Slit2 protects hearts against ischemia-reperfusion injury by inhibiting inflammatory responses and maintaining myofilament contractile properties. Slit2 protects hearts against ischemia-reperfusion injury by inhibiting inflammatory responses and maintaining myofilament contractile properties

Background The axon guidance cue Slit2 recently has been found to regulate calcium homeostasis and molecular signaling in various stress events in different organs. However, whether Slit2 plays a role in cardiac ischemia-reperfusion (IR) injury has not been reported. Here, we aimed to investigate the role of Slit2 and the underlying mechanisms in cardiac IR injury. Methods Langendorff-perfused isolated hearts from Slit2-overexpressing (Slit2-Tg) mice and their background strain C57BL/6J mice were subjected to 20 min of global ischemia followed by 40 min of reperfusion. Left ventricular function of isolated hearts was monitored. Infarct size of post-IR hearts was determined by staining with 2,3,5-triphenyltetrazolium chloride (TTC) and histological changes of cardiac tissues and cells were determined with hematoxylin-eosin (HE) staining and transmission electron microscopy. Transcriptomic analysis was used to predict the biological processes and signaling pathways affected by Slit2 overexpression in the post-IR myocardium. Pro-Q staining and Western blotting was used to assess the phosphorylation levels of cardiac myofilaments and expression levels of myofilament-associated protein kinase and phosphatases. Results Slit2 overexpression increased post-IR left ventricular developed pressure (LVDP) by 35% and reduced infarct size by 53%, along with decreased myofibrillar disruption, mitochondrial swelling, and mitochondrial cristae dissolution. Slit2 overexpression significantly changed post-IR gene expression profiles. Functional products of these genes include regulation of cation transmembrane transport, cation homeostasis, collagen fibril organization, and regulation of heart rate. And post-IR myocardial KEGG pathways upregulated by Slit2 overexpression include ECM-receptor interaction, PI3K-Akt signaling pathway, and adrenergic signaling. Slit2 overexpression impacted myofilament phosphorylation together with myofilament-associated protein kinase C (PKC) isoforms and protein phosphatases (PPs). IR in C57BL/6J hearts upregulated phosphorylation of cardiac troponin-I (cTnI), which was suppressed by Slit2 overexpression. Myofilament‐associated PKCε, PKCδ, and PP2A were significantly increased post‐IR in C57BL/6J hearts, but in Slit2‐Tg hearts, myofilament‐associated PKCε and PP2A were increased and PKCδ was suppressed. Conclusions Our results demonstrate that Slit2 overexpression protects cardiac function and reduces IR injury, which is associated with Slit2‐induced gene profile shifts. The suppression of MyBP‐C and troponin‐I phosphorylation, and myofilament‐associated PKCδ levels induced by Slit2 overexpression could contribute to the cardioprotection of Slit2 in post-IR myocardium. Overall design: Total RNAs from C57BL/6J and Slit2-overexpressing (Slit2-Tg) hearts after IR or non-IR were isolated using TRIzol® Reagent. Each group contains three independent samples.Subsequently, a total amount of 3 µg RNA per sample were used for transcriptome sequencing.

背景：轴突导向因子Slit2近期被发现可在不同器官的多种应激事件中调控钙稳态与分子信号通路。然而，Slit2是否参与心肌缺血再灌注（ischemia-reperfusion, IR）损伤的调控尚未见报道。本研究旨在探讨Slit2在心肌IR损伤中的作用及其潜在分子机制。
方法：本研究取过表达Slit2的小鼠（Slit2-Tg）及其背景品系C57BL/6J小鼠的Langendorff灌流离体心脏，建立20 min全心缺血后再灌注40 min的IR模型。监测离体心脏的左心室功能；采用2,3,5-三苯基氯化四氮唑（TTC）染色法测定IR后心脏的梗死面积；通过苏木精-伊红（HE）染色与透射电子显微镜观察心肌组织及细胞的组织学变化。采用转录组学分析预测IR后心肌中Slit2过表达所影响的生物学过程与信号通路；采用Pro-Q染色与蛋白质印迹（Western blotting）检测心肌肌丝的磷酸化水平，以及肌丝相关蛋白激酶与磷酸酶的表达水平。
结果：Slit2过表达可使IR后小鼠心脏的左心室发展压（LVDP）提升35%，并使梗死面积减少53%，同时可减轻肌丝断裂、线粒体肿胀及线粒体嵴溶解程度。Slit2过表达显著改变了IR后心肌的基因表达谱，这些差异基因的功能富集涉及阳离子跨膜转运调控、阳离子稳态维持、胶原纤维组织构建以及心率调控等生物学过程。Slit2过表达上调的IR后心肌京都基因与基因组百科全书（KEGG）通路包括细胞外基质-受体相互作用、PI3K-Akt信号通路以及肾上腺素能信号通路。Slit2过表达可影响心肌肌丝的磷酸化水平，同时调控肌丝相关蛋白激酶C（PKC）亚型与蛋白磷酸酶（PPs）的表达。在C57BL/6J小鼠心脏中，IR可上调心肌肌钙蛋白I（cTnI）的磷酸化水平，而该现象可被Slit2过表达抑制。IR后C57BL/6J小鼠心脏的肌丝相关PKCε、PKCδ及PP2A水平显著升高；但在Slit2-Tg小鼠心脏中，肌丝相关PKCε与PP2A水平升高，而PKCδ的表达被抑制。
结论：本研究结果表明，Slit2过表达可保护心肌功能并减轻IR损伤，这与Slit2诱导的基因表达谱改变密切相关。Slit2过表达所介导的肌丝结合蛋白C（MyBP-C）与肌钙蛋白I磷酸化水平的抑制，以及肌丝相关PKCδ水平的下调，可能是Slit2对IR后心肌产生心脏保护作用的重要机制。
整体实验设计：本研究从IR处理及未处理的C57BL/6J小鼠与Slit2过表达（Slit2-Tg）小鼠心脏中提取总RNA，所用试剂为TRIzol®试剂；每组设置3个独立生物学重复。随后，每个样本取3 μg总RNA用于转录组测序。