Decreased serotonin transporter activity in the mitral valve contributes to progression of degenerative mitral regurgitation

Abstract

Degenerative mitral valve (MV) regurgitation (MR) is a highly prevalent heart disease that requires surgery in severe cases. Here, we show that a decrease in the activity of the serotonin transporter (SERT) accelerates MV remodeling and progression to MR. Through studies of a population of patients with MR, we show that selective serotonin reuptake inhibitor (SSRI) use and SERT promoter polymorphism 5-HTTLPR LL genotype were associated with MV surgery at younger age. Functional characterization of 122 human MV samples, in conjunction with in vivo studies in SERT^-/- mice and wild-type mice treated with the SSRI fluoxetine, showed that diminished SERT activity in MV interstitial cells (MVICs) contributed to the pathophysiology of MR through enhanced serotonin receptor (HTR) signaling. SERT activity was decreased in LL MVICs partially because of diminished membrane localization of SERT. In mice, fluoxetine treatment or SERT knockdown resulted in thickened MV leaflets. Similarly, silencing of SERT in normal human MVICs led to up-regulation of transforming growth factor β1 (TGFβ1) and collagen (COL1A1) in the presence of serotonin. In addition, treatment of MVICs with fluoxetine not only directly inhibited SERT activity but also decreased SERT expression and increased HTR2B expression. Fluoxetine treatment and LL genotype were also associated with increased COL1A1 expression in the presence of serotonin in MVICs, and these effects were attenuated by HTR2B inhibition. These results suggest that assessment of both 5-HTTLPR genotype and SERT-inhibiting treatments may be useful tools to risk-stratify patients with MV disease to estimate the likelihood of rapid disease progression.

Figure 1:. Phenotype of degenerative MR MV leaflets includes SERT expression downregulation.

(A) Representative echocardiography images of a patient with MR prior to MV surgery; LA, left atrium; LV, left ventricle. Arrow 1 (top panel, green) indicates the thickened and prolapsed posterior mitral leaflet. Arrow 2 (top panel, green) indicates the thickened anterior mitral leaflet with shallowed coaptation depth. Arrow 3 (bottom panel, black) indicates torrential anterior directed MR jet. (B) Representative IHC staining of normal MV samples from heart donors (top two rows) and MR MV samples recessed during surgery (bottom two rows); a, zona atrialis; f, zona fibrosa; s, zona spongiosa. Movat’s stain shows accumulation of elastic fibers (stained in black, black arrow) and an increase in glycosaminoglycans (stained in blue, black arrowheads) intertwined with collagen (stained in yellow). IHC of αSMA shows areas with increased MV interstitial cell activation in MR MV (green arrows). IHC of Ki67 shows occasional areas of cell proliferation in MR MV (blue arrow). Right column shows IHC for the matricellular protein osteopontin (OPN). Volcano plots of gene expression results by RT² profiler PCR panels of (C) TGFβ-related genes and (D) serotonin-related genes in MR tissue (n=44) in comparison with normal MV tissue (n=20). Horizontal dotted lines denote the genes with −log10 > 1.3 (corresponding to a p value = 0.05 by Student t test, purple dots), vertical dotted lines define genes with a log2 fold change > 1 (red dots) or < −1 (blue dots), corresponding to fold change = 2 and 0.5 versus normal, respectively. (E) Scatter dot plot of SERT expression from RT² profiler PCR in normal (n=20) and MR MV tissue (n=44). Re-analysis of data included in panel D. Error bars indicate standard error. P value by Mann Whitney U test. (F) Representative immunofluorescence staining of SERT in normal and MR MV tissue (green). Within each sample, yellow dotted box area is shown with higher agnification to the right of the main image. Nuclei are visualized by DAPI stain (blue). All IHC/immunofluorescence were n≥4/group. All gene expression results were calculated by the 2−ΔΔCT method and normalized to housekeeping genes.

Figure 2:. SSRI use and 5-HTTLPR genotype LL impact the timing and need for MV surgery in degenerative MR patients.

(A) Pie chart illustrating the distribution of 5-HTTLPR genotypes in a local cohort of MR patients (n=225). (B-D) Multivariate Cox model analysis results of patients with degenerative MR undergoing cardiac surgery. Data were assessed employing Cox’s proportional hazards model where the response variable was the age at MV surgery. Results corresponding to (B) our local cohort (n=225) and (C) Optum database population (n=9441). (D) Sub-analysis of the local cohort (n=225) showing interactions with 5-HTTLPR genotype LL. Values are shown as hazard ratio ± lower and upper 95^th percentile confidence limits, reflecting the hazard of requiring surgery at younger ages (hazard ratio>1.0) compared to older age at surgery (hazard ratio<1.0). AV, aortic valve; CAD, coronary artery disease; HF, heart failure, NYHA, New York Heart association; SSRI, serotonin reuptake inhibitor; PAD, peripheral artery disease; male-female refers to “male over female” variable in the multivariate model.

Figure 3:. 5-HTTLPR genotype LL and the SSRI fluoxetine decrease the activity of SERT in MVICs.

(A) Gene expression in a sub-cohort of MV tissue from human normal MV tissue (n=9) and MV from patients with degenerative MR undergoing mitral valve surgery (n=32). (B) Gene expression of SERT at baseline in MVIC isolated from normal and MR MV (n≥8/group). (C) Gene expression of SERT in MVIC (normal and MR combined) after seven-day treatment with 1μM fluoxetine (n≥21/group, biological replicates corresponding to MVIC subpopulations from 13 MR patients). (D) Gene expression of SERT in MR MVIC stratified by 5-HTTLPR genotype after seven-day treatment with 1μM fluoxetine (n≥4/group). Re-analysis of data included in panel C. All gene expression results calculated by the 2−ΔΔCT method and presented as % of the mean of corresponding control (normal or untreated) normalized to GAPDH. (E) SERT activity by FFN246 uptake assay at baseline in MVIC isolated from normal and MR MV (n≥10/group, biological replicates corresponding to MVIC subpopulations from 3 normal subjects and 13 MR patients). (F) SERT activity in MVIC (normal and MR combined) with or without 30-minute pre-treatment with 1μM fluoxetine (n=65/group, biological replicates corresponding to MVIC subpopulations from 13 MR patients). (G) SERT activity in MR MVIC stratified by 5-HTTLPR genotype with or without 30-minute pre-treatment with 1μM fluoxetine (n≥24 biological replicates corresponding to MVIC subpopulations from ≥ 4 MR patients/group. Re-analysis of data included in panel F). (H) Representative images of visualization of FFN246 uptake (green) in MVIC by confocal microscopy. Propidium iodide (PI) stain was performed to aid in cell localization (red). (I) Representative confocal microscopy of extracellular SERT in cultured MR MVIC (green), performed with antibody incubation before fixation and without cell permeabilization. Staining performed in control MVIC with silencing RNA-mediated knockdown of SERT is shown to indicate non-specific signal (right panel). All immunofluorescence, n≥4/group. (J) Quantification of Western blot of SERT in the extracellular cytosolic membrane fraction in MR MVIC following surface biotinylation pull-down assay, and representative images from blots with different samples in each (n=3/group, estimated chance or type-I error = 0.027). All data are shown as % of the mean of all samples in each blot. Error bars indicate standard error. In vitro assays were run a minimum of three times. P values by Mann Whitney U test when two groups were compared (panels A, B, C, E, F and G), two-way ANOVA with Bonferroni (panel D), or Kruskal Wallis with Dunn’s multiple comparison test (panels G and J), when appropriate.

Figure 4:. SERT downregulation alters HTR2B expression and increases ECM component gene expression in the MV.

(A) Representative trichrome staining of heart sections of 8-week-old wild type and SERT^−/− mice, showing the MV leaflets; AL, anterior leaflet; PL, posterior leaflet. Bottom row shows colorimetric thresholding to highlight collagen in red. (B) Gene expression of COL1A1, TGFβ1, αSMA, HTR2A and HTR2B and in MV leaflets from 8-week-old wild type (WT) and SERT^−/− mice (n =7/group). (C) Representative confocal microscopy of SERT (green) in human control MVIC with silencing RNA (siRNA)-mediated knockdown of SERT or non-targeting (NT) RNA, performed with cell permeabilization. Phalloidin (red) was used to visualize cell structure and nuclei are visualized by DAPI stain (blue). Bottom row images have been overexposed to show SERT signal in knocked-down MVIC. (D) Gene expression of SERT, COL1A1, HTR2B, TGFβ1 and αSMA in human control MVIC with SERT or NT siRNA, treated for 24 hours with or without 10μM of serotonin (5HT, n=5/group). (E) Representative Picrosirius red staining of heart sections of 120-day-old wild type mice treated with or without 20 mg/kg/day fluoxetine for 60 days, showing the thickened MV leaflets (green arrows). Bottom row shows colorimetric thresholding to highlight collagen in red. (F) Gene expression of COL1A1, TGFβ1, αSMA, HTR2A and HTR2B in MV leaflets from wild type mice treated with or without fluoxetine (n≥4/group). (G) Representative immunofluorescence staining of HTR2B (green) in MV leaflets from wild type mice treated with or without fluoxetine. DAPI used to denote nuclei (blue). Green signal in the myocardium (top left and bottom right corners of left panel) includes unspecific tissue autofluorescence. All IHC, n≥4/group. All gene expression results calculated by the 2−ΔΔCT method and presented as % of the mean of corresponding control (WT or non-treated) normalized to GAPDH. Assays were run a minimum of three times. Error bars indicate standard error. P values by Mann Whitney U test (panel B) or Student’s T-test (panel F) when two groups were compared, two-way ANOVA with Bonferroni multiple comparison test (panel D), when appropriate.

Figure 5:. SERT downregulation increases COL1A1 expression through a HTR2B-mediated mechanism.

(A) Gene expression of COL1A1 in human MVIC (normal and MR combined) after seven-day treatment with 1μM fluoxetine (n≥8/group, biological replicates corresponding to MVIC from 8 normal subjects and 10 MR patients). (B) COL1A1 expression in control and MR MVIC after 24-hour treatment of serotonin (5HT, 10μM), TGFβ1 (10ng/ml) or vehicle (n≥5/group, biological replicates corresponding to MVIC from 5 normal subjects and 17 MR patients). (C) Gene expression of COL1A1 in MR MVIC after 24-hour treatment of serotonin (10μM), TGFβ1 (10ng/ml) or vehicle, stratified by 5-HTTLPR genotype (n=5/group. Re-analysis of data included in panel B). P values correspond to comparisons versus corresponding untreated. (D) Phosphorylated (P-) ERK (normalized to total ERK and GAPDH) in MR MVIC treated for 10 minutes with 1μM serotonin or vehicle. Quantification above and representative western blots below (n=4/group). (E) Representative confocal microscopy of P-ERK (green) in MR MVIC treated for 10 minutes with 1μM serotonin or vehicle. Phal indicates phalloidin (red), nuclei are visualized by DAPI stain (blue). All IHC, n≥4/group. (F) COL1A1 expression in MR MVIC after 24-hour treatment of serotonin (10μM), TGFβ1 (10ng/ml) with or without HTR2B-inhibitor LY272015 (100nM), HTR2A-inhibitor ketanserin (10μM), or vehicle (n≥6/group). (G) COL1A1 expression in MR MVIC after 24-hour treatment of serotonin with or without inhibitor LY272015 (LY) or ketanserin (ket), stratified by 5-HTTLPR genotype (n≥4/group. Re-analysis of data included in panel F). (H) HTR2B expression at baseline in MVIC isolated from normal and MR MV(n≥8/group). (I) HTR2B expression in human MVIC (normal and MR combined) after seven-day treatment with 1μM fluoxetine (n≥10/group, biological replicates corresponding to MVIC subpopulations from 4 normal subjects and 12 MR patients). (J) HTR2B expression in MR MVIC after seven-day treatment with fluoxetine, stratified by 5-HTTLPR genotype (n≥4/group. Re-analysis of data included in panel I). (K) COL1A1 expression in LL MR MVIC after seven-day treatment with 1μM fluoxetine, with or without subsequent 24-hour treatment of serotonin (10μM) and LY272015 (LY, 100nM) (n≥4/group). All gene expression results calculated by the 2−ΔΔCT method and presented as % of the mean of corresponding control normalized to GAPDH. Assays were run a minimum of three times, with a minimum of two cell-lines per assay. Error bars indicate standard error. P values by Student’s T-test (panel A) or Mann Whitney U test (panels D, H and I) when two groups were compared, two-way ANOVA with Bonferroni multiple comparison test (panels B, C, F, G, J, and K), when appropriate.