Human NR4A2 adenoviral particles

Sustained increases in sympathetic nerve activity are important contributors to pathological cardiac hypertrophy, ventricular arrhythmias, and left ventricular systolic dysfunction in chronic heart failure. The orphan nuclear receptor NR4A2 is an immediate early response gene activated in the heart upon β-adrenergic stimulation. Here, treatment of adult rat ventricular myocytes with isoproterenol resulted in a rapid (<4 hours) increase in NR4A2 levels accompanied by a transient (<24 hours) increase in nuclear localization of the transcription factor. Adenovirus-mediated overexpression of NR4A2 to similar levels modulated the expression of genes associated with adrenergic receptor signaling, calcium signaling, cell growth, and proliferation, and counteracted the increases in protein synthesis rate and cell surface area mediated by chronic isoproterenol stimulation. Consistent with these findings, NR4A2 overexpression also blocked phosphorylation activation of the growth-associated kinases ERK1/2, Akt, and p70 S6 kinase. Prominent among the NR4A2-induced transcriptional changes was the upregulation of the dual-specificity phosphatases DUSP2 and DUSP14, known inhibitors of ERK1/2. Taken together, these results suggest that NR4A2 functions as a novel negative feedback regulator of β-adrenoceptor-mediated growth responses in cardiomyocytes, at least in part through DUSP-mediated inhibition of ERK1/2 signaling.

To determine the transcriptional remodeling events caused by increased NR4A2 expression in adult cardiomyocytes, ARVMs were transduced with an adenovirus containing the full-length open reading frame of human NR4A2 (Ad-h-NR4A2) under the transcriptional control of a cytomegalovirus promoter. Cells transduced with Ad-h-NR4A2 increased total NR4A2 mRNA and protein levels 100-fold and 15-fold, respectively, compared with cells transduced with a control recombinant adenovirus encoding green fluorescent protein (Ad-GFP) (Figure 1A and B). The overexpressed protein was primarily localized to the nucleus as shown by immunofluorescence (Figure 1C). Thus, transgenic expression of NR4A2 in cardiomyocytes recapitulated the short-term effects of isoproterenol stimulation on the induction and localization of endogenously expressed transcription factors.

Figure 1. NR4A2 modulates expression of genes encoding proteins involved in adrenergic signaling, cell growth, and calcium signaling. (Ashraf S, et al., 2019)