Analysis of miR-411 downstream targets uncovers contrasting roles of serotonin signalling in cardiomyocyte regeneration and post-ischaemic remodelling

Abstract Stimulation of adult cardiomyocytes (CMs) to re-enter the cell cycle has emerged as a promising strategy for repairing the heart after myocardial infarction (MI). Our previous work showed that miR-411 enhances CM proliferation and improves cardiac function post-MI by activating YAP, a key effector of the Hippo pathway (1). However, how exactly miR-411 suppresses Hippo signalling remains unresolved. Here, we aimed to define the downstream actions of miR-411 and assess whether modifying its target genes could enhance cardiac repair. MicroRNA target screening identified the serotonin transporter gene (SERT) as a key miR-411 target in CMs and in hearts overexpressing miR-411. SERT expression was significantly reduced both in vitro (cultured CMs) and in vivo (mouse hearts) following mir-411 stimulation. Furthermore, a luciferase-based miRNA binding assay confirmed direct interaction between miR-411 and the exon 4 of SERT. Consistent with findings from the miR-411 overexpression model, siRNA-mediated SERT knockdown increased YAP activity and promoted CM proliferation and survival. To understand how SERT influences YAP, we drew on evidence that loss of SERT raises extracellular serotonin (5-HT), leading to increased 5-HT receptor activation (2). Since previous evidence has shown that most 5-HT receptors are G protein-coupled (3) and can enhance YAP signalling by reducing LATS1 phosphorylation through ROCK1 (4), we proposed that SERT depletion triggers this pathway. Consistent with this, SERT-deficient CMs displayed elevated phosphorylation of MYPT1, a downstream ROCK1 target, indicating that SERT might regulate YAP via ROCK1-dependent signalling. We then examined whether inhibiting SERT expression in vivo benefits recovery following cardiac ischaemia–reperfusion (I/R) injury. AAV9 vectors encoding shRNAs against SERT under the cTnT promoter (AAV9-shSERT) were given intravenously to mice following I/R. Unexpectedly, AAV9-shSERT treatment resulted in impaired cardiac function five weeks post I/R, with reduced ejection fraction and fractional shortening. Immunofluorescence staining showed increased proliferation markers in both CMs and cardiac fibroblasts (CFs). Histological analysis also revealed enlarged CMs and increased fibrosis, indicating that CM-specific SERT knockdown worsened, rather than improved, left ventricular remodelling. Taken together, our data suggest that SERT has a more complex role in cardiac remodelling than previously recognised. Although its reduction activates YAP and appears beneficial in isolated CMs, the in vivo environment allows additional serotonin-responsive cell types, particularly CFs, to contribute to the outcome. Excess 5-HT following SERT knockdown may therefore intensify ROCK1-YAP signalling in CFs, promoting fibrosis and worsening ventricular function. Understanding how these cell-specific responses diverge will be essential for refining strategies aimed at harnessing YAP activity for cardiac regeneration.