Analysis of the evolution and causes of groundwater chemistry after ecological water replenishment of the Jialu River, China.

Ecological water replenishment is an important measure for conserving water sources and improving the water environment. To explore the evolution and causes of groundwater chemistry after ecological water replenishment in the Jialu River, this study utilized groundwater monitoring data from 2015 to 2019 following ecological water replenishment. Various methods, including Piper's trilinear diagram, Gibbs diagram, principal component analysis, and ion ratio analysis, were employed for research purposes. The results indicate that (1) since the implementation of ecological water replenishment in the Jialu River, there has been a general downwards trend in total dissolved solids (TDS) in groundwater. The dominant cation in groundwater is Ca2+, whereas HCO3- is the dominant anion. The concentration of cations in groundwater has generally decreased, with noticeable reductions in SO42- and Cl- concentrations in the upper reaches of the recharge river contributing to improved groundwater quality. (2) A comparison with 2015 reveals a gradual transition at sampling points from chemical types such as HCO3-Ca·Mg and HCO3·Cl-Ca·Mg to an ecological water replenishment chemical type (HCO3-Ca).

Soil water dynamics and deep percolation in an agricultural experimental area of the North China Plain over the past 50 years: Based on field monitoring and numerical modeling.

The unregulated irrigation systems used in the late 20th century have led to increasingly severe deep percolation (DP) in the agricultural irrigation areas of the North China Plain. This has become an important factor limiting the efficient utilization of water resources and sustainable environmental development in these irrigation areas. However, the thick vadose zone is hydrodynamically exceptionally complex. The soil hydrological cycle is constantly changing under the influence of major climate change and human activity, thereby causing changes in DP that are difficult to quantify accurately. Here, the Luancheng Agricultural Irrigation District in North China was selected for a continuous 20-year in situ experiment. Soil-water dynamics were monitored using neutron probes and tensiometers, to determine the complete annual soil-water cycle and the hydrodynamic properties of the thick vadose zone irrigation district. For 1971-2021, DP was simulated using the HYDRUS-1D model and was verified by fitting observed values. Soil water content (SWC) exhibited similar trends in years that differed in terms of the amounts of irrigation and precipitation. The 0-100 cm soil layer was significantly affected by precipitation and other factors, and recharge >60 mm/d caused percolation. DP occurred mostly after irrigation or during the period of intensive precipitation in July-October. The maximum percolation rate was 16.9 mm/d under the present irrigation method. The main factors leading to DP were soil water storage capacity (R2 = 0.86) and precipitation (R2 = 0.54). Under the evolution of irrigation measures in the last 50 years, the average DP has gradually decreased from 574.2 mm (1971-1990) to 435.5 mm (2005-2021). However, a substantial amount of precipitation and irrigation water infiltrated the soil and percolated into the deep soil layer without being utilized by the crop. Therefore, there is an urgent need to consider measures to reduce DP to improve water-use efficiency in agriculture.

Heterogeneity of left ventricular signal characteristics in response to acute vagal stimulation during ventricular fibrillation in dogs.

Studies have shown that long-term vagal stimulation is protective against ventricular fibrillation; however, the effects of acute vagal stimulation during ventricular fibrillation in the normal heart have not been investigated. We examined the effects of acute vagal stimulation on ventricular fibrillation in a canine model. In 4 dogs, we induced 30-second periods of ventricular fibrillation by means of intraventricular pacing. During 2 of the 4 periods of fibrillation that we analyzed, vagal stimulation was delivered through electrodes in the caudal ends of the vagus nerves. Noncontact unipolar electrograms were recorded from 3 ventricular regions: the basal septum, apical septum, and lateral free wall. We then computed the most frequent cycle length, mean organization index, and mean electrogram amplitude for each region. During fibrillation, vagal stimulation shortened the most frequent cycle lengths in the basal septum (P=0.02) and apical septum (P=0.0001), but not in the lateral wall (P=0.46). In addition, vagal stimulation significantly reduced the mean organization indices in the apical septum (P <0.001) and lateral wall (P <0.001), but not in the basal septum (P=0.19). Furthermore, vagal stimulation raised the mean electrogram amplitude in the basal septum (P <0.01) but lowered it substantially in the apical septum (P=0.00005) and lateral wall (P=0.00003). We conclude that vagal stimulation acutely affects the characteristics of ventricular fibrillation in canine myocardium in a spatially heterogeneous manner. This nonuniformity of response may have implications with regard to manipulating the autonomic system as a means of modifying the substrate for ventricular dysrhythmias.

Functional characterization of hyperpolarization-activated cyclic nucleotide-gated channels in rat pancreatic beta cells.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate pacemaker activity in some cardiac cells and neurons. In the present study, we have identified the presence of HCN channels in pancreatic beta-cells. We then examined the functional characterization of these channels in beta-cells via modulating HCN channel activity genetically and pharmacologically. Voltage-clamp experiments showed that over-expression of HCN2 in rat beta-cells significantly increased HCN current (I(h)), whereas expression of dominant-negative HCN2 (HCN2-AYA) completely suppressed endogenous I(h). Compared to control beta-cells, over-expression of I(h) increased insulin secretion at 2.8 mmol/l glucose. However, suppression of I(h) did not affect insulin secretion at both 2.8 and 11.1 mmol/l glucose. Current-clamp measurements revealed that HCN2 over-expression significantly reduced beta-cell membrane input resistance (R(in)), and resulted in a less-hyperpolarizing membrane response to the currents injected into the cell. Conversely, dominant negative HCN2-AYA expression led to a substantial increase of R(in), which was associated with a more hyperpolarizing membrane response to the currents injected. Remarkably, under low extracellular potassium conditions (2.5 mmol/l K(+)), suppression of I(h) resulted in increased membrane hyperpolarization and decreased insulin secretion. We conclude that I(h) in beta-cells possess the potential to modulate beta-cell membrane potential and insulin secretion under hypokalemic conditions.

Age-dependent differences in the inhibition of HCN2 current in rat ventricular myocytes by the tyrosine kinase inhibitor erbstatin.

Previously, we have shown that murine HCN2 channels over-expressed in newborn and adult cardiac myocytes produce currents with different biophysical characteristics. To investigate the role of tyrosine kinase modulation in these age-dependent differences, we employed the broad spectrum tyrosine kinase inhibitor erbstatin. Our results demonstrated distinct and separable effects of erbstatin on channel gating and current amplitude and a marked age dependence to these effects. In newborn myocytes, erbstatin decreased current amplitude, shifted the activation relation negative, and slowed activation kinetics. The effect on activation voltage but not that on amplitude was absent when expressing a cAMP-insensitive mutant (HCN2R/E), while a C-terminal truncated form of HCN2 (HCN2DeltaCx) exhibited only the voltage dependent but not the amplitude effect of erbstatin. Thus, the action of erbstatin on the activation relation and current amplitude are distinct and separable in newborn myocytes, and the effect on activation voltage depends on the cAMP status of HCN2 channels. In contrast to newborn myocytes, erbstatin had no effect on HCN2 under control conditions in adult myocytes but induced a negative shift with no change in amplitude when saturated cAMP was added to the pipette solution. We conclude that erbstatin's effects on HCN2 current magnitude and voltage dependence are distinct and separable, and there are fundamental developmental differences in the heart that affect channel function and its modulation by the tyrosine kinase inhibitor erbstatin.

Wild-type and mutant HCN channels in a tandem biological-electronic cardiac pacemaker.

BACKGROUND: Biological pacemakers (BPM) implanted in canine left bundle branch function competitively with electronic pacemakers (EPM). We hypothesized that BPM engineered with the use of mE324A mutant murine HCN2 (mHCN2) genes would improve function over mHCN2 and that BPM/EPM tandems confer advantage over either approach alone. METHODS AND RESULTS: In cultured neonatal rat myocytes, activation midpoint was -46.9 mV in mE324A versus -66.1 mV in mHCN2 (P < 0.05). mE324A manifested a positive shift of voltage dependence of gating kinetics of activation and deactivation compared with mHCN2 (P < 0.05) in myocytes as well as Xenopus oocytes. In intact dogs in complete atrioventricular block, saline (control), mHCN2, or mE324A virus was injected into left bundle branch, and EPM were implanted (VVI 45 bpm). Twenty-four-hour ECGs were monitored for 14 days. With EPM discontinued, there was no difference in duration of overdrive suppression among groups. However, basal heart rates in controls were less than those in mHCN2, which did not differ from those in E324A (45 versus 57 versus 53 bpm; P < 0.05). When spontaneous rate fell below 45 bpm, EPM intervened at that rate, triggering 83% of beats in control, contrasting (P < 0.05) with 26% (mHCN2) and 36% (mE324A). On day 14, epinephrine (1 microg/kg per minute IV) induced a 50% heart rate increase in all mE324A, one third of mHCN2, and one fifth of control (P < 0.05 mE324A versus control or mHCN2). CONCLUSIONS: mE324A induces faster, more positive pacemaker current activation than mHCN2 and stable, catecholamine-sensitive rhythms in situ that compete with EPM comparably but more catecholamine responsively than mHCN2. BPM/EPM tandems function reliably, reduce the number of EPM beats, and confer sympathetic responsiveness to the tandem.

The cAMP response element binding protein modulates expression of the transient outward current: implications for cardiac memory.

OBJECTIVE: Long-term cardiac memory (LTCM), expressed as a specific pattern of T-wave change on ECG, is associated with 1) reduced transient outward potassium current (I(to)), 2) reduced mRNA for the pore-forming protein of I(to), Kv4.3, 3) reduced cAMP response element binding protein (CREB), and 4) diminished binding to its docking site on the DNA, the cAMP response element (CRE). We hypothesized a causal link between the decrease of the transcription factor CREB and down-regulation of I(to) and one of its channel subunits, KChIP2, in LTCM. METHODS: After three weeks of left ventricular pacing to induce LTCM (8 paced, 7 sham control dogs), epicardial KChIP2 mRNA and protein levels were assessed by real-time PCR and Western blotting. Mimicking the CREB down-regulation in LTCM, CREB was knocked down in situ in other dogs using adenoviral anti-sense. Effects on the action potential notch, reflecting I(to), were investigated in situ using monophasic action potential (MAP) recordings and at the cellular level by the whole-cell patch clamp technique. CREB binding in the KChIP2 promoter region was ascertained by electrophoretic mobility-shift assays. RESULTS: In LTCM, epicardial KChIP2 mRNA and protein were reduced by 62% and 76%, respectively, compared to shams (p < 0.05). CREB binding by the canine KChIP2 promoter region was demonstrated. CREB knockdown led to disappearance of the phase1 notch in MAP and ablation of I(to). CONCLUSIONS: These results strengthen the hypothesis that down-regulation of CREB-mediated transcription underlies the attenuation of epicardial I(to) in LTCM. They also emphasize that ventricular pacing exerts effects at a subcellular level contributing to memory and conceivably to other forms of cardiac remodeling.

MiRP1 modulates HCN2 channel expression and gating in cardiac myocytes.

MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotide-gated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between -60 and -95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a beta subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.

Cardiac ion channel expression and regulation: the role of innervation.

The expression and function of numerous cardiac ion channels change with development and disease. Whereas multiple regulatory processes and molecular mechanisms are certainly involved, one factor, sympathetic innervation, contributes to many of the developmental changes and is suggested to play a role in pathology. The onset of cardiac sympathetic innervation of the mammalian ventricle during early post-natal life has been associated with functional alterations in several ionic currents, including Na(+), L-type Ca(2+), pacemaker, inward rectifier and transient outward K(+) currents. The neural signaling molecule is not the same in each case, with evidence pointing to contributions from sustained activation of myocardial neuropeptide Y receptors, alpha-adrenergic receptors and beta-adrenergic receptors, as well as additional, but as yet unidentified, targets. Knowledge of the mechanisms by which innervation regulates ion channel expression and function during normal development may aid efforts to reverse remodel the diseased heart and to target pharmacologic agents to remodeled channels.

Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers.

We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker to the heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by electroporation expressed high levels of Cs+-sensitive current (31.1+/-3.8 pA/pF at -150 mV) activating in the diastolic potential range with reversal potential of -37.5+/-1.0 mV, confirming the expressed current as I(f)-like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation. Acetylcholine had no direct effect, but in the presence of isoproterenol, shifted activation 15 mV negative. Transfected hMSCs influenced beating rate in vitro when plated onto a localized region of a coverslip and overlaid with neonatal rat ventricular myocytes. The coculture beating rate was 93+/-16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161+/-4 bpm when hMSCs were expressing both EGFP+mHCN2 (P<0.05). We next injected 10(6) hMSCs transfected with either control plasmid or mHCN2 gene construct subepicardially in the canine left ventricular wall in situ. During sinus arrest, all control (EGFP) hearts had spontaneous rhythms (45+/-1 bpm, 2 of right-sided origin and 2 of left). In the EGFP+mHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rate=61+/-5 bpm; P<0.05). Moreover, immunostaining of the injected regions demonstrated the presence of hMSCs forming gap junctions with adjacent myocytes. These findings demonstrate that genetically modified hMSCs can express functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart or other electrical syncytia.

Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates.

BACKGROUND: We hypothesized that administration of the HCN2 gene to the left bundle-branch (LBB) system of intact dogs would provide pacemaker function in the physiological range of heart rates. METHODS AND RESULTS: An adenoviral construct incorporating HCN2 and green fluorescent protein (GFP) as a marker was injected via catheter under fluoroscopic control into the posterior division of the LBB. Controls were injected with an adenoviral construct of GFP alone or saline. Animals were monitored electrocardiographically for up to 7 days after surgery, at which time they were anesthetized and subjected to vagal stimulation to permit emergence of escape pacemakers. Hearts were then removed and injection sites visually identified and removed for microelectrode study of action potentials, patch clamp studies of pacemaker current, and/or immunohistochemical studies of HCN2. For 48 hours postoperatively, 7 of 7 animals subjected to 24-hour ECG monitoring showed multiple ventricular premature depolarizations and/or ventricular tachycardia attributable to injection-induced injury. Thereafter, sinus rhythm prevailed. During vagal stimulation, HCN2-injected dogs showed rhythms originating from the left ventricle, the rate of which was significantly more rapid than in the controls. Excised posterior divisions of the LBB from HCN2-injected animals manifested automatic rates significantly greater than the controls. Isolated tissues showed immunohistochemical and biophysical evidence of overexpressed HCN2. CONCLUSIONS: A gene-therapy approach for induction of biological pacemaker activity within the LBB system provides ventricular escape rhythms that have physiologically acceptable rates. Long-term stability and feasibility of the approach remain to be tested.

Neuropeptide Y is an essential in vivo developmental regulator of cardiac ICa,L.

Cell culture studies demonstrate an increase in cardiac L-type Ca2+ current (ICa,L) density on sympathetic innervation in vitro and suggest the effect depends on neurally released neuropeptide Y (NPY). To determine if a similar mechanism contributes to the postnatal increase in ICa,L in vivo, we prepared isolated ventricular myocytes from neonatal and adult mice with targeted deletion of the NPY gene (Npy-/-) and matched controls (Npy+/+). Whole-cell voltage clamp demonstrates ICa,L density increases postnatally in Npy+/+ (by 56%), but is unchanged in Npy-/-. Both ICa,L density and action potential duration are significantly greater in adult Npy+/+ than Npy-/- myocytes, whereas ICa,L density is equivalent in neonatal Npy+/+ and Npy-/- myocytes. These data indicate NPY does not influence ICa,L prenatally, but the postnatal increase in ICa,L density is entirely NPY-dependent. In contrast, there is a similar postnatal negative voltage shift in the I-V relation in Npy+/+ and Npy-/-, indicating NPY does not influence the developmental change in ICa,L voltage-dependence. Immunoblot analyses and measurements of maximally activated ICa,L (in presence of forskolin or BayK 8644) show that the differences in current density between Npy+/+ and Npy-/- cannot be attributed to altered Ca2+ channel alpha1C subunit protein expression. Rather, these results suggest that the in vivo NPY-dependent postnatal increase in ICa,L density in cardiac myocytes results from regulation ICa,L properties by NPY.

Expression and function of a biological pacemaker in canine heart.

BACKGROUND: We hypothesized that localized overexpression of the hyperpolarization-activated, cyclic nucleotide-gated (HCN2) pacemaker current isoform in canine left atrium (LA) would constitute a novel biological pacemaker. METHODS AND RESULTS: Adenoviral constructs of mouse HCN2 and green fluorescent protein (GFP) or GFP alone were injected into LA, terminal studies performed 3 to 4 days later, hearts removed, and myocytes examined for native and expressed pacemaker current (I(f)). Spontaneous LA rhythms occurred after vagal stimulation-induced sinus arrest in 4 of 4 HCN2+GFP dogs and 0 of 3 GFP dogs (P<0.05). Native I(f) in nonexpressed atrial myocytes was 7+/-4 pA at -130 mV (n=5), whereas HCN2+GFP LA had expressed pacemaker current (I(HCN2)) of 3823+/-713 pA at -125 mV (n=10) and 768+/-365 pA at -85 mV. CONCLUSIONS: HCN2 overexpression provides an I(f)-based pacemaker sufficient to drive the heart when injected into a localized region of atrium, offering a promising gene therapy for pacemaker disease.

Functional comparison of HCN isoforms expressed in ventricular and HEK 293 cells.

Pacemaker current (I(f)) encoded by the HCN gene family contributes importantly to cardiac rhythm. That contribution depends on the biophysical characteristics of I(f), such as voltage dependence, which vary markedly with cardiac region, development and disease. Heterologous expression studies of individual HCN isoforms have failed to account for the diverse functionality of the native current. To investigate the influence of cellular environment on the gating of HCN channels, we compared the functional characteristics of HCN2 and HCN4, the two major ventricular isoforms, when over-expressed in a normal context (neonatal myocytes) and in a heterologous context (HEK 293 cells). Independent of cell type, HCN4 activates substantially slower than HCN2 and with a half-maximum activation voltage approximately equal 10 mV less negative. However, both isoforms activate more positively in myocytes than in HEK 293 cells. The latter result suggests a context dependence (i.e. cell-type specificity) to HCN voltage dependence that exerts a comparable influence on these two isoforms. This is distinct from the inherent difference in the biophysical properties of HCN2 and HCN4.