In vivo transfection of manganese superoxide dismutase gene or nuclear factor κB shRNA in nodose ganglia improves aortic baroreceptor function in heart failure rats

Abstract

Arterial baroreflex sensitivity is attenuated in chronic heart failure (CHF) state, which is associated with cardiac arrhythmias and sudden cardiac death in patients with CHF. Our previous study showed that CHF-induced sodium channel dysfunction in the baroreceptor neurons was involved in the blunted baroreflex sensitivity in CHF rats. Mitochondria-derived superoxide overproduction decreased expression and activation of the sodium channels in the baroreceptor neurons from CHF rats. However, the molecular mechanisms responsible for the sodium channel dysfunction in the baroreceptor neurons from CHF rats remain unknown. We tested the involvement of nuclear factor κB (NFκB) in the sodium channel dysfunction and evaluated the effects of in vivo transfection of manganese superoxide dismutase gene and NFκB shRNA on the baroreflex function in CHF rats. CHF was developed at 6 to 8 weeks after left coronary artery ligation in adult rats. Western blot and chromatin immunoprecipitation data showed that phosphorylated NFκB p65 and ability of NFκB p65 binding to the sodium channel promoter were increased in the nodose ganglia from CHF rats. In vivo transfection of adenoviral manganese superoxide dismutase gene or lentiviral NFκB p65 shRNA into the nodose ganglia partially reversed CHF-reduced sodium channel expression and cell excitability in the baroreceptor neurons and improved CHF-blunted arterial baroreflex sensitivity. Additionally, transfection of adenoviral manganese superoxide dismutase also inhibited the augmentation of phosphorylated NFκB p65 in the nodose neurons from CHF rats. The present study suggests that superoxide-NFκB signaling contributes to CHF-induced baroreceptor dysfunction and resultant impairment of baroreflex function.

Keywords: baroreflex; heart failure; pressoreceptors; sodium channels; superoxides.

Figure 1

Representative (A) and grouped data showing expression and phosphorylation of IKK, IκB, and NFκB proteins in nodose ganglia from sham and CHF rats. IKK: IκB kinase; IκB: NFκB inhibitory protein. Data are mean ± SEM; n=5 rats in each group. *P<0.05 vs. sham rats.

Figure 2

Ability of NFκB p65 binding to Na_v1.7 promoter (A), and expression (B) and currents (C) of Na_v 1.7 channels in sham and CHF rats. A, Ability of NFκB p65 binding to Na_v1.7 promoter was measured using chromatin immunoprecipitation assay. Nodose ganglia from 4 rats were pooled to obtain sufficient tissue for assay. N=12 rats in each group. B, Na_v1.7 channel protein in nodose ganglia was measured by Western blotting. N=5 rats in each group. C, Na_v1.7 current density in aortic baroreceptor neurons was measured by whole cell patch-clamp technique. N=12 cells from 5 rats in each group. Data are mean ± SEM. *P<0.05 vs. sham.

Figure 3

Effect of in-vivo lentiviral NFκB p65 shRNA transfection (2 μl, 3 × 10⁶ ifu/ml) on expression of NFκB and Na_v1.7 channel proteins in the nodose ganglia (A), and Na_v1.7 currents (B) and cell excitability (C) in the aortic baroreceptor neurons from sham and CHF rats with and without NFκB p65 shRNA transfection into nodose ganglia. In panel A, n=5 rats in all experimental groups. In panels B and C, n=10 neurons from 5 rats in each group. Data are mean ± SEM. *P<0.05 vs. sham; ^#p<0.05 vs. CHF.

Figure 4

Effect of in-vivo adenoviral manganese superoxide dismutase (Ad.MnSOD, 2 μl, 2 × 10¹⁰ pfu/ml) transfection on expression of MnSOD, NFκB and Na_v1.7 channel proteins (A) and superoxide production (B) in the nodose ganglia, and Na_v1.7 currents (C) and cell excitability (D) in the aortic baroreceptor neurons from sham and CHF rats with and without Ad.MnSOD transfection into nodose ganglia. In panel A, n=5 rats in all experimental groups. In panel B, total superoxide level (left) was measured in 5 rats per group, and mitochondrial-derived superoxide level (right) was analyzed in 20 nodose neurons from 5 rats per group. In panels C and D, n=10 neurons from 5 rats in each group. Data are mean ± SEM. *P<0.05 vs. sham; ^#p<0.05 vs. CHF.

Figure 5

Effect of in-vivo lentiviral NFκB p65 shRNA (left panel) or Ad.MnSOD (right panel) transfection on reflex ΔMAP (mean arterial pressure) and ΔHR (heart rate) in response to different frequencies of ADN (aortic depressor nerve) stimulation in anesthetized sham and CHF rats with and without lentiviral NFκB p65 shRNA or Ad.MnSOD transfection. Data are mean ± SEM, n=5 rats in each group. *P<0.05 vs. sham; ^#p<0.05 vs. CHF.