Cardiac Ventricular Myocytes Research Articles

Inhibitory effect of YM-244769, a novel Na+/Ca2+ exchanger inhibitor on Na+/Ca2+ exchange current in guinea pig cardiac ventricular myocytes.

Recently, YM-244769 (N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) has been reported as a new potent and selective Na+/Ca2+ exchange (NCX) inhibitor by using various cells transfected with NCX using the 45Ca2+ fluorescent technique. However, the electrophysiological study of YM-244769 on NCX had not been performed in the mammalian heart. We examined the effects of YM-244769 on NCX current (INCX) in single cardiac ventricular myocytes of guinea pigs by using the whole-cell voltage clamp technique. YM-244769 suppressed the bidirectional INCX in a concentration-dependent manner. The IC50 values of YM-244769 for the bidirectional outward and inward INCX were both about 0.1μM. YM-244769 suppressed the unidirectional outward INCX (Ca2+ entry mode) with an IC50 value of 0.05μM. The effect on the unidirectional inward INCX (Ca2+ exit mode) was less potent, with 10μM of YM-244769 resulting in the inhibition of only about 50%. At 5mM intracellular Na+ concentration, YM-244769 suppressed INCX more potently than it did at 0mM [Na+]i. Intracellular application of trypsin via the pipette solution did not change the blocking effect of YM-244769. In conclusion, YM-244769 inhibits the Ca2+ entry mode of NCX more potently than the Ca2+ exit mode, and inhibition by YM-244769 is [Na+]i-dependent and trypsin-insensitive. These characteristics are similar to those of other benzyloxyphenyl derivative NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The potency of YM-244769 as an NCX1 inhibitor is higher than those of KB-R7943 and SN-6 and is similar to that of SEA0400.

Modeling Na+-Ca2 + exchange in the heart: Allosteric activation, spatial localization, sparks and excitation-contraction coupling

The cardiac sodium (Na+)/calcium (Ca2+) exchanger (NCX1) is an electrogenic membrane transporter that regulates Ca2+ homeostasis in cardiomyocytes, serving mainly to extrude Ca2+ during diastole. The direction of Ca2+ transport reverses at membrane potentials near that of the action potential plateau, generating an influx of Ca2+ into the cell. Therefore, there has been great interest in the possible roles of NCX1 in cardiac Ca2+-induced Ca2+ release (CICR). Interest has been reinvigorated by a recent super-resolution optical imaging study suggesting that ~18% of NCX1 co-localize with ryanodine receptor (RyR2) clusters, and ~30% of additional NCX1 are localized to within ~120nm of the nearest RyR2. NCX1 may therefore occupy a privileged position in which to modulate CICR. To examine this question, we have developed a mechanistic biophysically-detailed model of NCX1 that describes both NCX1 transport kinetics and Ca2+-dependent allosteric regulation. This NCX1 model was incorporated into a previously developed super-resolution model of the Ca2+ spark as well as a computational model of the cardiac ventricular myocyte that includes a detailed description of CICR with stochastic gating of L-type Ca2+ channels and RyR2s, and that accounts for local Ca2+ gradients near the dyad via inclusion of a peri-dyadic (PD) compartment. Both models predict that increasing the fraction of NCX1 in the dyad and PD decreases spark frequency, fidelity, and diastolic Ca2+ levels. Spark amplitude and duration are less sensitive to NCX1 spatial redistribution. On the other hand, NCX1 plays an important role in promoting Ca2+ entry into the dyad, and hence contributing to the trigger for RyR2 release at depolarized membrane potentials and in the presence of elevated local Na+ concentration. Whole-cell simulation of NCX1 tail currents are consistent with the finding that a relatively high fraction of NCX1 (~45%) resides in the dyadic and PD spaces, with a dyad-to-PD ratio of roughly 1:2. Allosteric Ca2+ activation of NCX1 helps to “functionally localize” exchanger activity to the dyad and PD by reducing exchanger activity in the cytosol thereby protecting the cell from excessive loss of Ca2+ during diastole.

Circulation Research “In This Issue” Anthology

Circulation Research “In This Issue” Anthology

Blocking effect of carvedilol, a beta‐blocker on Na+/Ca2+ exchange current in isolated cardiac ventricular cells of guinea pig

BackgroundCarvedilol has a1, b1, and b2‐adrenergic blocking effects together with the effects such as vasodilating, anti‐ischemic, anti‐oxidant, anti‐apoptotic, anti‐proliferate and anti‐inflammatory activities. Carvedilol inhibited IKr, IKs, ICa‐L and Ito in rabbit cardiac ventricular myocytes. On the other hand, metoprolol, a nonselective b‐blocker also has blocking several channels, including IK1, ICa and Ito in hearts. Murayama et al. (2013) reported that carvedilol and its analogues inhibit delayed after depolarizations (DADs) in Langendorff‐perfused rabbit hearts. It is well known that DADs are caused by the Na+/Ca2+ exchange current (INCX).MethodsWe examined the effects of carvedilol and metoprolol, two b‐blockers, on INCX by using the whole‐cell voltage‐clamp method in isolated guinea pig ventricles and CCL39 fibroblast cells expressing dog cardiac Na+/Ca2+ exchanger (NCX1).ResultsCarvedilol suppressed INCX in a concentration‐dependent manner but metoprolol did not in isolated guinea pig cardiac ventricular myocytes. IC50 values for the Ca2+ influx (outward) and efflux (inward) components of INCX were 69.7 μM and 61.5 μM, respectively. Carvedilol at 100 μM inhibited INCX in CCL39 cells expressing wild type NCX1 similarly to mutant NCX1 without the intracellular regulatory loop. Carvedilol at 30 μM abolished ouabain‐induced DADs in isolated cardiac ventricular myocytes.ConclusionsCarvedilol suppressed bi‐directional INCX in a concentration‐dependent manner with the IC50 value of approximately 65 μM. We conclude that carvedilol inhibits NCX1 at supratherapeutic concentrations.

Inorganic polyphosphate in cardiac myocytes: from bioenergetics to the permeability transition pore and cell survival.

Inorganic polyphosphate (polyP) is a linear polymer of Pi residues linked together by high-energy phosphoanhydride bonds as in ATP. PolyP is present in all living organisms ranging from bacteria to human and possibly even predating life of this planet. The length of polyP chain can vary from just a few phosphates to several thousand phosphate units long, depending on the organism and the tissue in which it is synthesized. PolyP was extensively studied in prokaryotes and unicellular eukaryotes by Kulaev's group in the Russian Academy of Sciences and by the Nobel Prize Laureate Arthur Kornberg at Stanford University. Recently, we reported that mitochondria of cardiac ventricular myocytes contain significant amounts (280±60pmol/mg of protein) of polyP with an average length of 25 Pi and that polyP is involved in Ca(2+)-dependent activation of the mitochondrial permeability transition pore (mPTP). Enzymatic polyP depletion prevented Ca(2+)-induced mPTP opening during ischaemia; however, it did not affect reactive oxygen species (ROS)-mediated mPTP opening during reperfusion and even enhanced cell death in cardiac myocytes. We found that ROS generation was actually enhanced in polyP-depleted cells demonstrating that polyP protects cardiac myocytes against enhanced ROS formation. Furthermore, polyP concentration was dynamically changed during activation of the mitochondrial respiratory chain and stress conditions such as ischaemia/reperfusion (I/R) and heart failure (HF) indicating that polyP is required for the normal heart metabolism. This review discusses the current literature on the roles of polyP in cardiovascular health and disease.

Modeling the Role of NCX in the Regulation of Cardiomyocyte Microdomain Ca2+ Dynamics

The sodium (Na+)/calcium (Ca2+) exchanger (NCX) is an electrogenic membrane transporter that mainly serves as a Ca2+ removal mechanism during relaxation and maintains Ca2+ homeostasis in cardiomyocytes. The direction of transport reverses at membrane potentials near action the potential plateau, generating an influx of Ca2+ into the cell. Therefore, there has been great interest in the role NCX plays in cardiac Ca2+-induced Ca2+-release (CICR), with some controversial findings questioning whether NCX has any role at all. A recent super-resolution optical imaging study suggested that ∼17% of NCX co-localize within the domain occupied by ryanodine receptor (RyR) clusters and ∼30% of NCX are within 100nm of the dyad boundary. Therefore, NCX may play a critical role in modulating Ca2+ spark activity and CICR as a result of its spatial distribution in the T-tubule. Here we examine the potential role of NCX using computational modeling. We have developed a novel mechanistic and biophysically-detailed model of NCX that describes both NCX transport kinetics and dynamics of Ca2+-dependent allosteric regulation. We incorporated this new NCX model into a previously developed super-resolution Ca2+ spark model to examine effects of local NCX spatial distribution on properties of Ca2+ sparks. Dyadic NCX reduces spark frequency but has minimal effects on spark amplitude and half-width. Furthermore, we incorporate NCX into a previously developed computational model of the cardiac ventricular myocyte that includes a detailed description of CICR simulated with stochastic gating of L-Type Ca2+ channels and RyRs in the dyad and accounts for local Ca2+ gradients via inclusion of peri-dyadic compartments. We use this model to investigate the role of NCX and its localization on whole-cell Ca2+ dynamics, the Ca2+ levels in the junctional sarcoplasmic reticulum, and APs.

Restricted Diffusion of cAMP is Mediated by Mitochondrial Localized PKA Buffering in Living Cells

Different G-protein coupled receptors can elicit unique functional responses by selectively utilizing specific subsets of spatially restricted cAMP signaling domains. Previous studies have concluded that this can only be achieved if the rate of cAMP movement within cells is much slower than the rate of free diffusion. However, this prediction has not been demonstrated experimentally. In this study, we directly test the hypothesis that the cytoplasmic diffusion of cAMP is indeed slow and then address the long standing question of what regulates its movement. We used Raster Image Correlation Spectroscopy (RICS) to directly measure the diffusion coefficients of various molecules in intact HEK 293 cells and adult ventricular cardiac myocytes to evaluate the effect of particle size and cell morphology on cAMP movement. Fluorescein and pharos-450 (φ450), fluorescent molecules similar in size to cAMP, exhibited diffusion coefficients 3-4 fold slower than their expected rates of free diffusion. The diffusion coefficient of φ450-labeled cAMP (φ450-cAMP) was >6 fold slower than fluorescein or φ450 free dye alone. This extremely slow rate of diffusion could not be explained by its size. In cardiac myocytes, φ450-cAMP co-localized with the type II regulatory (RII) subunit of protein kinase A (PKA). In addition, disruption of RII subunit anchoring to A kinase anchoring proteins (AKAPs) significantly increased the diffusion coefficients of both RII subunits and φ450-cAMP. Furthermore, RII subunits and φ450-cAMP co-localized with mitochondria. These results demonstrate that the cytosolic movement of cAMP is indeed much slower than the predicted rate of free diffusion and that this is due to interactions with PKA regulatory subunits associated specifically with mitochondria. These findings have important implications with respect to cAMP signaling in all cell types.

Isolated neonatal rat papillary muscles: a new model to translate neonatal rat myocyte signaling into contractile mechanics.

Isolated cardiac tissue allows investigators to study mechanisms underlying normal and pathological conditions, which would otherwise be difficult or impossible to perform invivo. Cultured neonatal rat ventricular cardiac myocytes (NRVM) are widely used to study signaling and growth mechanisms in the heart, primarily due to the versatility, economy, and convenience of this invitro model. However, the lack of a well-defined longitudinal cellular axis greatly hampers the ability to measure contractile function in these cells, and therefore to associate signaling with mechanical function. In these methods, we demonstrate that this limitation can be overcome by using papillary muscles isolated from neonatal rat hearts. In the methods we describe procedures for isolation of right ventricular papillary muscles from 3-day-old neonatal rats and effects of mechanical and humoral stimuli on contraction and relaxation properties of these tissues.

Detachment of surface membrane invagination systems by cationic amphiphilic drugs.

Several cell types develop extensive plasma membrane invaginations to serve a specific physiological function. For example, the megakaryocyte demarcation membrane system (DMS) provides a membrane reserve for platelet production and muscle transverse (T) tubules facilitate excitation:contraction coupling. Using impermeant fluorescent indicators, capacitance measurements and electron microscopy, we show that multiple cationic amphiphilic drugs (CADs) cause complete separation of the DMS from the surface membrane in rat megakaryocytes. This includes the calmodulin inhibitor W-7, the phospholipase-C inhibitor U73122, and anti-psychotic phenothiazines. CADs also caused loss of T tubules in rat cardiac ventricular myocytes and the open canalicular system of human platelets. Anionic amphiphiles, U73343 (a less electrophilic U73122 analogue) and a range of kinase inhibitors were without effect on the DMS. CADs are known to accumulate in the inner leaflet of the cell membrane where they bind to anionic lipids, especially PI(4,5)P2. We therefore propose that surface detachment of membrane invaginations results from an ability of CADs to interfere with PI(4,5)P2 interactions with cytoskeletal or BAR domain proteins. This establishes a detubulating action of a large class of pharmaceutical compounds.

PP2A Level in Colorectal Cancer Cells Predicts the Response of p38 Targeted Therapy

PP2A Level in Colorectal Cancer Cells Predicts the Response of p38 Targeted Therapy

Altered Na/Ca exchange distribution in ventricular myocytes from failing hearts.

In mammalian cardiac ventricular myocytes, Ca efflux via Na/Ca exchange (NCX) occurs predominantly at T tubules. Heart failure is associated with disrupted t-tubular structure, but its effect on t-tubular function is less clear. We therefore investigated t-tubular NCX activity in ventricular myocytes isolated from rat hearts ∼18 wk after coronary artery ligation (CAL) or corresponding sham operation (Sham). NCX current (INCX) and l-type Ca current (ICa) were recorded using the whole cell, voltage-clamp technique in intact and detubulated (DT) myocytes; intracellular free Ca concentration ([Ca]i) was monitored simultaneously using fluo-4. INCX was activated and measured during application of caffeine to release Ca from sarcoplasmic reticulum (SR). Whole cell INCX was not significantly different in Sham and CAL myocytes and occurred predominantly in the T tubules in Sham myocytes. CAL was associated with redistribution of INCX and ICa away from the T tubules to the cell surface and an increase in t-tubular INCX/ICa density from 0.12 in Sham to 0.30 in CAL myocytes. The decrease in t-tubular INCX in CAL myocytes was accompanied by an increase in the fraction of Ca sequestered by SR. However, SR Ca content was not significantly different in Sham, Sham DT, and CAL myocytes but was significantly increased by DT of CAL myocytes. In Sham myocytes, there was hysteresis between INCX and [Ca]i, which was absent in DT Sham but present in CAL and DT CAL myocytes. These data suggest altered distribution of NCX in CAL myocytes.

Modeling Local X-ROS and Calcium Signaling in the Heart

Stretching single ventricular cardiac myocytes has been shown experimentally to activate transmembrane nicotinamide adenine dinucleotide phosphate oxidase type 2 to produce reactive oxygen species (ROS) and increase the Ca2+ spark rate in a process called X-ROS signaling. The increase in Ca2+ spark rate is thought to be due to an increase in ryanodine receptor type 2 (RyR2) open probability by direct oxidation of the RyR2 protein complex. In this article, a computational model is used to examine the regulation of ROS and calcium homeostasis by local, subcellular X-ROS signaling and its role in cardiac excitation-contraction coupling. To this end, a four-state RyR2 model was developed that includes an X-ROS-dependent RyR2 mode switch. When activated, [Ca2+]i-sensitive RyR2 open probability increases, and the Ca2+ spark rate changes in a manner consistent with experimental observations. This, to our knowledge, new model is used to study the transient effects of diastolic stretching and subsequent ROS production on RyR2 open probability, Ca2+ sparks, and the myoplasmic calcium concentration ([Ca2+]i) during excitation-contraction coupling. The model yields several predictions: 1) [ROS] is produced locally near the RyR2 complex during X-ROS signaling and increases by an order of magnitude more than the global ROS signal during myocyte stretching; 2) X-ROS activation just before the action potential, corresponding to ventricular filling during diastole, increases the magnitude of the Ca2+ transient; 3) during prolonged stretching, the X-ROS-induced increase in Ca2+ spark rate is transient, so that long-sustained stretching does not significantly increase sarcoplasmic reticulum Ca2+ leak; and 4) when the chemical reducing capacity of the cell is decreased, activation of X-ROS signaling increases sarcoplasmic reticulum Ca2+ leak and contributes to global oxidative stress, thereby increases the possibility of arrhythmia. The model provides quantitative information not currently obtainable through experimental means and thus provides a framework for future X-ROS signaling experiments.

Tissue-Specific Cell Cycle Indicator Reveals Unexpected Findings for Cardiac Myocyte Proliferation.

Discerning cardiac myocyte cell cycle behavior is challenging owing to commingled cell types with higher proliferative activity. To investigate cardiac myocyte cell cycle activity in development and the early postnatal period. To facilitate studies of cell type-specific proliferation, we have generated tissue-specific cell cycle indicator BAC transgenic mouse lines. Experiments using embryonic fibroblasts from CyclinA2-LacZ-floxed-EGFP, or CyclinA2-EGFP mice, demonstrated that CyclinA2-βgal and CyclinA2-EGFP were expressed from mid-G1 to mid-M phase. Using Troponin T-Cre;CyclinA2-LacZ-EGFP mice, we examined cardiac myocyte cell cycle activity during embryogenesis and in the early postnatal period. Our data demonstrated that right ventricular cardiac myocytes exhibited reduced cell cycle activity relative to left ventricular cardiac myocytes in the immediate perinatal period. Additionally, in contrast to a recent report, we could find no evidence to support a burst of cardiac myocyte cell cycle activity at postnatal day 15. Our data highlight advantages of a cardiac myocyte-specific cell cycle reporter for studies of cardiac myocyte cell cycle regulation.

Cardiac remodeling in Gαq and Gα11 knockout mice

BackgroundAlthough both Gαq- and Gα11-protein signaling are believed to be involved in the regulation of cardiac hypertrophy, their detailed contribution to myocardial function remains elusive. Methods and resultsWe studied remodeling processes in healthy transgenic mice with genetically altered Gαq/Gα11-expression, in particular a global Gα11-knockout and a novel inducible cardiac specific Gαq-knockout, as well as a combined double knockout (dKO) mouse line. Echocardiography and telemetric ECG recordings revealed that compared with wild type mice, hearts of dKO mice showed an increased ejection fraction and a decreased heart rate, irrespective of age resulting in a maintained cardiac output. We attributed these findings to the lack of Gα11, which the absence was associated with a decreased afterload. Histological analysis of the extracellular matrix in the heart depicted a diminished presence of collagen in aging hearts of dKO mice compared to wild-type mice. The results of a transcriptome analysis on isolated ventricular cardiac myocytes revealed alterations of the activity of genes involved in the Gαq/Gα11-dependent regulation of the extracellular matrix, such as the matricellular protein Cyr61. ConclusionsFrom our data we conclude that Gαq/Gα11 signaling pathways play a pivotal role in maintaining gene activity patterns. For the heart we revealed their importance in modulating the properties of the extracellular matrix, a mechanism that might be an important contributor and mechanistic basis for the development of pressure-overload induced cardiac hypertrophy.

A multiscale computational model of spatially resolved calcium cycling in cardiac myocytes: from detailed cleft dynamics to the whole cell concentration profiles.

Mathematical modeling of excitation-contraction coupling (ECC) in ventricular cardiac myocytes is a multiscale problem, and it is therefore difficult to develop spatially detailed simulation tools. ECC involves gradients on the length scale of 100 nm in dyadic spaces and concentration profiles along the 100 μm of the whole cell, as well as the sub-millisecond time scale of local concentration changes and the change of lumenal Ca2+ content within tens of seconds. Our concept for a multiscale mathematical model of Ca2+ -induced Ca2+ release (CICR) and whole cardiomyocyte electrophysiology incorporates stochastic simulation of individual LC- and RyR-channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell. We developed a novel computational approach to resolve the concentration gradients from dyadic space to cell level by using a quasistatic approximation within the dyad and finite element methods for integrating the partial differential equations. We show whole cell Ca2+-concentration profiles using three previously published RyR-channel Markov schemes.

Effects of aconitine, mesaconitine and hypaconitine on calcium release in isolated adult rat cardiac myocytes

Objective To investigate the effects of aconitine, mesaconitine and hypaconitine on calcium release in isolated adult rat cardiac myocytes. Methods The left ventricular cardiac myocytes isolated from adult Sprague-Dawley rats were perfused with acnitine, mesaconitine and hypaconitine at 0.3 μmol/L, 1 μmol/L, 3 μmol/L for 12 min. The spontaneous calcium release (SCR) rate, the end-diastolic [Ca2+] (F0) and the calcium transient amplitude (ΔF) were detected 4 min, 8 min and 12 min after the perfusion. 12 min after the perfusion with acnitine, mesaconitine and hypaconitine at 0.3 μmol/L, the changes of systolic dynamics and calcium transient were detected for the positive inotropic effect. Results Any of aconitine, mesaconitine and hypaconitine induced SCR, mesconitine- induced SCR rate was highest at low concentration (0.3 μmol/L), and aconitine-induced SCR rate highest at high concentration (3 μmol/L). Compared with the control, 12 min after the perfusion with acnitine, mesaconitine and hypaconitine at 3 μmol/L elevated F0 (1.459 ± 0.379, 1.585 ± 0.493, 1.213 ± 0.254 vs. 1.079 ± 0.108, all P<0.05) and ΔF(1.615 ± 0.455, 2.210 ± 0.756, 1.528 ± 0.422 vs. 1.036 ± 0.125, all P<0.05), mesaconitine with ΔF higher than aconitine and hypaconitine. At low concentration (0.3 μmol/L), compared with control, aconitine, mesaconitine and hypaconitine increased ΔF (0.409 ± 0.127, 0.423 ± 0.107, 0.414 ± 0.118 vs. 0.260 ± 0.065; P<0.05 or P<0.01) and contraction amplitudes (5.464% ± 2.239%, 7.449% ± 2.548%, 5.524% ± 1.645% vs. 3.428% ± 0.911%; P<0.05 or P<0.01), prolonged the time to peak of calcium transient (0.041 ± 0.016 s, 0.039 ± 0.009 s, 0.038 ± 0.011 s vs. 0.032 ± 0.007 s; P<0.05 or P<0.01); compared with aconitine, mesaconitine and hypaconitine decreased calcium transient time constant (0.301 ± 0.054 s, 0.324 ± 0.064 s vs. 0.361 ± 0.076 s; P<0.05 or P<0.01) and diastolic t50 (0.124 ± 0.035 s, 0.126 ± 0.040 s vs. 0.157 ± 0.056 s; P<0.05 or P<0.01). Conclusions Aconitine, mesaconitine and hypaconitine reveal the positive inotropic effects couple with the toxic effects. Increased [Ca2+] in cardiac myocytes is the key factor for the positive inotropic effects, but also the risk factor for SCR. Key words: Calcium; Myocytes, cardiac; Drugs, Chinese herbal; Aconitine; Rats; Mesaconitine; Hypaconitine

Altered distribution of ICa impairs Ca release at the t-tubules of ventricular myocytes from failing hearts

In mammalian cardiac ventricular myocytes, Ca influx and release occur predominantly at t-tubules, ensuring synchronous Ca release throughout the cell. Heart failure is associated with disrupted t-tubule structure, but its effect on t-tubule function is less clear. We therefore investigated Ca influx and release at the t-tubules of ventricular myocytes isolated from rat hearts ~18weeks after coronary artery ligation (CAL) or corresponding Sham operation. L-type Ca current (ICa) was recorded using the whole-cell voltage-clamp technique in intact and detubulated myocytes; Ca release at t-tubules was monitored using confocal microscopy with voltage- and Ca-sensitive fluorophores. CAL was associated with cardiac and cellular hypertrophy, decreased ejection fraction, disruption of t-tubule structure and a smaller, slower Ca transient, but no change in ryanodine receptor distribution, L-type Ca channel expression, or ICa density. In Sham myocytes, ICa was located predominantly at the t-tubules, while in CAL myocytes, it was uniformly distributed between the t-tubule and surface membranes. Inhibition of protein kinase A with H-89 caused a greater decrease of t-tubular ICa in CAL than in Sham myocytes; in the presence of H-89, t-tubular ICa density was smaller in CAL than in Sham myocytes. The smaller t-tubular ICa in CAL myocytes was accompanied by increased latency and heterogeneity of SR Ca release at t-tubules, which could be mimicked by decreasing ICa using nifedipine. These data show that CAL decreases t-tubular ICa via a PKA-independent mechanism, thereby impairing Ca release at t-tubules and contributing to the altered excitation–contraction coupling observed in heart failure.

The Study of Synthesis, Accumulation and Release Processes of Atrial and Brain Natriuretic Peptides in Experimental Renovascular Hypertension

DOI: 10.17691/stm2015.7.2.04 Received November 10, 2014М.V. Galkina, PhD, Senior Researcher, Electron Microscopy unit, Central Scientific Research laboratory;О.s. baskina, PhD, Senior Researcher, Morphology Department, Central Scientific Research laboratory;М.L. bugrova, PhD, Associate Professor, Head of Electron Microscopy unit, Central Scientific Research laboratory Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russian federationThe aim of the investigation was to study the dynamics of synthesis, accumulation and release processes of atrial (ANP) and brain (BNP) natriuretic peptides on day 60 of experimental renovascular hypertension.Materials and Methods. The experiments were carried put on white non-pedigree male rats. we studied renovascular hypertension using a two-kidney model with the left renal artery ligation. Arterial pressure (AP) was measured via caudal artery using an invasive technique. we analyzed left ventricular cardiac myocyte areas by light optics. Electron microscopic investigation was applied to study the left ventricular and right atrial tissue samples. ANP and BNP accumulation and release processes were assessed using a quantitative analysis of immunolabeled granules of atrial myocytes (А-type, “mature, storing”, and B-type, “dissolving”) under the transmission electron microscope. we performed a morphometric analysis of morphological types of the left ventricular and right atrial mitochondria.Results. The number of granules containing ANP and BNP in atrial myocytes on day 60 after the left renal artery ligation in rats was found to be no significantly different from that in the intact group. we revealed AP decrease up to the initial level. ANP and BNP content in atrial myocytes and AP recovered against the background of cardiac myocyte hypertrophy, mitochondrial de-energization and destructive changes in microcirculatory bloodstream.Conclusion. In a long-term period of experimental renovascular hypertension, ANP and BNP have a positive effect on AP level falling up to the initial one due to ANP and BNP release on day 30 of the experiment. The efficiency of natriuretic peptides acting as a regulatory component in primary hypertension formation depends on arterial hypertension genesis.Key words: аtrial natriuretic peptide; brain natriuretic peptide; renovascular hypertension.For contacts: Galkina Maria Vladimirovna, e-mail: maryrax82@gmail.comAtrial and Brain Natriuretic Peptides in Experimental Renovascular Hypertension

PI4P Hydrolysis Represents a General Mechanism for DAG Generation and PKC/PKD Activation

Receptor stimulated hydrolysis of phosphatidylinositol 4,5‐bisphosphate (PIP­2­) is critical for many physiological processes, such as neurotransmission and proliferation. Recently our laboratory identified a novel pathway in neonatal rat ventricular cardiac myocytes (NRVMs), where agonist stimulation triggers PLC dependent hydrolysis of the PIP­2­ precursor phosphatidylinositol 4‐phosphate (PI4P) leading to DAG production, nuclear PKD activation and cardiac hypertrophy. We have seen that this newly identified pathway exists in other cell types as well, such as Mouse Embryo Fibroblasts (MEFs) and PANC‐1 cells. To determine if PLC dependent PI4P hydrolysis represents a general mechanism for DAG generation and PKC/PKD activation we examined PI4P depletion and PKD activation in other cell types. In our studies we used a PI 4‐kinase inhibitor, phenylarsine oxide (PAO) or expression of a Golgi specific 4‐phosphatase, Sac1‐K2A (Sac1) to deplete PI4P. PAO treatment or Sac1 expression in NRVMs and MEFs, showed a dramatic reduction of inositol phosphate accumulation after endothelin‐1 (ET‐1) stimulation. PAO treatment completely blocked ET‐1 dependent global PKD activation in NRVMs and MEFs but not neurotensin (NT) dependent PKD activation in PANC‐1 cells. Sac1 expression showed reduced ET‐1 dependent global PKD activation in NRVMs and MEFs but did not change the NT dependent PKD activation in PANC‐1 cells. These results suggest that though Golgi PI4P hydrolysis occurs in multiple cell types, NRVMs and MEFs use PI4P hydrolysis as a source for DAG to promote PKD activation. This shows that PI4P hydrolysis occurs upon receptor stimulation and provides a novel signaling mechanism with numerous implications in physiology and disease.

Effect of acute and prolonged alcohol administration on Mg2+ homeostasis in cardiac cells

Alcoholic cardiomyopathy represents a major clinical complication in chronic alcoholics. Previous studies from our laboratory indicate that acute and chronic exposure of liver cells to ethanol results in a major loss of cellular Mg2+ as a result of alcohol oxidation. We investigated whether exposure to ethanol induces a similar Mg2+ loss in cardiac cells. The results indicate that chronic exposure to a 6% ethanol-containing diet depleted cardiac myocytes of >25% of their cellular Mg2+ content. Acute ethanol exposure, instead, induced a time- and dose-dependent manner of Mg2+ extrusion from perfused hearts and collagenase-dispersed cardiac ventricular myocytes. Pretreatment with chlormethiazole prevented ethanol-induced Mg2+ loss to a large extent, suggesting a role of ethanol oxidation via cyP4502E1 in the process. Magnesium extrusion across the sarcolemma occurred via the amiloride-inhibited Na+/Mg2+ exchanger. Taken together, our data indicate that Mg2+ extrusion also occurs in cardiac cells exposed to ethanol as a result of alcohol metabolism by cyP4502E1. The extrusion, which is mediated by the Na+/Mg2+ exchanger, only occurs at doses of ethanol ≥0.1%, and depends on ethanol-induced decline in cellular ATP. The significance of Mg2+ extrusion for the onset of alcoholic cardiomyopathy remains to be elucidated.