The Na+,K+-ATPase (NKA) is essential for neuronal excitability. The neuron-specific α3 isoform differs from the ubiquitous α1 isoform by its low affinity for intracellular Na+, weak voltage dependence, and slightly reduced ATP sensitivity, yet the functional relevance of these features remains unclear. Using biophysically detailed models of stretch receptor neurons, we examined how isoform-specific pump kinetics influence firing behavior. Substitution of α1 for α3 NKA abolished sustained spike trains and reduced high-frequency entrainment. In contrast, modifying α3 NKA to exhibit α1-like voltage or ATP dependence did not affect neuronal excitability. These results indicate that Na+ affinity, rather than voltage or ATP dependence, is the critical determinant of α3 NKA specialization. Our findings provide a mechanistic explanation for the selective expression of α3 NKA in muscle spindle afferents and other high-frequency neurons, highlighting that NKA isoform properties are tuned to the discharge demands of distinct neuronal populations.

Heart failure of various etiologies, including diabetic cardiomyopathy (DCM), is a global problem. However, the features of DCM pathogenesis, including the role of the T-tubular system in this process, are not fully understood. The available literature data on T-system remodeling in diabetic rodent models are contradictory, with changes in this system being demonstrated in some of them already at the prediabetic stage. So, this work was aimed to assess changes in the T-system in a rat model of metabolic syndrome (MS) that precedes type 2 diabetes mellitus. MS was induced in Wistar rats by a high-carbohydrate (HC) and a combined high-carbohydrate high-fat (HCHF) diets for 10 weeks. The parameters to be finally estimated were body, abdominal fat, and heart weights, as well as fasting plasma glucose levels measured by a glucose tolerance test (GTT). The structure of the T-system was examined via confocal microscopy in isolated hearts stained with DI-8-ANEPPS. HCHF-fed rats developed more severe MS since their body and abdominal fat weights, as well as fasting plasma glucose levels, were significantly higher compared to the controls; moreover, GTT revealed the signs of glucose tolerance. HC-fed rats were characterized by a moderate MS, as manifested only in an increase in abdominal fat weight. The structural correlates of T-system remodeling were also found in HCHF-fed rats only, as manifested in a significant increase in the average interval between the rows of T-tubules. These findings differ from our results obtained in a model of type 1 prediabetes and diabetes. Thus, T-system remodeling in rat cardiomyocytes begins already at the stage of HCHF-induced MS and proceeds similarly to that described for type 2 diabetes, but differs from that in type 1 prediabetes and diabetes, suggesting different pathways of DCM pathogenesis in different diabetic types.

Impaired cardiovascular function in metabolic syndrome (MS) and type 2 diabetes mellitus (T2DM) is not adequately studied, and data on changes in electrocardiogram (ECG) parameters, including in rodent models of MS and T2DM, are contradictory and ambiguous. Aim of the study was to investigate ECG parameters in models of MS and T2DM and to identify possible correlations between impaired glucose tolerance and the severity of changes in ECG parameters. Material and methods. Male Wistar rats were used in the experiments. MS was induced by a high-fat diet (HFD) for 17 weeks; T2DM was induced by a combination of HFD and streptozotocin injection (HFD + STZ): 11 weeks of HFD followed by an injection of streptozotocin and continued HFD for another 6 weeks. Control animals were kept on a standard diet. Results. The glucose tolerance test confirmed the development of impaired glucose tolerance to the prediabetic level in the HFD group and to the diabetic level in the HFD+STZ group. ECG changes were found only in the HFD+STZ group: a significant (compared to the control and HFD group) increase in the amplitude and area T wave on the ECG was shown. Conclusions. Pronounced ECG abnormalities develop only in the model of T2DM, but not in the MS model.

Rats kept on a high-fat/high-sucrose diet (HFSD) for 10–12 weeks demonstrated the development of hyperglycemia and signs of visceral obesity. Compared to the control, extracellular action potentials (eAP) of subepicardial myocytes of the left ventricle (LV) of HFSD rats characterized by a significantly increased fraction of signals with a pronounced afterhyperpolarization (AHP) phase and an accelerated decline. Local delivery of apamin (a blocker of small conductance Ca2+-activated K+ channels (IKCa, SK channels) to the eAP registration cite at a concentration of 500 nM in the solution inside the pipette was accompanied by suppression of the AHP phase and prolongation of the eAP decline. The obtained data suggest that HFSD leads to an increase in the expression and/or activity of SK channels and, as a result, to the development of AHP and shortening of eAP in epicardial cardiomyocytes of the LV of the rat heart.

The role of small-conductance Ca2+-activated K+-channels (SK channels) in the pathogenesis of cardiomyopathies of various etiologies remains poorly understood. The purpose of this work was to evaluate the effect of the blocker of SK channels, apamin, on the extracellularly recorded action potentials (eAPs) of subepicardial myocytes in the left ventricles of sham-operated rats and rats with myocardial infarction caused by ischemia-reperfusion. It was found that local delivery of the SK channel blocker apamin at a concentration of 500 nM to the eAP recording area did not affect the eAP profiles in the group of sham-operated rats but caused a significant slowdown in the repolarization time and a decrease in the afterhyperpolarization phase of eAPs in the group of rats with myocardial infarction. These data suggest that changes in the waveform of eAPs after infarction are associated with increased expression and/or activity of SK channels in subepicardial myocytes. The possible role of these channels in the structural and functional remodeling of the myocardium of the left ventricle of the heart after ischemia-reperfusion is discussed.

One of the complications of diabetes mellitus (DM) is diabetic cardiomyopathy (DCM), whose molecular mechanisms of pathogenesis have not been fully studied. Previously, the involvement of Na+/K+-ATPase and components of the Ca2+ transport system in cardiomyocytes in the development of DCM was shown. The aim of the work was to study the expression and activity of Na+/K+-ATPase and Ca2+-ATPase (SERCA2) in the myocardium of male Wistar rats in a model of streptozotocin (STZ)-induced prediabetes and overt type 1 diabetes (T1DM). STZ was administered at once i.p. in doses of 30–35 mg/kg. Rats with glucose levels above 11 mM were considered diabetic (STZ-D1 group), and those with moderate hyperglycemia were considered prediabetic (STZ-preD1 group). The activity of Na+/K+-ATPase and Ca2+-ATPase was determined (by the rate of release of inorganic phosphate, Pi), and the expression of the genes α1- and α2-isoforms of Na+/K+-ATPase, SERCA2, and Kir6.1, Kv7.1, and Kv2.1 potassium channels was also determined. In the control (C) group, the activity of ouabain (1 mM) -sensitive Mg2+-dependent ATPase was 6.03 ± 0.6 mmol Pi/g/h. In the STZ-D1 and STZ-preD1 groups, Na+/K+-ATPase activity did not differ from group C. The level of gene expression of α1- and α2- subunits of Na+/K+-ATPase in the STZ-D1 group decreased by more than 45 STZ-preD1 group increased by 64 and 81 sensitivity of expression to insulinopenia. The activity of Ca2+-ATPase and the expression of the SERCA2 gene did not differ between the groups, which might be because the 4-week period after STZ administration is not sufficient for the development of Ca2+-ATPase deficiency in the rat heart. The level of expression of the genes of the potassium channel subtypes Kv2.1, Kir6.1, and Kv7.1 increased in the STZ-preD1 group, which may indicate a potential contribution of the studied potassium channel subtypes to the adaptation mechanism to moderate hyperglycemia.

A number of systemic heart diseases leading to the development of heart failure (aortic stenosis, hypertension, diabetic cardiomyopathy, reperfusion injury etc.) are accompanied by a pronounced reorganization of the T-system of cardiomyocytes, both in humans and animals. However, structural-functional changes within this membrane compartment of cardiomyocytes following ischemia-reperfusion (IR) have not been thoroughly studied. The aim of the work was to study the remodeling of the T-system in the subepicardial cardiomyocytes of the left ventricle (LV) of the rat heart after IR injury using confocal microscopy and extracellular recording methods. The study was carried out after 24 hours, two weeks, and four weeks following IR. A remodeling of extracellular action potentials, recorded in the cardiomyocyte membrane patches that are devoid of t-tubule entrances (a single negative peak, type 1 eAP), was observed. Starting from 24 hours up to 4 weeks after IR, there was an increase in the duration of their decline time ( T 90 ) and the formation of eAP after-hyperpolarization phase, reaching maximum values by the fourth week after IR. A decrease in the amplitude of the second peak of eAPs, recorded in the cardiomyocyte membrane patches that contained t-tubule openings (a double negative peak, type 2 eAP), was also noticed four weeks after IR. However, in this investigation, no observable changes in the structural organization of the T-system were found. These data suggest that functional modifications of the epicardial cardiomyocyte T-system after IR injury may precede its structural modifications.

Overexposure to Mn causes a neurological disorder—manganism—with motor symptoms that overlap closely with disorders associated with haploinsufficiency in the gene encoding for α3 isoform of Na+,K+-ATPase (NKA). The present study was designed to test the hypothesis that behavioral changes in the mouse model of manganism may be associated with changes in the expression and activity of α3 NKA in the cerebellum (CB) and striatum (STR)—the key brain structures responsible for motor control in adult mice. C57Bl/6 mice were exposed to MnCl2 at 0.5 g/L (in drinking water) for up to eight weeks. After four weeks of Mn consumption, Mn levels were increased in the CB only. Behavioral tests demonstrated decreased performance of Mn-treated mice in the shuttle box test (third through sixth weeks), and the inclined grid walking test (first through sixth weeks), suggesting the development of learning impairment, decreased locomotion, and motor discoordination. The activity of NKA significantly decreased, and the expression of α1-α3 isoforms of NKA increased in the second week in the CB only. Thus, signs of learning and motor disturbances developing in this model of manganism are unlikely to be directly linked to disturbances in the expression or activity of NKA in the CB or STR. Whether these early changes may contribute to the pathogenesis of later behavioral deficits remains to be determined.

The role of small conductance (SK) Ca2+-dependentpotassium channels in pathogenesis of diabetic cardiomyopathy remainsuncertain. In our studies in the rat model of streptozotocin-inducedtype 1 diabetess mellitus (STZ-DM1) a significant increase abovecontrol in the prevalence of extracellular action potentials (eAP)recorded from isolated heart left ventricles and terminating witha prominent phase of after-hyperpolarization was observed. In experimentswith local delivery to the site of recording of several tested potasssiumchannel blockers suppression of this phase of eAP after hyperpolarizationwas achieved only with application of apamin, the selective SK channelinhibitor. These data suggest that eAP afterhyperpolarization phaseis associated with activation of SK channels and that activity and/orexpression of these channels is increased in epicardial cardiomyocytesof left ventricles of STZ-DM1 rat hearts.

Morphine-6-O-sulfate (M6S), a polar, zwitterionic sulfate ester of morphine, is a powerful and safe analgesic in several rat models of pain. A sensitive liquid chromatography-tandem mass spectrometry bioanalytical method was developed and validated for the simultaneous determination of M6S and morphine (MOR) in rat plasma and brain after M6S administration. Morphine-d(6) was used as internal standard. Multiple reaction monitoring was used for detection and quantitation of M6S, MOR, and morphine-d(6) in the turbo ion spray positive mode. The chromatographic separation was carried out on an Alltech Altima C18 column. The analytical method was validated for linearity, precision, accuracy, specificity, and stability over a concentration range of 3-8000 ng/ml in rat plasma and 10-10,000 ng/ml in brain samples for both M6S and MOR. The validated method was applied to determine the PK profile of M6S in plasma after i.v., i.p., and oral dosing in male Sprague-Dawley rats. Rats were administered M6S by i.p. administration (5.6 and 10.0 mg/kg) or orally (10 and 30 mg/kg) and bioavailability compared to an i.v. injection (1 mg/kg) of M6S. The in vivo results indicate that M6S is not a prodrug of morphine, since M6S is not biotransformed into MOR in plasma after either i.p. or oral administration, and MOR was not detected in brain. The bioavailability of M6S was >93% and about 5% after i.p. and oral dosing, respectively. The low oral bioavailability of M6S may be due to poor permeation of the intestinal epithelial membrane. After i.p.-administration, M6S appears to reach brain tissues in low, but significant, concentrations.

Deep-space missions may alter immune cell phenotype in the primary (e.g., thymus) and secondary (e.g., spleen) lymphoid organs contributing to the progression of a variety of diseases. In deep space missions, astronauts will be exposed to chronic low doses of HZE radiation while being in microgravity. Ground-based models of long-term uninterrupted exposures to HZE radiation are not yet available. To obtain insight in the effects of concurrent exposure to microgravity and chronic irradiation (CIR), mice received a cumulative dose of chronic 0.5 Gy gamma rays over one month +/- simulated microgravity (SMG). To obtain insight in a dose rate effect, additional mice were exposed to single acute irradiation (AIR) at 0.5 Gy gamma rays. We measured proportions of immune cells relative to total number of live cells in the thymus and spleen, stress level markers in plasma, and change in body weight, food consumption, and water intake. CIR affected thymic CD3+/CD335+ natural killer T (NK-T) cells, CD25+ regulatory T (Treg) cells, CD27+/CD335+ natural killer (NK1) cells and CD11c+/CD11b- dendritic cells (DCs) differently in mice subjected to SMG than in mice with normal loading. No such effects of CIR on SMG as compared to normal loading were observed in cell types from the spleen. Differences between CIR and AIR groups (both under normal loading) were found in thymic Treg and DCs. Food consumption, water intake, and body weight were less after coexposure than singular or no exposure. Compared to sham, all treatment groups exhibited elevated plasma levels of the stress marker catecholamines. These data suggest that microgravity and chronic irradiation may interact with each other to alter immune cell phenotypes in an organ-specific manner and appropriate strategies are required to reduce the health risk of crewmembers.

Left ventricles (LV) of isolated hearts of the control rats (n = 7) and rats injected with streptozotocin (STZ; n = 21) were studied on the four-week post-injection term using 5-µm tip (O.D.) glass extracellular microelectrodes. STZ-rats that maintained hyperglycemia by the time of the study (random glucose > 11 mM; n = 3) were designated as diabetic animals (diabetes mellitus group; STZ-DM). Remaining STZ-rats (n = 18) were designated as normoglycemic rats (STZ-NG group). In control and in STZ-rat hearts two main types of sinus rhythm action potentials (AP) of epicardial ventricular myocytes could be recorded: signals characterized by a single (AP1) or double negative peaks (AP2). Respective time parameters of AP1 (rise time, and 50% and 90% decay times (T50 and T90) not differed between studied groups of rats. However, T50 of AP2 of LV myocytes of STZ-NG and STZ-DM rats were statistically significantly longer than T50 of AP2 recorded in control rat hearts. Furthermore, in 28% of STZ-NG and in 67% of STZ-DM rat hearts additional type of AP (AP3) featuring a complex, multi-peak negative phase and prominent positive phase of after-hyperpolarization were recorded. Appearance of AP3 was associated with a proportional decrease in the relative frequency of AP2 recordings (from 80-85% to 55-60% of all recordings). Frequency of AP1 recordings remained stable, 15-20% in all experiments in all groups of rats. Tentatively, observed in this study modulation in electrical activity of ventricular myocytes of STZ-rat hearts results from structure-functional changes in organization of the T-system of these myocytes. Regardless of validity of this suggestion, it is important that changes in electrogenesis of cardiomyocytes develop fast (1 month) not only in STZ-DM rats, but also in hearts of STZ-rats with normal or moderately increased blood glucose levels, which may be considered as the state equivalent to the state of early prediabetes.

Acute exposure to hypoxic conditions is a frequent natural event during the development of bird eggs. However, little is known about the effect of such exposure on the ability of young embryos in which cardiovascular regulation is not yet developed to maintain a normal heart rate (HR). To address this question, we studied the effect of 10–20 min of exposure to moderate or severe acute hypoxia (10% or 5% O2, respectively) on the HR of day 4 (D4) chicken embryos. In ovo, video recording of the beating embryo heart inside the egg revealed that severe, but not moderate, hypoxia resulted in significant HR changes. The HR response to severe hypoxia consisted of two phases: the first phase, consisting of an initial decrease in HR, was followed by a phase of partial HR recovery. Upon the restoration of normoxia, after an overshoot period of a few minutes, the HR completely recovered to its basal level. In vitro (isolated heart preparation), the first phase of the HR response to severe hypoxia was strengthened (nearly complete heart silencing) compared to that in ovo, and the HR recovery phase was greatly attenuated. Furthermore, neither an overshoot period nor complete HR recovery after hypoxia was observed. Thus, the D4 chicken embryo heart can partially maintain its rhythm during hypoxia in ovo, but not in vitro. Some factors from the egg, such as catecholamines, are likely to be critical for avian embryo responding to hypoxic condition and survival.

Disorganization of the T-system of cardiomyocytes is considered an early and critical step in the development of Diabetic Cardiomyopathy (DCM). To test this suggestion, male Wistar rats were injected with streptozotocin (STZ, 30 or 45 mg/kg) and studied one month later. STZ-rats that developed and maintained hyperglycemia (random blood glucose > 11 mM) were designated as hyperglycemic (STZ-HG) rats, while the remaining STZ-rats – as normoglycemic (STZ-NG) animals. The structural integrity of the T-system was investigated using an analysis of confocal images of the left ventricle (LV) sub-epicardium of isolated hearts, stained with the Di-8-ANEPPS. In control, T-system was organized into regular networks of t-tubules aligned with Z-discs of cardiomyocyte’s sarcomeres. Accordingly, the frequency distributions of intervals between neighboring t-tubules (INT, measured along the major cell axis) peaked at a 2 µm value with not more than 21% of INT (per cell) exceeding the 3 µm cut-off. Only 4±3% of the control cardiomyocytes (274 cells, 4 rats) could be considered as deficient, according to this parameter (>21% occurrence of long INT). Compared to control, in the hearts of STZ-NG and STZ-HG rats, the fractions of such deficient cardiomyocytes were statistically significantly higher: 48±13% (STZ-NG, 8 rats, 573 cells) and 76±8% (STZ-HG, 4 rats, 247 cells). Thus, structural changes in the T-system of the rat heart LV cardiomyocytes develop early during chronic hyperglycemia (overt diabetes) as well as during near-normoglycemic stages of diabetes (prediabetes). The relevance of these changes to the development of DCM in subjects with prediabetes remains to be studied.

Cardiac disruption and development of a diabetic cardiomyopathy (DCM) are a direct consequence of diabetes. In work activity Na/K-ATPase in a myocardium of rats with different extent of diabetic impairment was studied. Suppression of activity of Na/K-ATPase in the cardiomyocytes the left ventricle of rats was revealed at strongly developed diabetes, but not at a prediabetes stage. It was concluded, that development of DCM breaks transport processes in a membrane of rats cardiomyocytes, and the expressiveness of observed changes depends on diabetes degree.

Action potentials (APs) were recorded extracellularly from ventricular and atrial cardiomyocytes using isolated rat heart preparations and narrow-tipped (5-µm O.D.) recording pipettes. The negative phase of the AP waveform in ventricular cardiomyocytes exhibited a prolonged and, in about 60% of records, double-peak AP deflection, while that of APs in atrial cardiomyocytes regularly consisted of a single and relatively short peak. Confocal imaging and Monte-Carlo simulations suggest a stochastic variation in the number of t-tubule mouths under the lumen of randomly positioned extracellular recording pipette as the leading determinant of both, between and within, ventricular/atrial AP waveform variations.