NOR-1 is an upstream regulator of Ca2+ homeostasis and calmodulin kinase II-dependent arrhythmogenesis

Dysregulation of NOR-1 has been reported in cardiovascular diseases associated with altered calcium handling. We therefore investigated how NOR-1 affects calcium homeostasis and arrhythmogenesis to identify the underlying mechanisms.

We assessed the expression of NOR-1 and calcium regulatory proteins in myocardial samples from humans and mice that overexpress human NOR-1 in the heart (TgNOR-1). Gene promoter activity was determined by transient co-transfection assays. Calcium imaging, patch-clamp recordings and immunofluorescent labeling was performed in mouse cardiomyocytes. Experimental data were used for mathematical modeling. Arrhythmias were induced in vivo by an epinephrine/caffeine challenge.

In human cardiac samples, mRNA levels of genes coding for SERCA2a, CASQ2, TN-C1 and PLB correlated with those of NOR-1, while TgNOR-1 mice exhibited higher cardiac levels of these calcium regulatory proteins than wild-type animals (WT). NOR-1 increased luciferase activity driven by the proximal promoter of both human and mouse genes. Calcium transients were smaller in TgNOR-1 cardiomyocytes while L-type calcium current and SR calcium content were unchanged. Mathematical modeling revealed that more calcium binds to CASQ2 and is released from the SR in TgNOR-1 cardiomyocytes, but the resulting calcium transient is smaller because more calcium is buffered by TN-C1. Furthermore, NOR-1 transgenesis increased the level of reactive oxygen species and CAMKII oxidation, as well as RyR2 phosphorylation at Ser-2814, which led to a higher incidence of calcium waves and afterdepolarizations in resting TgNOR-1 cardiomyocytes. In electrically paced myocytes, NOR-1 transgenesis promoted extra-transients at low frequencies and calcium alternans at high frequencies. Importantly, these derangements in intracellular calcium handling concurred with a dramatic increase in epinephrine/caffeine-induced bidirectional ventricular tachycardia in TgNOR-1 mice. Moreover, the antioxidant N-acetylcysteine and the CAMKII inhibitor KN-93 attenuated arrythmia in TgNOR-1 mice and arrhythmogenic calcium release in isolated myocytes.

Our findings highlight NOR-1 as an upstream regulator of CAMKII-oxidation underlying arrhythmia induced by defective calcium homeostasis, pointing to therapies targeting CAMKII as a means to attenuate arrhythmia triggered by pathological upregulation of NOR-1.