Metastasis in breast cancer carries a disproportionately worse prognosis than localized primary disease. To identify microRNAs (miRNA) involved in metastasis, the expression of 254 miRNAs was measured across the following cell lines using microarray analysis: MDA-MB-231 breast cancer cells, cells that grew as a tumor in the mammary fat pad of nude mice (TMD-231), metastatic disease to the lungs (LMD-231), bone (BMD-231) and adrenal gland (ADMD-231). A brain-seeking variant of this cell line (231-BR) was used additionally in validation studies. Twenty miRNAs were upregulated and seven were downregulated in metastatic cancer cells compared with TMD-231 cells. The expression of the tumor suppressor miRNAs let-7 and miR-22 was consistently downregulated in metastatic cancer cells. These metastatic cells expressed higher levels of putative/proven miR-22 target oncogenes ERBB3, CDC25C and EVI-1. Introduction of miR-22 into cancer cells reduced the levels of ERBB3 and EVI-1 as well as phospho-AKT, an EVI-1 downstream target. The miR-22 primary transcript is located in the 5 0 -untranslated region of an open reading frame C17orf91, and the promoter/enhancer of C17orf91 drives miR-22 expression. We observed elevated C17orf91 expression in non-basal subtype compared with basal subtype breast cancers. In contrast, elevated expression of EVI-1 was observed in basal subtype and was associated with poor outcome in estrogen receptor-negative breast cancer patients. These results suggest that metastatic cancer cells increase specific oncogenic signaling proteins through downregulation of miRNAs. Identifying such metastasisspecific oncogenic pathways may help to manipulate tumor behavior and aid in the design of more effective targeted therapies.

Background Cervical vagal nerve (CVN) stimulation may improve left ventricular ejection fraction in patients with heart failure. Objectives To test the hypothesis that sympathetic structures are present in the CVN and to describe the location and quantitate these sympathetic components of the CVN. Methods We performed immunohistochemical studies of the CVN from 11 normal dogs and simultaneously recorded stellate ganglion nerve activity, left thoracic vagal nerve activity, and subcutaneous electrocardiogram in 2 additional dogs. Results A total of 28 individual nerve bundles were present in the CVNs of the first 11 dogs, with an average of 1.87 ± 1.06 per dog. All CVNs contain tyrosine hydroxylase-positive (sympathetic) nerves, with a total cross-sectional area of 0.97 ± 0.38 mm2. The sympathetic nerves were nonmyelinated, typically located at the periphery of the nerve bundles and occupied 0.03%–2.80% of the CVN cross-sectional area. Cholineacetyltransferase-positive nerve fibers occupied 12.90%–42.86% of the CVN cross-sectional areas. Ten of 11 CVNs showed tyrosine hydroxylase and cholineacetyltransferase colocalization. In 2 dogs with nerve recordings, we documented heart rate acceleration during spontaneous vagal nerve activity in the absence of stellate ganglion nerve activity. Conclusions Sympathetic nerve fibers are invariably present in the CVNs of normal dogs and occupy in average up to 2.8% of the cross-sectional area. Because sympathetic nerve fibers are present in the periphery of the CVNs, they may be susceptible to activation by electrical stimulation. Spontaneous activation of the sympathetic component of the vagal nerve may accelerate the heart rate.

Background Renal sympathetic denervation (RD) is a promising method of neuromodulation for the management of cardiac arrhythmia. Objective We tested the hypothesis that RD is antiarrhythmic in ambulatory dogs because it reduces the stellate ganglion nerve activity (SGNA) by remodeling the stellate ganglion (SG) and brain stem. Methods We implanted a radiotransmitter to record SGNA and electrocardiogram in 9 ambulatory dogs for 2 weeks, followed by a 2nd surgery for RD and 2 months SGNA recording. Cell death was probed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results Integrated SGNA at baseline, 1 and 2 months after RD were 14.0±4.0, 9.3±2.8 and 9.6±2.0 μV, respectively (p=0.042). The SG from RD but not normal control (N=5) dogs showed confluent damage. An average of 41±10% and 40±16% of ganglion cells in the left and right SG, respectively, were TUNEL-positive in RD dogs compared with 0% in controls dogs (p= 0.005 for both). Left and right SG from RD dogs had more tyrosine hydroxylase-negative ganglion cells than left SG of control dogs (p= 0.028 and 0.047 respectively). Extensive TUNEL positive neurons and glial cells were also noted in the medulla, associated with strongly positive glial fibrillary acidic protein staining. The distribution was heterogeneous, with more cell death in the medial than lateral aspects of the medulla. Conclusion Bilateral RD caused significant central and peripheral sympathetic nerve remodeling and reduced SGNA in ambulatory dogs. These findings may in part explain the antiarrhythmic effects of RD.

Background The effects of intermittent open loop vagal nerve stimulation (VNS) on ventricular rate (VR) during atrial fibrillation (AF) remain unclear. Objective To test the hypothesis that VNS damages the stellate ganglion (SG) and improves VR control during persistent AF. Methods We performed left cervical VNS in ambulatory dogs while simultaneously recording the left SG nerve activity (SGNA) and vagal nerve activity. Tyrosine hydroxylase (TH) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess neuronal cell death in SG. Results We induced persistent AF by atrial pacing in 6 dogs, followed by intermittent VNS with short ON-time (14 s) and long OFF-time (66 s). The integrated SGNA (iSGNA) and VR during AF were 4.84 mV-s [95% confidence interval, CI, 3.08 to 6.60] and 142 bpm [CI, 116 to 168], respectively. VNS reduced iSGNA and VR, respectively, during AF to 3.74 mV-s [CI, 2.27 to 5.20; p=0.021] and 115 bpm [CI, 96 to 134; p=0.016] during 66-s OFF-time, and to 4.07 mV-s [CI, 2.42 to 5.72; p=0.037] and 114 bpm [CI, 83 to 146; p=0.039] during 3-min OFF-time. VNS increased the frequencies of prolonged (>3 s) pauses during AF. TH staining showed large confluent areas of damage in the left SG, characterized by pyknotic nuclei, reduced TH staining, increased percentage of TH-negative ganglion cells and positive TUNEL staining. Occasional TUNEL-positive ganglion cells were also observed in the right SG. Conclusions VNS damaged the SG, leading to reduced SGNA and better rate control during persistent AF.

BackgroundPurkinje cells (PCs) are important in cardiac arrhythmogenesis. Whether small‐conductance calcium‐activated potassium (SK) channels are present in PCs remains unclear. We tested the hypotheses that subtype 2 SK (SK2) channel proteins and apamin‐sensitive SK currents are abundantly present in PCs.Methods and ResultsWe studied 25 normal rabbit ventricles, including 13 patch‐clamp studies, 4 for Western blotting, and 8 for immunohistochemical staining. Transmembrane action potentials were recorded in current‐clamp mode using the perforated‐patch technique. For PCs, the apamin (100 nmol/L) significantly prolonged action potential duration measured to 80% repolarization by an average of 10.4 ms (95% CI, 0.11–20.72) (n=9, P=0.047). Voltage‐clamp study showed that apamin‐sensitive SK current density was significantly larger in PCs compared with ventricular myocytes at potentials ≥0 mV. Western blotting of SK2 expression showed that the SK2 protein expression in the midmyocardium was 58% (P=0.028) and the epicardium was 50% (P=0.018) of that in the pseudotendons. Immunostaining of SK2 protein showed that PCs stained stronger than ventricular myocytes. Confocal microscope study showed SK2 protein was distributed to the periphery of the PCs.Conclusions SK2 proteins are more abundantly present in the PCs than in the ventricular myocytes of normal rabbit ventricles. Apamin‐sensitive SK current is important in ventricular repolarization of normal PCs.