Non-coding RNA-activated by DNA damage promotes proliferation and inhibits apoptosis of induced pluripotent stem cell-derived cardiomyocytes

Abstract

BACKGROUND: Although cell transplantation offers a promising approach for the treatment of myocardial infarction, the low transplantation rate limits its application. Therefore, promoting the proliferation of transplanted cells and reducing apoptosis are the key issues to be solved urgently to improve the therapeutic effect. OBJECTIVE: To investigate the effects of non-coding RNA-activated by DNA damage (NORAD) on the proliferation of human induced pluripotent stem cell-derived cardiomyocytes and their apoptosis induced by oxygen-glucose deprivation/reoxygenation, as well as the effects of transplanting NORAD-overexpressing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-NORADOECMs) on cardiac function in a murine model of myocardial infarction. METHODS: The expression of NORAD in the hearts of mice at different ages (3 days old and 8 weeks old) was measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). A cellular model of hiPSC-NORADOECMs was established by infecting human induced pluripotent stem cells with a lentiviral vector designed to specifically upregulate NORAD, followed by directed differentiation into cardiomyocytes. The overexpression efficiency was confirmed by RT-qPCR. Proliferation was assessed by immunofluorescence staining for Ki67. Apoptosis was induced by oxygen-glucose deprivation/reoxygenation (OGD/R). Intracellular reactive oxygen species (ROS) levels were measured by flow cytometry, and the expression of apoptosis-related proteins (Bax, Bcl-2, Cleaved Caspase-3) was detected by western blot. In vivo, hiPSC-NORADOECMs were transplanted into the infarcted myocardium of mice, and cardiac function was evaluated by echocardiography after 4 weeks. RESULTS AND CONCLUSION: NORAD expression was significantly higher in 3-day-old neonatal mouse hearts compared with 8-week-old adult hearts. hiPSC-NORADOECMs were successfully generated, showing increased Ki67 expression compared with control cells. Overexpression of NORAD inhibited OGD/R-induced ROS production, decreased Bax and Cleaved Caspase-3 protein levels, and increased Bcl-2 levels. Transplantation of hiPSC-NORADOECMs significantly improved cardiac function in myocardial infarction mice. These findings indicate that NORAD overexpression promotes hiPSC-CM proliferation and inhibits apoptosis by reducing ROS production, thereby enhancing the reparative capacity of hiPSC-CMs in myocardial infarction.

1. Introduction

Myocardial infarction is primarily caused by coronary artery occlusion, leading to prolonged hypoxia of cardiomyocytes, massive cell death, and ultimately cardiac dysfunction [1-3]. In mammals, cardiomyocytes exit the cell cycle shortly after birth, rendering them unable to effectively self-repair and restore cardiac function [4-6]. Currently, cell therapy using exogenous cells to fill the infarcted scar area is one of the most promising cardiac repair strategies [7-9]. In 2007, human somatic cells were successfully reprogrammed into induced pluripotent stem cells (hiPSCs), which are similar to human embryonic stem cells in morphology, proliferation, surface antigen expression, and gene expression profiles [10]. hiPSCs have the ability to differentiate into multiple cell types, such as cardiomyocytes, endothelial cells, vascular smooth muscle cells, and neurons, making them a valuable tool for cell therapy, drug screening, and disease modeling [11-14]. Scientists have already used disease-specific hiPSC-derived cardiomyocytes (hiPSC-CMs) to establish models of arrhythmia, dilated cardiomyopathy, and hypertrophic cardiomyopathy, laying the foundation for personalized treatment [15-16]. hiPSC-CMs exhibit electrophysiological properties similar to primary cardiomyocytes [17], display action potentials similar to those of cardiomyocytes [18], and can be derived from the patient's own hiPSCs, avoiding immune rejection during transplantation, making them an important cell source for myocardial infarction cell therapy [19]. However, the survival rate of transplanted cells is generally low [20-21]. Therefore, studying the proliferation mechanism of hiPSC-CMs, promoting their proliferation and inhibiting apoptosis, and thereby improving the transplantation rate, are key issues to be solved for the use of hiPSC-CMs in cardiac repair.

In mammalian cells, approximately 98% of gene transcripts are non-coding RNAs, among which long non-coding RNAs (lncRNAs) are a group of non-coding RNAs longer than 200 nucleotides [22-23]. LncRNAs play crucial roles in various cellular processes, including cell cycle, apoptosis, and differentiation [24-25]. Unlike protein-coding genes, lncRNAs exhibit tissue- and cell-type-specific expression patterns, and their dysregulation has been implicated in many diseases. Among them, non-coding RNA-activated by DNA damage (NORAD) has been identified as a key regulator of genomic stability and mitosis. However, its role in cardiac regeneration and repair remains largely unexplored. This study aims to investigate the effects of NORAD on the proliferation and apoptosis of hiPSC-CMs, and to evaluate the therapeutic potential of NORAD-overexpressing hiPSC-CMs in a mouse model of myocardial infarction.