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

AIMTo create a model for studying ionic channels by means of the expressing human HCN2 and G418-resistant HEK293 cell lines established.

METHODSpcDNA3-hHCN2 was transfected with Lipofectin2000 into HEK293 cell line. The transfected cells would be survived in the further culture medium containing G418 antibiotic as the hHCN2 gene could express a G418 resistant products. Whole-cell patch clamp investigated that hHCN2 gene was transfected into HEK293 cells.

RESULTSThe G418 resistant (600 ug/ml) HEK293 cell line was established successfully and whole-cell patch clamp recorded ionic currents of transfected hHCN2.

CONCLUSIONThe G418 resistant HEK293 cell line was successfully established with transfection of plasmid pcDNA3-hHCN2 by Lipofectin, which might be useful for studying the relationship between the structure and function of cloned ionic channels.

Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; Ion Channels ; genetics ; Patch-Clamp Techniques ; Plasmids ; Potassium Channels ; Transfection

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

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

OBJECTIVETo construct plasmid expressing pacemaker gene pIRES2-EGFP-HCN2 and study its effects in transfected atrial myocytes in vitro and in canine model of sick sinus syndrome (SSS).

METHODSmHCN2 gene was isolated from PTR plasmids and cloned into eukaryotic expression plasmid pIRES2-EGFP. Recombinant plasmids pIRES2-EGFP-HCN2 was transfected with by electroporation into neonatal atrial cardiomyocytes or injected to the sinoatrial (SA) region of canines with SSS induced by catheter and chemical ablation. pIRES2-EGFP-HCN2 expression was detected under fluorescence microscope and confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Spontaneous beating rate in atrial cardiomyocytes was detected with light microscope.

RESULTSEGFP expression was seen in transfected atrial cardiomyocytes 24 to 48 hours after transfection and the spontaneous beating rate was significantly increased than that in non-transfected atrial cardiomyocytes [(180 +/- 11) bpm vs (140 +/- 14) bpm, P < 0.05]. Heart rate was significantly increased 24 hours post recombinant plasmids pIRES2-EGFP-HCN2 injection compared to saline injection in canines with SSS [(150 +/- 13) bpm vs (105 +/- 17) bpm, P < 0.05]. Green fluorescence was also detected in frozen SA tissue sections of canines injected with recombinant plasmids pIRES2-EGFP-HCN2 and the production amplified by RT-PCR was about 300 bp which is consistent with mHCN2 gene fragment.

CONCLUSIONThe recombinant eukaryotic expression plasmid pIRES2-EGFP-HCN2 can improve pacing function in atrial myocytes and in canine model of SSS.

Animals ; Disease Models, Animal ; Dogs ; Gene Expression ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; In Vitro Techniques ; Ion Channels ; genetics ; Myocytes, Cardiac ; metabolism ; Plasmids ; Sick Sinus Syndrome ; therapy

OBJECTIVETo analysis the effect of amiodarone on funny current (I(f)) and hyperpolarization-activated cation channel (HCN) gene expressions of the neonatal rat ventricular myocytes.

METHODSVentricular myocytes of 1 - 3 days-old rats were isolated and cultured. The cardiomyocytes were treated by amiodarone (0.01, 0.1, 1, 10, 100 micromol/L) for 3 hours or amiodaron (10 micromol/L) for 0, 0.5, 1, 3, 6 hours. The I(f) and HCN 1 - 4 gene expressions were measured through the whole-cell configuration of the patch-clamp technique and real-time quantitative polymerase chain reaction (real-time PCR) using SYBR Green PCR kit.

RESULTS(1) HCN1, HCN2, HCN3 and HCN4 represented (0.23 +/- 0.01)%, (83.58 +/- 0.04)%, (0.79 +/- 0.01)% and (15.44 +/- 0.01)% of total HCN mRNA, respectively. (2) Amiodaron resulted in a dose-dependent I(f) [(3.1 +/- 0.9)%, (9.7 +/- 2.4)%, (36.7 +/- 5.8)%, (80.3 +/- 1.8)% and (85.9 +/- 3.1)%, respectively at -145 mV, IC(50) (1.32 +/- 0.28) micromol/L], HCN2 [(2.1 +/- 0.8)%, (8.9 +/- 3.6)%, (30.1 +/- 4.2)%, (78.3 +/- 3.6)% and (81.1 +/- 1.9)%, respectively] and HCN4 decrease [(0.5 +/- 0.2)%, (2.1 +/- 2.6)%, (8.8 +/- 3.2)%, (60.1 +/- 4.6)% and (59.6 +/- 6.5)%, respectively]. (3) Amiodaron (10 micromol/L) also induced a time-dependent I(f) [(1.1 +/- 0.1)%, (12.6 +/- 2.3)%, (80.6 +/- 2.2)% and (80.1 +/- 2.1)%, respectively], HCN2 [(1.0 +/- 0.1)%, (9.8 +/- 3.9)%, (82.9 +/- 4.6)% and (83.9 +/- 1.7)%, respectively] and HCN4 decrease [(0.1 +/- 0.1)%, (1.9 +/- 1.1)%, (59.4 +/- 7.8)% and (60.9 +/- 3.1)%, respectively]. However, HCN1 and HCN3 expressions were not affected by amiodaron treatment.

CONCLUSIONCurrent density of I(f) and the expression of HCN2 and HCN4 were decreased by amiodaron which might be the possible antiarrhythmic working mechanisms of amiodaron.

Amiodarone ; pharmacology ; Animals ; Animals, Newborn ; Cells, Cultured ; Cyclic Nucleotide-Gated Cation Channels ; genetics ; metabolism ; Female ; Gene Expression ; Heart Ventricles ; metabolism ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; Male ; Myocytes, Cardiac ; drug effects ; metabolism ; Patch-Clamp Techniques ; Potassium Channels ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

BACKGROUNDExtensive research toward creating a biological pacemaker by enhancement of inward depolarizing current has been performed. However, studies have mainly focused on inducing spontaneous activity and have not adequately addressed ways to improve pacemaker function. In this study we attempted to improve pacemaker function by altering connexin expression in rat mesenchymal stem cells (MSCs) to a phenotype similar to native sinus node pacemaker cells.

METHODSTo generate a biological pacemaker, MSCs were transduced with a cardiac pacemaker gene-hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), via transfection with a lentiviral vector. Funny current (I(f)) in HCN4(+) MSCs was recorded by voltage-clamp. Overexpression of connexin 45 (gene Gja7) in MSCs was achieved by transfection with the plasmid pDsRED2-N1-Gja7-RFP. Double-immunolabelling with anti-connexin 43 and anti-connexin 45 antibodies were used to identify the gap junction channels. The effects of the genetically modified MSCs on cardiomyocyte excitability were determined in MSCs cocultured with neonatal rat ventricular myocytes. Spontaneous action potentials of neonatal rat ventricular myocytes were recorded by current-clamp.

RESULTSHigh level time- and voltage-dependent inward hyperpolarization current that was sensitive to 4 mmol/L Cs(+) was detected in HCN4(+) MSCs, confirming that HCN4 acted as I(f) channels in MSCs. Connexin 43 and connexin 45 were simultaneously detected in CX45(+) MSCs. Beating frequency was (82 +/- 8) beats per minute (n = 5) in myocytes cocultured with non-transfected control MSCs, versus (129 +/- 11) beats per minute (n = 5) in myocytes cocultured with HCN4(+) MSCs. Myocytes cocultured with MSCs cotransfected with HCN4 and connexin 45 had the highest beating frequency at (147 +/- 9) beats per minute (n = 5).

CONCLUSIONThese findings demonstrate that overexpression of connexin 45 and subsequent formation of heteromeric connexin 45/connexin 43 gap junction channels in HCN4 expressing MSCs can improve their function as cardiac biological pacemakers in vitro.

Animals ; Animals, Newborn ; Biological Clocks ; physiology ; Cells, Cultured ; Connexins ; genetics ; metabolism ; Electrophysiology ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; Mesenchymal Stromal Cells ; cytology ; metabolism ; physiology ; Myocytes, Cardiac ; cytology ; metabolism ; physiology ; Potassium Channels ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction