KLF15-Wnt-Dependent Cardiac Reprograming Up-Regulates SHISA3 in the Mammalian Heart

Sustained cardiac stress promotes the transition from an adaptive response to heart failure. Understanding of mechanisms governing this transition will assist in identifying targets that prevent this progression. Our study revealed age-specific transcriptional functions mediated by KLF15 that are crucial for cardiac homeostasis. We report that postnatally, KLF15 continuously activates cardiac metabolism, but represses pathological, hypertrophic pathways associated with cardiomyocyte de-differentiation and endothelial cell (EC) remodeling in an age-dependent manner. Our integrative genomic and transcriptomic analyses identified novel target genes directly bound, and either activated or repressed by KLF15 in vivo in the adult heart. We identified a cooperative program inducing aberrant EC remodeling, caused by a reduction of KLF15 and a concomitant activation of Wnt signaling. Within this program, we further identified a so far uncharacterized cardiac gene - Shisa3, which is expressed in the developing heart and is upregulated in cardiac hypertrophy, ischemia and failure. Importantly, we demonstrate that the KLF15- and Wnt co-dependent, SHISA regulation occurs also in the human myocardium. Altogether, our results unraveled and characterized a previously unknown cardiac gene Shisa3, and attributed its significance to EC homeostasis of the adult heart, controlled by KLF15-Wnt dynamics. Purpose: The aim of this study was to compare transcriptome profiles (RNA-seq) of heart tissue with a WT or KO Klf15 locus at different murine ages - postnatal day10 (p10), 4-week-old and 20-week-old mice. Methods: Cardiac tissue total RNA profiles for different groups were obtained using deep sequencing, in triplicates, using Illumina HiSeq4000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat, followed by DESeq2. qPCR validation was performed using TaqMan and SYBR Green assays. Conclusions: Our study represents the first detailed analysis of the processes triggered upon Klf15 loss in hearts of different murine postnatal ages, which was so far not investigated. We report that this Klf15 loss first triggers Wnt canonical pathway activation, followed by activation of the non-canonical Wnt components, culminating in heart failure. Gene expression profiling from cardiac ventricles of P10, 4-week-old and 20-week-old mice with Klf15 wild type and loss of function.持续的心脏应激可促使适应性心脏应答向心力衰竭转化。阐明调控这一疾病转化的分子机制，将有助于识别阻断疾病进展的干预靶点。本研究揭示了由Kruppel样因子15（KLF15）介导的、对心脏稳态至关重要的年龄依赖性转录调控功能。我们发现，在出生后阶段，KLF15持续激活心脏代谢通路，但以年龄依赖的方式抑制与心肌细胞去分化及内皮细胞（endothelial cell, EC）重塑相关的病理性肥大通路。本研究的整合基因组学与转录组学分析，鉴定出成年心脏体内由KLF15直接结合并激活或抑制的新型靶基因。我们还发现了由KLF15表达下调伴随Wnt信号通路（Wnt signaling）激活所诱导的异常内皮细胞重塑协同调控程序。在该调控程序中，我们进一步鉴定出一个此前未被表征的心脏基因——Shisa3，其在发育中的心脏中表达，并在心肌肥大、心肌缺血及心力衰竭中表达上调。重要的是，我们证实了KLF15与Wnt共同调控的Shisa3表达机制同样存在于人类心肌组织中。综上，本研究解析并表征了此前未知的心脏基因Shisa3，并阐明其在成年心脏内皮细胞稳态中的重要作用，该过程受KLF15-Wnt信号动态平衡调控。 研究目的：本研究旨在比较不同月龄小鼠（出生后第10天（p10）、4周龄及20周龄）中，野生型（WT）与Klf15基因敲除（KO）小鼠心脏组织的转录组谱（RNA测序（RNA-seq））。 实验方法：本研究通过Illumina HiSeq4000平台，对各组小鼠心脏组织的总RNA进行三次生物学重复的深度测序以获取转录组数据。对通过质量过滤的测序读段，采用TopHat进行转录本异构体水平分析，随后使用DESeq2开展差异表达分析。采用TaqMan探针法与SYBR Green染料法进行qPCR验证。 研究结论：本研究首次详细分析了不同出生后年龄小鼠心脏中Klf15缺失所触发的生物学过程，这一研究主题此前尚未被探索。我们发现，Klf15缺失首先激活Wnt经典通路，随后激活非经典Wnt通路组分，最终诱发心力衰竭。本研究获取了P10、4周龄及20周龄Klf15野生型与功能缺失型小鼠心室的基因表达谱数据。