs recruited 2009–2010) with simultaneous ECG monitoring. All echocardiographic examinations were performed and evaluated by either of 2 echocardiographers (ILj and JH). Dogs were gently restrained in right and left lateral recumbency on an ultrasound examination table.
Standardized M-mode images and 2D loops were digitally stored. Assessment of mitral valve structures was conducted from right parasternal long-axis views and left apical 4-chamber view. The same views were used to assess the degree of MR by color Doppler mapping. Mitral regurgitation was subjectively assessed as the area of regurgitant jet relative to the area of the LA, as described elsewhere with slight modifications according to the following: regurgitation scores were recorded as (1) none or minimal (only observed at a point close to the mitral valve leaflet coaptation); (2) mild (<30%); (3) moderate (>30–50%); and (4) severe (>50%). The left atrial to aortic root (LA/Ao) ratio was measured as previously described. Measurements were made on 3 consecutive cardiac cycles and the mean value from each dog was used in the statistical analysis. Screening of potential regurgitations through the tricuspid, aortic, and pulmonic valves was performed routinely using color Doppler echocardiography. M-mode measurements of the LV were performed using standard techniques on images obtained from the right parasternal short axis view on 5 consecutive cardiac cycles. The mean value for each variable was used in the statistical analyses. Values for the percent increases of end-diastolic left ventricular internal dimension (LVIDdinc) and end-systolic left ventricular internal dimension (LVIDsinc) were calculated as follows: [observed dimension − expected normal dimension]/expected normal dimension × 100. Expected normal dimensions were calculated as previously described: LVIDd (body weight0.294 × 1.53), and LVIDs (body weight0.315 × 0.95).
Diagnosis of MMVD was exclusively based on color Doppler and 2D echocardiographic findings, and diagnostic criteria included characteristic valvular lesions of the mitral valve apparatus (thickened or prolapsing mitral valve leaflets or both) and demonstrated MR on color Doppler echocardiogram, as previously described.[42, 46] Estimation of MMVD severity was based on the obtained LA/Ao ratio and the MR jet size, and dogs were classified into one of the following groups: Healthy; dogs of a breed predisposed to early onset of MMVD, but without 2D echocardiographic evidence (thickened or prolapsing mitral valve leaflets or both) of the disease (LA/Ao <1.5 and none or minimal MR jet only observed at a point close to the mitral valve leaflet coaptation), mild (LA/Ao ≤1.5 and MR jet <30%), moderate (LA/Ao >1.5 and <1.8 and MR jet <50%), or severe (LA/Ao ≥1.8 and MR jet >50%) MMVD.
Whereas a separate classification of MMVD severity on the basis of the MR jet area and LA/Ao ratio would result in the dog being put into different categories, eg, a dog with a jet area of >50% and a LA/Ao of 1.7, MMVD severity would be classified on the basis of LA/Ao ratio alone.
Blood was collected from the jugular vein into 5-mL serum tubes and 5-mL EDTA tubes.
Serum samples were centrifuged within 30 minutes after collection. Serum was harvested and transferred into 1.5-mL plastic cryotubes, and samples were stored at −80°C for subsequent analysis. Samples obtained in 2007–2008 were analyzed in August 2009, and samples obtained in 2009–2010 were analyzed in February 2011. Before analysis, the frozen serum was thawed slowly at room temperature, and then assayed in duplicate for 5-HT concentrations. Serum 5-HT concentrations were analyzed using an enzyme-linked immunosorbent assay,5 according to the manufacturer's instructions. The in-house validation, which was performed by an experienced laboratory technician, showed an average recovery of 5-HT in spiked serum samples of 98.2% (range 95.2–106.2%) and a lower detection limit of 0.088 ng/mL. The intra-assay coefficient of variance (CV) was 2.7% in the samples analyzed in 2009 and 7.5% in the samples analyzed in 2011.
The EDTA blood samples were analyzed within 2 hours in a subset of the dogs using a hematology analyzer.6 The blood is stained with acridine orange and platelets form the upper layer of the buffy coat after centrifugation. The width of this layer of platelets in the standardized tubes indicates the total platelet volume (plateletcrit, PCT).
The presence of macrothrombocytosis was determined in a subset of the dogs by a board-certified veterinary clinical pathologist (ILi). The evaluation was based on platelet characteristics (ie, platelet count, mean platelet volume, and cytogram pattern) obtained from a hematology analyzer.7,8 Dogs were divided into 3 groups: (1) dogs with platelets of normal size and normal numbers; (2) dogs with mild–moderate macrothrombocytosis; and (3) dogs with pronounced macrothrombocytosis and few platelets.
Statistical analyses were performed using a commercially available software program.9 Group data are presented as medians and interquartile range (IQR). A value of P < .05 was considered significant for the analyses, unless otherwise indicated.
The nonparametric Kruskal–Wallis test was used to investigate overall associations between serum 5-HT concentration and the 4 MMVD severity groups. If a significant association (P < .05) was detected, a pair-wise comparison was also performed by use of the Mann–Whitney U-test with Bonferroni adjustment, for which a value of P < .008 was considered significant.
Unilinear regression analyses were used to evaluate associations between serum 5-HT concentration, dog characteristics (recruitment period: 2007–2008 or 2009–2010, age, sex, body weight, CKCS: yes or no), HR obtained from the echocardiogram, SBP, PCT, presence of macrothrombocytosis (3 groups), storage time of the serum samples, and echocardiographic measurements (LA/Ao ratio, FS%, LVIDdinc, and LVIDsinc).
Variables with P < .2 in the unilinear regression analysis were included in a multiple regression analysis. Because of the high covariance between LA/Ao ratio and LVIDdinc, only LA/Ao was included in the multiple regression model. A logarithmic transformation was performed for the multiple regression analyses to achieve a normal distribution for skewed variables (serum 5-HT concentrations and PCT). Analyses were performed in a backward stepwise manner, starting with all variables included in the model and then removing the variable with the highest P value until all the remaining variables had a value of P < .05. All variables were assessed only as main effects; no interaction terms were considered in the model. The distribution of residuals in the multiple-regression analysis was tested for normality using Shapiro-Wilk W-test. The adjusted R2 is defined as the percentage of the total sum of squares that can be explained by the regression and it also considers the degrees of freedom for variables added.
A total of 120 dogs (68 females and 52 males) with a median age of 8.0 (IQR 6.0–10.3) years and a median body weight of 9.6 (7.8–10.8) kg were included in the study. The most commonly recruited breed was CKCS (n = 92), followed by Dachshund (n = 17), mixed breed (n = 3), Jack Russell (n = 2), and Shih-tzu (n = 2). Four other breeds with 1 dog each also were represented in the study. Twenty-six dogs had unremarkable echocardiograms or very mild MR (<5% jet) and were considered healthy, 47 dogs had mild MMVD, 16 dogs had moderate MMVD, and 31 dogs had severe MMVD. A total of 66 dogs were enrolled during the period 2007–2008, and 54 dogs were enrolled during the period 2009–2010. Twenty of the severe MMVD dogs were diagnosed with CHF at the time of inclusion or had previously been diagnosed with CHF but stabilized by heart failure therapy. Eighteen of these dogs were receiving cardiac medication at the time of inclusion: furosemide (17 dogs), pimobendan (8 dogs), ACE inhibitor (6 dogs), spironolactone (1 dog), and digoxin (3 dogs). The PCT was analyzed in 56 of the dogs, and the presence of macrothrombocytosis was evaluated in 61 of the dogs. Summary statistics for the different MMVD severity groups are shown in Table 1.
|Age (years)||4.5 (3.4–6.3)a||6.9 (6.1–10)ab||9.1 (7.5–11)bc||9.9 (8.6–11)c|
|Weight (kg)||8.8 (7.4–9.9)a||9.2 (7.7–11)ab||10 (8.1–11)ab||11 (8.8–13)b|
|HR (bpm)||118 (103–131)a||107 (97–130)a||119 (105–136)a||143 (121–168)b|
|SBP (mmHg)||132 (121–142)a||136 (127–151)ab||142 (129–152)ab||129 (116–139)ac|
|LA/Ao||1.2 (1.1–1.2)||1.2 (1.2–1.3)||1.6 (1.5–1.7)||2.1 (1.9–2.4)|
|LVIDd (cm)||2.8 (2.7–3.3)a||3.2 (2.9–3.5)a||3.8 (3.6–4.3)b||4.5 (4.1–4.8)c|
|LVIDdinc (%)||3.1 (−5.3 to 11.6)a||8.0 (1.2–16)b||28 (21–45)b||45 (38–61)c|
|LVIDs (cm)||2.0 (1.8–2.4)a||2.2 (2.0–2.4)ab||2.4 (2.1–2.8)bc||2.7 (2.4–3.1)c|
|LVIDsinc (%)||6.1 (0.0–23)a||12 (4.7–24)a||24 (8.8–45)ab||37 (24–58)b|
|FS (%)||29 (26–33)a||30 (28–34)a||36 (34–42)b||38 (34–45)b|
|5-HT (ng/mL)||657 (501–864)a||645 (430–1068)a||585 (400–725)ab||513 (307–632)b|
|PCT||321 (223–370)a||312 (250–396)a||318 (289–396)a||307 (251–404)a|
Detectable serum 5-HT concentrations were found in all dogs. An overall significant difference (P = .0035) was found between the 4 MMVD severity groups and 5-HT concentration. Lower serum 5-HT concentrations were found in dogs with severe MMVD, compared with concentrations in healthy dogs (P = .0025) and in dogs with mild MMVD (P = .0011) (Fig 1).
Serum 5-HT concentrations decreased with increasing LA/Ao ratio (R2 = 0.12, P < .0001), LVIDdinc (R2 = 0.09, P = .0009), and LVIDsinc (R2 = 0.06, P = 0.0099) and age (R2 = 0.08, P = .0015). In addition, higher 5-HT concentration was found in CKCS dogs compared with the dogs of other breeds than CKCS (P = .0005), and in female dogs compared with male dogs (P = .0010) (Fig 2).
Modeling serum 5-HT concentration as outcome variable and including LA/Ao, LVIDsinc, CKCS: yes or no, age, sex, recruitment period, and storage time in the model, confirmed a major effect of LA/Ao (P = .0067), sex (P = .027), and CKCS Y/N (P = 0.037) on 5-HT concentration (Fig 2). The final model had an adjusted model R2 of 0.18.
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.
The skillful technical assistance of Marta Kot, Robert Kruse, and Gunilla Drugge is greatly appreciated. Supported by the Agria Research Foundation. Presented as an oral presentation at the European College of Veterinary Internal Medicine – Companion Animals (ECVIM-CA) congress, Maastricht, Netherland, September 2012.
Conflict of Interest: Authors disclose no conflict of interest.
Oscillometric Krutech VET420A, Jorgen Kruuse A/S, Marslev, Denmark
Vet HDO monitor, S +B medVet GmbH, Babenhausen, Germany
GE Vivid 3 ultrasound unit, General Electric Co, Stockholm, Sweden
iE33, Philips Ultrasound, Bothell, WA
Serotonin ELISA (RE59121), IBL-Hamburg GmbH, Hamburg, Germany
QBC VetAutoread, IDEXX Laboratories, Westbrook, ME
Sysmex XT2000iV, Sysmex, Kobe, Japan
Advia 2120, Siemens Healthcare Diagnostics, Deerfield, IL
JMP, version 9.0.1, SAS Institute Inc, Cary, NC