Human NF2 adenoviral particles

β-Adrenergic receptor (β-AR) stimulation induces cardiomyocyte apoptosis in vitro and in vivo. Neurofibromin 2 (NF2) is a member of the ezrin/radixin/moesin (ERM) family of proteins. Post-translational modifications, such as phosphorylation and sumoylation, affect NF2 activity, subcellular localization, and function. Here, treatment of adult rat ventricular myocytes (ARVMs) with a β-AR agonist (isoproterenol) for 15 minutes increased NF2 phosphorylation (serine-518) and sumoylation. Specific inhibition of β1-AR and protein kinase A (PKA) reduced the β-AR-stimulated increase in NF2 post-translational modifications, whereas inhibition of β2-AR had no effect. Adenoviral stimulation of β-AR and expression of wild-type (WT)-NF2 increased phosphorylation of mammalian sterile-like kinase 1/2 (MST1/2) and YAP activating protein (YAP), downstream targets of NF2. Knockdown of NF2 in H9C2 cardiomyocytes using siRNA reduced the β-AR-stimulated increase in NF2 and YAP phosphorylation. siRNA-mediated knockdown of NF2 reduced the β-AR-stimulated increase in apoptosis, whereas expression of WT-NF2 induced apoptosis in ARVM cells. Expression of WT-NF2 stimulated the mitochondrial death pathway, as indicated by activation of c-Jun N-terminal kinase (JNK) and increased cytosolic cytochrome c levels and Bax expression.

In ARVMs, β-AR-stimulated apoptosis occurs through the engagement of the JNK and mitochondrial death pathways. To investigate the involvement of the mitochondrial death pathway, researchers measured JNK activation, Bax and Bcl2 expression, and cytoplasmic cytochrome c levels in ARVMs infected with adenovirus expressing WT-NF2 for 48 hours. Western blot analysis of cell lysates using a phospho-specific JNK antibody revealed a significant increase (approximately 1.6-fold) in phosphorylation of JNK (46 and 54 KDa) compared to cells expressing GFP (Figure 1A). NF2 expression also increased the protein level of the pro-apoptotic protein Bax by approximately 1.5-fold (Figure 1B). The protein level of Bcl2 remained unchanged. The Bcl2/Bax ratio was significantly lower in cells expressing NF2 (Figure 1C). The level of cytoplasmic cytochrome c was significantly higher in cells expressing WT-NF2 compared with cells expressing GFP (approximately 1.6-fold; Figure 1D).

Figure 1. Adenoviral-mediated expression of NF2 activates mitochondrial death pathway in ARVMs. (Dalal S, et al., 2018)