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.

An integrated review on new targets in the treatment of neuropathic pain

Neuropathic pain is a complex chronic pain state caused by the dysfunction of somatosensory nervous system, and it affects the millions of people worldwide. At present, there are very few medical treatments available for neuropathic pain management and the intolerable side effects of medications may further worsen the symptoms. Despite the presence of profound knowledge that delineates the pathophysiology and mechanisms leading to neuropathic pain, the unmet clinical needs demand more research in this field that would ultimately assist to ameliorate the pain conditions. Efforts are being made globally to explore and understand the basic molecular mechanisms responsible for somatosensory dysfunction in preclinical pain models. The present review highlights some of the novel molecular targets like D-amino acid oxidase, endoplasmic reticulum stress receptors, sigma receptors, hyperpolarization-activated cyclic nucleotide-gated cation channels, histone deacetylase, Wnt/β-catenin and Wnt/Ryk, ephrins and Eph receptor tyrosine kinase, Cdh-1 and mitochondrial ATPase that are implicated in the induction of neuropathic pain. Studies conducted on the different animal models and observed results have been summarized with an aim to facilitate the efforts made in the drug discovery. The diligent analysis and exploitation of these targets may help in the identification of some promising therapies that can better manage neuropathic pain and improve the health of patients.

Rebound depolarization of substantia gelatinosa neurons and its modulatory mechanisms in rat spinal dorsal horn / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the rebound depolarization of substantia gelatinosa (SG) neurons in rat spinal dorsal horn and explore its modulatory mechanisms to provide better insights into rebound depolarization-related diseases.</p><p><b>METHODS</b>Parasagittal slices of the spinal cord were prepared from 3- to 5-week-old Sprague-Dawley rats. The electrophysiologic characteristics and responses to hyperpolarization stimulation were recorded using whole-cell patch-clamp technique. The effects of hyperpolarization-activated cyclic nucleotide gated cation (HCN) channel blockers and T-type calcium channel blockers on rebound depolarization of the neurons were studied.</p><p><b>RESULTS</b>A total of 63 SG neurons were recorded. Among them, 23 neurons showed no rebound depolarization, 19 neurons showed rebound depolarization without spikes, and 21 neurons showed rebound depolarization with spikes. The action potential thresholds of the neurons without rebound depolarization were significantly higher than those of the neurons with rebound depolarization and spikes (-28.7∓1.6 mV vs -36.0∓2.0 mV, P<0.05). The two HCN channel blockers CsCl and ZD7288 significantly delayed the latency of rebound depolarization with spike from 45.9∓11.6 ms to 121.6∓51.3 ms (P<0.05) and from 36.2∓10.3 ms to 73.6∓13.6 ms (P<0.05), respectively. ZD7288 also significantly prolonged the latency of rebound depolarization without spike from 71.9∓35.1 ms to 267.0∓68.8 ms (P<0.05). The T-type calcium channel blockers NiCl2 and mibefradil strongly decreased the amplitude of rebound depolarization with spike from 19.9∓6.3 mV to 9.5∓4.5 mV (P<0.05) and from 26.1∓9.4 mV to 15.5∓5.0 mV (P<0.05), respectively. Mibefradil also significantly decreased the amplitude of rebound depolarization without spike from 14.3∓3.0 mV to 7.9∓2.0 mV (P<0.05).</p><p><b>CONCLUSION</b>Nearly two-thirds of the SG neurons have rebound depolarizations modulated by HCN channel and T-type calcium channel.</p>

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.

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.

Cyclic nucleotide-gated channels and sperm function / 中华男科学杂志

The cyclic nucleotide-gated (CNG) channel is a nonselective cation channel and one of the main entrances of Ca2+ influxion into cells. CNG channels are opened by direct binding of cyclic nucleotides. Six different genes encode the CNG protein, 4 A subunits and 2 B subunits. The activity of CNG channels can be regulated by Ca2+/Ca(2+)-binding proteins (CaM) and phosphorylation/dephosphorylation. Recently, extensive attention has been drawn to the researches on CNG channels in the reproductive system, and many studies show that CNG channels play a pivotal role in sperm motility, capacitation and acrosome reaction. This article focuses on the relationship of CNG channels with sperm function.

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.

Endothelin-1 stimulates the expression of pacemaker channel I(f) in cardiomyocytes through a p38 MAPK-independent signaling pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the transcriptional regulation of pacemaker channel I(f) mediated by vasoactive peptide endothelin-1 (ET-1) in neonatal rat ventricular myocytes and its mechanism.</p><p><b>METHODS</b>Neonatal rat ventricular myocytes were enzymatically isolated. I(f) current was recorded using the whole-cell patch-clamp technique. The expression of hyperpolarization-activated cyclic nucleotide-gated channel (HCN) isoforms HCN2 and HCN4 were measured by quantitative RT-PCR.</p><p><b>RESULTS</b>ET-1 increased the expression of HCN2 and HCN4 mRNA in a dose- and time-dependent manner. These effects were blocked by specific ETA receptor antagonist BQ-123 but not the ETB receptor antagonist BQ-788. The effects of ET-1 on HCN2 and HCN4 mRNA expression were not affected by the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB-203580).</p><p><b>CONCLUSION</b>These findings indicate that ET-1 stimulates the expression of pacemaker channel I(f) in cardiomyocytes via ETA receptor through a p38 MAPK-independent signaling pathway, which might be linked to the intrinsic arrhythmogenic potential of ET-1.</p>

Conservation and divergence of Grb7 family of Ras-binding domains

Ras proteins are signal-transducing GTPases that cycle between inactive GDP-bound and active GTP-bound forms. Ras is a prolific signaling molecule interacting with a spectrum of effector molecules and acting through more than one signaling pathway. The Ras-effector proteins contain a Ras-associating (RA) domain through which these associate with Ras in a GTP-dependent manner. The RA domain is highly conserved among the members of the growth factor receptor-bound (Grb) 7 family of proteins which includes Grb7, Grb10 and Grb14. Our laboratory has reported an unusual observation that RA domain of Grb14 binds to the C-terminal nucleotide binding site of cyclic nucleotide gated channel (CTRCNGA1) and inhibits the channel activity. Molecular modeling of the CTR-CNGA1 displays 50%-70% tertiary structural similarity towards Ras proteins. We named this region as Ras-like domain (RLD). The interaction between RA-Grb14 and RLD-CNGA1 is mediated through a simple protein-protein interaction temporally and spatially regulated by light and cGMP. It is interesting to note that Grb14 binds to GTPase-mutant Rab5, a Ras-related small GTPase whereas Grb10 binds only to GTP-bound form of active Rab5 but not to GTPase-defective mutant Rab5. These results suggest that Grb14 might have been evolved later in the evolution that binds to both Ras and nucleotide binding proteins such as CNGA1. Our studies also suggest that eukaryotic CNG channels could be evolved through a gene fusion between prokaryotic ion channels and cyclic nucleotide binding proteins, both of which might have undergone several sequence variations for functional adaptation during evolution.

Mechanism involved in the modulation of photoreceptor-specific cyclic nucleotidegated channel by the tyrosine kinase adapter protein Grb14

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide-gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In the current study we show that this RA domain-mediated inhibition of rod CNG channel is electrostatic in nature. Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg(559) through a negatively charged β-turn at its RA domain. Moreover, the three Glu residues (180-182) in Grb14 are absolutely critical for electrostatic interaction with the cGMP binding pocket and resultant inhibition. Our study also demonstrates that substitution of Lys140 for Ala or in combination with polyglutamte mutants of Grb14 results in a significantly reduced binding with CNGA1. These results suggest that in addition to Glu(180-182) and Lys(140), other residues in Grb14 may be involved in the electrostatic interaction with CNGA1. The RA domain is highly conserved among the members of Grb7 family of proteins, which includes Grb7, Grb10 and Grb14. Further, only Grb14 is able to modulate the channel activity, but not Grb7 and Grb10. All together, it suggests the existence of a divergence in RA domains among the members of the Grb7 family.

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.

Effect of terbutaline on sodium transport in alveolar type I and type II cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect of terbutaline on sodium transport in rat alveolar type I (ATI) and type II (ATII) cells of rats.</p><p><b>METHODS</b>The whole cell currents were recorded from ATII cells isolated from rat lungs perfused with or without amiloride (inhibitor of epithelial sodium channel) and ZnCl(2) (inhibitor of cyclic nucleotide-gated cation channel) in the whole cell recording mode using the patch-clamp technique. The effect of terbutaline on the currents was examined.</p><p><b>RESULTS</b>The main currents recorded from ATII cells were amiloride-sensitive and Zn(2+)-sensitive. The amiloride-sensitive and Zn(2+)-sensitive current shared a similar proportion (P>0.05). Both currents could be significantly increased by terbutaline (P<0.05), and the proportion of amiloride-sensitive current was 1.7 times that of Zn(2+)-sensitive current (P<0.05).</p><p><b>CONCLUSION</b>There are functional epithelial sodium channels (ENaC) and cyclic nucleotide-gated cation channels (CNG) on freshly isolated ATII cells, both serving as the main channels for sodium transport. Terbutaline increases the absorption of alveolar fluid primarily by increasing sodium transport of ENaC and CNG on ATI and AT II cells.</p>

Involvement of hyperpolarization-activated, cyclic nucleotide-gated cation channels in dorsal root ganglion in neuropathic pain / 生理学报

Dorsal root ganglion (DRG) neurons have peripheral terminals in skin, muscle, and other peripheral tissues, and central terminals in the spinal cord dorsal horn. Hyperpolarization-activated current (I(h)) of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are present in the DRG. The genes encoding HCN channels have four subtypes named HCN1 to HCN4. HCN channels are permeable to both K(+) and Na(+). They underlie the depolarization that modulates the rhythmic generations of action potentials (APs), contribute to the resting membrane potential, and modify the waveform of propagated synaptic and generator potentials. Neuropathic pain is characterized by spontaneous pain, hyperalgesia and allodynia. After spinal nerve injury, the cell bodies of the primary sensory neurons in segmental DRG become hyperexcitable, characterized for some neurons by the presence of spontaneous firing (or ectopic discharge). In the following, we summarize our observations on the role of HCN channels in DRG neurons in neuropathic pain. 1 HCN subtypes and I(h) in DRG neurons Immunohistochemical staining revealed a subgroup of neurons in the DRG that were stained with rabbit polyclonal antibodies specific for HCN1, 2, 3 and 4. The most prominently expressed HCN subtype was HCN1. HCN1-positive cells in DRG were medium to large in size and doubly labeled with neurofilament-200 (NF-200), and were not labeled with isolectin B4 (IB4), a C fiber marker. In contrast, HCN2, 3 or 4 was expressed in all DRG neurons at a lower level. HCN4 was confined to small neurons. DRG neurons expressed I(h). When membrane was hyperpolarized, the channel was activated, mediating a slowly activated, inward current. I(h) was distributed mainly in large and medium-sized DRG neurons. 2 Changes in expression of HCN in DRG after spinal nerve ligation Western blotting was used to detect the changes in the expression of HCN subtypes in the DRG after spinal nerve ligation. HCN1 mRNA and protein were reduced in the DRG whose spinal nerve had been ligated. HCN1 expression was decreased to the lowest level at day 14 and restored at day 28 after spinal nerve ligation. HCN2 mRNA and medium molecular weight protein was also decreased in spinal-nerve ligated DRG. HCN3 and 4 in the same ganglion remained unchanged as evidenced by immunohistochemical staining, until day 28 when they became significantly decreased. HCN4 mRNA in DRG did not change, and protein expression slightly increased. Interestingly, abundant axonal accumulation of HCN channel protein at the injured sites in chronic constriction injury (CCI) rats. Electron immunomicroscopy showed strong positive immunolabeling on the axolemma of myelinated thick axons. 3 Role of I(h) in neuronal excitability and ectopic discharges after spinal nerve ligation ZD7288, a specific I(h) blocker, inhibited I(h) in a time- and concentration-dependent manner. With patch-clamp recording on acutely isolated DRG neurons, it was found that ZD7288 perfusion resulted in a decrease of both I(h) activity and the activation time constant. ZD7288 decreased the number of repetitive APs and caused an increase in AP rise time, accompanied by a small hyperpolarization of the membrane resting potential. The results demonstrated that I(h) was involved in AP firing, and possessed the physiological functions to facilitate neuronal excitability and ectopic firing. Extracellular electrophysiological recording from dorsal root fibers associated with the spinal nerve-ligated ganglion revealed three different firing patterns of ectopic discharges: tonic or regular, bursting and irregular. The average frequency of ectopic discharges and the proportions of active filaments also changed rapidly, both parameters reaching a peak within 24 h then declining gradually in the following days. It was also found that proportions of three different firing patterns changed dynamically over time. The tonic and bursting types were dominant patterns in the first 24 h, while the irregular became the only pattern at day 14. We found that all three firing patterns (tonic, bursting and irregular) were dose- and time-dependently inhibited by local application of ZD7288 to DRG. The rate of suppression was negatively related to the frequency of firing prior to the application of ZD7288. We also found that, while the tonic firing pattern was gradually transformed to bursting type by application of 100 mumol/L ZD7288, it could be transformed to integer multiples firing by 1000 mumol/L ZD7288. 4 Effects of administration of ZD7288 on mechanical allodynia after spinal nerve ligation or CCI After spinal nerve ligation, i.t. injection of 30 mug ZD7288 significantly increased the 50% paw withdrawal threshold, ipsilateral to the ligated nerve. ZD7288 had no effect if the dose was lower than 15 mug, but resulted in motor deficits if the dose was higher than 60 mug. ZD7288 produced much better effects in the early stage (5 or 14 days after spinal nerve ligation) than that in the late stage (28 days after spinal nerve ligation). In CCI rats, ZD7288 application to the injured sited also significantly suppressed the ectopic discharges from injured nerve fibers with no effect on impulse conduction. Moreover, mechanical allodynia was inhibited. In conclusion, these results demonstrated that I(h) participated in the development and maintenance of peripheral sensitivity associated with neuropathic pain and that it is a potential target for the design of novel analgesics in the future.

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>

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>

Gene Expression Profiles in Cervical Cancer with Radiation Therapy Alone and Chemo-radiation Therapy / 대한방사선종양학회지

PURPOSE: To analyze the gene expression profiles of uterine cervical cancer, and its variation after radiation therapy, with or without concurrent chemotherapy, using a cDNA microarray. MATERIALS AND METHODS: Sixteen patients, 8 with squamous cell carcinomas of the uterine cervix, who were treated with radiation alone, and the other 8 treated with concurrent chemo-radiation, were included in the study. Before the starting of the treatment, tumor biopsies were carried out, and the second time biopsies were performed after a radiation dose of 16.2~27 Gy. Three normal cervix tissues were used as a control group. The microarray experiments were performed with 5 groups of the total RNAs extracted individually and then admixed as control, pre-radiation therapy alone, during-radiation therapy alone, pre-chemoradiation therapy, and during-chemoradiation therapy. The 33P-labeled cDNAs were synthesized from the total RNAs of each group, by reverse transcription, and then they were hybridized to the cDNA microarray membrane. The gene expression of each microarrays was captured by the intensity of each spot produced by the radioactive isotopes. The pixels per spot were counted with an Arrayguage(R), and were exported to Microsoft Excel(R). The data were normalized by the Z transformation, and the comparisons were performed on the Z-ratio values calculated. RESULTS: The expressions of 15 genes, including integrin linked kinase (ILK), CDC28 protein kinase 2, Spry 2, and ERK 3, were increased with the Z-ratio values of over 2.0 for the cervix cancer tissues compared to those for the normal controls. Those genes were involved in cell growth and proliferation, cell cycle control, or signal transduction. The expressions of the other 6 genes, including G protein coupled receptor kinase 6, were decreased with the Z-ratio values of below -2.0. After the radiation therapy, most of the genes, with a previously increase expressions, represented the decreased expression profiles, and the genes, with the Z-ratio values of over 2.0, were cyclic nucleotide gated channel and 3 Expressed sequence tags (EST). In the concurrent chemo-radiation group, the genes involved in cell growth and proliferation, cell cycle control, and signal transduction were shown to have increased expressions compared to the radiation therapy alone group. The expressions of genes involved in angiogenesis (angiopoietin-2), immune reactions (formyl peptide receptor-like 1), and DNA repair (cAMP phosphodiesterase) were increased, however, the expression of gene involved in apoptosis (death associated protein kinase) was decreased. CONCLUSION: The different kinds of genes involved in the development and progression of cervical cancer were identified with the cDNA microarray, and the proposed theory is that the proliferation signal starts with ILK, and is amplified with Spry 2 and MAPK signaling, and the cellular mitoses are increased with the increased expression of Cdc 2 and cell division kinases. After the radiation therapy, the expression profiles demonstrated the evidence of the decreased cancer cell proliferation. There was no significant difference in the morphological findings of cell death between the radiation therapy alone and the chemo-radiation groups in the second time biopsy specimen, however, the gene expression profiles were markedly different, and the mechanism at the molecular level needs further study.

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.