Role of T-Box 3 in Cardiomyocyte Apoptosis

Abstract

Background: T-box 3 (Tbx3) is a transcription factor that plays a key role in the embryonic development of multiple organs, such as mammary glands, limbs and heart. Although Tbx3 is selectively expressed in the cardiac conduction system in healthy hearts, our recent observations suggest that Tbx3 is also expressed in ventricular tissues in response to injury. This study aims to investigate if expression of Tbx3 in ventricular myocytes affects cell survival. Methods and Results: Adenovirus was used to express Tbx3 in primary culture of neonatal rat ventricular myocytes (NRVMs). Flow cytometry was performed to quantify cells in early-phase apoptosis (i.e., Annexin V positive cells), which started to increase at 48 h after Ad-Tbx3 virus transduction (24.7% vs. 11.8% in control Ad-GFP), and at 72 h the apoptotic cells were increased to 59.1% (vs. 21.3% in Ad-GFP). To further investigate the effect of Tbx3 on apoptosis, TUNEL staining was used to detect DNA cleavage and fragmentation, an event of late-phase apoptosis. TUNEL-positive cells in the Ad-Tbx3 group were 2.5%, 15.0% and 24.9% at 24, 48, and 72 h, respectively, while the control Ad-GFP group had a very low level of TUNEL-positive cells (0.4%, 1.7% and 3.7%, respectively). The P2x1 gene, encoding an ATP-gated cation channel, was identified by RNA sequencing as a top upregulated gene by Tbx3 in NRVMs. Flow cytometry demonstrated that Tbx3-induced apoptosis was further increased by a P2X1 agonist but was attenuated by a P2X1 antagonist. RNA sequencing revealed that several other apoptosis-related genes, such as Fas, were also updated in Tbx3- expressed NRVMs. Summary: Our study demonstrated a pro-apoptotic role of Tbx3 in ventricular myocytes with P2X1 as one of the mediators.