Expression and localization of TRPC proteins in rat ventricular myocytes at various developmental stages.

Growing evidence indicates that transient receptor potential canonical (TRPC) channels play important roles in various Ca(2+)-mediated physiological and pathophysiological processes, including development. Many types of TRPC proteins are expressed in the heart. However, limited data are available comparing the expression and localization among TRPC proteins in the ventricular myocyte at various developmental stages. Our purpose is to investigate the expression and localization profile of TRPC proteins in ventricular myocytes of fetal (18.5 days), neonatal (< 24 h after birth) and adult (8 week old) rats. Western blotting, immunofluorescence and confocal laser scanning microscopy were employed. TRPC1/3-6 proteins were expressed in the rat ventricle throughout the three developmental stages. The expression profile of TRPC1/3/4 in the ventricle followed an upward trend from the fetus to the adult. By contrast, TRPC6 in the ventricle was expressed at the highest level in the fetal group and was sharply down-regulated immediately after birth. TRPC5 expression in the ventricle did not change significantly during the three stages. TRPC1/3/5/6 proteins were localized to the T-tubule and TRPC1/3/4/6 to intercalated disks in adult myocytes. The wide spatiotemporal overlap and dynamic regulation of TRPC expression in ventricular myocytes indicates potential complex combinations and redundancy of native TRPC proteins in the heart and gives important clues for further investigations into the exact subunit compositions and functional properties of native TRPC channels in the heart.

ASIC2 Synergizes with TRPV1 in the Mechano-Electrical Transduction of Arterial Baroreceptors.

Mechanosensitive ion channels (MSCs) are key molecules in the mechano-electrical transduction of arterial baroreceptors. Among them, acid-sensing ion channel 2 (ASIC2) and transient receptor potential vanilloid subfamily member 1 (TRPV1) have been studied extensively and documented to play important roles. In this study, experiments using aortic arch-aortic nerve preparations isolated from rats revealed that both ASIC2 and TRPV1 are functionally necessary, as blocking either abrogated nearly all pressure-dependent neural discharge. However, whether ASIC2 and TRPV1 work in coordination remained unclear. So we carried out cell-attached patch-clamp recordings in HEK293T cells co-expressing ASIC2 and TRPV1 and found that inhibition of ASIC2 completely blocked stretch-activated currents while inhibition of TRPV1 only partially blocked these currents. Immunofluorescence staining of aortic arch-aortic adventitia from rats showed that ASIC2 and TRPV1 are co-localized in the aortic nerve endings, and co-immunoprecipitation assays confirmed that the two proteins form a compact complex in HEK293T cells and in baroreceptors. Moreover, protein modeling analysis, exogenous co-immunoprecipitation assays, and biotin pull-down assays indicated that ASIC2 and TRPV1 interact directly. In summary, our research suggests that ASIC2 and TRPV1 form a compact complex and function synergistically in the mechano-electrical transduction of arterial baroreceptors. The model of synergism between MSCs may have important biological significance beyond ASIC2 and TRPV1.

Spatio-temporal niche of major fish species in Pishan waters off Zhejiang Province, China.

Based on seasonal bottom trawl surveys in November 2015 (autumn), February (winter), May (spring) and August (summer) in 2016, the index of relative importance (IRI), Shannon index and Pianka index were used to analyze the breadth and overlap of spatio-temporal niche of major fish species in the Pishan waters off Zhejiang Province. Redundancy analysis and interspecific competition coefficient were used to examine the competition relationship and niche differentiation of those major fish species. The results showed that a total of 61 fish species were recorded throughout the year, which belonged to 13 orders, 29 families and 48 genera. A total of 19 species with IRI>100 were identified as major fish species. The temporal niche overlap value between Chelidonichthys kumu and Atule kalla was largest, indicating high temporal synchronization. Both the spatial niche overlap value and the spatio-temporal niche overlap value between Trpauchen vagina and Cynoglossus interruptus were largest, indicating that their spatial homology was high and that the use of spatial and temporal two-dimensional resource of both species were consistent. The percentage of species pairs with spatio-temporal niche overlap at the significant level (Qik>0.6) was only 5.8% in Pishan waters, indicating that the spatial and temporal distribution of those species were quite different. The overlap of spatio-temporal niche significantly varied across different seasons. The results of the interspecific competition coefficient were basically consistent with the niche overlap. Results of the redundancy analysis revealed the relationships between major fish species and environmental factors (temperature, salinity, dissolved oxygen), and further explained the niche diffe-rentiation among species.

Mechanosensitivity of STREX-lacking BKCa channels in the colonic smooth muscle of the mouse.

Stretch sensitivity of Ca²(+)-activated large-conductance K(+) channels (BK(Ca)) has been observed in a variety of cell types and considered to be a potential mechanism in mechanoelectric transduction (MET). Mechanical stress is a major stimulator for the smooth muscle in the gastrointestinal (GI) tract. However, much about the role and mechanism of MET in GI smooth muscles remains unknown. The BK(Ca) shows a functional diversity due to intensive Slo I alternative splicing and different α/ß-subunit assembly in various cells. The stress-regulated exon (STREX) insert is suggested to be an indispensable domain for the mechanosensitivity of BK(Ca). The purpose of this study was to determine whether the BK(Ca) in colonic myocytes of the adult mouse is sensitive to mechanical stimulation and whether the STREX insert is a crucial segment for the BK(Ca) mechanosensitivity. The α- and ß1-subunit mRNAs and the α-subunit protein of the BK(Ca) channels were detected in the colonic muscularis. We found that the BK(Ca) STREX-lacking variant was abundantly expressed in the smooth muscle, whereas the STREX variant was not detectable. We demonstrated that the STREX-lacking BK(Ca) channels were also sensitive to membrane stretch. We suggest that in addition to the STREX domain, there are other additional structures in the channel responsible for mechanically coupling with the cell membrane.

A simple automated stimulator of mechanically induced arrhythmias in the isolated rat heart.

Transient stretching of the ventricle can trigger arrhythmias and evoke ventricular fibrillation, especially when the stimulation occurs in the vulnerable period. To explore the sensitivity of small hearts we used a commercial pressure servo to study the kinetic relationship of left ventricular pressure to excitability and arrhythmias in the rat heart. Stimulation protocols were readily composed on the computer and programmed to vary the stimulus amplitude and timing relative to pacing. The pressure-induced premature ventricular excitations were similar to those observed in larger hearts, but the convenience of using small hearts allows the use of inexpensive transgenic animals to explore the molecular basis of transduction.

Pharmacological and biophysical properties of swelling-activated chloride channel in mouse cardiac myocytes.

The present study was designed to observe the properties of swelling-activated chloride channel (ICl.swell) in mouse cardiac myocytes using patch clamp techniques. In whole-cell recordings, hypotonic solution activated a chloride current that exhibited outward rectification, weak voltage-dependent inactivation, and anion selectivity with permeability sequence of I- > Br- > Cl-. The current was sensitive to Cl- channel blockers tamoxifen, NPPB and DIDS. In single-channel recordings, cell swelling activated a single channel current which showed outward rectification with open probability of 0.76 +/- 0.08 and conductance of 38.1 +/- 2.5 pS at +100 mV under [Cl-] symmetrical condition. I-V relation revealed the reversal potential as expected for a Cl(-)-selective channel. These results suggested that in mouse cardiac myocytes, swelling-activated, outward rectifying chloride channel with a single channel conductance of 38.1 +/- 2.5 pS (at +100 mV under [Cl-] symmetrical condition) underlies the volume regulatory Cl- channel.

A ventricular pressure-clamping system for the study of mechano-electrical feedback.

It is necessary to control the mechanical stimuli precisely in the studies of cardiac mechano-electrical feedback (MEF). In the present study a ventricular pressure-clamping system has been developed, which can be applied to isolated-perfused rabbit hearts. Controlled by a computer, this system not only can make the left ventricle follow a command defining the same pressure wave as that during a beating cycle under physiological condition, but also deliver mechanical stimuli with a proper waveform to the ventricle at a particular time phase. This system integrates multiple functions, including perfusing, pacing, recording of electrocardiogram and monophasic action potentials, and clamping and measuring of ventricular pressures in isolated-perfused hearts. Thus, it is a distinct system for investigating the phenomena and mechanisms of cardiac MEF at organ level.

Dynamic properties of stretch-activated K+ channels in adult rat atrial myocytes.

The effect of mechanical stress on the heart's electrical activity has been termed mechanoelectric feedback. The response to stretch depends upon the magnitude and the waveform of the stimulus, and upon the timing relative to the cardiac cycle. Stretch-activated ion channels (SACs) have been regarded as the most likely candidates for serving as the primary transducers of mechanical stress. We explored the steady state and dynamic responses of single channels in adult rat atrial cells using the patch clamp with a pressure clamp. Surprisingly, we only observed K(+)-selective SACs, probably of the 2P domain family. The channels were weakly outward rectifying with flickery bursts. In cell attached mode, the mean conductance was 74+/-14 and 65+/-16 pS for +60 and -60 mV, respectively (140 mM [K(+)](out), 2mM [Mg(2+)](out) and 0mM [Ca(2+)](out)). The latency of the response to pressure steps was 50-100 ms and the time to peak approximately 400 ms. About half of the channels in cell-attached patches showed adaptation/inactivation where channel activity declined to a plateau of 20-30% of peak in approximately 1s. The time dependent behavior of these SACs is generally consistent with whole-cell currents observed in chick and rat ventricular cells, although the net current was outward rather than inward.

[Effects of calcitonin gene-related peptide on repolarization process in isolated guinea-pig atrial myocardium at the physiological temperature].

The purpose of this study was to investigate the effects of calcitonin gene-related peptide (CGRP) on the repolarization process in isolated guinea-pig atrial cells and to determine the contribution of K(+) channels to the CGRP-induced changes in action potential using conventional microelectrode method at the physiological temperature. We found that: (1) CGRP (16 nmol/L) antagonized the influences of potassium channel blockers, 4-AP and BaCl2, on action potential; (2) CGRP (16 nmol/L) increased the amplitude and maximum depolarizing velocity of slow action potential and shortened the conducting time in guinea pig atrial myocardium at extracellular K(+) concentration of 18.5 mmol/L; (3) CGRP (16 nmol/L) alleviated triggered activity induced by superfusion with solution containing CsCl and no potassium ion; and (4) the effects of CGRP on the configuration of action potential were temperature-dependent. At the temperature of 36.5+/-0.5 degrees C, CGRP (5, 16, and 50 nmol/L) increased the amplitude of the action potential and shortened APD(20), APD(50) and APD(90). The CGRP effects on APD(20) and APD(50) were dose-dependent and reversible. On the contrary, CGRP prolonged APD(20), APD(50) and APD(90) at the temperature of 25.5+/-2.1 degrees C. The present study suggests that CGRP possesses multiple effects on various ionic channels. Among them the effects on potassium currents are major determinants in the changes in action potential induced by CGRP under physiological temperature. It is necessary to further study the influences of CGRP on different types of potassium channels.

Mechanical effects on KATP channel gating in rat ventricular myocytes.

Cardiac KATP channels link metabolism with electrical activity. They are implicated in arrhythmias, secretion of atrial natriuretic peptide and protection of the heart from hypertrophy and failure. These processes may involve mechanosensitivity. KATP channels can be activated by mechanical stimulation and disrupting the cortical actin increases the activity. We propose that KATP channels are modulated by local bilayer tension and this tension is affected by cortical F-actin. Here we measured KATP background activity and stretch sensitivity with inside-out patches of rat ventricular myocytes before and after disrupting F-actin. Disrupting F-actin potentiated background activity but did not influence the slope sensitivity in the semilog relationship of NPo vs. suction that is a measure of the change in dimensions between closed and open states. Thus actin alters prestress on the channel probably by parallel elastic sharing of mean cortical tension with the bilayer.

[Improved methods for researching isolated carotid sinus baroreceptors automatically controlling for sinus pressure].

OBJECTIVE: To develop a system for automatically controlling carotid sinus pressure in the study on baroreceptors. METHODS: The preparation containing carotid sinus with parts of the connected vessels and carotid sinus nerve (CS-CSN) were isolated and perfused. A critical pressure controlling component (PRE-U, Hoerbiger, Deutschland) dictated by a computer was integrated into the system to clamp the intrasinus pressure. The pressure command and the relevant intrasinus pressure were compared to evaluate the validity of the pressure controlling system. RESULTS: A variety of sinus pressure-controlling patterns, including pulsation, ramp and step pressures, could be achieved accurately by using the system, and the pressure-dependent discharge activities of sinus nerve were confirmed. CONCLUSION: This system for clamping carotid sinus pressure could realize multiple pressure-controlling patterns and is a useful and flexible pressure controlling method that could applied in the study on mechano-electric transduction of baroreceptors.

The effects of allitridi and amiodarone on the conduction system and reverse use-dependence in the isolated hearts of rats with myocardial infarction.

ETHNOPHARMACOLOGICAL RELEVANCE: Allium sativum L. (DaSuan in Mandarin) is a traditional Chinese herb that has been used to prevent and heal cardiovascular diseases. OBJECTIVE: To study the effects of allitridi (an active constituent of Allium sativum L.) and amiodarone on the conduction system and on reverse use-dependence in the isolated hearts of normal rats and rats with myocardial infarction (MI). MATERIALS AND METHODS: Male Sprague Dawley rats, with a ligated left anterior descending coronary artery, were used as myocardial infarction models to investigate the biological effects of the traditional Chinese herb. A single-phase electrode assay and isolated heart perfusion administration methods were employed to study and compare the electrophysiological effects of allitridi and amiodarone on normal and MI rats. Monophasic action potential (MAP) in vitro, effective refractory period (ERP) and monophasic action potential duration (MAPD)/ERP were measured to investigate reverse use-dependence (RUD) with allitridi and amiodarone. Moreover, bundle maps and heart rates were analyzed to evaluate the electrophysiological effects of allitridi on the conduction system of the cardiac muscles. Coronary flow was used to study the beneficial effects of the two drugs on the bundle of His in myocardial infraction. RESULTS: (1) Allitridi and amiodarone can reduce the infarction model of the His bundle (A-H, H-V) conduction and cardiac sinus rhythm in normal rats and isolated rat hearts. After washing in physiological solution (AK-H) for 15 min, the allitridi group partially recovered, but the amiodarone group did not recover. (2) Allitridi and amiodarone had no significant effects on the change of MAPD(90) or ERP in normal and MI rat hearts at different pacing frequencies (200, 250 and 300 beats/min), which indicated no RUD. In addition, the effects of allitridi on prolonging MAPD(90) and ERP were weaker than those of amiodarone (P<0.01). The effects of allitridi on myocardial repolarization and its variation rate were also weaker than those of amiodarone (P<0.01). However, the prolonged administration of allitridi still did not cause RUD. Allitridi and amiodarone can significantly increase the ERP/APD(90) rate of the isolated heart ventricles of normal rats and rats with MI. CONCLUSION: We propose that allitridi and amiodarone have similar effects on the cardiac conduction system and on the electrophysiology without RUD, which may be the result of the use of multi-channel blockers, such as calcium channel blockers and IKr and IKs channel blockers. Allitridi may be a promising antiarrythmic drug.

[Arrhythmia triggered by stretching rabbit left ventricles and the block effect of streptomysin].

AIM: To observe the effect of stretching left ventricles in the end of action potential on rabbit cardiac activity, and to investigate its possible mechanisms. METHODS: Stretch (120 mmHg, 50 ms) was applied in the end of action potential by the pressure-clamp technique to observe if there would be any changes in rabbit cardiac activity and streptomycin (500 micromol/L) was used to identify the mechanism. RESULTS: Stretch in the end of action potential caused arrhythmia (P < 0.05) and streptomycin blocked the above effect (P < 0.05). CONCLUSION: Streptomycin could block the effect of stretching left ventricles in the end of action potential on rabbit cardiac activity, which indicates that stretch-activated ion channels involve it.

Stretch-activated potassium channels in hypotonically induced blebs of atrial myocytes.

Stress in the lipids of the cell membrane may be responsible for activating stretch-activated channels (SACs) in nonspecialized sensory cells such as cardiac myocytes, where they are likely to play a role in cardiac mechanoelectric feedback. We examined the influence of the mechanical microenvironment on the gating of stretch-activated potassium channels (SAKCs) in rat atrial myocytes. The goal was to examine the role of the cytoskeleton in the gating process. We recorded from blebs that have minimal cytoskeleton and cells treated with cytochalasin B (cyto-B) to disrupt filamentous actin. Histochemical and electron microscopic techniques confirmed that the bleb membrane was largely free of F-actin. Channel currents showed mechanosensitivity and potassium selectivity and were activated by low pH and arachidonic acid, similar to properties of TREK-1. Some patches showed a time-dependent decrease in current that may be adaptation or inactivation, and since this decrease appeared in control cells and blebs, it is probably not the result of adaptation in the cytoskeleton. Cyto-B treatment and blebbing caused an increase in background channel activity, suggesting a transfer of stress from actin to bilayer and then to the channel. The slope sensitivity of gating before and after cyto-B treatment was similar to that of blebs, implying the characteristic change of dimensions associated with channel gating was the same in the three mechanical environments. The mechanosensitivity of SAKCs appears to be the result of interaction with membrane lipids and not of direct involvement of the cytoskeleton.

[The effects of repetitive limb ischemia on the systemic concentration of NO, NOS in plasma of healthy humans].

AIM: To investigate the effects of RLI on plasma nitric oxide (NO) and NO synthase (NOS) isoforms of healthy humans. METHODS: 30 healthy human subjects (aged from 40 - 70 years old) were recruited. RLI was induced by five 5 min cycles of ischemia of non dominant arm (200 mmHg, 5 min interval). Blood pressure, heart rate, and the feelings of ischemic arm were continuously monitored. Venous plasma was collected in contralateral arm at Pre, Post-0 h, Post-4 h, and Post-24 h. Plasma level of NO was measured by Griess reaction, and NOS was measured by chemical method. RESULTS: Blood pressure and heart rate varied in normal range. The uncomfortable feeling was decreased with the increasing numbers of ischemic cycles. Plasma level of NO, and iNOS in plasma were significantly increased at Post-0 h, Post-4 h, and Post-24 h compared to Pre (P < 0.05). tNOS was also significantly increased at Post-0 h and Post-4 h compared to Pre (P < 0.05). No significant change in plasma cNOS was shown at following three time points than Pre. CONCLUSION: These findings suggest that RLI can elevate plasma level of NO, tNOS, and iNOS in healthy humans. RLI might be a safe method as a rIPC, and it would have important possibility to be performed in clinic.