Targeting 5-HT_2B Receptor Signaling Prevents Border Zone Expansion and Improves Microstructural Remodeling after Myocardial Infarction

Abstract

Background:

Myocardial infarction (MI) induces an intense injury response which ultimately generates a collagen-dominated scar. While required to prevent ventricular rupture, the fibrotic process is often sustained in a manner detrimental to optimal recovery. Cardiac myofibroblasts are the cells tasked with depositing and remodeling collagen and are a prime target to limit the fibrotic process post-MI. Serotonin 2B receptor (5-HT_2B) signaling has been shown to be harmful in a variety of cardiopulmonary pathologies and could play an important role in mediating scar formation after MI.

Methods:

We employed two pharmacologic antagonists to explore the effect of 5-HT_2B inhibition on outcomes post-MI and characterized the histological and microstructural changes involved in tissue remodeling. Inducible, 5-HT_2B ablation driven by Tcf21^MCM and Postn^MCM were used to evaluate resident cardiac fibroblast- and myofibroblast-specific contributions of 5-HT_2B, respectively. RNA sequencing was used to motivate subsequent in vitro analyses to explore cardiac fibroblast phenotype.

Results:

5-HT_2B antagonism preserved cardiac structure and function by facilitating a less fibrotic scar, indicated by decreased scar thickness and decreased border zone area. 5-HT_2B antagonism resulted in collagen fiber redistribution to thinner collagen fibers which were more anisotropic, enhancing left ventricular contractility, while fibrotic tissue stiffness was decreased, limiting the hypertrophic response of uninjured cardiomyocytes. Using a tamoxifen-inducible Cre, we ablated 5-HT_2B from Tcf21-lineage resident cardiac fibroblasts and saw similar improvements to the pharmacologic approach. Tamoxifen-inducible Cre-mediated ablation of 5-HT_2B after onset of injury in Postn-lineage myofibroblasts also improved cardiac outcomes. RNA sequencing and subsequent in vitro analyses corroborate a decrease in fibroblast proliferation, migration, and remodeling capabilities through alterations in Dnajb4 expression and Src phosphorylation.

Conclusions:

Together, our findings illustrate that 5-HT_2B expression in either cardiac fibroblasts or activated myofibroblasts directly contributes to excessive scar formation, resulting in adverse remodeling and impaired cardiac function after MI.

Introduction

Cardiac fibroblasts (CFs) comprise approximately 11% of cells in the healthy adult heart.¹ Quiescent CFs reside in healthy ventricles and maintain homeostasis through low-level extracellular matrix (ECM) turnover and organization.² Injurious environmental stimuli, such as hypoxia, altered tissue mechanics, and cytokines like TGF-β, signal the phenotypic switch of CFs to a more proliferative, hypercontractile, and hypersecretory state.³ In the case of myocardial infarction (MI), Tcf21 lineage-traced resident CFs transdifferentiate into myofibroblasts, which can then be traced through their expression of the marker periostin – a secreted matricellular protein produced in adults exclusively following injury.^4–6 Myofibroblasts localize to both infarcted tissue and the surrounding border zone (BZ) to potentiate the reparative fibrotic injury response due to the lack of intrinsic regenerative capacity of the myocardium.^7–9

Following MI, myofibroblasts play the indispensable role of stabilizing and reinforcing the left ventricle (LV) via formation of a collagen-dominated scar.³ Insufficient ECM deposition can lead to LV rupture or aneurysm, while excessive ECM deposition leads to tissue stiffening, scar expansion, and arrhythmias.¹⁰ Furthermore, replacement of contractile myocardium with a collagenous scar creates a local increase in mechanical strain at the BZ of surviving myocardium and scar tissue. To compensate for biomechanical alterations, connective tissue often expands beyond the original injury, creating a subsequent decline in tissue compliance and cardiac output.^11,12 Most chronic myocardial conditions are associated with excessive deposition of fibrotic tissue, making the myofibroblast a desirable therapeutic target to limit fibrotic overactivity.^13,14

Serotonergic dysfunction has long been understood to contribute to a myriad of cardiopulmonary pathologies. Specifically, signaling through the serotonin 2B receptor (5-HT_2B) is particularly involved in valvular heart disease and pulmonary hypertension.^15,16 In response to TGF-β, fibroblast-like valve interstitial cells undergo myofibroblast transformation, which can be prevented with 5-HT_2B antagonism.¹⁷ In a murine model of experimental pulmonary hypertension, it has been shown that 5-HT_2B in the myeloid compartment is necessary to develop disease and instigate the stiffening of arterioles.^18,19 5-HT_2B has also been shown to play an integral role in mediating isoproterenol-induced cardiac hypertrophy through modulating the inflammatory milieu in a CF-dependent manner.²⁰ There are multiple downstream 5-HT_2B signaling pathways, but the mitogen-activated protein kinase effector p38 and the tyrosine kinase Src are two of the most commonly observed. Both of which are known regulators of cell contractility, ECM deposition, and ECM stiffness.^21,22

Here, we hypothesized that 5-HT_2B antagonism could hinder excessive fibrotic remodeling after MI and create a post-MI scar that preserved cardiac function. We successfully limited adverse remodeling following MI using two independent 5-HT_2B antagonists. Using newly developed, tamoxifen-inducible Cre recombinase mouse models for either resident fibroblast- or myofibroblast-targeted ablation of 5-HT_2B, we illustrate that CFs have a 5-HT_2B- mediated deleterious effect on cardiac structure and function after infarct. We show that targeted ablation of 5-HT_2B only from myofibroblasts after injury is sufficient to improve cardiac outcomes. These results are attributed to decreased scar thickness, limited BZ expansion into healthy myocardium, and reduced stiffness of scar tissue. RNA sequencing pointed to a limited proliferative and remodeling capacity in myofibroblasts lacking 5-HT_2B driven by an increased expression of Dnajb4, which encodes a heat shock protein with known anti-proliferative and anti-invasive properties, and decreased activation of the tyrosine kinase Src.

Methods

The data that support the reported findings and code used for analyses in this study are available from the corresponding author upon reasonable request. All mouse experiments were approved by the Vanderbilt Institutional Animal Care and Use Committee before their commencement. MI was induced via permanent coronary artery occlusion with consistent infarct size confirmed via TTC staining (Figure IA in the Supplement). Serial echocardiography (example image in Figure IB in the Supplement) was used to assess cardiac structure and function on mice receiving a 5-HT_2B antagonist or mice with cell-specific 5-HT_2B ablation. Htr2b^fl/flTcf21^MCM/+ and Htr2b^fl/flPostn^MCM/+ mice (inducible resident fibroblast- and myofibroblast-specific 5-HT_2B knockouts, respectively) were generated to compare with Htr2b^fl/fl littermate controls; all mice were crossed with Rosa26-stop-tdTomato reporter mice to visually verify Cre activation. Tissue was collected for analysis to determine properties of the scar, BZ, and uninjured myocardium. Picrosirius red staining was used to identify ECM deposition and associated fibrotic measurements (scar thickness, BZ area, and interstitial fibrosis). Polarized light imaging was used to determine collagen fiber thickness and second-harmonic generation imaging to quantify fiber orientation. Atomic force microscopy (AFM) was used to acquire mechanical properties of the tissue and wheat germ agglutinin to identify cardiomyocyte borders for cross-sectional area measurements. CFs (PDGFRα+) were isolated one week after MI from Htr2b^fl/fl and Htr2b^fl/flPostn^MCM/+ animals to perform bulk RNA sequencing. CFs were isolated from adult WT and 5-HT_2B knockout mice for in vitro studies. An expanded methods section is available in the Online Data Supplement.

Results

5-HT_2B antagonism preserves cardiac structure and function following MI

To test our hypothesis that 5-HT_2B regulates scar formation following MI, we first confirmed there was a marked increase in Htr2b expression three days after injury which was sustained into the fibrotic healing phase seven days after infarction (Figure II in the Supplement). Htr2a, which encodes the only other member of the 5-HT₂ receptor subfamily expressed in the cardiovascular system,23 is not upregulated until seven days post-MI, and it is still only induced to a fraction of Htr2b levels. These findings motivated our exploration into 5-HT_2B signaling. To determine if 5-HT_2B signaling influences ventricular remodeling after MI, wild-type (WT) mice underwent permanent coronary artery ligation and were administered either vehicle control (dimethyl sulfoxide, DMSO) or the 5-HT_2B antagonist SB204741 (SB) at the time of injury (Figure 1A). Interestingly, female mice showed no response to SB treatment (Figure IIIA–C in the Supplement) which may be due to sex-specific differences of 5-HT_2B governed cardiac activity, so subsequent antagonist studies were conducted in male mice.

Figure 1. Antagonism of 5-HT_2B preserves cardiac structure and function following myocardial infarction (MI) and prevents deterioration of myocardial contractile capability.

A, Experimental approach. 12-week-old mice were subjected to MI surgery and coincidentally treated with dimethyl sulfoxide (DMSO) control or the 5-HT_2B antagonist, SB204741 (SB). Treatment was ceased three weeks following injury, and serial echocardiography was performed at times shown. B, Left ventricular ejection fraction (LV EF). C, Left ventricular fractional shortening (LV FS). D,E, Left ventricular internal dimension at end-diastole (LVID;d) and end-systole (LVID;s). F,G, Left ventricular volume at end-diastole (LV Vol;d) and end-systole (LV Vol;s). H, Vector diagram showing magnitude and direction of myocardial deformation in systole and quantified global longitudinal strain (GLS). B-H, Mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 between DMSO and SB treatments, #P<0.05 between timepoints within treatment group following 2-way ANOVA and Holm-Sidak post hoc test. Number of mice denoted in B applies to subsequent groups except H where explicitly labeled.

We observed a preservation of cardiac function indicated by decreased reduction in ejection fraction (EF) and fractional shortening (FS) one week after injury in mice treated with 5-HT_2B antagonist, highlighting a slowed LV impairment following MI. This effect was maintained for the six-week period after injury, even with the removal of the antagonist three weeks post-MI (Figure 1B–C). The observed effects were due to the improved systolic inner dimension and volume of the LV (Figure 1D–G). We employed a speckle-tracking algorithm to obtain the global longitudinal strain (GLS) of the LV as another sensitive measure of LV function. While GLS was identically reduced in both groups the first week after injury, cardiac contractility of SB-treated animals stabilized, while the contractility of DMSO-treated control animals continued to deteriorate over the duration of the experiment (Figure 1H). In order to confirm these results, the experiment was repeated using another selective, high affinity 5-HT_2B antagonist RS127445 (RS) which provided a comparable effect (Figure IVA–H in the Supplement). Heart rate was not affected by inhibition of 5-HT_2B signaling (Table I in the Supplement). These data suggest increased 5-HT_2B signaling following ischemic injury plays a detrimental role in tissue healing.

Fibrotic injury response is altered by 5-HT_2B antagonism

Using a custom-built image processing pipeline,³ short-axis tissue sections were analyzed six weeks following MI (unless otherwise noted) to identify the role of 5-HT_2B signaling in the healing process post-MI. Tissue thickness was calculated and picrosirius red staining demarcated the viable myocardium (yellow) from collagenous scar tissue (red; Figure 2A–B). The BZ (green dots; Figure 2B) was defined as the transition region between collagen-dominated scar tissue (stained >85% red) and myocardium-dominated tissue (stained >85% yellow) (Figure 2C). In SB-treated mice, the deposition of fibrotic tissue was diminished as indicated by the formation of a thinner scar than control mice (Figure 2D). Scars in the SB-treated group appear to form thinner in the first week after injury and retain this thinness from week one to week six. This composition did not compromise scar integrity as there was no difference in mortality between the two groups (Figure V in the Supplement).

Figure 2. Diminished 5-HT_2B signaling decreases fibrotic scar formation and border zone (BZ) expansion after myocardial infarction (MI) through alterations in collagen composition.

A-C, Analytical approach of calculating tissue thickness (A), demarcation of scar vs. healthy myocardium (B), and (C) mathematical definition of BZ as the transition region between scar dominated (>85% collagen stained red with picrosirius red; PSR) and myocardium dominated (>85% myocardium stained yellow) with inset illustrating a representative curve from each treatment. D, Thickness of the formed scar is decreased with SB204741 (SB) treatment but does not thin over time (N=3–10). E, Decreased BZ infiltration with 5-HT_2B antagonism as indicated by the rapid transition from scar to myocardium (N=8–10). F, No difference in interstitial fibrosis was observed (N=8–10). G-H, PSR stain imaged under polarized light in the BZ and scar revealed an increased proportion of thinner, less mature collagen fibers in the BZ of SB-treated mice (N=9–10). I, Analysis of collagen fiber orientation in the BZs of DMSO- (31 FOVs across 4 mice) and SB- (35 FOVs across 5 mice) treated animals to quantify the distribution of orientations to classify as isotropic or anisotropic. All data collected 6 weeks post-MI except where noted in D. D-F, H, Mean ± SEM, *P<0.05, **P<0.01 (color denotes difference between corresponding color proportion in H) (D,H) 2-way ANOVA and Holm-Sidak post hoc test or (E,F) 2-tailed Student t test.

We then calculated the BZ transition rate (indicated by the slope of the green dot region plotted in Figure 2C) to quantify the area of material mismatch between the contractile myocardium and stiff, collagenous scar. We observed an increased rate of change from collagen to myocardium (i.e. a shorter BZ region) with 5-HT_2B antagonism revealing decreased BZ infiltration and scar disruption of viable myocardium (Figure 2E). There were no differences in the area fraction of collagen in the remote uninjured myocardium to indicate tissue-wide fibrosis (Figure 2F).

Since we observed differences in fibrotic response in both the scar and BZ of SB-treated animals, picrosirius red staining was imaged under polarized light to observe collagen fiber thickness. While there was no shift in the distribution of collagen fiber thickness in the scar, a higher percentage of thin collagen fibers was observed in response to 5-HT_2B antagonism within the BZ (Figure 2G–H). We also used second-harmonic generation (SHG) imaging to analyze collagen fiber orientation (Figure VI in the Supplement). SB-treated mice had a higher fraction of fields of view (FOV) dominated by strongly aligned, anisotropic collagen fibers in the BZ, which would impart an improved contractility (Figure 2I). Mice treated with RS also demonstrated similar alterations in the scar formation process (Figure VIIA–C in the Supplement). Histological analyses indicate that treatment with a 5-HT_2B antagonist is capable of controlling scar formation after MI and limiting BZ expansion through alterations in collagen fiber formation and organization.

Microstructural changes in response to impaired 5-HT_2B signaling

We next explored how alterations in the fibrotic healing process affects tissue stiffness. AFM was used to investigate mechanical changes of the fibrotic tissue area to determine if tissue compliance could play a role in preserving cardiac function in response to SB treatment. While there were no differences in tissue stiffness in the early stages of scar formation, antagonist-treated groups showed a decreased tissue stiffness in both the scar and BZ at six weeks following injury (Figure 3A–B, Figure VIID in the Supplement). There were no changes in the myocardial stiffness in uninjured myocardium (Figure VIII in the Supplement).

Figure 3. Tissue stiffening and concomitant increase in cardiomyocyte size are prevented with 5-HT_2B blockade.

Atomic force microscopy (AFM) was employed to analyze scar and BZ stiffness after MI. Each heart was arrested in diastole via submersion in 3M potassium chloride upon dissection. A, Each row illustrates the distribution of stiffness values within an individual mouse with representative histogram in the middle. B, Mean tissue stiffness of scar and BZ (N=3–9). C, qPCR analysis of Nppb, the gene encoding the heart failure marker natriuretic peptide B (N=4). D-E, Wheat germ agglutinin (WGA) staining of short-axis cardiomyocytes reveals increased cross-sectional area in control treated animals after MI compared to sham operation is prevented with 5-HT_2B antagonism (N=3–12). B-C, E, Mean ± SEM, *P<0.05, **P<0.01 (B,E) 2-way ANOVA and Holm-Sidak post hoc test or (C) 2-tailed Student t test.

Since increased tissue stiffening can lead to a hypertrophic response in cardiomyocytes,²⁴ we quantified the expression of the gene encoding the cardiomyocyte injury marker natriuretic peptide B (Nppb). Six weeks after MI, there was a significant reduction in Nppb in the SB group (Figure 3C). To further characterize the cardiomyocyte response to diminished fibrotic remodeling, short-axis cross-sectional area of cardiomyocytes distant to the infarcted tissue was quantified. We observed a significant increase in cardiomyocyte area six weeks after MI in vehicle treated animals compared to SB-treated animals (Figure 3D–E); these results again held with RS treatment (Figure VIIE in the Supplement). There was also a significant increase in cardiomyocyte area of vehicle-treated animals over their sham counterparts which was prevented with SB treatment (Figure 3E). These results indicate a biophysical alteration in cardiac composition downstream of the scar formation process influences remote cardiomyocyte hypertrophy after MI.

Deletion of 5-HT_2B in resident fibroblasts improves cardiac response to MI

We next examined potential cell populations responsible for the effects seen following systemic administration of a 5-HT_2B antagonist. While it is known 5-HT_2B influences cardiomyocyte development and mitogenesis,^25,26 we did not observe alterations in cardiac structure or function in sham-operated animals given antagonist (Figure IXA–C in the Supplement). Therefore, we ruled out that 5-HT_2B antagonism in the absence of injury affects the resident cell populations (primarily cardiomyocytes) with regards to LV dimensions or output. In the context of MI, we investigated Htr2b expression via quantitative polymerase chain reaction (qPCR) in three cell populations. First, adult cardiomyocytes were isolated from infarcted LVs seven days after injury. Compared to sham operation, there was approximately a 5-fold increase in Htr2b expression (Figure XA in the Supplement). However, since we observed a 17-fold increase in bulk tissue at this time point (Figure II in the Supplement), we sought other contributing cell types. The next cell population investigated was endothelial cells. WT mice were subjected to MI, and CD31-expressing cells were isolated via fluorescence-activated cell sorting (FACS). qPCR revealed that CD31+ cells had a negligible contribution to the 17-fold induction of Htr2b following MI (Figure XB–C in the Supplement). Furthermore, there was not an alteration in the expression for the gene encoding E-selectin (Sele) in scar tissue of mice treated with a 5-HT_2B antagonist, indicating blocking 5-HT_2B signaling does not mediate endothelial cell activation (Figure XE in the Supplement). While there was no increase in Htr2b expression in CD45+ cells seven days after MI compared to sham operation (Figure XB,D in the Supplement), there was the possibility that the immune cells, which infiltrate early after injury, had already been cleared from the injury. To test this hypothesis, we used a bone marrow transplant model based on previous findings showing that mice lacking 5-HT_2B in the bone marrow compartment exhibit decreased tissue remodeling and arteriole stiffness in an experimental model of pulmonary hypertension.^18,19 Age- and sex-matched donors (WT or Htr2b^−/−) were transplanted into WT mice with engraftment success of approximately 95% (Figure XIA–B in the Supplement). We observed no differences in cardiac structure or function between the transplant groups after MI, indicating a cell type of non-hematopoietic origin mediated the results observed in the 5-HT_2B antagonist studies (Figure XIIA–D in the Supplement).

To determine if 5-HT_2B signaling in the remaining major cardiac cell population, resident CFs, impacts healing after MI, we deleted the gene encoding 5-HT_2B in resident CFs using a tamoxifen-inducible, Tcf21^MerCreMer (Tcf21^MCM) transgene (Figure 4A). The tdTomato fluorescent reporter was observed in all Htr2b^fl/flTcf21^MCM/+ mice indicating successful recombination in the CFs without affecting survival (Figure XIII and XIVA in the Supplement). Following MI, Htr2b^fl/flTcf21^MCM/+ mice exhibited significantly improved EF and FS (Figure 4B–C). These mice also had preserved LV inner dimension and volume in both diastole and systole six weeks post-MI (Figure 4D–G). GLS steadily decreased over time in Htr2b^fl/fl mice lacking the Tcf21^MCM transgene and was significantly lower than the stabilized contractility of mice harboring the Tcf21^MCM transgene (Figure 4H). Six weeks after MI, morphometric analysis revealed increased heart weight normalized to tibia length in Htr2b^fl/fl mice indicative of cardiac hypertrophy (Figure 4I). These results show that ablation of the 5-HT_2B receptor from resident CFs is effective in improving cardiac outcomes after MI in a manner similar to that achieved with 5-HT_2B antagonism.

Figure 4. Htr2b deletion in resident cardiac fibroblasts (CFs) abates impact of myocardial infarction (MI).

A, Left Schematic illustrating the Tcf21 locus harboring a tamoxifen-inducible MerCreMer (MCM) cDNA, tdTomato reporter in the Rosa26 locus preceded by a loxP-flanked stop codon, and a loxP-flanked Htr2b target allele. Middle Experimental timeline for 16–17-week-old mice. Right Key. B, Left ventricular ejection fraction (LV EF). C, Left ventricular fractional shortening (LV FS). D,E, Left ventricular internal dimension at end-diastole (LVID;d) and end-systole (LVID;s). F,G, Left ventricular volume at end-diastole (LV Vol;d) and end-systole (LV Vol;s). H, Global longitudinal strain (GLS). I, Heart weight (HW) normalized to tibia length (TL). B-I, Mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 between Htr2b^fl/fl and Htr2b^fl/flTcf21^MCM/+ animals, #P<0.05, ##P<0.01 between timepoints within genotype following (B-H) 2-way ANOVA and Holm-Sidak post hoc test or (I) 2-tailed Student t test. Number of mice analyzed denoted in B applies to all subsequent data.

Myofibroblast-specific deletion of 5-HT_2B improves cardiac response to MI

Tcf21^MCM driven ablation of 5-HT_2B targets residential CFs present even in the absence of injury. We wanted to assess the functionality of ablating 5-HT_2B after the induction of an MI. We utilized a mouse model which expressed the MCM cDNA driven by the periostin promoter (Postn^MCM) (Figure 5A). This mouse model has been well-characterized to demonstrate the marking of nearly all newly activated fibroblasts (myofibroblasts) following an injury, without induction prior to injury.^5,27 Following MI, all Htr2b^fl/flPostn^MCM/+ hearts exhibited signal from the tdTomato reporter, indicating successful recombination without affecting survival (Figure XIVB and XV in the supplement). Htr2b^fl/flPostn^MCM/+ mice demonstrated a significant improvement in the functional metrics of EF and FS one week after MI compared to Htr2b^fl/fl animals which were maintained six weeks following the injury (Figure 5B–C). Myofibroblast-specific 5-HT_2B ablation resulted in improved LV inner dimension and volume in both diastole and systole six weeks after MI, indicating a preserved cardiac structure (Figure 5D–G). Ventricular deformation measured by GLS was preserved in the Htr2b^fl/flPostn^MCM/+ group while the control group continued to deteriorate over the six-week experiment (Figure 5H). Finally, morphometric analyses revealed a decreased heart weight with 5-HT_2B ablation (Figure 5I). As it has been shown that tissue-resident fibroblasts of the Tcf21 lineage are the primary source of subsequent injury-activated, periostin-expressing myofibroblasts ⁵, these results confirm 5-HT_2B expression in the CF population worsens the injury response to MI and that blocking 5-HT_2B signaling after injury is sufficient to improve cardiac outcomes.

Figure 5. Htr2b deletion in activated myofibroblasts abates impact of myocardial infarction (MI).

A, Left Schematic illustrating the Postn locus harboring a tamoxifen-inducible MerCreMer (MCM) cDNA, tdTomato reporter in the Rosa26 locus preceded by a loxP-flanked stop codon, and a loxP-flanked Htr2b target allele. Middle Experimental timeline for 11–14-week-old mice. Right Key. B, left ventricular ejection fraction (LV EF). C, left ventricular fractional shortening (LV FS). D,E, Left ventricular internal dimension at end-diastole (LVID;d) and end-systole (LVID;s). F,G, Left ventricular volume at end-diastole (LV Vol;d) and end-systole (LV Vol;s). H, Global longitudinal strain (GLS). I, Heart weight (HW) normalized to tibia length (TL). B-I, Mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 between Htr2b^fl/fl and Htr2b^fl/flPostn^MCM/+, #P<0.05, between timepoints within genotype following (B-H) 2-way ANOVA and Holm-Sidak post hoc test or (I) 2-tailed Student t test. Number of mice analyzed denoted in B applies to all subsequent data.

5-HT_2B knockout decreases CF proliferative and remodeling capacity

In order to investigate how periostin-driven ablation of 5-HT_2B imparts beneficial outcomes following MI, we performed RNA sequencing on PDGFRα+ cells isolated via FACS from the scar tissue of infarcted hearts seven days after MI (Figure XVI in the Supplement). Despite only about 20% of PDGFRα+ cells being tdTomato+ in the Htr2b^fl/flPostn^MCM/+ group (indicating 5-HT_2B knockout), we observed differential expression of 63 genes which, following Gene Ontology (GO) analysis, were overwhelmingly associated with biological processes linked to the control of cell cycle and mitosis as well as individual genes (i.e. Nexn, Robo4, Fgf23, and Arhgap5) governing cell migration and remodeling (Figure 6A, Table II in the Supplement). One such gene of interest that was upregulated in the Htr2b^fl/flPostn^MCM/+ group was the tumor suppressor Dnajb4 which reduces the invasive and metastatic behavior cellular phenotype in lung cancer.^28,29 In isolated 5-HT_2B^−/− CFs, it was observed that loss of 5-HT_2B resulted in an increased expression of Dnajb4 (Figure 6B). As previous reports have shown Dnajb4 controls the activation of Src²⁸, we probed Src phosphorylation in CFs and observed a decreased phosphorylation of Src in 5-HT_2B^−/− CFs (Figure 6C, Figure XVII in the Supplement). The decrease in these molecular mediators of cell proliferation and invasion led us to investigate the capabilities of CFs to populate the wounded tissue after MI. We observed a decreased area fraction of periostin staining in the infarct of SB-treated mice one week after injury, indicative of decreased myofibroblast proliferation and infiltration (Figure 6D, Figure XVIII in the Supplement). Similarly, in vitro analysis of 5-HT_2B^−/− CFs revealed a decreased proliferative capacity as seen by fewer cells incorporating BrdU (Figure 6E). CFs lacking 5-HT_2B demonstrated an approximate 50% decrease in migratory capacity in an in vitro wound healing assay (Figure 6F). Finally, CFs were seeded into free-floating collagen gels in order to test their ability to remodel a collagen matrix. In gels seeded with 5-HT_2B^−/− CFs, gel contraction after 72 hours was significantly hindered (Figure 6G). These results point to a mechanism of 5-HT_2B-mediated myofibroblast proliferation and matrix remodeling leading to adverse scar formation following MI.

Figure 6. Htr2b deletion impairs proliferative and remodeling capabilities of cardiac fibroblasts (CFs).

A, Volcano plot highlighting differentially regulated genes in PDGFRα+ cells isolated from Htr2b^fl/flPostn^MCM/+ (N=4) compared to Htr2b^fl/fl animals (N=2). B, Increased expression of Dnajb in 5-HT_2B-knockout CFs (N=3). C, Phosphorylated Src is decreased in isolated CFs lacking 5-HT_2B (normalized to total Src and loading control α-tubulin, N=4). D, Periostin immunostaining reveals a decrease in myofibroblast presence in damaged tissue one week after MI (N=4). E, 5-HT_2B-knockout CFs exhibit decreased proliferation measured by BrdU incorporation (N=3). F, 5-HT_2B-knockout CFs are less migratory than WT counterparts (N=3). G, Free-floating collagen gel contraction assay reveals a decrease in collagen matrix remodeling by 5-HT_2B-knockout CFs (N=6). B-G, Mean ± SEM, *P<0.05, following (B-E) 2-tailed Student t test or (F,G) 2-way ANOVA and Holm-Sidak post hoc test.

Discussion

Experimental MI induced by permanent coronary artery occlusion triggers the expansion and activation of resident CFs from their quiescent, homeostatic state. CFs transdifferentiate into highly active myofibroblasts following injury, migrating to and proliferating at the site of tissue damage to secrete ECM and contract scar tissue.³⁰ The quality of initial scar formation has lasting effects on cardiac outcomes. A structurally sufficient scar is necessary to reinforce the LV wall, but persistent fibrotic activity leads to chronic cardiac deterioration.^10,31 Therefore, it is desirable to properly tune the fibrotic response following MI such that a functional scar is able to form but is dampened before the activity of myofibroblasts becomes deleterious.

Prior studies have shown anti-fibrotic effects of 5-HT_2B disruption in various cardiopulmonary pathologies.^{18–20,32,33} To our knowledge, this study is the first investigation to report the direct contribution of 5-HT_2B signaling in wound healing after MI. Through the implementation of two pharmacological inhibitors and two models of genetically targeted ablation, we have shown that the 5-HT_2B receptor is an effective target to limit fibrosis following MI injury in mice. Due to advances in identification of genetic markers for CFs, we were able to isolate myofibroblasts as the cell population responsible for the improved recovery after MI seen in animals treated with a 5-HT_2B antagonist.

In the present study, echocardiographic analysis revealed global 5-HT_2B antagonism improves cardiac structure and function one week after MI versus vehicle treatment. While there were not significant changes in these metrics between one and six weeks after injury, GLS deteriorated from week one to six in the control groups whereas 5-HT_2B inhibition stabilized this measure of cardiac contractility. GLS has been shown to be an independent predictor of adverse remodeling after ST-elevated MI in humans and can be a more sensitive functional output than the traditionally used EF.³⁴

We further explored changes to tissue architecture which led to an improvement in cardiac function and alterations in scar formation. A heavier collagen burden increases passive tissue stiffness, heightening afterload and hindering systolic function.³⁵ 5-HT_2B blockade successfully decreased collagen burden, indicated by decreased scar thickness, without negatively affecting survival rate. The BZ is a vulnerable region of tissue which experiences heightened wall stress and where scar expansion occurs, further damaging cardiomyocytes not directly affected by the initial ischemic event.^12,36 We found that targeting 5-HT_2B resulted in an increased transition rate from scar tissue to surviving myocardium (i.e. less BZ region), revealing decreased intrusion of collagen fibers from the scar into uninjured myocardium. The prevention of scar expansion can minimize the disruption of the cardiac syncytium, providing a more coordinated systolic cycle.³⁷ Similar to reports linking 5-HT_2B to collagen content and composition through activation of lung and valve fibroblasts, ^33,38 our results show a redistribution of collagen fiber thickness in the BZ, favoring less mature, more compliant collagen fibers. This difference was not seen in the scar, further suggesting the formation of a mechanically sound scar with limited capacity to expand beyond the BZ. The collagen fibers in BZs of SB-treated animals exhibited more frequent regions of anisotropic collagen fiber distributions which are able to undergo elastic deformation along with cardiomyocyte contraction and increase LV contraction.³⁹ This effect is enhanced by the softer scar and BZ. Since these effects prevented early hypertrophic signs observed in control animals, 5-HT_2B antagonism reveals a desirable mechanical and biophysical outcome after MI achieved by a muted initial fibrotic response that provides adequate scar formation without hindering the systolic capabilities of the heart.

It is clear that the translational approach of pharmacological inhibition of 5-HT_2B signaling is effective to establish a therapeutic effect on scar formation and gives insight into the mechanistic alterations of collagen deposition and remodeling, but the lack of specificity renders it insufficient to identify the cell population which mediates the observed effect. Therefore, we implemented several models to manipulate 5-HT_2B in different cell populations. Cardiomyocytes express 5-HT_2B, and blockade of this receptor can partially mitigate noradrenaline overload-induced hypertrophy.⁴⁰ Cardiomyocytes isolated seven days after infarct do show an increase in Htr2b expression, but only to a fraction of the degree to which expression is increased in bulk tissue. CD31+ cells neither exhibited increased Htr2b gene expression after infarct nor altered gene expression for the marker of endothelial activation, E-selectin. Similarly, Htr2b expression in CD45+ cells did not change after MI, and bone-marrow transplants from 5-HT_2B-knockout mice did not recapitulate the results from antagonist studies, pointing to a mechanism different from previous reports exploring fibrotic remodeling in pulmonary disease.^18,19 Therefore, we developed a novel model of CF-specific 5-HT_2B ablation using Tcf21-driven Cre expression. This approach circumvented developmental defects seen with gestational ablation of 5-HT_2B;²⁶ however, it could also have imparted benefits prior to MI. Regardless, eliminating 5-HT_2B from resident, Tcf21-expressing CFs resulted in a vastly improved cardiac phenotype compared to control animals. Similar improvements as the antagonist studies were achieved with even more pronounced structural benefits observed in the improvement in both LV systolic and diastolic metrics, as well as a decreased heart weight. The enhanced effects of the genetic approach over the pharmacological approach are most likely due to a sufficient recombination of the Htr2b gene locus to convey a stronger effect than what is achieved by transient binding of the antagonist which is subject to metabolic processing and removal from the system.

To implement an approach which more closely replicates the pharmacological strategy, we utilized a Postn-driven model of 5-HT_2B ablation. Myofibroblasts, which are overwhelmingly derived from a Tcf21 lineage,⁵ are absent in healthy tissue and populate the infarct zone 2–4 days after injury.¹³ Targeting this specialized cell type achieved nearly identical results as the Tcf21-driven model and further hones in on the population affected by 5-HT_2B antagonism. Knowing that myofibroblasts are derived from resident fibroblasts, the overlapping results importantly demonstrate that targeting injury-responsive myofibroblasts without affecting their progenitor cells is sufficient to affect scar formation after MI. The mechanistic implication of this result is that 5-HT_2B-dependent infiltration and scar remodeling occurs after the transdifferentiation of resident CFs. Furthermore, it demonstrates the redundancy in the two genetic models where targeting either resident CFs in the absence of injury or myofibroblasts derived from resident CFs achieves the same result.

While only the contribution of bone marrow-derived populations was explicitly eliminated, initial evidence reveals that the contribution of cardiomyocyte or endothelial expression of 5-HT_2B plays a negligible role in improving scar formation after MI. We reported a dramatic increase in Htr2b expression after MI which cannot be accounted for by cardiomyocytes, CD31+, and/or CD45+ cells. Considering the utility of the CF-specific genetic ablation of 5-HT_2B, we are confident that this increase is due to CFs. Pericytes cannot be excluded as potential contributors as these cells express Tcf21 and function in a multiplicity of roles involved in MI healing.^6,8,41 The periostin-driven model of 5-HT_2B ablation in myofibroblasts should circumvent pericyte contribution, and so we are confident that cells which produce and organize collagen are the primary effector cells. While cardiomyocyte expression of Htr2b is increased after MI, it accounts for a small fraction of the total increase. In healthy hearts, cardiomyocytes are bountiful with CFs making up a small portion of cells.¹ While these populations shift after injury, it is still conceivable that the contribution of cardiomyocyte 5-HT_2B is minimal since a more abundant cell population is a minor contributor with mesenchymal cells accounting for the vast majority of Htr2b expression. In addition, reports show blocking 5-HT_2B signaling after injury may reduce cardiomyocyte hypertrophy,^40,42 providing additional protection in the antagonist studies. Regardless, cardiomyocyte expression of 5-HT_2B is intact with the Tcf21- and Postn-driven 5-HT_2B ablation models, further supporting that the alteration in scar formation and cardiac outcomes is mediated by a fibroblast cell population. Because of this evidence, utilization of 5-HT_2B antagonists provides an approach to further investigate the mechanism behind improved cardiac outcomes. Since myofibroblasts are not immediately present after MI, it may not be necessary to begin treatment instantly after injury. Furthermore, since echocardiographic readouts were improved as early as seven days after injury, it is possible that 5-HT_2B inhibition during the acute healing phase of initial scar formation is sufficient to preserve cardiac performance.

It is interesting that 5-HT_2B antagonism was ineffective in females. It has been shown that global knockout of 5-HT_2B results in more severe histopathological lesions and a stronger systolic dysfunction in adult male mice compared to females as a result of impaired cardiac development.²⁶ Furthermore, sex-specific differences in hepatocellular carcinoma has been attributed to increased serotonin production in males compared to females, resulting in a 5-HT_2B-mediated increase of fibrogenic hepatic stellate cells density.⁴³ It must be noted that both males and females were included in both genetic models, and both sexes responded similarly to 5-HT_2B ablation. While genetic manipulation is sufficient to improve outcomes in females, these observations indicate that there is a biological difference in the way that female mice respond to 5-HT_2B antagonism, potentially through metabolic processing of the molecule or ligand/receptor interactions.

RNA sequencing revealed 5-HT_2B as a regulator of proliferation and matrix remodeling in CFs. We employed a surrogate CF marker PDGFRα to select for CFs, and despite only 20% of these cells displaying the tdTomato fluorescent reporter in the Htr2b^fl/flPostn^MCM/+ group, we still observed differential regulation of multiple genes in myofibroblasts with 5-HT_2B ablation. Supported by differences in associated GO terms, we primarily investigated CF proliferation as well as ECM remodeling. We saw upregulation of genes such as Plagl1 which inhibits cell growth and proliferation, potentially through PPARγ,⁴⁴ and Nexn whose loss is associated with dilated cardiomyopathy.⁴⁵ Several interesting transcripts were also downregulated: Lrp2 which increases proliferation of epicardial cells (CF precursors),⁴⁶ Fgf23 which demonstrates broad mitogenic and cell survival actions specifically in the heart,⁴⁷ and Robo4 which is associated with increased matrix metalloproteinase expression and predisposition for aortic valve disease.⁴⁸ Of note, we have shown that in CFs lacking 5-HT_2B, Dnajb4 is upregulated. It is known that the heat shock protein encoded by this gene is highly expressed in human CFs.⁴⁹ There is a direct link to increased expression of this protein and the inhibition of Src,²⁸ and consistent with this prior report, we found decreased phosphorylation of Src in 5-HT_2B^−/− CFs. Dnajb4 is known to control cell proliferation and migration, supporting our observations in which myofibroblasts were sparser in damaged tissue of SB-treated animals and 5-HT_2B knockout decreased proliferation, migration, and collagen matrix remodeling. The known link between Dnajb4, Src phosphorylation, and cell proliferation and remodeling capacity provides a mechanistic insight into the improved phenotype observed with 5-HT_2B inhibition. Blocking 5-HT_2B signal transduction in the beginning stages of scar formation hinders the proliferation and migration of myofibroblasts into the infarct zone through increasing Dnajb4 expression, which, in turn, inhibits endogenous activation of Src. This approach allowed myofibroblasts to form a functional scar while avoiding a mechanical environment that predisposes the tissue to scar expansion and hypertrophy.

Due to the complexities associated with the intricate coordination involved in the inflammatory response post-MI, we set out to target the effector cells of fibrosis (i.e. CFs) to control the initial reparative response and limit adverse fibrotic remodeling. Targeting 5-HT_2B on CFs can control scar mechanics and limit fibrosis without affecting scar stability.⁵⁰ Further, inhibiting the activity of 5-HT_2B has an acute benefit that is sustained well beyond the initial healing phase and demonstrates that cardiac hypertrophy subsequent to an ischemic event can be curtailed. Taken together, this work has identified 5-HT_2B as a potential therapeutic target for muting the over activity of myofibroblasts following MI to preserve cardiac phenotype and prevent the initiation and progression of cardiac fibrosis and heart failure.

Clinical Perspective.

What is new?

• Antagonism of the serotonin 2B receptor (5-HT_2B) improves outcomes after myocardial infarction through limiting the fibrotic process of scar formation.

• Biomechanical characterization of the scar and adjacent border zone provides useful insight into cardiac fibroblast-mediated fibrosis which results in the associated echocardiographic metrics of tissue structure and function.

• Using pharmacological and genetic approaches, this study pinpoints 5-HT_2B expression in myofibroblasts as a regulator of cell proliferation and invasion after myocardial infarction.

What are the clinical implications?

• This study suggests that early inhibition of 5-HT_2B signaling after myocardial infarction is sufficient to optimize scar formation, resulting in a functional scar which is less likely to expand beyond the initial infarct and cause long-term remodeling.

• Prolonged presence of the antagonist was not required to maintain the benefits observed in the early stages after injury, indicating that acute treatment can alter chronic remodeling.

• 5-HT_2B blockade does not negatively affect contribution to the healing response after MI provided by non-cardiac fibroblast cell types.

Nonstandard Abbreviations and Acronyms

5-HT_2B

serotonin 2B receptor

AFM

atomic force microscopy

BZ

border zone

CFs

cardiac fibroblasts

DMSO

dimethyl sulfoxide; vehicle control

ECM

extracellular matrix

FOV

field of view

GLS

global longitudinal strain

Htr2b^fl/fl

floxed Htr2b control

Htr2b^fl/flPostn^MCM/+

inducible myofibroblast ablation of Htr2b

Htr2b^fl/flTcf21^MCM/+

inducible fibroblast ablation of Htr2b

Nppb

natriuretic peptide B

RS

RS127445; 5-HT_2B antagonist

SB

SB204741; 5-HT_2B antagonist

SHG

second harmonic generation