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Myotrophin-induced activation of NF-[kappa]B has been shown to be associated with cardiac hypertrophy (CH) that progresses to heart failure (HF). In the present study, we examined the cause-and-effect relationship between myotrophin and NF-[kappa]B activation using small hairpin RNA (shRNA) against myotrophin both in vitro (using neonatal rat myocytes) and in vivo [using myotrophin transgenic (Myo-Tg) mice, which overexpress myotrophin in the heart, develop CH, and gradually progress to HF]. Among several lentiviral vectors expressing myotrophin shRNAs, L-sh-109 showed the best silencing effect at both the mRNA (155.3 [+ or -] 5.9 vs. 32.5 [+ or -] 5.5, P < 0.001) and protein levels associated with a significant reduction of atrial natriuretic factor (ANF) and NF-[kappa]B. In vivo, when L-sh-109 was delivered directly into the hearts of 10-wk-old Myo-Tg mice, we observed a significant regression of cardiac mass (8.0 vs. 5.7 mg/g, P < 0.001) and myotrophin gene expression (54.5% over untreated Myo-Tg mice, P < 0.001) associated with a reduction in ANF and NF-[kappa]B signaling components. Our data suggest that using RNA interference to silence the myotrophin gene prevents NF-[kappa]B activation, associated with an attenuation of CH. This strategy could be an excellent therapeutic means for the treatment of CH and HF. nuclear factor-[kappa]B; heart failure

The transcription factor nuclear factor (NF)-[kappa]B plays a leading role in cardiac hypertrophy associated with heart failure, but whether it is involved in cardiac mass reduction is not known. We evaluated whether inhibiting the NF-[kappa]B cascade with pyrrolidine dithiocarbamate (PDTC) in spontaneously hypertensive rats (SHRs) and age-matched WistarKyoto rats (WKYs) affected hypertrophy. We measured NF-[kappa]B signaling components [NF[kappa]B translocation, I[kappa]B[alpha], p65, mRNA and protein levels, and I[kappa]B kinase-[beta] (IKK[beta]) activity] at 12 and 36 wk in WKYs and SHRs and at 10 wk in PDTC-treated rats (n = 9). NF-[kappa]B activation was also evaluated in rats treated for 10 wk with captopril or hydralazine alone or with either drug plus PDTC. All components were increased in SHRs compared with WKYs. After PDTC treatment, NF-[kappa]B activity was inhibited, and heart weight-to-body weight ratio in SHRs was significantly attenuated (3.52 [+ or -] 0.04 to 3.32 [+ or -] 0.05 mg/kg). Captopril treatment significantly reduced cardiac mass (3.5 vs. 3.05 mg/kg; n = 9) and inhibited NF-[kappa]B activity (169.71 [+ or -] 5.70 to 106.7 [+ or -] 12.44). Hydralazine had no effect on cardiac mass (3.5 vs. 3.42 mg/kg) or NF-[kappa]B activity (169.71 [+ or -] 5.70 to 155.52 [+ or -] 6.11). Hydralazine plus PDTC reduced blood pressure (191.16 [+ or -] 1.7 to 158.5 [+ or -] 2.36 mmHg) and inhibited NF-[kappa]B activity (169.71 [+ or -] 5.70 to 97.29 [+ or -] 3.65). Our data suggest that 1) cardiac hypertrophy in SHRs is partly due to NF-[kappa]B activation, 2) inhibition of NF-[kappa]B activity by PDTC parallels regression of hypertrophy, and 3) regression of hypertrophy is partly due to inhibition of NF-[kappa]B activity, independent of hypertension. The relationship between NF-[kappa]B activity and cardiac remodeling is causal, not coincidental. nuclear factor-[kappa]B; pyrrolidine dithiocarbamate; captopril; hydralazine

The transcription factor nuclear factor-[kappa]B (NF-[kappa]B) regulates expression of a variety of genes involved in immune responses, inflammation, proliferation, and programmed cell death (apoptosis). Here, we show that in rat neonatal ventricular cardiomyocytes, activation of NF-[kappa]B is involved in the hypertrophic response induced by myotrophin, a hypertrophic activator identified from spontaneously hypertensive rat heart and cardiomyopathic human hearts. Myotrophin treatment stimulated NF-[kappa]B nuclear translocation and transcriptional activity, accompanied by I[kappa]B-[alpha] phosphorylation and degradation. Consistently, myotrophin-induced NF-[kappa]B activation was enhanced by wild-type I[kappa]B kinase (IKK) [beta] and abolished by the dominant-negative IKK[beta] or a general PKC inhibitor, calphostin C. Importantly, myotrophin-induced expression of two hypertrophic genes (atrial natriuretic factor [ANF] and c-myc) and also enhanced protein synthesis were partially inhibited by a potent NF-[kappa]B inhibitor, pyrrolidine dithio-carbamate (PDTC), and calphostin C. Expression of the dominantnegative form of I[kappa]B-[alpha] or IKK[beta] also partially inhibited the transcriptional activity of ANF induced by myotrophin. These findings suggest that the PKC-IKK-NF-[kappa]B pathway may play a critical role in mediating the myotrophin-induced hypertrophic response in cardiomyocytes.

Abstract Background The bZIP class Abscisic acid Responsive Element (ABRE)-binding factor, OSBZ8 (38.5 kD) has been considered to regulate ABA-mediated transcription in the suspension cultured cells of japonica rice. Still, nothing is known about the expression of OSBZ8 at protein level in vegetative tissue of salt sensitive and salt tolerant rice plants. In our previous study, Electrophoretic Mobility Shift Assay (EMSA) of [32P]ABRE-DNA and nuclear extracts prepared from the lamina of Pokkali rice plants has detected the presence of an ABRE-binding factor. Northern analysis has also detected salinity stress induced accumulation of transcripts for bZIP class of factor. Therefore, OSBZ8 was considered to play an important role in the regulation of transcription in the vegetative tissue of rice. The aim of this study is to find out whether OSBZ8 has any role in regulating the NaCl-stress induced gene expression in vegetative tissue and whether the expression of OSBZ8 factor directly correlates with the stress tolerance of different varieties of indica type rice. Results Northern analysis of total RNA from roots and lamina of salt-sensitive M-I-48 and salt-tolerant Nonabokra, when probed with the N-terminal unique region of OSBZ8 (OSBZ8p, without the highly conserved basic region), a transcript of 1.3 kb hybridized and its level was much higher in tolerant cultivar. EMSA with Em1a, the strongest ABA Responsive Element till reported from the upstream of EmBP1, and the nuclear extracts from laminar tissue of untreated and salt-treated seedlings of three salt sensitive, one moderately sensitive and two salt tolerant indica rice cultivars showed specific binding of nuclear factor to ABRE element. Intensity of binding was low and inducible in salt sensitive rice cultivars while high and constitutive in salt tolerant cultivars. EMSA with 300 bp 5'upstream region of Rab16A gene, a well known salt stress and ABA-inducible gene of rice, showed formation of two complexes, again very weak in salt sensitive and strong in salt tolerant rice cultivar. Conclusion The bZIP factor OSBZ8 was found to be present in the ABRE-DNA: protein complex as shown by the supershift of the complex by the purified antiserum raised against OSBZ8p. Treatment of the seedlings with NaCl was found to enhance the complex formation, suggesting the regulation of OSBZ8 gene at both transcriptional and post-translational steps. Comparative EMSA with different varieties of rice suggests a positive correlation with the expression pattern of OSBZ8 and salt tolerance in rice cultivars.