Na/K Pump-Induced [Na]i Gradients in Rat Ventricular Myocytes Measured with Two-Photon Microscopy

AbstractVia the Na/Ca and Na/H exchange, intracellular Na concentration ([Na]i) is important in regulating cardiac Ca and contractility. Functional data suggest that [Na]i might be heterogeneous in myocytes that are not in steady state, but little direct spatial information is available. Here we used two-photon microscopy of SBFI to spatially resolve [Na]i in rat ventricular myocytes. In vivo calibration yielded an apparent Kd of 27±2mM Na. Similar resting [Na]i was found using two-photon or single-photon ratiometric measurements with SBFI (10.8±0.7 vs. 11.1±0.7mM). To assess longitudinal [Na]i gradients, Na/K pumps were blocked at one end of the myocyte (locally pipette-applied K-free extracellular solution) and active in the rest of the cell. This led to a marked increase in [Na]i at sites downstream of the pipette (where Na enters the myocyte and Na/K pumps are blocked). [Na]i rise was smaller at upstream sites. This resulted in sustained [Na]i gradients (up to ∼17 mM/120μm cell length). This implies that Na diffusion in cardiac myocytes is slow with respect to trans-sarcolemmal Na transport rates, although the mechanisms responsible are unclear. A simple diffusion model indicated that such gradients require a Na diffusion coefficient of 10–12μm2/s, significantly lower than in aqueous solutions

SERCA Activity Controls the Systolic Calcium Increase in the Nucleus of Cardiac Myocytes

In cardiomyocytes, nuclear calcium is involved in regulation of transcription and, thus, remodeling. The cellular mechanisms regulating nuclear calcium, however, remain elusive. Therefore, the aim of this study was to identify and characterize the factors that regulate nuclear calcium in cardiomyocytes. We focused on the roles of (1) the cytoplasmic calcium transient (CaT), (2) the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and (3) intracellular calcium stores for nuclear calcium handling. Experiments were performed on rat ventricular myocytes loaded with Fluo-4/AM. Subcellularly resolved CaTs were visualized using confocal microscopy. The cytoplasmic CaT was varied by reducing extracellular calcium (from 1.5 to 0.3 mM) or by exposure to isoprenaline (ISO, 10 nM). SERCA was blocked by thapsigargin (5 μM). There was a strict linear dependence of the nucleoplasmic CaT on the cytoplasmic CaT over a wide range of calcium concentrations. Increasing SERCA activity impaired, whereas decreasing SERCA activity augmented the systolic calcium increase in the nucleus. Perinuclear calcium store load, on the other hand, did not change with either 0.3 mM calcium or ISO and was not a decisive factor for the nucleoplasmic CaT. The results indicate, that the nucleoplasmic CaT is determined largely by the cytoplasmic CaT via diffusion of calcium through nuclear pores. They identify perinuclear SERCA activity, which limits the systolic calcium increase in the nucleus, as a novel regulator of the nuclear CaT in cardiac myocytes

The European Network for Translational Research in Atrial Fibrillation (EUTRAF): objectives and initial results.

Atrial fibrillation (AF) is the most common sustained arrhythmia in the general population. As an age-related arrhythmia AF is becoming a huge socio-economic burden for European healthcare systems. Despite significant progress in our understanding of the pathophysiology of AF, therapeutic strategies for AF have not changed substantially and the major challenges in the management of AF are still unmet. This lack of progress may be related to the multifactorial pathogenesis of atrial remodelling and AF that hampers the identification of causative pathophysiological alterations in individual patients. Also, again new mechanisms have been identified and the relative contribution of these mechanisms still has to be established. In November 2010, the European Union launched the large collaborative project EUTRAF (European Network of Translational Research in Atrial Fibrillation) to address these challenges. The main aims of EUTRAF are to study the main mechanisms of initiation and perpetuation of AF, to identify the molecular alterations underlying atrial remodelling, to develop markers allowing to monitor this processes, and suggest strategies to treat AF based on insights in newly defined disease mechanisms. This article reports on the objectives, the structure, and initial results of this network

Automated detection and analysis of Ca(2+) sparks in x-y image stacks using a thresholding algorithm implemented within the open-source image analysis platform ImageJ.

Previous studies have used analysis of Ca(2+) sparks extensively to investigate both normal and pathological Ca(2+) regulation in cardiac myocytes. The great majority of these studies used line-scan confocal imaging. In part, this is because the development of open-source software for automatic detection of Ca(2+) sparks in line-scan images has greatly simplified data analysis. A disadvantage of line-scan imaging is that data are collected from a single row of pixels, representing only a small fraction of the cell, and in many instances x-y confocal imaging is preferable. However, the limited availability of software for Ca(2+) spark analysis in two-dimensional x-y image stacks presents an obstacle to its wider application. This study describes the development and characterization of software to enable automatic detection and analysis of Ca(2+) sparks within x-y image stacks, implemented as a plugin within the open-source image analysis platform ImageJ. The program includes methods to enable precise identification of cells within confocal fluorescence images, compensation for changes in background fluorescence, and options that allow exclusion of events based on spatial characteristics

The European Network for Translational Research in Atrial Fibrillation (EUTRAF): objectives and initial results

Atrial fibrillation (AF) is the most common sustained arrhythmia in the general population. As an age-related arrhythmia AF is becoming a huge socio-economic burden for European healthcare systems. Despite significant progress in our understanding of the pathophysiology of AF, therapeutic strategies for AF have not changed substantially and the major challenges in the management of AF are still unmet. This lack of progress may be related to the multifactorial pathogenesis of atrial remodelling and AF that hampers the identification of causative pathophysiological alterations in individual patients. Also, again new mechanisms have been identified and the relative contribution of these mechanisms still has to be established. In November 2010, the European Union launched the large collaborative project EUTRAF (European Network of Translational Research in Atrial Fibrillation) to address these challenges. The main aims of EUTRAF are to study the main mechanisms of initiation and perpetuation of AF, to identify the molecular alterations underlying atrial remodelling, to develop markers allowing to monitor this processes, and suggest strategies to treat AF based on insights in newly defined disease mechanisms. This article reports on the objectives, the structure, and initial results of this networ

Knowledge acquisition in the domain of CNC machining centers: the Moltke approach

MOLTKE is a research project dealing with a complex technical application. After describing the domain of CNCmachining centers and the applied KA methods, we summarize the concrete KA problems which we have to handle. Then we describe a KA mechanism which supports an engineer in developing a diagnosis system. In chapter 6 weintroduce learning techniques operating on diagnostic cases and domain knowledge for improving the diagnostic procedure of MOLTKE. In the last section of this chapter we outline some essential aspects of organizationalknowledge which is heavily applied by engineers for analysing such technical systems (Qualitative Engineering). Finally we give a short overview of the actual state of realization and our future plans

Activation of the cAMP–protein kinase A pathway facilitates Na+ translocation by the Na+–K+ pump in guinea-pig ventricular myocytes

The effects of the adenylyl cyclase activator forskolin on steady-state and transient currents generated by the Na+-K+ pump were studied in guinea-pig ventricular myocytes by means of whole-cell voltage clamp at 30 °C.In external solution containing 144 mM Na+ (Nao+) and 10 mM K+ (Ko+), steady-state Na+-K+ pump current (Ip) activated by 5 mM pipette Na+ ( Napip+) at -20 mV was reversibly augmented by forskolin (4 μM) to 133 ± 4 % of the control current (n = 15). The forskolin analogue 1,9-dideoxyforskolin (10 μM), which does not activate adenylyl cyclases, did not increase Ip (n = 2). Application of the protein kinase A (PKA) inhibitor H-89 (10 μM) in the continued presence of forskolin reversed the forskolin-induced elevation of Ip (n = 3).The forskolin effect on Ip persisted in the presence of 50 mM Napip+ which ensured that the internal Na+-binding sites of the Na+-K+ pump were nearly saturated. Under these conditions, the drug increased Ip to 142 ± 3 % of the control Ip when the pipette free Ca2+ concentration ([Ca2+]pip) was 0·013 nM (n = 5) and to 138 ± 4 % of the control Ip when free [Ca2+]pip was 15 nM (n = 9).In Na+-free external solution, Ip activated by 50 mM Napip+ and 1·5 mM Ko+ was likewise increased by forskolin but to a lesser extent than in Na+-containing medium (116 ± 3 % of control, n = 10).In order to investigate exclusively partial reactions in the Na+ limb of the pump cycle, transient pump currents under conditions of electroneutral Na+-Na+ exchange were studied. Transient pump currents elicited by voltage jumps displayed an initial peak and then decayed monoexponentially. Moved charge (Q) and the rate constant of current decay varied with membrane potential (V). The Q-V relationship followed a Boltzmann distribution characterized by the midpoint voltage (V0·5) and the maximum amount of movable charge (ΔQmax). Forskolin (2-10 μM) shifted V0·5 to more negative values while ΔQmax was not affected (n = 11). The effects of forskolin on transient pump currents were mimicked by 8-bromo-cAMP (500 μM; n = 2) and abolished by a peptide inhibitor of PKA (PKI, 10 μM; n = 5).We conclude that activation of the cAMP-PKA pathway in guinea-pig ventricular myocytes increases Na+-K+ pump current at least in part by modulating partial reactions in the Na+ limb of the pump cycle. Under physiological conditions, the observed stimulation of the cardiac Na+-K+ pump may serve to shorten the action potential duration and to counteract the increased passive sarcolemmal Na+ and K+ fluxes during sympathetic stimulation of the heart