Long‐term effect of neuronal nitric oxide synthase over‐expression on cardiac neurotransmission mediated by a lentiviral vector

Short-term over-expression of neuronal nitric oxide synthase (nNOS) with adenoviral gene transfer into peripheral cardiac autonomic neurons can facilitate cholinergic neurotransmission, and inhibit sympathetic transmission, by regulating cyclic nucleotide-dependent pathways coupled to neuronal calcium entry. We tested the idea whether cardiac neuromodulation by nNOS could be sustained by long-term over-expression of the enzyme following lentiviral gene transfer. We developed a lentiviral vector with an elongation factor 1 (EF1α) promoter to drive nNOS or enhanced green fluorescent protein (eGFP) expression. Lenti.EF1α-nNOS or Lenti.EF1α-eGFP was transferred to the right atrium of Spague–Dawley (SD) rats and acetylcholine (ACh) or noradrenaline (NA) release to field stimulation was measured 4 months after gene transfer. Atria transduced with Lenti.EF1α-nNOS had higher nNOS expression compared to the atria treated with Lenti.EF1α-eGFP (P < 0.05). We also detected significant increases (P < 0.05) in atrial cGMP and cAMP levels in the same tissue. Immunohistochemistry revealed co-localisation of eGFP in intrinsic cholinergic neurons (choline acetyltransferase positive) and intrinsic adrenergic neurons (tyrosine hydroxylase positive) following gene transfer. nNOS-transduced animals displayed enhanced ACh release (P < 0.05) and reduced NA release (P < 0.05) compared to the eGFP-treated group. nNOS-specific inhibition reversed the enhanced ACh release. Persistent nNOS over-expression mediated by a lentiviral vector can modulate sympatho-vagal control of cardiac excitability. This approach may provide a new tool to target impaired cardiac autonomic phenotypes that are disrupted by several cardiovascular pathologies.