Thalamocortical Circuit Controls Neuropathic Pain via Up-regulation of HCN2 in the Ventral Posterolateral Thalamus / 神经科学通报·英文版

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.

The effects of PDK1-Akt signaling pathway intervention on cardiomyocyte HCN4 ion channels / 中华心血管病杂志

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.

Facilitation of spinal α-motoneuron excitability by histamine and the underlying ionic mechanisms / 生理学报

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.

Cyclophosphamide-induced HCN1 channel upregulation in interstitial Cajal-like cells leads to bladder hyperactivity in mice

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are confirmed to be expressed in bladder interstitial Cajal-like cells (ICC-LCs), but little is known about their possible role in cystitis-associated bladder dysfunction. The present study aimed to determine the functional role of HCN channels in regulating bladder function under inflammatory conditions. Sixty female wild-type C57BL/6J mice and sixty female HCN1-knockout mice were randomly assigned to experimental and control groups, respectively. Cyclophosphamide (CYP)-induced cystitis models were successfully established in these mice. CYP treatment significantly enhanced HCN channel protein expression and I(h) density and significantly altered bladder HCN1 channel regulatory proteins. Carbachol (CCH) and forskolin (FSK) exerted significant effects on bladder ICC-LC [Ca²⁺]i in CYP-treated wild-type (WT) mice, and HCN1 channel ablation significantly decreased the effects of CCH and FSK on bladder ICC-LC [Ca²⁺]i in both naive and CYP-treated mice. CYP treatment significantly potentiated the spontaneous contractions and CCH (0.001-10 µM)-induced phasic contractions of detrusor strips, and HCN1 channel deletion significantly abated such effects. Finally, we demonstrated that the development of CYP-induced bladder overactivity was reversed in HCN1 -/- mice. Taken together, our results suggest that CYP-induced enhancements of HCN1 channel expression and function in bladder ICC-LCs are essential for cystitis-associated bladder hyperactivity development, indicating that the HCN1 channel may be a novel therapeutic target for managing bladder hyperactivity.

Dysfunctional hyperpolarization-activated cyclic nucleotide-gated ion channels in cardiac diseases

Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are reverse voltage-dependent, and their activation depends on the hyperpolarization of the membrane and may be directly or indirectly regulated by the cyclic adenosine monophosphate (cAMP) or other signal-transduction cascades. The distribution, quantity and activation states of HCN channels differ in tissues throughout the body. Evidence exhibits that HCN channels play critical roles in the generation and conduction of the electrical impulse and the physiopathological process of some cardiac diseases. They may constitute promising drug targets in the treatment of these cardiac diseases. Pharmacological treatment targeting HCN channels is of benefit to these cardiac conditions.

Changes of HCN4, Cx43 Expression in the Sinoatrial Node of Electric Shock Death / 法医学杂志

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.

Neuropathic pain enhances expression of HCN2 channel in rat cerebrospinal fluid-contacting nucleus / 生理学报

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.

HCN ion channel: biological characteristics and functions in pain / 生理学报

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in vertebrate are reverse voltage-dependent, and its activation depends on the hyperpolarization of cell and may be directly or indirectly regulated by the cyclic adenosine monophosphate (cAMP) or other signal transduction cascades. The distribution, quantity, and activation states of HCN channels differ in tissues throughout the body. By modulating If/If current, HCN channels may influence the resting membrane potential, and thus importantly regulate neuronal excitability, dendritic integration of synaptic potentials, and synaptic transmission. Evidence exhibits that HCN channels participate in pain and other physiological and pathological process. Pharmacological treatment targeting HCN channels is of benefit to relieve pain and other related diseases.

Role of HCN channels in the nervous system: membrane excitability and various modulations / 中国应用生理学杂志

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, distributing in a variety of tissues, especially in excitable cells such as heart cells and many kinds of neurons, have an important role in the modulation of heart rate and neuronal excitability. Different from typical voltage-gated sodium channels and potassium channels, HCN channels were evoked inward currents when the cell was hyperpolarized. More and more recent studies have disclosed that HCN channels play important roles in the nervous system, which were linked with its special electrophysiological features as well as its regulatory effect on the cellular membrane excitability. HCN channels could be modulated by many factors including both extracellular molecules and intracellular signaling cascades, which made its functions complicated in the different condition. Based on its role, HCN channels are presumed to be a promising target for chronic pain and brain disorders. In this paper, we will focus on the advancement of roles of HCN channels in the neural system as well as its complex modulator factors.

Study on the effect of Klotho gene interferred by plasmid-mediated short hairpin RNA (shRNA) on sinoatrial node pacing channel gene / 生物医学工程学杂志

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.

Inhibitory effects of propofol on supraoptic nucleus neurons of rat hypothalamus in vitro / 生理学报

To investigate the effects of novel intravenous general anesthetic propofol on membrane electrophysiological characteristics and action potential (AP) of the supraoptic nucleus (SON) neurons and possible ionic mechanisms, intracellular recordings were conducted in SON neurons from the coronal hypothalamic slice preparation of adult male Sprague Dawley (SD) rats. The results showed that bath application of 0.1 mmol/L propofol induced a significant decline in resting potential (P < 0.01), and higher concentrations of propofol (0.3 and 1.0 mmol/L) decreased time constant and slope resistance of cell membrane (P < 0.01). Under the hyperpolarizing current pulses exceeding 0.5 nA, an anomalous rectification was induced by hyperpolarization-activated cation channel (I(h) channel) in 11 out of 18 tested SON neurons. Bath of propofol reversibly decreased the anomalous rectification. Moreover, 0.1 mmol/L propofol elevated threshold level (P < 0.01) and decreased Max L. slope (P < 0.05) of the spike potential in SON neurons. Interestingly, 0.3 and 1.0 mmol/L propofol nullified APs in 6% (1/18) and 71% (12/17) tested SON neurons, respectively. In the SON neurons where APs were not nullified, propofol (0.3 mmol/L) decreased the amplitude of spike potential (P < 0.05). The higher concentrations of propofol (0.3 and 1.0 mmol/L) decreased firing frequencies evoked by depolarizing current pulses (0.1-0.7 nA), and shifted the current intensity-firing frequency relation curves downward and to the right. These results suggest that propofol decreases the excitability of SON neurons by inhibiting I(h) and sodium channels.

Overexpression of connexin 45 in rat mesenchymal stem cells improves the function as cardiac biological pacemakers / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Extensive 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.</p><p><b>METHODS</b>To 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.</p><p><b>RESULTS</b>High 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).</p><p><b>CONCLUSION</b>These 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.</p>

Hysteresis in human HCN4 channels: a crucial feature potentially affecting sinoatrial node pacemaking / 生理学报

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels modulate and regulate cardiac rhythm and rate. It has been suggested that, unlike the HCN1 and HCN2 channels, the slower HCN4 channel may not exhibit voltage-dependent hysteresis. We studied the electrophysiological properties of human HCN4 (hHCN4) channels and its modulation by cAMP to determine whether hHCN4 exhibits hysteresis, by using single-cell patch-clamp in HEK293 cells stably transfected with hHCN4. Quantitative real-time RT-PCR was also used to determine levels of expression of HCNs in human cardiac tissue. Voltage-clamp analysis revealed that hHCN4 current (I(h)) activation shifted in the depolarizing direction with more hyperpolarized holding potentials. Triangular ramp and action potential clamp protocols also revealed hHCN4 hysteresis. cAMP enhanced I(h) and shifted activation in the depolarizing direction, thus modifying the intrinsic hHCN4 hysteresis behavior. Quantitative PCR analysis of human sinoatrial node (SAN) tissue showed that HCN4 accounts for 75% of the HCNs in human SAN while HCN1 (21%), HCN2 (3%), and HCN3 (0.7%) constitute the remainder. Our data suggest that HCN4 is the predominant HCN subtype in the human SAN and that I(h) exhibits voltage-dependent hysteresis behavior that can be modified by cAMP. Therefore, hHCN4 hysteresis potentially plays a crucial role in human SAN pacemaking activity.

The changes of funny currents in the ventricular myocytes of neonatal rats and adult rats / 中国应用生理学杂志

<p><b>AIM</b>To record funny currents (If) of ventricular myocytes and to analysize hyperpolarization-activated cation channel(HCN) expression in the rats of different ages.</p><p><b>METHODS</b>Fresh ventricular myocytes were isolated from 3 days rats and adult rats.HCN expressions were measured by real-time quantitative polymerase chain reaction(real-time PCR). It was recorded through whole-cell patch clamp.</p><p><b>RESULTS</b>HCN1, HCN2, HCN3, HCN4 mRNA represented 0.23% +/- 0.01%, 83.58% +/- 0.04%, 0.79% +/- 0.01%, 15.44% +/- 0.01% of total HCN mRNA in the neonatal rats, respectively. If was recorded and the threshold for activation was -75 mV. In the adult rat, HCN1, HCN2, HCN3, HCN4 mRNA represented 0.72% +/- 0.02%, 91.58% +/- 0.08%, 0.27% +/- 0.02%, 7.12% +/- 0.02% of total HCN mRNA. The ratio of HCN2 to HCN4 was approximately (13.06 +/- 0.21):1. The threshold for activation of If was approximately -115 mV in the adult rats.</p><p><b>CONCLUSION</b>With the development of rats, the value of If is smaller. The threshold for activation of If is more negative. The ratio of HCN2 to HCN4 is bigger.</p>

Effects of amiodarone on funny current I(f) channel gene expression in neonatal rat ventricular myocytes / 中华心血管病杂志

<p><b>OBJECTIVE</b>To analysis the effect of amiodarone on funny current (I(f)) and hyperpolarization-activated cation channel (HCN) gene expressions of the neonatal rat ventricular myocytes.</p><p><b>METHODS</b>Ventricular 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.</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSION</b>Current 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.</p>

Distinct Regional and Cellular Localization of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 1 in Cerebellar Cortex of Rat

OBJECTIVE: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarizationactivated currents (Ih) that participate in regulating neuronal membrane potential and contribute critically to pacemaker activity, promoting synchronization of neuronal networks. However, distinct regional and cellular localization of HCN channels in the brain have not been precisely defined. Aim of this study was to verify the precise cellular location of HCN1 channels in rat cerebellum to better understand the physiological role these channels play in synaptic transmission between CNS neurons. METHODS: HCN1 expression in rat brain was analyzed using immunohistochemistry and electron-microscopic observations. Postsynaptic density-95 (PSD-95), otherwise known as locating and clustering protein, was also examined to clarify its role in the subcellular location of HCN1 channels. In addition, to presume the binding of HCN1 channels with PSD-95, putative binding motifs in these channels were investigated using softwaresearching method. RESULTS: HCN1 channels were locally distributed at the presynaptic terminal of basket cell and exactly corresponded with the location of PSD-95. Moreover, nine putative SH3 domain of PSD-95 binding motifs were discovered in HCN1 channels from motif analysis. CONCLUSION: Distinct localization of HCN1 channels in rat cerebellum is possible, especially when analyzed in conjunction with the SH3 domain of PSD-95. Considering that HCN1 channels contribute to spontaneous rhythmic action potentials, it is suggested that HCN1 channels located at the presynaptic terminal of neurons may play an important role in synaptic plasticity.

Pacemaker current gene expression of rat mesenchymal stem cells and identification of mesenchymal stem cells expressing human pacemaker current gene (hHCN2) / 中华心血管病杂志

<p><b>OBJECTIVE</b>To study pacemaker current gene expression of mesenchymal stem cells (MSCs) and the electrophysiological property of MSCs expressing human pacemaker current gene.</p><p><b>METHODS</b>Pacemaker 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.</p><p><b>RESULTS</b>mHCN1, 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.</p><p><b>CONCLUSIONS</b>1. 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.</p>

Recombinant plasmid pIRES2-EGFP-HCN2 improved pacing function in canine model of sick sinus syndrome / 中华心血管病杂志

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>mHCN2 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.</p><p><b>RESULTS</b>EGFP 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.</p><p><b>CONCLUSION</b>The recombinant eukaryotic expression plasmid pIRES2-EGFP-HCN2 can improve pacing function in atrial myocytes and in canine model of SSS.</p>

Establishment of a HEK293 cell line stably expressing human HCN2 gene / 中国应用生理学杂志

<p><b>AIM</b>To create a model for studying ionic channels by means of the expressing human HCN2 and G418-resistant HEK293 cell lines established.</p><p><b>METHODS</b>pcDNA3-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.</p><p><b>RESULTS</b>The G418 resistant (600 ug/ml) HEK293 cell line was established successfully and whole-cell patch clamp recorded ionic currents of transfected hHCN2.</p><p><b>CONCLUSION</b>The 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.</p>

Protective effects of hyperpolarizing cardioplegia with pinacidil on myocardium in rats / 华中科技大学学报（医学）（英德文版）

Whether the ATP-sensitive potassium channel opener pinacidil can provide myocardial protective effects in prolonged isolated global ischemic rat heart was investigated. On modified isolated rat working heart model, 40 hearts were divided into four groups randomly: Hyperpolarized arrest H-K solution containing pinacidil (50 mumol/L) (P1 and P2) and depolarized arrest St. Thomas' solution (S1 and S2) subjected to 15 degrees C hypothermia, 60 min (P1 and S1) or 120 min (P1 and S2) of ischemia and 30 min reperfusion. The experimental indices included cardioplegic efficiency, cardiac function, coronary blood flow, myocardial enzyme release, myocardial water and ATP content. Hyperpolarized arrest provided significantly better recovery of cardiac function than depolarized arrest. Postischemic coronary flow and myocardial ATP content were higher. The arrest time of electro-mechanical activities were longer than depolarized arrest. There were no differences among the groups in myocardial water contents. The hyperpolarized arrest solution containing pinacidil can provide a marked myocardial protective effect during prolonged hypothermic myocardial ischemia.