Hypoxia-responsive ZEB2 regulates a network of calcium handling genes in the injured heart
Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable improvement in managing the progression of heart disease, developing effective therapies for heart failure (HF) remains a challenge. A better understanding of molecular mechanisms that maintain proper Ca2+ levels and contractility in the injured heart could be of therapeutic value.  Here, we report that the transcription factor Zinc finger E-box-binding homeobox2 (ZEB2) is induced by Hypoxia-inducible factor 1-alpha (HIF1a) in hypoxic cardiomyocytes and regulates a network of genes involved in Ca2+-handling and contractility during ischemic heart disease. Gain- and loss-of-function studies in genetic mouse models revealed ZEB2 in cardiomyocytes to be necessary and sufficient to protect the heart against ischemia-induced diastolic dysfunction and structural remodeling. Moreover, RNA sequencing (RNA-seq) of ZEB2-overexpressing (Zeb2 cTg) hearts post-injury implicated ZEB2 in regulating numerous Ca2+-handling and contractility-related genes. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban (PLN) at both serine-16 and threonine-17, implying enhanced activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), thereby augmenting SR Ca2+ uptake and contractility. Furthermore, we observed a decrease in the activity of Ca2+-dependent calcineurin/NFAT signaling in Zeb2 cTg hearts, which is the main driver of pathological cardiac remodeling. On a post-transcriptional level, we showed that ZEB2 expression can be regulated by the cardiomyocyte-specific microRNA-208a (miR-208a). Blocking the function of miR-208a with antimiR-208a increased ZEB2 expression in the heart and effectively protected from the development of pathological cardiac hypertrophy. Together, we present ZEB2 as a central regulator of contractility and Ca2+-handling components in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in regulating Ca2+ homeostasis in cardiomyocytes is an essential step towards the development of improved therapies for HF. Overall design: To investigate the effect of ischemic injury on cardiac gene expression, we performed Ischemia/Reperfusion (IR) injury and isolated infarct area plus border zone tissue from hearts 14 days after injury. RNA-seq was performed on tissue from three animals per group, from Sham treated WT C57Bl/6J and 14 days post IR injury. To investigate the effect of ZEB2 overexpression, we performed IR injury on ZEB2 overexpressing hearts and wildtype littermate controls and harvested infarct area plus border zone 14 days after injury. RNA-seq was performed on tissue from three animals per group.