BACKGROUND:Recently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3’s relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress.METHODS:We performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling.RESULTS:We first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3’s hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction.CONCLUSIONS:Collectively, these results indicate a novel role for the SLIT3-ROBO1–signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

中文翻译：

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基质细胞-SLIT3/心肌细胞-ROBO1轴调节压力过载引起的心肌肥厚

背景：最近被证明可以调节心脏发育，分泌的轴突引导分子 SLIT3 在出生后的心脏中维持其表达。尽管已知 SLIT3 在出生后的心血管系统中表达，但 SLIT3 与出生后状态心血管功能的相关性仍不清楚。因此，本研究的目的是确定 SLIT3 的出生后心肌来源，并评估其在调节心脏对压力超负荷应激反应中的功能作用。 方法：我们对患者的心肌细胞和心肌组织样本进行了体外研究，并进行了使用接受横向主动脉缩窄的 SLIT3 和 ROBO1（roundabout 同源物 1）突变小鼠进行体内研究，以确定 SLIT3-ROBO1 在不良心脏重塑中的作用。 结果：我们首先发现先天性心脏病患者的心肌组织中 SLIT3转录增加导致心室压力超负荷的缺陷。对 WT（野生型）和报告小鼠心脏的免疫染色显示，SLIT3 由心脏内的心脏基质细胞（即成纤维细胞和血管壁细胞）分泌。来自心脏成纤维细胞和血管壁细胞的条件培养基均在体外刺激心肌细胞肥大，这种效应被抗SLIT3抗体部分抑制。此外，SLIT3 的 N 端而非 C 端片段和 SLIT3 的强制过度表达刺激心肌细胞肥大和肥大相关基因的转录。接下来我们确定 ROBO1 是心肌细胞中表达最高的迂回受体，并且 ROBO1 在体外介导 SLIT3 的肥大作用。在体内，小鼠中 Tcf21+ 成纤维细胞和 Tbx18+ 血管壁细胞特异性敲除 SLIT3 可减少主动脉横缩后的左心室肥厚和心脏纤维化。此外，在主动脉横缩后，α-MHC+心肌细胞特异性删除ROBO1也保留了左心室功能并消除了肥厚，但没有纤维化。结论：总的来说，这些结果表明SLIT3-ROBO1信号轴在调节产后的新作用压力超负荷引起的心肌细胞肥大。