Background Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. Based on our recent findings of increased Smad7 expression in surgically-resected bowel affected by NEC, we hypothesized that NEC macrophages undergo inflammatory activation because increased Smad7 expression renders these cells resistant to normal, gut-specific, transforming growth factor (TGF)-β-mediated suppression of inflammatory pathways. Methods We used surgically-resected human NEC tissue, murine models of NEC-like injury, bone marrow-derived and intestinal macrophages, and RAW264.7 cells. Smad7 and IκB kinase-beta (IKK-β) were measured by quantitative polymerase chain reaction (qPCR), Western blots, and immunohistochemistry. Promoter activation was confirmed in luciferase reporter and chromatin immunoprecipitation assays. Results NEC macrophages showed increased Smad7 expression, particularly in areas with severe tissue damage and high bacterial load. LPS-induced Smad7 expression suppressed TGF-β signaling and augmented NF-κB activation and cytokine production in macrophages. Smad7-mediated NF-κB activated was likely mediated via increased expression of IKK-β, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-β expression through direct binding to the IKK-β promoter and its transcriptional activation. Conclusions Smad7 expression in NEC macrophages interrupts TGF-β signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-β.

Hyperoxia exposure in mice leads to cardiac hypertrophy and voltage-gated potassium (Kv) channel remodeling. Because redox balance of pyridine nucleotides affects Kv function and hyperoxia alters cellular redox potential, we hypothesized that hyperoxia exposure leads to cardiac ion channel disturbances and redox changes resulting in arrhythmias. In the present study, we investigated the electrical changes and redox abnormalities caused by 72h hyperoxia treatment in mice. Cardiac repolarization changes were assessed by acquiring electrocardiogram (ECG) and cardiac action potentials (AP). Biochemical assays were employed to identify the pyridine nucleotide changes, Kv1.5 expression and myocardial injury. Hyperoxia treatment caused marked bradycardia, arrhythmia and significantly prolonged (ms) the, RR (186.2 ±10.7 vs. 146.4±6.2), PR (46.8±3.1 vs. 39.3±1.6), QRS (10.8±0.6 vs. 8.5±0.2), QTc (57.1±3.5 vs. 40±1.4) and JT (13.4±2.1 vs. 7.0±0.5) intervals, when compared with normoxia group. Hyperoxia treatment also induced significant increase in cardiac action potential duration (APD) (ex- APD90; 73.8±9.5 vs. 50.9±3.1 ms) and elevated levels of serum markers of myocardial injury; cardiac troponin I (TnI) and lactate dehydrogenase (LDH). Hyperoxia exposure altered cardiac levels of mRNA/protein expression of; Kv1.5, Kvβ subunits and SiRT1, and increased ratios of reduced pyridine nucleotides (NADH/NAD & NADPH/NADP). Inhibition of SiRT1 in H9C2 cells using Splitomicin resulted in decreased SiRT1 and Kv1.5 expression, suggesting that SiRT1 may mediate Kv1.5 downregulation. In conclusion, the cardiotoxic effects of hyperoxia exposure involve ion channel disturbances and redox changes resulting in arrhythmias.

Diabetes is a metabolic disorder that ultimately results in major pathophysiological complications in the cardiovascular system. Diabetics are predisposed to higher incidences of sudden cardiac deaths (SCD). Several studies have associated diabetes as a major underlying risk for heart diseases and its complications. The diabetic heart undergoes remodeling to cope up with the underlying changes, however ultimately fails. In the present study we investigated the changes associated with a key ion channel and transcriptional factors in a diabetic heart model. In the mouse db/db model, we identified key transcriptional regulators and mediators that play important roles in the regulation of ion channel expression. Voltage-gated potassium channel (Kv4.2) is modulated in diabetes and is down regulated. We hypothesized that Kv4.2 expression is altered by potassium channel interacting protein-2 (KChIP2) which is regulated upstream by NFkB and miR-301a. We utilized qRT-PCR analysis and identified the genes that are affected in diabetes in a regional specific manner in the heart. At protein level we identified and validated differential expression of Kv4.2 and KChIP2 along with NFkB in both ventricles of diabetic hearts. In addition, we identified up-regulation of miR-301a in diabetic ventricles. We utilized loss and gain of function approaches to identify and validate the role of miR-301a in regulating Kv4.2. Based on in vivo and in vitro studies we conclude that miR-301a may be a central regulator for the expression of Kv4.2 in diabetes. This miR-301 mediated regulation of Kv4.2 is independent of NFkB and Irx5 and modulates Kv4.2 by direct binding on Kv4.2 3′untranslated region (3′-UTR). Therefore targeting miR-301a may offer new potential for developing therapeutic approaches.

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