The Role Of Epicardial CCM Signaling In Cardiac Development And Regeneration

Ischemic heart disease remains the leading cause of death worldwide.Detailed knowledge of the roles of various cell types in the developing heart is key to achieving a proper understanding of cardiovascular development.The developing heart is divided into three layers,the endocardium,the myocardium and the epicardium.The epicardium plays a crucial role in embryonic heart development and adult heart repair.The epicardial cells differentiate into smooth muscle cells（SMCs）and cardiac fibroblasts（CF）,contributing to heart function.The epicardium also produce multiple growth factors（GFs）for cell communication and myocardium coronary vasculature development.Cerebral cavernous malformations（CCMs）are sporadic or autosomal dominantly inherited vascular malformations in the brain and loss-of-function mutations in three genes,CCM1,CCM2 and CCM3,have been identified to cause human CCMs.A role for CCM signaling in the developing heart was revealed by zebrafish embryos lacking ccm1,ccm2,ccm3 and heg that exhibited a characteristic dilated heart phenotype.Endocardial CCM signaling regulates the MEKK3-KLF-ADAMTS signaling pathway to control endothelial gene expression during cardiovascular development,loss of CCM signaling in endocardial cells results in heart failure associated with pronounced degradation of cardiac jelly.However,a role of CCM signaling in the epicardium has not been explored and the molecular events of epicardial CCM signaling underlying heart development remain unknown.This study aims to identify the role of epicardial CCM signaling in heart development and regeneration.Here,we analyzed the phenotypes and genotypes at different developmental stages of control and epicardial-specific Ccm1/2/3 knockout mice.Wt1^GFPCre-Mediated epicardial deletion of Ccm1/2/3 led to embryonic lethality during midgestation.Hematoxylin and eosin staining（H&E）of Ccm2 deleted hearts at E12.5 revealed thin myocardium and cardiac non-compaction compared with littermate controls.Employing an immunohistochemistry approaches,we demonstrated that deletion of Ccm2 leads to a reduction in epicardial cell differentiation and migration into myocardium.With isolation and culture of a population of epicardial cells during heart development,we identified Ccm2 play a role in epicardial differentiation and cell shape regulation.Transcriptomic and cell morphology analyses of epicardial cells from epicardial-specific Ccm2 deletion mice revealed a change of extracellular matrix assembly,cell-substrate adhesion,actin filament organization.A key mechanism for regeneration was the activation of the epicardium,leading to the establishment of a supporting scaffold for new cardiomyocytes,angiogenesis and cytokine secretion.Injury of the mammalian heart leads to irreversible loss of contractile myocardial tissue which is incapable of regeneration.The neonatal mouse can regenerate its heart after MI up to 7 days after birth.This acute episode culminates in sudden death of cardiomyocytes in the infarcted heart,which rapidly triggers innate immune pathways responsible for the clearance of dead cells,remodeling of extracellular matrix and the activation of collagen-producing myofibroblasts.The recovery of both the epicardium and the endocardium is achieved rapidly after injury and precedes myocardial regeneration.The activated epicardium differentiates into SMCs and CFs and secretes factors that promote proliferation of myocardium.In this study,we demonstrated a novel role for CCM signaling in post-MI epicardial derivatives through modulates extracellular matrix assembly,cell-substrate adhesion,actin filament organization to the injured myocardium.These studies were performed in adult and neonate mouse models following experimental myocardial infarction.To define the regeneration capacity of neonatal mouse heart,epicardial Ccm2 mutant mice demonstrated increase fibrosis and fail to regeneration.To elucidate the role of epicardial CCM signaling in post-MI recovery,we induced Ccm2 deletion in adult murine epicardium using the Wt1^{Cre ERT2} allele,with tamoxifen treatment following MI injury.Deletion of Ccm2 in epicardium decreased cardiac systolic function and increased fibrosis.Collectively,these results suggested that epicardial CCM signaling plays a key role in heart development and regeneration after MI.Ccm2 in epicardium augmented cardiomyocyte function,making it a promising adjuvant therapy in regenerative medicine.