Abstract 210: Silencing the NF-kB-p65 Gene by RNA Interference Resulted in Alteration of Cardiac Mass in Transgenic Mice Overexpressing Myotrophin
Activation of the NF-κB signaling pathway is associated with the hypertrophic process, particularly during its transition to heart failure (HF). Thus NF-κB is likely a good target for prevention/treatment of HF. RNA interference (RNAi), initiated by small interfering RNA (siRNA), is a powerful strategy for silencing gene transcripts. siRNA is a double-stranded RNA containing 21–23 bp, specific for the target sequence. To evaluate NF-κB signaling, we used transgenic mice (Myo-Tg), which overexpress myotrophin in the heart associated with elevated level of NF-κB activity. By 4 weeks of age, these mice exhibit hypertrophy that progresses to HF by 36 weeks. We determined various components, e.g., phosphorylation and total content of IκBα and p65 mRNA level, and IκB kinase-β activity of NF-κB in vivo. All components were elevated at 4 weeks and remained elevated up to 36 weeks. To examine the effect of inhibition of NF-κB activation on cardiac remodeling, we constructed short hairpin (sh)-RNA to specifically inhibit the NF-κB-p65 gene in lentiviral (L) vector (L-si-p65) using a U6 promoter. Significant inhibition of NF-κB activation was observed (68.4% over myotrophin-stimulated neonatal myocytes, p < 0.001) in myotrophin-induced myocytes hypertrophy. This inhibition was associated with inhibition of protein synthesis [76.6 ± 3.8 vs 31.1 ± 5.8 (% over control), p < 0.001] and ANF expression (168.8 ± 5.8 vs. 48.6 ± 4.3) in L-si-p65 transduced myocytes. To establish the effect of L-si-p65 further on cardiac mass in vivo, we delivered L-si-p65 directly to the hearts of 10-week-old in Myo-Tg mice. After 6 weeks of treatment, the Myo-Tg and L-si-65-Myo-Tg mice were sacrificed; all showed a significant regression of cardiac hypertrophy (heart-weight-to-body-weight ratio reduced from 8.5 to 6.5), associated with significant reduction in NF-κB activation (58.64 % over untreated Myo-Tg mice, p < 0.001) and ANF expression (32.3% over untreated Myo-Tg, p < 0.001). These data suggest, for the first time, that inhibition of NF-κB using direct gene delivery of sh-p65-RNA can bring about significant regression of cardiac hypertrophy and that NF-κB appeared to be a good target for inhibition as a means to prevent or treat hypertrophy/heart failure.