OBJECTIVE: To investigate the expression of hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) and connexin43 (Cx43) in the sinoatrial node of electric shock death. METHODS: As experimental group, 34 cases of electric shock death who had definite current mark evidence were selected from pathology department of Xuzhou Medical College from 2010 to 2013. As the control group, 20 cases of fatal severe craniocerebral injury in traffic accidents were chosen. The expressions of HCN4 and Cx43 in the sinoatrial node were observed by immunohistochemical technology. RESULTS: HCN4 positive cells expressed in the cell membrane and cytoplasm of the sinoatrial node. Cx43 positive cells expressed in the cell membrane and cytoplasm of T cells and myocardial cells. The expression of HCN4 was significantly higher than that of the control group (P < 0.05) and the expression of Cx43 was significantly lower than that of the control group (P < 0.05). CONCLUSION The changes of HCN4 and Cx43 expressions in the sinoatrial node illustrate electric shock death might be related to the abnormalities of cardiac electrophysiology and conduction.
Connexin 43/metabolism* ; Cyclic Nucleotide-Gated Cation Channels ; Heart Rate ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism* ; Immunohistochemistry/methods* ; Myocardium/metabolism* ; Myocytes, Cardiac ; Sinoatrial Node/physiopathology*

The thalamocortical (TC) circuit is closely associated with pain processing. The hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 channel is predominantly expressed in the ventral posterolateral thalamus (VPL) that has been shown to mediate neuropathic pain. However, the role of VPL HCN2 in modulating TC circuit activity is largely unknown. Here, by using optogenetics, neuronal tracing, electrophysiological recordings, and virus knockdown strategies, we showed that the activation of VPL TC neurons potentiates excitatory synaptic transmission to the hindlimb region of the primary somatosensory cortex (S1HL) as well as mechanical hypersensitivity following spared nerve injury (SNI)-induced neuropathic pain in mice. Either pharmacological blockade or virus knockdown of HCN2 (shRNA-Hcn2) in the VPL was sufficient to alleviate SNI-induced hyperalgesia. Moreover, shRNA-Hcn2 decreased the excitability of TC neurons and synaptic transmission of the VPL-S1HL circuit. Together, our studies provide a novel mechanism by which HCN2 enhances the excitability of the TC circuit to facilitate neuropathic pain.
Animals ; Mice ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics* ; Neuralgia ; RNA, Small Interfering ; Thalamus/metabolism* ; Up-Regulation

Objective: To explore the effects of 3-phosphate dependent protein kinase 1-protein kinase B (PDK1-Akt) signaling pathway on the transcription, expression and function of cardiac hyperpolarized activated cyclic nucleotide gated 4 (HCN4) ion channels. Methods: Atrial myocytes were obtained from healthy male wild-type C57 mice and heart-specific PDK1 knockout mice (PDK1-KO) by enzymolysis. Then the atrial myocytes were divided into blank control group and PDK1-KO group. In further studies, the isolated atrial myocytes were cultured and further divided into drug control group (treated with dimethyl sulfoxide (DMSO)) and PDK1 knockdown group (treated with 1 μg/ml PDK1 short hairpin RNA (shRNA) interference plasmid), SC79 group (treated with 8 μmol/ml SC79), GSK2334470 group (treated with 10 nmol/L GSK2334470) and PDK1 knockdown+SC79 group (8 μmol/ml SC79 and 1 μg/ml PDK1 shRNA interference plasmid). Real time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of PDK1 and HCN4, Western blot was used to detect the protein expression levels of PDK1, Akt and HCN4, the whole cell patch clamp was used to detecte the current density of HCN, and immunofluorescence was used to detecte the expression of HCN4 protein on atrial cells. Results: (1) the expression levels of HCN4 mRNA (1.46±0.03 vs. 0.99±0.01, P<0.001) and protein (1.14±0.02 vs. 1.00±0.06, P=0.017) in PDK1-KO group were higher than those in blank control group. The HCN current density in PDK1-KO group was higher than that in blank control group((-17.47±2.00) pA/pF vs. (-12.15±2.25) pA/pF, P=0.038). (2) The functions of PDK1 shRNA and specific Akt agonist SC79 were verified by comparing the PDK1 knockdown group and SC79 group with the drug control group. The results showed that the expression levels of PDK1 mRNA and protein in PDK1 knockdown group were lower than those in drug control group, and the expression level of phosphorylated Akt (Thr 308) protein in SC79 group was higher than that in drug control group. (3) The expression levels of HCN4 mRNA (3.61±0.46 vs. 1.00±0.08, P<0.001) and protein (2.33±0.11 vs. 1.00±0.05, P<0.001) in GSK2334470 group were higher than those in drug control group. (4) To reduce the effect of drug-miss target, the cultured atrial myocytes were transfected with shRNA plasmid of PDK1 and intervened with SC79. The results showed that the expression of HCN4 mRNA in PDK1 knockdown group was higher than that in the drug control group (1.76±0.11 vs. 1.00±0.06, P<0.001), and PDK1 knockdown+SC79 group (1.76±0.11 vs. 1.33±0.07, P=0.003). In PDK1 knockdown+SC79 group, the mRNA expression level was also higher than that in the drug control group (1.33±0.07 vs. 1.00±0.06, P<0.001). The expression level of HCN4 protein in PDK1 knockdown group was higher than that in drug control group (1.15±0.04 vs. 1.00±0.05, P=0.003). As for the The expression level of HCN4 protein, there was no significantly statistical difference between the PDK1 knockdown+SC79 group and the drug control group (P>0.05), but PDK1 knockdown+SC79 group was lower than PDK1 knockdown group (0.95±0.01 vs. 1.15±0.04, P<0.001). In patch clamp experiments, the results showed that the HCN current density was (-13.27±1.28) pA/pF in the drug control group, (-18.76±2.03) pA/pF in the PDK1 knockdown group, (-13.50±2.58) pA/pF in the PDK1 knockdown+SC79 group; the HCN current density of PDK1 knockdown group was higher than that of drug control group (P<0.001), but there was no significant difference between PDK1 knockdown+SC79 group and drug control group (P>0.05). (5) The results of immunofluorescence showed that the brightness of green fluorescence of PDK1 knockdown group was higher than that of drug control group, indicating that the expression of HCN4 localized on cell membrane was increased. However, the green fluorescence of PDK1 knockdown+SC79 group was lighter than that of PDK1 knockdown group, suggesting that the expression of HCN4 in PDK1-knockdown cell membrane decreased after further activating Akt. Conclusion: PDK1-Akt signaling pathway is involved in the regulation of HCN4 ion channel transcription, expression and function.
Animals ; Cyclic Nucleotide-Gated Cation Channels ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism* ; Male ; Mice ; Myocytes, Cardiac/metabolism* ; Potassium Channels/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction

Cell Line ; Cyclic Nucleotide-Gated Cation Channels ; drug effects ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Electrophysiology ; Humans ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; Muscle Proteins ; drug effects ; genetics ; metabolism ; Potassium Channels ; Transfection

OBJECTIVETo study pacemaker current gene expression of mesenchymal stem cells (MSCs) and the electrophysiological property of MSCs expressing human pacemaker current gene.

METHODSPacemaker current gene expression of MSCs were studied by real-time quantitative polymerase chain reaction (real-time PCR) and pcDNA3-hHCN2 was transfected with Lipofectin 2000 into MSCs. hHCN2 expression at mRNA and at protein levels in the transfected cells were identified by real-time PCR and Western blot, respectively. The ionic currents of cloned hHCN2 (IhHCN2) were recorded and the current characteristics were studied through the whole-cell patch clamp technique.

RESULTSmHCN1, mHCN2, mHCN3, mHCN4 represent (0.08+/-0.01)%, (77.16+/-0.03)%, (0.24+/-0.01)%, (22.53+/-0.02)% of total HCN mRNA in MSCs as determined by real-time PCR. Transfected hHCN2 ionic currents were recorded by whole-cell patch clamp and current density-voltage curves were obtained. The threshold for activation of IhHCN2 was approximately -80 mV and this current could be blocked by Cs+ (4 mmol/L). hHCN2 expression in transfected MSCs was detected both at mRNA and protein levels.

CONCLUSIONS1. mHCN2 and mHCN4 represent the major populations of total HCN mRNA in MSCs. 2. Plasmid pcDNA3-hHCN2 by Lipofectin could be successfully transfected into MSCs with IhHCN2 recorded by whole-cell patch clamp technique, this study provides a basis for future antiarrhythmic gene therapy.

Animals ; Cyclic Nucleotide-Gated Cation Channels ; Gene Expression ; Humans ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; Membrane Potentials ; physiology ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Polymerase Chain Reaction ; Potassium Channels ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection

Spinal α-motoneurons directly innervate skeletal muscles and function as the final common path for movement and behavior. The processes that determine the excitability of motoneurons are critical for the execution of motor behavior. In fact, it has been noted that spinal motoneurons receive various neuromodulatory inputs, especially monoaminergic one. However, the roles of histamine and hypothalamic histaminergic innervation on spinal motoneurons and the underlying ionic mechanisms are still largely unknown. In the present study, by using the method of intracellular recording on rat spinal slices, we found that activation of either H or H receptor potentiated repetitive firing behavior and increased the excitability of spinal α-motoneurons. Both of blockage of K channels and activation of Na-Ca exchangers were involved in the H receptor-mediated excitation on spinal motoneurons, whereas the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were responsible for the H receptor-mediated excitation. The results suggest that, through switching functional status of ion channels and exchangers coupled to histamine receptors, histamine effectively biases the excitability of the spinal α-motoneurons. In this way, the hypothalamospinal histaminergic innervation may directly modulate final motor outputs and actively regulate spinal motor reflexes and motor execution.
Animals ; Histamine ; pharmacology ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; metabolism ; Motor Neurons ; drug effects ; physiology ; Rats ; Receptors, Histamine H2 ; metabolism ; Sodium-Calcium Exchanger ; metabolism

Functional changes in synaptic transmission from the lateral entorhinal cortex to the dentate gyrus (LEC-DG) are considered responsible for the chronification of pain. However, the underlying alterations in fan cells, which are the predominant neurons in the LEC that project to the DG, remain elusive. Here, we investigated possible mechanisms using a rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain. We found a substantial increase in hyperpolarization-activated/cyclic nucleotide-gated currents (I_h), which led to the hyperexcitability of LEC fan cells of CFA slices. This phenomenon was attenuated in CFA slices by activating dopamine D2, but not D1, receptors. Chemogenetic activation of the ventral tegmental area -LEC projection had a D2 receptor-dependent analgesic effect. Intra-LEC microinjection of a D2 receptor agonist also suppressed CFA-induced behavioral hypersensitivity, and this effect was attenuated by pre-activation of the I_h. Our findings suggest that down-regulating the excitability of LEC fan cells through activation of the dopamine D2 receptor may be a strategy for treating chronic inflammatory pain.
Animals ; Chronic Pain ; Entorhinal Cortex/metabolism* ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; Neurons/metabolism* ; Rats ; Receptors, Dopamine D1/metabolism* ; Receptors, Dopamine D2

Animals ; Animals, Newborn ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; metabolism ; Male ; Microwaves ; adverse effects ; Random Allocation ; Rats, Wistar ; Sinoatrial Node ; metabolism ; radiation effects

The purpose of this research is to explore the distribution and expression of hyperpolarization-activated cyclic nucleotide-gated channels subtype 2 (HCN2) in cerebrospinal fluid (CSF)-contacting nucleus in neuropathic pain, and provide experimental evidence to reveal the biological function and regulation mechanisms of CSF-contacting nucleus in neuropathic pain. Neuropathic pain model was produced by chronic constriction injury (CCI) in Sprague-Dawley (SD) rats. Intracerebroventricular injection of cholera toxin subunit B (CTb) labeled with horseradish peroxidase (CB-HRP) was used to specifically mark distal CSF-contacting nucleus. The thermal withdrawal latency and mechanical withdrawal threshold of rats were recorded to detect the change of pain threshold. The expressions HCN2 channel and c-Fos proteins in CSF-contacting nucleus were detected by immunofluorescence and Western blot. The results showed that, compared with the control group, CTb-treated rats did not show any differences in the expressions of HCN2 channel and c-Fos proteins in CSF-contacting nucleus, as well as pain threshold. At 7, 14 d after CCI operation, the model rats showed not only significantly increased expressions of HCN2 channel and c-Fos in CSF-contacting nucleus, but also decreased pain threshold. ZD7288, a HCN2 channel blocker, could reverse the above changes in neuropathic pain model rats. These results suggest that the CSF-contacting nucleus may be involved in the process of neuropathic pain via the HCN2 channel.
Animals ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; metabolism ; Neuralgia ; metabolism ; Neurons ; metabolism ; Pain Threshold ; Potassium Channels ; metabolism ; Proto-Oncogene Proteins c-fos ; metabolism ; Pyrimidines ; pharmacology ; Rats ; Rats, Sprague-Dawley

The study was aimed to assess the effect of Klotho gene and sinoatrial node pacing channel gene (HCN4 and HCN2) for studying sick sinus syndrome, with Klotho gene under the interference of Plasmid-mediated short hairpin RNA. Twenty-five C57BL/6J mice were divided into four groups, i. e, plasmid shRNA 24h group, plasmid shRNA 12h group, sodium chloride 24h group and sodium chloride 12h group. Plasmid shRNA 50microL (1microg/microL) and sodium chloride 50microl were respectively injected according to mice vena caudalis into those in plasmid shRNA group and sodium chloride group. After 12h or 24h respectively, all mice were executed and their sinoatrial node tissues were cut. The mRNA of Klotho, HCN4 and HCN2 gene were detected by RT-PCR. The results of RT-PCR showed that Klotho, HCN4 and HCN2 mRNA levels were lower compared with those in sodium chloride 12h group after 12h interference interval. The results indicated that there might be the a certain relationship between Klotho gene and sinoatrial node pacing channel gene.
Animals ; Glucuronidase ; genetics ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Plasmids ; genetics ; Potassium Channels ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Sinoatrial Node ; metabolism ; physiology ; physiopathology