Lars Kaestner

Sustained activity of calcium release-activated calcium channels requires translocation of mitochondria to the plasma membrane.

A rise of the intracellular Ca2+ concentration has multiple signaling functions. Sustained Ca2+ influx across plasma membrane through calcium release-activated calcium (CRAC) channels is required for T-cell development in the thymus, gene transcription, and proliferation and differentiation of naïve T-cells into armed effectors cells. Intracellular Ca2+ signals are shaped by mitochondria, which function as a highly dynamic Ca2+ buffer. However, the precise role of mitochondria for Ca2+-dependent T-cell activation is unknown. Here we have shown that mitochondria are translocated to the plasma membrane as a consequence of Ca2+ influx and that this directed movement is essential to sustain Ca2+ influx through CRAC channels. The decreased distance between mitochondria and the plasma membrane enabled mitochondria to take up large amounts of inflowing Ca2+ at the plasma membrane, thereby preventing Ca2+-dependent inactivation of CRAC channels and sustaining Ca2+ signals. Inhibition of kinesin-dependent mitochondrial movement along microtubules abolished mitochondrial translocation and reduced sustained Ca2+ signals. Our results show how a directed movement of mitochondria is used to control important cellular functions such as Ca2+-dependent T-cell activation.

Irisin and exercise training in humans - results from a randomized controlled training trial.

BackgroundThe recent discovery of a new myokine (irisin) potentially involved in health-related training effects has gained great attention, but evidence for a training-induced increase in irisin remains preliminary. Therefore, the present study aimed to determine whether irisin concentration is increased after regular exercise training in humans.MethodsIn a randomized controlled design, two guideline conforming training interventions were studied. Inclusion criteria were age 30 to 60 years, <1 hour/week regular activity, non-smoker, and absence of major diseases. 102 participants could be included in the analysis. Subjects in the training groups exercised 3 times per week for 26 weeks. The minimum compliance was defined at 70%. Aerobic endurance training (AET) consisted of 45 minutes of walking/running at 60% heart rate reserve. Strength endurance training (SET) consisted of 8 machine-based exercises (2 sets of 15 repetitions with 100% of the 20 repetition maximum). Serum irisin concentrations in frozen serum samples were determined in a single blinded measurement immediately after the end of the training study. Physical performance provided positive control for the overall efficacy of training. Differences between groups were tested for significance using analysis of variance. For post hoc comparisons with the control group, Dunnett’s test was used.ResultsMaximum performance increased significantly in the training groups compared with controls (controls: ±0.0 ± 0.7 km/h; AET: 1.1 ± 0.6 km/h, P < 0.01; SET: +0.5 ± 0.7 km/h, P = 0.01). Changes in irisin did not differ between groups (controls: 101 ± 81 ng/ml; AET: 44 ± 93 ng/ml; SET: 60 ± 92 ng/ml; in both cases: P = 0.99 (one-tailed testing), 1−β error probability = 0.7). The general upward trend was mainly accounted for by a negative association of irisin concentration with the storage duration of frozen serum samples (P < 0.01, β = −0.33). After arithmetically eliminating this confounder, the differences between groups remained non-significant.ConclusionsA training-induced increase in circulating irisin could not be confirmed, calling into question its proposed involvement in health-related training effects. Because frozen samples are prone to irisin degradation over time, positive results from uncontrolled trials might exclusively reflect the longer storage of samples from initial tests.Trial registrationClinicaltrials.gov. Identifier: NCT01263522.

Calcium in red blood cells-a perilous balance.

Ca2+ is a universal signalling molecule involved in regulating cell cycle and fate, metabolism and structural integrity, motility and volume. Like other cells, red blood cells (RBCs) rely on Ca2+ dependent signalling during differentiation from precursor cells. Intracellular Ca2+ levels in the circulating human RBCs take part not only in controlling biophysical properties such as membrane composition, volume and rheological properties, but also physiological parameters such as metabolic activity, redox state and cell clearance. Extremely low basal permeability of the human RBC membrane to Ca2+ and a powerful Ca2+ pump maintains intracellular free Ca2+ levels between 30 and 60 nM, whereas blood plasma Ca2+ is approximately 1.8 mM. Thus, activation of Ca2+ uptake has an impressive impact on multiple processes in the cells rendering Ca2+ a master regulator in RBCs. Malfunction of Ca2+ transporters in human RBCs leads to excessive accumulation of Ca2+ within the cells. This is associated with a number of pathological states including sickle cell disease, thalassemia, phosphofructokinase deficiency and other forms of hereditary anaemia. Continuous progress in unravelling the molecular nature of Ca2+ transport pathways allows harnessing Ca2+ uptake, avoiding premature RBC clearance and thrombotic complications. This review summarizes our current knowledge of Ca2+ signalling in RBCs emphasizing the importance of this inorganic cation in RBC function and survival.

Prostaglandin E2 activates channel-mediated calcium entry in human erythrocytes: an indication for a blood clot formation supporting process

Prostaglandin E(2) (PGE(2)) is released from platelets when they are activated. Using fluorescence imaging and the patch-clamp technique, we provide evidence that PGE(2) at physiological concentrations (10(-10) M) activates calcium rises mediated by calcium influx through a non-selective cation-channel in human red blood cells. The extent of calcium increase varied between cells with a total of 45% of the cells responding. It is well known that calcium increases elicited the calcium-activated potassium channel (Gardos channel) in the red cell membrane. Previously, it was shown that the Gardos channel activation results in potassium efflux and shrinkage of the cells. Therefore, we conclude that the PGE(2) responses of red blood cells described here reveal a direct and active participation of erythrocytes in blood clot formation.

Regulation of phosphatidylserine exposure in red blood cells

The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for eryptosis, a mechanism for the RBC clearance from blood circulation. The process of PS exposure was investigated as function of the intracellular Ca2+ content and the activation of PKCα in human and sheep RBCs. Cells were treated with lysophosphatidic acid (LPA), 4-bromo-A23187, or phorbol-12 myristate-13 acetate (PMA) and analysed by flow cytometry, single cell fluorescence video imaging, or confocal microscopy. For human RBCs, no clear correlation existed between the number of cells with an elevated Ca2+ content and PS exposure. Results are explained by three different mechanisms responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by LPA, 4-bromo-A23187, and PMA; (ii) PKC activation by LPA and PMA; and (iii) enhanced lipid flop caused by LPA. In sheep RBCs, only the latter mechanism occurs suggesting absence of scramblase activity.

Reduced Cardiac L-Type Ca2+ Current in Cavβ2−/− Embryos Impairs Cardiac Development and Contraction With Secondary Defects in Vascular Maturation

Cardiac myocyte contraction depends on transmembrane L-type Ca2+ currents and the ensuing release of Ca2+ from the sarcoplasmic reticulum. Here we show that these L-type Ca2+ currents are essential for cardiac pump function in the mouse at developmental stages where the functional significance of the heart becomes imperative to blood flow and to the continuing growth and survival of the embryo. Disruption of the Cavβ2 gene, which encodes for the predominant ancillary β subunit of cardiac Ca2+ channels, resulted in diminished L-type Ca2+ currents in cardiomyocytes of embryonic day 9.5 (E9.5). This led to a functionally compromised heart, causing defective remodeling of intra- and extraembryonic blood vessels and embryonic death following E10.5. The defects in vascular remodeling were also observed when the Cavβ2 gene was selectively targeted in cardiomyocytes, demonstrating that they are secondary to cardiac failure rather than a result of the lack of Cavβ2 proteins in the vasculature. Partial rescue of the Ca2+ channel currents by a Ca2+ channel agonist significantly postponed embryonic death in Cavβ2−/− mice. Taken together, these data strongly support the essential role of L-type Ca2+ channel activity in cardiomyocytes for normal heart development and function and that this is a prerequisite for proper maturation of the vasculature.

Cardiac Rac1 overexpression in mice creates a substrate for atrial arrhythmias characterized by structural remodelling

AIMS The small GTPase Rac1 seems to play a role in the pathogenesis of atrial fibrillation (AF). The aim of the present study was to characterize the effects of Rac1 overexpression on atrial electrophysiology. METHODS AND RESULTS In mice with cardiac overexpression of constitutively active Rac1 (RacET), statin-treated RacET, and wild-type controls (age 6 months), conduction in the right and left atrium (RA and LA) was mapped epicardially. The atrial effective refractory period (AERP) was determined and inducibility of atrial arrhythmias was tested. Action potentials were recorded in isolated cells. Left ventricular function was measured by pressure-volume analysis. Five of 11 RacET hearts showed spontaneous or inducible atrial tachyarrhythmias vs. 0 of 9 controls (P < 0.05). In RacET, the P-wave duration was significantly longer (26.8 +/- 2.1 vs. 16.7 +/- 1.1 ms, P = 0.001) as was total atrial activation time (RA: 13.6 +/- 4.4 vs. 3.2 +/- 0.5 ms; LA: 7.1 +/- 1.2 vs. 2.2 +/- 0.3 ms, P < 0.01). Prolonged local conduction times occurred more often in RacET (RA: 24.4 +/- 3.8 vs. 2.7 +/- 2.1%; LA: 19.1 +/- 6.3 vs. 1.2 +/- 0.7%, P < 0.01). The AERP and action potential duration did not differ significantly between both groups. RacET demonstrated significant atrial fibrosis but only moderate systolic heart failure. RacET and statin-treated RacET were not significantly different regarding atrial electrophysiology. CONCLUSION The substrate for atrial arrhythmias in mice with Rac1 overexpression is characterized by conduction disturbances and atrial fibrosis. Electrical remodelling (i.e. a shortening of AERP) does not play a role. Statin treatment cannot prevent the structural and electrophysiological effects of pronounced Rac1 overexpression in this model.

Red cell investigations: Art and artefacts

Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology. Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards. Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy.

Functional NMDA receptors in rat erythrocytes

N-methyl-d-aspartate (NMDA) receptors are ligand-gated nonselective cation channels mediating fast neuronal transmission and long-term potentiation in the central nervous system. These channels have a 10-fold higher permeability for Ca(2+) compared with Na(+) or K(+) and binding of the agonists (glutamate, homocysteine, homocysteic acid, NMDA) triggers Ca(2+) uptake. The present study demonstrates the presence of NMDA receptors in rat erythrocytes. The receptors are most abundant in both erythroid precursor cells and immature red blood cells, reticulocytes. Treatment of erythrocytes with NMDA receptor agonists leads to a rapid increase in intracellular Ca(2+) resulting in a transient shrinkage via Gardos channel activation. Additionally, the exposure of erythrocytes to NMDA receptor agonists causes activation of the nitric oxide (NO) synthase facilitating either NO production in l-arginine-containing medium or superoxide anion (O(2)(.-)) generation in the absence of l-arginine. Conversely, treatment with an NMDA receptor antagonist MK-80, or the removal of Ca(2+) from the incubation medium causes suppression of Ca(2+) accumulation and prevents attendant changes in cell volume and NO/O(2)(.-) production. These results suggest that the NMDA receptor activity in circulating erythrocytes is regulated by the plasma concentrations of homocysteine and homocysteic acid. Moreover, receptor hyperactivation may contribute to an increased incidence of thrombosis during hyperhomocysteinemia.

Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion

Red blood cells (RBCs) are a major component of blood clots, which form physiologically as a response to injury or pathologically in thrombosis. The active participation of RBCs in thrombus solidification has been previously proposed but not yet experimentally proven. Holographic optical tweezers and single-cell force spectroscopy were used to study potential cell-cell adhesion between RBCs. Irreversible intercellular adhesion of RBCs could be induced by stimulation with lysophosphatidic acid (LPA), a compound known to be released by activated platelets. We identified Ca(2+) as an essential player in the signaling cascade by directly inducing Ca(2+) influx using A23187. Elevation of the internal Ca(2+) concentration leads to an intercellular adhesion of RBCs similar to that induced by LPA stimulation. Using single-cell force spectroscopy, the adhesion of the RBCs was identified to be approximately 100 pN, a value large enough to be of significance inside a blood clot or in pathological situations like the vasco-occlusive crisis in sickle cell disease patients.