MBD1 Contributes to the Genesis of Acute Pain and Neuropathic Pain by Epigenetic Silencing of Oprm1 and Kcna2 Genes in Primary Sensory Neurons.

The transmission of normal sensory and/or acute noxious information requires intact expression of pain-associated genes within the pain pathways of nervous system. Expressional changes of these genes after peripheral nerve injury are also critical for neuropathic pain induction and maintenance. Methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, regulates gene transcriptional activity. We report here that MBD1 in the primary sensory neurons of DRG is critical for the genesis of acute pain and neuropathic pain as DRG MBD1-deficient mice exhibit the reduced responses to acute mechanical, heat, cold, and capsaicin stimuli and the blunted nerve injury-induced pain hypersensitivities. Furthermore, DRG overexpression of MBD1 leads to spontaneous pain and evoked pain hypersensitivities in the WT mice and restores acute pain sensitivities in the MBD1-deficient mice. Mechanistically, MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 is likely a key player under the conditions of acute pain and neuropathic pain.SIGNIFICANCE STATEMENT In the present study, we revealed that the mice with deficiency of methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, in the DRG displayed the reduced responses to acute noxious stimuli and the blunted neuropathic pain. We also showed that DRG overexpression of MBD1 produced the hypersensitivities to noxious stimuli in the WT mice and rescued acute pain sensitivities in the MBD1-deficient mice. We have also provided the evidence that MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 may participate in the genesis of acute pain and neuropathic pain likely through regulating DNMT3a-controlled Oprm1 and Kcna2 gene expression in the DRG neurons.

Long Noncoding RNA Kcna2 Antisense RNA Contributes to Ventricular Arrhythmias via Silencing Kcna2 in Rats With Congestive Heart Failure.

BACKGROUND: Congestive heart failure (CHF) is a common cardiovascular disease that is often accompanied by ventricular arrhythmias. The decrease of the slow component of the delayed rectifier potassium current (IKs) in CHF leads to action potential (AP) prolongation, and the IKs is an important contributor to the development of ventricular arrhythmias. However, the molecular mechanisms underlying ventricular arrhythmias are still unknown. METHODS AND RESULTS: Kcna2 and Kcna2 antisense RNA (Kcna2 AS) transcript expression was measured in rat cardiac tissues using quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. There was a 43% reduction in Kcna2 mRNA in the left ventricular myocardium of rats with CHF. Kcna2 knockdown in the heart decreased the IKs and prolonged APs in cardiomyocytes, consistent with the changes observed in heart failure. Conversely, Kcna2 overexpression in the heart significantly attenuated the CHF-induced decreases in the IKs, AP prolongation, and ventricular arrhythmias. Kcna2 AS was upregulated ≈1.7-fold in rats with CHF and with phenylephrine-induced cardiomyocyte hypertrophy. Kcna2 AS inhibition increased the CHF-induced downregulation of Kcna2. Consequently, Kcna2 AS mitigated the decrease in the IKs and the prolongation of APs in vivo and in vitro and reduced ventricular arrhythmias, as detected using electrocardiography. CONCLUSIONS: Ventricular Kcna2 AS expression increases in rats with CHF and contributes to reduced IKs, prolonged APs, and the occurrence of ventricular arrhythmias by silencing Kcna2. Thus, Kcna2 AS may be a new target for the prevention and treatment of ventricular arrhythmias in patients with CHF.

The transcription factor C/EBPß in the dorsal root ganglion contributes to peripheral nerve trauma-induced nociceptive hypersensitivity.

Changes in gene transcription in the dorsal root ganglion (DRG) after nerve trauma contribute to the genesis of neuropathic pain. We report that peripheral nerve trauma caused by chronic constriction injury (CCI) increased the abundance of the transcription factor C/EBPß (CCAAT/enhancer binding protein ß) in the DRG. Blocking this increase mitigated the development and maintenance of CCI-induced mechanical, thermal, and cold pain hypersensitivities without affecting basal responses to acute pain and locomotor activity. Conversely, mimicking this increase produced hypersensitivity to mechanical, thermal, or cold pain. In the ipsilateral DRG, C/EBPß promoted a decrease in the abundance of the voltage-gated potassium channel subunit Kv1.2 and µ opioid receptor (MOR) at the mRNA and protein levels, which would be predicted to increase excitability in the ipsilateral DRG neurons and reduce the efficacy of morphine analgesia. These effects required C/EPBß-mediated transcriptional activation of Ehmt2 (euchromatic histone-lysine N-methyltransferase 2), which encodes G9a, an epigenetic silencer of the genes encoding Kv1.2 and MOR. Blocking the increase in C/EBPß in the DRG improved morphine analgesia after CCI. These results suggest that C/EBPß is an endogenous initiator of neuropathic pain and could be a potential target for the prevention and treatment of this disorder.

Cardiotoxic effect of levofloxacin and ciprofloxacin in rats with/without acute myocardial infarction: Impact on cardiac rhythm and cardiac expression of Kv4.3, Kv1.2 and Nav1.5 channels.

Prolongation of QT interval is possible with fluoroquinolones, yet the underlying contributing factors have not been elucidated. Two widely used fluoroquinolone drugs were at the focus of this study in rats with/without acute myocardial dysfunction (AMI) induced by isoproterenol. The effects of levofloxacin and ciprofloxacin on the cardiac mRNA expression of rat Kv4.3, Kv1.2 and Nav1.5 mRNAs were determined. Administration of the two antibiotics produced dose-dependent changes in ECG parameters that were more prominent in rats with AMI than healthy rats; this was accompanied by elevations in serum lactate dehydrogenase and creatine kinase-MB. Histopathological examination indicated some loss of striations, edema and fibrotic changes in rats with AMI; however the two antibiotics did not further exacerbate the cardiac histopathology. mRNA expression of the ion channels was altered in rats with AMI and healthy rats. In conclusion, long-term administration of levofloxacin and ciprofloxacin produced deleterious effects on the ECG pattern of rats with/without AMI. The effect was generally baseline-dependent and therefore, rats with AMI showed greater ECG disturbances and increases in cardiac enzymes. Taken together, these data make it advisable to monitor patients with a history of acute AMI requiring treatment with these antibiotics until data from human studies are available.

Noncoding RNAs: new players in chronic pain.

Chronic pain, a common clinical symptom, is often treated inadequately or ineffectively in part due to the incomplete understanding of molecular mechanisms that initiate and maintain this disorder. Newly identified noncoding RNAs govern gene expression. Recent studies have shown that peripheral noxious stimuli drive expressional changes in noncoding RNAs and that these changes are associated with pain hypersensitivity under chronic pain conditions. This review first presents current evidence for the peripheral inflammation/nerve injury-induced change in the expression of two types of noncoding RNAs, microRNAs, and Kcna2 antisense RNA, in pain-related regions, particularly in the dorsal root ganglion. The authors then discuss how peripheral noxious stimuli induce such changes. The authors finally explore potential mechanisms of how expressional changes in dorsal root ganglion microRNAs and Kcna2 antisense RNA contribute to the development and maintenance of chronic pain. An understanding of these mechanisms may propose novel therapeutic strategies for preventing and/or treating chronic pain.

K+ channel alterations in the progression of experimental autoimmune encephalomyelitis.

Voltage-gated K(+) (Kv) channels play critical roles not only in regulating synaptic transmission and intrinsic excitability of neurons, but also in controlling the function and proliferation of other cells in the central nervous system (CNS). The non-specific Kv channel blocker, 4-AminoPyridine (4-AP) (Dalfampridine, Ampyra®), is currently used to treat multiple sclerosis (MS), an inflammatory demyelinating disease. However, little is known how various types of Kv channels are altered in any inflammatory demyelinating diseases. By using established animal models for MS, experimental autoimmune encephalomyelitis (EAE), we report that expression and distribution patterns of Kv channels are altered in the CNS correlating with EAE severity. The juxtaparanodal (JXP) targeting of Kv1.2/Kvß2 along myelinated axons is disrupted within demyelinated lesions in the white matter of spinal cord in EAE. Moreover, somatodendritic Kv2.1 channels in the motor neurons of lower spinal cord significantly decrease correlating with EAE severity. Interestingly, Kv1.4 expression surrounding lesions is markedly up-regulated in the initial acute phase of both EAE models. Its expression in glial fibrillary acidic protein (GFAP)-positive astrocytes further increases in the remitting phase of remitting-relapsing EAE (rrEAE), but decreases in late chronic EAE (chEAE) and the relapse of rrEAE, suggesting that Kv1.4-positive astrocytes may be neuroprotective. Taken together, our studies reveal myelin-dependent and -independent alterations of Kv channels in the progression of EAE and lay a solid foundation for future study in search of a better treatment for MS.

Expression of glutamate receptors and potassium channels in vestibular nuclei neurons during development of signal processing.

The principal cells of the chick tangential vestibular nucleus offer a simple neuron model to study signal processing in second-order, vestibular reflex projection neurons. The principal cells represent a relatively uniform population of vestibular nuclei neurons which receive a major input from the primary vestibular fibers and send axons to targets mainly involved in the vestibuloocular reflexes. Here, studies performed on ion channels involved in the emergence and establishment of signal processing in this morphologically-identified subset of vestibular nuclei neurons are reviewed, including the AMPA glutamate receptor subunits GluR1, GluR2, GluR3, and GluR4 and the potassium channel subunits Kv1.1 and Kv1.2.

Voltage-gated delayed rectifier K v 1-subunits may serve as distinctive markers for enteroglial cells with different phenotypes in the murine ileum.

Due to entangled results concerning K(v)1 subunit distribution in the gastrointestinal wall, we aimed to unravel the expression of the delayed rectifier potassium subunits K(v)1.1 and K(v)1.2 in the murine ileum. Presence and distribution of both subunits were determined in cryosections and whole-mount preparations of the ileum of three different murine strains by indirect immunofluorescence, and analysed by conventional fluorescence and confocal microscopy. Distribution of both subunits was similar in the ileum of the three strains. K(v)1.1 immunoreactivity (IR) was found in some S100-expressing enteroglial cells (EGC) located at the periphery of myenteric ganglia, in S100-positive EGC along interganglionic, intramuscular and vascular nerve fibres, and in S100-positive EGC of the submucous plexus. K(v)1.1 IR was also observed in some GFAP-expressing EGC at the periphery of myenteric ganglia, and in GFAP-positive EGC of submucous ganglia. K(v)1.2 IR was detected in some intramuscular S100-positive EGC, in almost all submucous S100-expressing EGC, and in a few GFAP-expressing EGC. K(v)1.2 IR was also expressed in a majority of enteric neurons. Coding of these neurons showed that all cholinergic and most nitrergic neurons express K(v)1.2. In conclusion, the results showed that K(v)1.1 and K(v)1.2 were predominantly expressed in distinct EGC phenotypes. K(v)1.2 was also observed in distinct neuron subpopulations. Our results support the active role of EGC with distinct phenotypes in intestinal functions, which is relevant in view of their modulating role on intestinal barrier and inflammatory responses.

Changes in Na(+) channel expression and nodal persistent Na(+) currents associated with peripheral nerve regeneration in mice.

Patients with peripheral neuropathy frequently suffer from positive sensory (pain and paresthesias) and motor (muscle cramping) symptoms even in the recovery phase of the disease. To investigate the pathophysiology of increased axonal excitability in peripheral nerve regeneration, we assessed the temporal and spatial expression of voltage-gated Na(+) channels as well as nodal persistent Na(+) currents in a mouse model of Wallerian degeneration. Crushed sciatic nerves of 8-week-old C57/BL6J male mice underwent complete Wallerian degeneration at 1 week. Two weeks after crush, there was a prominent increase in the number of Na(+) channel clusters per unit area, and binary or broad Na(+) channel clusters were frequently found. Excess Na(+) channel clusters were retained up to 20 weeks post-injury. Excitability testing using latent addition suggested that nodal persistent Na(+) currents markedly increased beginning at week 3, and remained through week 10. These results suggest that axonal regeneration is associated with persistently increased axonal excitability resulting from increases in the number and conductance of Na(+) channels.

Kv1 potassium channel C-terminus constant HRETE region: arginine substitution affects surface protein level and conductance level of subfamily members differentially.

We have shown previously that truncating all of the variable cytoplasmic C-terminus of Kv1.1 potassium channels to G421stop had only a small inhibitory effect on their cell surface conductance density levels and cell surface protein levels. Here we investigated the role of a highly conserved cytoplasmic C-terminal charged region of five amino acids (HRETE) of the S6 transmembrane domain in the protein and conductance expression of Kv1.1, Kv1.2, and Kv1.4 channels. For Kv1.1 we found that E420stop, T419stop, and E418stop showed cell surface conductance densities and cell surface protein levels similar to full length control, whereas R417stop and H416stop exhibited essentially no conductance but their surface protein levels were similar to full length control. A bulky non-negatively charged hydrophilic amino acid at position 417 appeared to be critical for wild type gating of Kv1.1 because R417K and R417Q rescued conductance levels whereas R417A or R417E did not. The R417A mutation in the full length Kv1.1 also exhibited surface protein levels similar to control but it did not exhibit significant conductance. In contrast, mutation of the equivalent arginine to alanine in full length Kv1.2 and Kv1.4 appeared to have little or no effect on channel conductance but rather decreased cell surface protein levels by inducing partial high ER retention. These findings are consistent with the notion that the arginine amino acid in the HRETE region plays a different role in affecting conductance levels or cell surface protein levels of very closely related Kv1 potassium channels.

Intracerebral hemorrhage elicits aberration in cardiomyocyte contractile function and intracellular Ca2+ transients.

BACKGROUND AND PURPOSE: The sequelae of intracerebral hemorrhage involve multiple organ damage including electrocardiographic alteration, although the mechanism(s) behind myocardial dysfunction is unknown. The aim of this study was to examine the impact of intracerebral hemorrhage on cardiomyocyte contractile function, intracellular Ca2+ handling, Ca2+ cycling proteins, I kappa B beta protein (IkappaB) phosphorylation, hypoxia-inducible factor 1alpha (HIF-1alpha), and nitrosative damage within 48 hours of injury. METHODS: Mechanical and intracellular Ca2+ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (+/-dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), fura-2 fluorescence intensity (FFI), and intracellular Ca2+ decay. RESULTS: Myocytes from intracerebral hemorrhage rats exhibited depressed PS, +/-dL/dt, prolonged TPS and TR90, as well as declined baseline FFI and slowed intracellular Ca2+ decay between 12 and 24 hours after injury. Most of these aberrations returned to normal levels 48 hours after hemorrhage with the exception of -dL/dt and TR90. Myocytes from 24-hour posthemorrhage rats exhibited a stepper negative staircase in PS with increased stimulus frequency. Cardiac expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2a and phospholamban was enhanced, whereas that of Na+-Ca2+ exchanger and voltage-dependent K+ channel was decreased. IkappaB phosphorylation, HIF-1alpha, inducible NO synthase, and 3-nitrotyrosine were enhanced 12 hours after injury. CONCLUSIONS: These data demonstrated that intracerebral hemorrhage initiates cardiomyocyte contractile and intracellular Ca2+ dysregulation possibly related to altered expression of Ca2+ cycling proteins, nitrosative damage, and myocardial phosphorylation of IkappaB.

Age-related molecular reorganization at the node of Ranvier.

In myelinated axons, action potential conduction is dependent on the discrete clustering of ion channels at specialized regions of the axon, termed nodes of Ranvier. This organization is controlled, at least in part, by the adherence of myelin sheaths to the axolemma in the adjacent region of the paranode. Age-related disruption in the integrity of internodal myelin sheaths is well described and includes splitting of myelin sheaths, redundant myelin, and fluctuations in biochemical constituents of myelin. These changes have been proposed to contribute to age-related cognitive decline; in previous studies of monkeys, myelin changes correlate with cognitive performance. In the present study, we hypothesize that age-dependent myelin breakdown results in concomitant disruption at sites of axoglial contact, in particular at the paranode, and that this disruption alters the molecular organization in this region. In aged monkey and rat optic nerves, immunolabeling for voltage-dependent potassium channels of the Shaker family (Kv1.2), normally localizing in the adjacent juxtaparanode, were mislocalized to the paranode. Similarly, immunolabeling for the paranodal marker caspr reveals irregular caspr-labeled paranodal profiles, suggesting that there may be age-related changes in paranodal structure. Ultrastructural analysis of paranodal segments from optic nerve of aged monkeys shows that, in a subset of myelinated axons with thick sheaths, some paranodal loops fail to contact the axolemma. Thus, age-dependent myelin alterations affect axonal protein localization and may be detrimental to maintenance of axonal conduction.