First quantitative dosages: Strong correlations between non-5-HT2Rs serotonin receptors on normal human heart valves

Abstract

Objectives: Although critical in animal and human development and pathology, a measurement of the quantitative expression of 5-HTR serotonin receptors on animal or human valvular tissues has never been performed.

Methods: Quantification of the most frequent 5-HTRs reported as being present in human peripheral tissue was performed using radiolabeled agonists/antagonists. A membrane protein extract from normal human valves (aortic/mitral/tricuspid and some pulmonary) and associated diseased left myocardium, all unusable in clinics, were obtained from the Homograft bank.

Results: We analyzed 5-HT_1AR/5-HT_1B/DR/5-HT_2AR/5-HT_2BR/5-HT _2CR/5-HT₄R/5-HT₇R from 28 hearts. We confirmed the presence of tissue and measured the quantitative content for respective proteins in femtomol/mg of protein extracts: for 5-HT_2AR (35.9+/-0.7), 5-HT_2BR (28.8+/-1.3) but also a newly observed and robust expression for 5-HT₄R (38+/-4.2). We identified one, 5-HT_1ARs (4.9+/-0.3), and the possible expression, but at a very low level, of previously reported 5-HT_1B/DRs (1.3+/-0.5) as well as the new 5-HT₇Rs (3.5+/0.1) and 5-HT_2CRs (1.2+/-0.1). Interestingly, by using univariate analysis, we were able to observe many correlations between the different 5-HTR levels of expression especially between 5-HT_1AR/5-HT_1B/DR and also between 5-HT₄R/5-HT₇R, but none were observed between 5-HT_2AR and 5-HT_2BR. Using multivariate analyses for a specific 5-HTR level of expression, after adjustment for implantation sites and other 5-HTRs, we found that 5-HT_1AR was correlated with 5-HT_1B/DR;5-HT₄R with 5-HT₇R and 5-HT_1AR;5-HT_2BR with 5-HT_2AR only. For 5-HT₂C, no correlation was observed.

Conclusion: 5-HT_2AR/5-HT_2BR and 5-HT₄R were all observed to have a high and equal level of expression on human valves, but that of 5-HT_1AR was more limited. Since these non-5-HT₂Rs are coupled with different G-proteins, with specific signaling, theoretically they may control the main 5-HT₂R signaling (i.e., PLC/DAG-PKC-ERK/Ras/Src signaling) involved in valvular fibrosis and degeneration.

Keywords: heart valve disease; human heart valves; quantitative expression; serotonin 5-HT; serotonin receptor 5-HTRs; valvular degeneration.

Figure 1

Quantitative expressions of 5-HTR receptors on human “normal” heart valves by radioligand binding affinity with the human left ventricle (i.e., diseased left cardiomyopathy) as a control. This figure shows the quantitative measurements of receptor expression for aortic, mitral, tricuspid, and pulmonary valves with the left ventricle as a control. Specific quantification of the protein expression for 5-HTRs (5-HT_1AR, 5-HT1_B/DR, 5-HT_2AR, 5-HT_2BR, 5-HT_2CR, 5-HT₄R, and 5-HT₇R) on valvular tissue protein extract was performed with specific radioligands. 5-HT_1AR and 5-HT_1B/DR are known to be associated with a Gi/Go protein and thus decrease cAMP levels. On the other hand, 5-HT₄R and 5-HT₇R are associated with a Gs protein and thus increase cAMP levels. 5-HT₂Rs are associated with another G-type protein (Gq/G₁₁) and thus activate PLC, Src, and Ras. 5-HTR₇R was the most recent receptor to be identified. Up until now, no quantification of 5-HTRs has been conducted on human heart valves. We had access to a total of 28 transplant hearts and had mitral and tricuspid sites for all of them. For 14 hearts, we had the three main sites (aortic/mitral/tricuspid), and for three hearts, we had four sites at the same time (including pulmonary site). All measurements of expression levels were performed in triplicate. As shown in this figure, all types of receptors we tested, known to be expressed in peripheral tissues, are expressed in valvular tissues, with the main receptors being quantitatively 5-HT_2AR, 5HT_2BR, and the unreported 5-HT₄R. Interestingly, we did not observe any difference in terms of the level of expression between the different sides and sites investigated. Each bar diagram in the figure is based on 28 samples for tricuspid and mitral valves, with left ventricle as control, and 14 samples for the aortic position. P-values correspond to the results of ANOVA tests. No particular difference was observed between the right sides (i.e., tricuspid, pulmonary) and left sides (i.e., aortic, mitral) (ANOVA test). The pulmonary positions (i.e., only 3 samples available) tended to have the same level of expression as the other sites.

Figure 2

Comparison of the levels of expression of different pairs of 5-HTRs. This figure corresponds to this figure. As shown in this figure, there were strong correlations between several 5-HTRs (A1–D). The strongest correlations were found between the 5-HT_1AR and 5-HT_1B/DR quantitative protein levels (A1) and between 5-HT₄R and 5-HT₇R levels (A2). 5-HT_1AR and 5-HT_1B/DR are coupled with the same G protein (i.e., Gi/Go), while 5-HT₄R and 5-HT₇R are coupled with G protein Gs. We observed that 5-HT_2CR levels were negatively correlated with all other 5-HTRs (C). The negative correlation between 5-HT_2CR levels and other 5-HT2Rs (i.e., 5-HT_2AR or 5-HT_2BR) was less marked (B4). We also observed positive correlations between receptors coupled with different G proteins: 5-HT₄R levels were negatively correlated with 5-H_1AR levels (C1) and 5-HT_1AR levels with 5-HT₇R levels (C2). There was no correlation between 5-HT_2AR levels and 5-HT_2BR levels after univariate analysis (D). Respective correlations following univariate analyses are shown in different panels. All correlations between 5-HTRs following univariate analysis are presented in Table 2. Spearman's Rank Correlation analysis (n = 70 samples for each analysis).

Figure 3

Most significant statistical associations based on multivariate regression analyses for a specific 5-HTR level of expression with explicative variables being all other 5-HTR receptors and the implantation sites. This figure is based on Table 4. Seventy samples were included in each statistical analysis. See also the corresponding Supplementary Figure S2 adapted from Table 3 corresponding to the most significant statistical associations after multivariate regression analysis between 5-HTR levels with the explicative variables being another 5-HTR and the site of implantation. The yellow boxes show the main classical signal transduction pathways that are possibly activated after stimulation from specific 5-HTRs: 5-HT_1AR, 5-HT_1B/DR, 5-HT_2CR, 5-HT_2AR, 5-HT_2BR, 5-HT₄R, and 5-HT₇R especially with respect to the main signaling cAMP, but other signaling may also be present. The width of the arrows is proportional to the level of statistical correlation found following multivariate regression analysis. Only those comparisons between 5-HTRs with a P-value < 0.1 following multivariate regression analysis are shown in this figure. Blue arrows correspond to a correlation between 5-HTRs where the coefficient has an absolute value between 0 and 1 and is positive. Red arrows correspond to a correlation where the coefficient has an absolute value between 0 and 1 and is negative. In summary, as shown in this figure, statistically significant correlations were observed between levels for 5-HT_1AR and 5-HT_1B/DR and levels for 5-HT₄R and 5-HT₇R. For 5-HT4R levels, there was also a negative correlation with 5-HT1AR levels. Interestingly, we observed a negative correlation between 5-HT_2AR levels and 5-HT_2BR levels (coef. = +0.20 [−0.02; 0.54]; P = 0.037). The yellow boxes show the main signaling pathways for each 5-HTR if their specific G protein is involved. These signaling pathways are just hypothetical in heart valves and need confirmation.

Figure 4

Most significant study results evaluating expression of 5-HTRS, TPH1, or SCC6 (i.e., the gene for SERT) by PCR on human samples in normal, prolapse, myxomatous degenerative valves, or under mechanical stimulation. n = number of samples analyzed. For PCR quantification for mitral prolapse and myxomatous valve degeneration, the values are compared to the normal. Ref. references for this figure/ 1) Levy et al. (5); 2) Driesbaugh et al. (2); 3) Hulin et al. (25); 4) Roy et al. (27); 5) Sainger et al. (28). See also for review: Ayme-Dietrich et al. (3) and Barnes et al. (11, 23).