Klaus Zorn-Pauly

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein‐coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA1, LPA2, and LPA3 on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal‐regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA‐induced global alterations in protein expression patterns a 2‐D DIGE/LC‐ESI‐MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.

If blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes

Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS–HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.

Dipeptidyl peptidase-4 independent cardiac dysfunction links saxagliptin to heart failure

ABSTRACT Saxagliptin treatment has been associated with increased rate of hospitalization for heart failure in type 2 diabetic patients, though the underlying mechanism(s) remain elusive. To address this, we assessed the effects of saxagliptin on human atrial trabeculae, guinea pig hearts and cardiomyocytes. We found that the primary target of saxagliptin, dipeptidyl peptidase‐4, is absent in cardiomyocytes, yet saxagliptin internalized into cardiomyocytes and impaired cardiac contractility via inhibition of the Ca2+/calmodulin‐dependent protein kinase II‐phospholamban‐sarcoplasmic reticulum Ca2+‐ATPase 2a axis and Na+‐Ca2+ exchanger function in Ca2+ extrusion. This resulted in reduced sarcoplasmic reticulum Ca2+ content, diastolic Ca2+ overload, systolic dysfunction and impaired contractile force. Furthermore, saxagliptin reduced protein kinase C‐mediated delayed rectifier K+ current that prolonged action potential duration and consequently QTc interval. Importantly, saxagliptin aggravated pre‐existing cardiac dysfunction induced by ischemia/reperfusion injury. In conclusion, our novel results provide mechanisms for the off‐target deleterious effects of saxagliptin on cardiac function and support the outcome of SAVOR‐TIMI 53 trial that linked saxagliptin with the risk of heart failure.

Cardioprotective effects of 5‐hydroxymethylfurfural mediated by inhibition of L‐type Ca2+ currents

The antioxidant 5‐hydroxymethylfurfural (5‐HMF) exerts documented beneficial effects in several experimental pathologies and is currently tested as an antisickling drug in clinical trials. In the present study, we examined the cardiovascular effects of 5‐HMF and elucidated the mode of action of the drug.

Functional impairment of endothelial cells by the antimycotic amphotericin B.

We set out to determine the membrane potential (Vm) of the endothelial cell line EA.hy926 and its sensitivity to the antimycotic amphotericin B (AmB), a commonly used antifungal component in cell culture media. We measured the endothelial Vm under various experimental conditions by patch clamp technique and found that Vm of AmB-treated cells is (-12.1 ± 9.3) mV, while in AmB-untreated (control) cells it is (-57.1 ± 4.1) mV. In AmB-free extracellular solutions, Vm recovered toward control levels and this gain in Vm rapidly dissipated upon re-addition of AmB, demonstrating a rapid and reversible effect of AmB on endothelial Vm. The consequences of AmB dependent alterations in endothelial transmembrane potential were tested at the levels of Ca(2+) signaling, of nucleotide concentrations, and energy metabolism. In AmB-treated cells we found substantially reduced Ca(2+) entry (to about 60% of that in control cells) in response to histamine induced endoplasmic reticulum (ER) Ca(2+) depletion, and diminished the ATP-to-ADP ratio (by >30%). Our data demonstrate a marked and experimentally relevant dependence of basic functional parameters of cultured endothelial cells on the presence of the ionophoric antimycotic AmB. The profound and reversible effects of the widely used culture media component AmB need careful consideration when interpreting experimental data obtained under respective culture conditions.

Sinoatrial Beat to Beat Variability Assessed by Contraction Strength in Addition to the Interbeat Interval

Beat to beat variability of cardiac tissue or isolated cells is frequently investigated by determining time intervals from electrode measurements in order to compute scale dependent or scale independent parameters. In this study, we utilize high-speed video camera recordings to investigate the variability of intervals as well as mechanical contraction strengths and relative contraction strengths with nonlinear analyses. Additionally, the video setup allowed us simultaneous electrode registrations of extracellular potentials. Sinoatrial node tissue under control and acetylcholine treated conditions was used to perform variability analyses by computing sample entropies and Higuchi dimensions. Beat to beat interval variabilities measured by the two recording techniques correlated very well, and therefore, validated the video analyses for this purpose. Acetylcholine treatment induced a reduction of beating rate and contraction strength, but the impact on interval variability was negligible. Nevertheless, the variability analyses of contraction strengths revealed significant differences in sample entropies and Higuchi dimensions between control and acetylcholine treated tissue. Therefore, the proposed high-speed video camera technique might represent a non-invasive tool that allows long-lasting recordings for detecting variations in beating behavior over a large range of scales.

Letter to the editor: Accurate cell capacitance determination from a single voltage step: a reminder to avoid unnecessary pitfalls

to the editor: Cell membrane capacitance C m is a fundamental property of excitable cells. It scales with the cell surface area and is therefore used for cell size estimation and most important for normalizing currents and conductances. Consequently, proper knowledge of C m is also crucial for