Purpose:

Cardiovascular diseases (CVDs) remain the leading cause of death globally and cause a heavy socio-economic burden. Current therapies targeting CVDs are mostly symptomatic or supportive treatments which have little effects to actually treat or reverse the diseases; therefore, novel therapeutic strategies are urgently needed. Long non-coding RNAs (lncRNAs) have gained increasing attention as powerful regulators of the gene expression network in both heart development and heart disease. However, current knowledge about lncRNA biology, especially regarding the underlying mechanisms in the development of CVDs and their clinical value is limited. The aim of this study is to identify and validate lncRNAs that are involved in postnatal heart development and that may serve as a potential therapeutic target.

Methods:

By high-throughput RNA sequencing we identified a novel lncRNA named Foxo6os which is regulated during mouse heart development and cardiac diseases. SiRNA-mediated knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 activation (CRISPRa)-based overexpression were applied to investigate the loss- and gain-of-function of Foxo6os in vitro. Based on the in vitro data, a Cre/loxP-based Foxo6os knockout (KO) mouse strain was generated to study the in vivo function of Foxo6os. To investigate the underlying mechanism of Foxo6os, RNA pulldown and mass spectrometry analysis were performed to screen for Foxo6os binding proteins. Furthermore, a human functional-conserved transcript AC093151.2 was identified to translate our study into clinical application.

Results and Conclusion:

LncRNA Foxo6os is enriched in cardiomyocytes (CMs) and is highly up-regulated during cardiac development. In contrast to physiological conditions, Foxo6os is down-regulated in various murine cardiac disease models. Knockdown of Foxo6os reduced the viability and ratio of α/β-MHC, and increased the apoptotic rate in CMs, while overexpression of Foxo6os by CRISPRa system showed protective effects. AAV6-mediated overexpression of the putative human functional homolog, AC093151.2, significantly alleviated the apoptosis rate of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured under hypoxic conditions.

Mechanistically, we showed Foxo6os to react to global stress conditions including hypoxia through interaction with the cold-inducible RNA-binding protein (Cirbp). Foxo6os whole body KO mice (Foxo6os^-/-) showed a higher mortality rate in the early developmental stage. A pathological electrophysiological remodeling and impaired cardiac function was found in Foxo6os^-/- mice. Moreover, the Cirbp and Kcnj3 (Potassium inwardly rectifying channel subfamily J member 3) protein expression was lower in the Foxo6os^-/- mice compared to Foxo6os^fl/fl mice, suggesting the pathological regulation of cardiac function through the Foxo6os-Cirbp-Kcnj3 axis.

Collectively, these findings suggested that lncRNA Foxo6os is cardio-protective and essential for CM homeostasis and survival. Its potential human homolog AC093151.2 represents an encouraging therapeutic target for CVDs treatment in the future.