Simulated Microgravity and Hypergravity Affect the Expression Level of Soluble Guanylate Cyclase, Adenylate Cyclase, and Phosphodiesterase Genesin Rat Ventricular Cardiomyocytes.

Ion channels activity is regulated through soluble guanylate cyclase (sGC) and adenylate cyclase (AC) pathways, while phosphodiesterases (PDE) control the intracellular levels of cAMP and cGMP. Here we applied RNA transcriptome sequencing to study changes in the gene expression of the sGC, AC, and PDE isoforms in isolated rat ventricular cardiomyocytes under conditions of microgravity and hypergravity. Our results demonstrate that microgravity reduces the expression of sGC isoform genes, while hypergravity increases their expression. For a subset of AC isoforms, gene expression either increased or decreased under both microgravity and hypergravity conditions. The expression of genes encoding 10 PDE isoforms decreased under microgravity, but increased under hypergravity. However, under both microgravity and hypergravity, the gene expression increased for 7 PDE isoforms and decreased for 3 PDE isoforms. Overall, our findings indicate specific gravity-dependent changes in the expression of genes of isoforms associated with the studied enzymes.

Simulated Microgravity Changes the Number of Mechanically Gated and Mechanosensitive Ion Channels Genes Transcripts in Rat Ventricular Cardiomyocytes.

The mechanoelectrical feedback in the heart is based on the work of mechanically gated (MGCs) and mechanosensitive (MSCs) channels. Since microgravity alters the heart's morphological and physiological properties, we hypothesized that the expression of both MGCs and MSCs would be affected. We employed RNA transcriptome sequencing to investigate changes in the gene transcript levels of MGCs and MSCs in isolated rat ventricular cardiomyocytes under control conditions and in a simulated microgravity environment. For the first time, our findings demonstrated that simulated microgravity induces alterations in the gene transcript levels of specific MGCs, such as TRPM7, TRPV2, TRPP1, TRPP2, Piezo1, TMEM63A, TMEM36B, and known MSCs, including K2P2.1, K2P3.1, Kir6.1, Kir6.2, NaV1.5, CaV1.2, KV7.1. However, other voltage-gated channels and channels lacking a voltage sensor remained unaffected. These findings suggest that the altered expression of MGCs and MSCs could lead to changes in the net currents across the membrane, ultimately impacting the heart's function.

Hypergravity Increases the Number of Gene Transcripts of Mechanically Gated and Mechanosensitive Ion Channels in Rat Ventricular Cardiomyocytes.

Since hypergravity changes the morphological and physiological properties of the heart, it was assumed that the expression of ion channels that respond to cell stretching or compressing, mechanically gated channels (MGC) and mechanosensitive channels (MSC), would be affected. Using RNA transcriptome sequencing, the change in the number of transcripts for MGC and MSC genes was studied in isolated rat ventricular cardiomyocytes under 4g hypergravity for 5 days. It was shown for the first time that hypergravity induces changes in the number of transcripts of MGC genes: an increase for TRPC1, TRPC3, TRPM7, TRPP1 (PKD1), TRPP2 (PKD2), TMEM63A, TMEM63B, but a decrease for TRPV2, Piezo1, Piezo2. The number of MSC gene transcripts increases: TREK-1, Kir6.2, Nav1.5, Cav1.2, Cav1.3, Kv7.1, and Kv1.2. This potentially leads to an increase in the expression of MGC and MSC proteins leading to an increase in the net current and, as a result, pathological changes in the heart function.

VIP/PACAP signaling as an alternative target during hyperoxic exposure in preterm newborns.

The use of oxygen therapy (high doses of oxygen - hyperoxia) in the treatment of premature infants results in their survival. However, it also results in a high incidence of chronic lung disease known as bronchopulmonary dysplasia, a disease in which airway hyper-responsiveness and pulmonary hypertension are well known as consequences. In our previous studies, we have shown that hyperoxia causes airway hyper-reactivity, characterized by an increased constrictive and impaired airway smooth muscle relaxation due to a reduced release of relaxant molecules such as nitric oxide, measured under in vivo and in vitro conditions (extra- and intrapulmonary) airways. In addition, the relaxation pathway of the vasoactive intestinal peptide (VIP) and/or pituitary adenylate cyclase activating peptide (PACAP) is another part of this system that plays an important role in the airway caliber. Peptide, which activates VIP cyclase and pituitary adenylate cyclase, has prolonged airway smooth muscle activity. It has long been known that VIP inhibits airway smooth muscle cell proliferation in a mouse model of asthma, but there is no data about its role in the regulation of airway and tracheal smooth muscle contractility during hyperoxic exposure of preterm newborns.

Gadolinium as an Inhibitor of Ionic Currents in Isolated Rat Ventricular Cardiomyocytes.

The whole-cell patch-clamp technique was used to examine the effect of gadolinium Gd3+ (a non-specific blocker of mechanically gated current IMGCh, a component of late current IL) on ionic currents in insolated rat ventricular cardiomyocytes alone and in combination with the blockers of L-type calcium currents (ICaL) nifedipine (10 µM) or verapamil (1 µM). In K+in/K+out or Cs+in/Cs+out media, blockade of ICaL produced no effect on IL at negative potentials, but inhibited IL at positive ones. In K+in/K+out medium, Gd3+ (5 µM) decreased the net persistent current (Inp) at -45 mV from 198.6±6.4 to 96.7±9.5 pA over 15 min. Gd3+ alone or in combination with ICaL blockers shifted the reversal potential of IL to more negative values. At negative potentials, Gd3+ decreased IK1 and inward current including IMGCh. At positive potentials, Gd3+ alone or in combination with ICaL blockers decreased IL. When applied for 15 min in Cs+in/Cs+out medium at -45 mV, Gd3+ produced no effect on net current and inward and outward components of IL. Thus, Gd3+ can be viewed as a specific blocker of IMGCh only in Cs+ medium.

[Correlations of IL-18 and IL-6 with sodium consumption in patients with arterial hypertension and diabetes mellitus].

AIM: To determine correlations of AH-associated interleukins (IL-18, IL-6) with sodium consumption in AH patients with and without DM. MATERIALS AND METHODS: The study included AH patients with and without DM (n=63) who were managed at the Municipal Clinic #64, Moscow Department of Health Care, Branch 1. Plasma levels of IL-6 and IL-18 were measured using ELISA kits (Bender Med-Systems). Salt consumption was determined using a Charlton: SaltScreener questionnaire. Statistical analyses were performed using the Statistica 10.0 software. RESULTS: Four groups were formed: Group 1, grade 2 AH and DM (n=19); Group 2, grade 2 AH and no DM (n=4); Group 3, grade 3 AH and no DM (n=28); and Group 4, grade 3 AH and DM (n=12). Group 2 was small and was excluded from further analysis due to impossibility of statistical treatment. All patients consumed more than 6 g of salt per day (approximately 10 g). Analysis of intergroup differences in selected parameters showed differences between groups in levels of cholesterol, triglycerides, LDL, and GFR. The following correlations were identified in the groups: Group 1, positive correlation of IL-18 with sodium consumption (r=0.65) and CRP level (r=0.52) and of IL-6 with LDL level (r=0.48); Group 3, positive correlation of IL-18 with IL-6 (r=0.66) and of IL-6 with CRP (r=0.52); Group 4, positive correlation of IL-18 with GFR (r=0.82) and of IL-6 with waist circumference (WC) (r=0.84) and IL-6 (r=0.73). CONCLUSION: Patients consuming more than 6 g of salt daily (approximately 10 g) with AH and DM had more pronounced inflammation, which promoted progression of kidney disease.

Discrete Stretch Eliminates Electrophysiological Dose-Dependent Effects of Nitric Oxide Donor SNAP in Rat Atrium.

Depolarization of cardiomyocytes triggered by stretch and activation of mechanically gated ion channels can lead to serious arrhythmias. However, stretch-induced signaling activating these channels remain little studied. This study tested the hypothesis on implication of NO in shaping the electrical abnormalities provoked by stretch of the right atrial myocardium in rat via a mechanism engaging a signaling cascade, where NO plays a significant role. This approach showed that in isolated right atrial preparation, NO donor SNAP induces the electrical abnormalities similar to those provoked by stretch, and the latter results from activation of NO synthase.

Kinetics of Mechanical Stretch-Induced Nitric Oxide Production in Rat Ventricular Cardiac Myocytes.

Discrete mechanical stretch of isolated spontaneously contracting cardiac myocytes was employed to examine the kinetics of NO production in these cells. NO oscillations were detected with fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. The mechanisms underlying stretch-induced changes in NO concentration remain unclear and further studies are needed to evaluate the role of NO oscillation in the regulation of cardiomyocyte function.

Cytokine Effects on Mechano-Induced Electrical Activity in Atrial Myocardium.

The role of cytokines as regulators of stretch-related mechanisms is of special importance since mechano-sensitivity plays an important role in a wide variety of biological processes. Here, we elucidate the influence of cytokine application on mechano-sensitivity and mechano-transduction. The atrial myocardial stretch induces production of interleukin (IL)-2, IL-6, IL-13, IL-17A, and IL-18 with exception of tumor necrosis factor α (TNF-α), IL-1ß, and vascular endothelial growth factor B (VEGF-B). Positive ionotropic effect was specific for VEGF-B, negative ionotropic effects were specific for TNF-α, IL-1ß, IL-2, IL-6, IL-13, IL-17A and IL-18, while IL-1α doesn't show direct ionotropic effect. The IL-2, IL-6, IL-17A, IL-18, and VEGF-B cause elongation of the APD, in comparison with the reduced APD caused by the IL-13. The TNF-α, IL-1ß, and IL-18 influences L-type Ca2+ channels, IL-2 has an inhibitory effect on the fast Na+ channels while IL-17A and VEGF-B were specific for Kir channels. With exception of the IL-1α, IL-2, and VEGF-B, all analyzed cytokines include nitric oxide dependent signaling with resultant combined effects on mechano-gated and Ca2+ channels. The relationships between these pathways and the time-dependence of their activation are of important considerations in the evaluation of cytokine-induced electrical abnormality, specific for cardiac dysfunctions. In general, the discussion presented in this review covers research devoted to counterbalance between different cytokines in the regulation of stretch-induced effects in rat atrial myocardium. ABBREVIATIONS: APs: action potentials; APD25: action potential durations to 25% of re-polarization; APD50: action potential durations to 50% of repolarization; APD90: action potential durations to 90% of repolarization; MGCs: mechanically gated channels.

[Factors associated with levels of interleukins -18, -8, and -6 in hypertensive patients at high and very high cardiovascular risk].

AIM: To identify the most significant factor influencing blood levels of cytokines in patients at high and very high cardiovascular risk. MATERIALS AND METHODS: A patient base from the "Management of chronic patients with multiple diseases" project was analyzed. 523 patients (mean age, 87±17.8) were included. Plasma samples were analyzed for concentrations of sodium, creatinine, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, and NT-proBNP. GFR was calculated using the CKD-EPI formula. Time-related CHF progression was assessed in one year; the time-related progression was considered an increase in CHF stage. Salt consumption was determined using the Charlton: SaltScreener questionnaire at the baseline visit and at one year. Low-salt diet containing 5 g of salt per day was recommended to all patients; 3.5 g of salt per day was recommended to patients with a documented diagnosis of CHF. Statistical analysis was performed using the Statistica 10.0 software. RESULTS: 52.2 % of included patients consumed 6-10 g of salt per day; 43.4 % of patients consumed 10 g of salt or more per day; and only 4.4 % of patients consumed 5 g of salt or less per day. 21 % of included patients were at high risk of cardiovascular complications whereas for the vast majority of patients (79 %), the risk was stratified as very high. Two clusters of patients were formed based on the grade of hypertension, one-year CHF progression, and plasma levels of IL-6, -8, and -18. The one-year progression of CHF most significantly influenced the levels of IL-18, -8, and -6. The IL-6 level was correlated with the NT-proBNP level; an approximately similar degree of correlation was found for NT-proBNP and BP. CONCLUSION: Therefore, the performed statistical analysis determined correlations between the following factors: IL-6 level, NTproBNP level, and one-year CHF progression.

Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease.

In this study, we were focused on the differences between certain circulating cytokine levels in patients with or without sinus arrhythmia, according to the median IL-6 level. All patients were stable with regards to symptoms and therapy for at least one month prior to the measurements conducted within this study.Exclusion criteria were: patients with sleep apnea, asthma, respiratory insufficiency of any genesis, active infection, allergy, inflammatory diseases, cancer, diabetes of any type and treatment with anti-inflammatory drugs. The study was approved by the Institutional Review Board. All recruited patients gave their verbal and written consent for participation in the study. The study group consisted of 74 patients divided into two groups: with (38) and without sinus arrhythmia but with diagnosed coronary artery disease (36). Sinus arrhythmia was confirmed by 24h Holter monitoring. From all test parameters only cytokines IL-2, IL-8, IL-10, IL-17 and IL-18, showed statistically significant increasing in patients with statistically higher IL-6 levels. It is possible that IL-6 may not be a marker for the selection of patients with sinus arrhythmia or coronary artery disease. The findings indicate that IL-6 represents a reliable indicator for increased expression of IL-2, IL-8, IL-10, IL-17 and IL-18 in patients with sinus arrhythmia or coronary artery disease. Further studies in a large number of patients would be necessary to confirm our observations.

IL-1 provokes electrical abnormalities in rat atrial myocardium.

Using a micro-electrode technique we studied the effects of interleukin 1α and interleukin 1ß on bio-electric activity of rat atrial myocardium under normal conditions and after gradual stretching. Perfusion with interleukin 1α increased the duration of the action potential at the level of 90% re-polarization. Stretch induced tachy-arrhythmia in the presence of interleukin 1α is mainly regulated via stretch increased nitric oxide production, while the ionotropic effect of the interleukin-1α during stretching is not pronounced. The perfusion with interleukin 1ß did not change the values of the duration of the action potentials at the levels of 25, 50 and 90% repolarization. The interleukin lß caused an appearance of extra-systolic patterns which turned into normal rhythm, alternating with periods of normal activity. The total intracellular nitric oxide level induced by both interleukin 1ß and stretching is balanced by interleukin-1ß induced cation influx.

Effects of interleukin-6 on the bio-electric activity of rat atrial tissue under normal conditions and during gradual stretching.

Using the micro-electrode technique we studied the effects of interleukin-6 on bio-electric activity of rat atrial tissue under normal conditions and after gradual stretching. It was shown that IL-6 caused increasing of the duration of the action potential at the levels of 25, 50, and 90% re-polarization. The hump-like depolarization at APD90 appeared 7-10 min after initial stretching and transformed into single extra-potentials after tension removing. Perfusion with IL-6 for more than 20 min led to the appearance of atrial fibrillation even with the application of slight tension. Close observation of the IL-6 induced mechanisms and stretch induced APD alteration, confirmed the existence of a tight link between examined cytokine and stretch induced mechanisms.

Effects of interleukin-18 on bioelectric activity of rat atrial cardiomyocytes under normal conditions and during gradual stretching of the tissue.

Effects of IL-18 (50 ng/ml) on bioelectric activity of rat atrial cardiomyocytes under normal conditions and after gradual stretching of the tissue was studied using microelectrode technique. It was shown that in 85% experiments, IL-18 increased the duration of action potential at the level of 25, 50, and 90% repolarization without changing the magnitude of the resting potential, amplitude and repetition rate of action potentials, and cardiomyocyte contraction force. In addition, IL-18 abolished mechanically induced changes in the shape of action potentials during stretching.

[Mechanically gated cardiac ion channels and their regulation by cytokines].

The publication presents discussion of the modern vision of mechanisms of mechanoelectric feedback in heart as well as most recent findings regarding possible regulation of cardiomyocyte mechanically gated ion channels by endogenous compounds of immune origin--cytokines. Special attention is devoted to description of cytokine action on cardiac cells, in particular to nitrogen oxide effects on ionic currents, which contribute to generation of the action potential of the cardiomyocyte. We hypothesize that cytokines can potentially trigger such mechano-dependent cardiac pathologies as arrhythmias and fibrillation.

[Influence of interleukin-13 on bioelectrical activity of rat atrial cardiomyocytes in normal and stretch conditions].

We studied the effect of anti-inflammatory ininterleukin-13 (IL-13; 50 ng/ml) on bioelectrical activity of rat atrial cardiomyocytes under control conditions and on the background of stretch by means of microelectrode technique. IL-13 did not lead to alterations of the resting membrane potential and action potential amplitude during 35 minutes. However APD25, APD50, APD90 in 50% of cases significantly decreased, while in other 50% of cases it increased. In case when IL-13 decreased APD cellular stretch by 1.7 mN caused an increase in APD frequency by 120% and caused decrease in APD25, APD50, APD90, which happened on the background of modest cellular depolarization in the range of 5 mV. When IL-13 increased APD, tissue stretching just by 0.8 mN caused depression of the frequency by 10% and increase in APD25, APD50, APD90. This happened on the background of small cellular depolarization.

Effect of nitric oxide on mechanoelectric feedback in rat right atrium.

In situ microelectrode examination of rat right atrium showed that in physiologically prestretched tissue, NO donor SNAP modifies the repolarization phase of cardiomyocyte AP in a "hump-like" way provoking the development of arrhythmia. Gadolinium both prevents and eliminates this effect attesting to involvement of stretch-activated channels in the development of NO-induced abnormalities. Elevation of SNAP concentration or further stretch of the tissue (presumably, it increases NO concentration) eliminated the hump depolarization induced by moderate SNAP stimulation. Thus, low NO opens the stretch-activated channels while high NO inactivates them.

Role of nitric oxide in activity control of mechanically gated ionic channels in cardiomyocytes: NO-donor study.

Whole-cell ionic currents through mechanically gated channels (MGC) were recorded in isolated cardiomyocytes under voltage clamp conditions. In unstrained cells, NO donors SNAP and DEA-NO activated MGC and induced MG-like currents. In contrast, in stretched cells with activated MGC, these NO-donors inactivated and inhibited MGC.

Single mechano-gated channels activated by mechanical deformation of acutely isolated cardiac fibroblasts from rats.

AIM: Mechanosensitive conductances were reported in cardiac fibroblasts, but the properties of single channels mediating their mechanosensitivity remain uncharacterized. The aim of this work was to investigate single mechano-gated channels (MGCs) activated by mechanical deformations of cardiac fibroblasts. METHODS: Currents through single MGCs and mechanosensitive whole-cell currents were recorded from isolated rat atrial fibroblasts using the cell-attached and whole-cell patch-clamp configurations respectively. Defined mechanical stress was applied via the patch pipette used for the whole-cell recordings. RESULTS: Under resting conditions occasional short openings of two types of single MGCs with conductances of 43 and 87 pS were observed. Both types of channels displayed a linear current-voltage relationship with the reversal potential around 0 mV. Small (1 microm) mechanical deformations affected neither single nor whole-cell mechano-gated currents. Cell compressions (2, 3 and 4 microm) augmented the whole-cell currents and increased the frequency and duration of single channel openings. Cell stretches (2, 3 and 4 microm) inactivated the whole-cell currents and abolished the activity of single MGCs. Gd(3+) (8 microm) blocked the whole-cell currents within 5 min. No single channel activity was observed in the cell-attached mode when Gd(3+) was added to the intrapipette solution. Cytochalasin D and colchicine (100 microm each) completely blocked both the whole-cell and single channel currents. CONCLUSIONS: These findings show that rat atrial fibroblasts express two types of MGCs whose activity is governed by cell deformation. We conclude that fibroblasts can sense the direction of applied stress and contribute to mechano-electrical coupling in the heart.

Role of nitric oxide in the regulation of mechanosensitive ionic channels in cardiomyocytes: contribution of NO-synthases.

The role of NO in the regulation of currents passing through ion channels activated by cell stretching (mechanically gated channels, MGC), particularly through cation-selective K(+)-channels TRPC6, TREK1 (K(2P)2.1), and TREK2 (K(2P)10.1), was studied on isolated mouse, rat, and guinea pig cardiomyocytes using whole-cell patch-clamp technique. In non-deformed cells, binding of endogenous NO with PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-1-oxy-3-oxide) irreversibly shifted the diastolic membrane potential towards negative values, modulates K(ir)-channels by reducing I(K1), and blocks MGC. Perfusion of stretched cells with PTIO solution completely blocked MG-currents. NO-synthase inhibitors L-NAME and L-NMMA completely blocked MGC. Stretching of cardiomyocytes isolated from wild type mice and from NOS1(-/-)- and NOS2(-/-)- knockout mice led to the appearance in MG-currents typical for the specified magnitude of stretching, while stretching of cardiomyocytes from NOS3(-/-)- knockout mice did not produce in MG-current. These findings suggest that NO plays a role in the regulation of MGC activity and that endothelial NO-synthase predominates as NO source in cardiomyocyte response to stretching.