Saxagliptin but Not Sitagliptin Inhibits CaMKII and PKC via DPP9 Inhibition in Cardiomyocytes

Some oral anti-hyperglycemic drugs, including gliptins that inhibit dipeptidyl peptidase 4 (DPP4), have been linked to the increased risk of heart failure (HF) in type-2 diabetic patients. While the cardiovascular safety trial, TECOS, revealed no link between sitagliptin and the risk of HF, a substantial 27% increase in the hospitalization for HF was observed in type-2 diabetic patients treated with saxagliptin within the SAVOR-TIMI 53 trial. A previous in vitro study revealed that saxagliptin impairs the Ca2+/calmodulin-dependent protein kinase II (CaMKII)-phospholamban (PLB)-sarcoplasmic reticulum Ca2+-ATPase 2a axis and protein kinase C (PKC) activity in cardiomyocytes leading to impaired cardiac contractility and electrophysiological function. However, the link between saxagliptin and its target proteins (CaMKII and PKC) remains to be explored. Since DPP8 and DPP9 (but not DPP4) are expressed by cardiomyocytes and saxagliptin is internalized by cardiomyocytes, we investigated whether DPP8/9 contribute to saxagliptin-mediated inhibition of CaMKII and PKC activity. Structural analysis revealed that the DPP4-saxagliptin interaction motif (S630, Y547) for the cyanopyrrolidine group is conserved in DPP8 (S755, Y669) and DPP9 (S730, Y644). Conversely, F357 that facilitates binding of the anchor lock domain of sitagliptin in the S2 extensive subsite of DPP4 is not conserved in DPP8/9. In parallel, unlike saxagliptin, sitagliptin did not affect phosphorylation of CaMKII/PLB or activity of PKC in HL-1 cardiomyocytes. These findings were recapitulated by pharmacological inhibition (TC-E-5007, a DPP8/9 antagonist) and knock-down of DPP9 (but not DPP8). In primary mouse ventricular cardiomyocytes, saxagliptin (but not sitagliptin) impaired Ca2+ transient relaxation and prolonged action potential duration (APD). These results suggest that saxagliptin-DPP9 interaction impairs the CaMKII-PLB and PKC signaling in cardiomyocytes. We reveal a novel and potential role of DPP9 in cardiac signaling. The interaction of saxagliptin with DPP9 may represent an underlying mechanism for the link between saxagliptin and HF. Elucidation of saxagliptin-DPP9 interaction and downstream events may foster a better understanding of the role of gliptins as modulators of cardiac signaling

Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo

Changes in intra-and extracellular potassium ion (K+) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K+ changes is severely limited by the lack of practicable detection methods. We developed K+-sensitive genetically encoded, Forster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K+ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K+ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K+ signals. Hitherto unknown Ca2+-triggered, organelle-specific K+ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K+ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K+ assays and open new avenues for live-cell K+ imaging

EXTERNE EVALUATION ALS WESENTLICHER BEITRAG ZUR QUALITÄTSSICHERUNG AN PÄDAGOGISCHEN HOCHSCHULEN

Pädagogische Hochschulen sind im Kreis der tertiären bzw. postsekundären Bildungseinrichtungen Österreichs die jüngsten Institutionen. Mit dem Hochschulgesetz 2005 erhielten neben den öffentlichen Universitäten, Fachhochschulen und privaten Universitäten auch Pädagogische Hochschulen den Hochschulstatus, sodass 2007 aus drei Vorgängerinstitutionen – den Pädagogischen Akademien, den Pädagogischen Instituten und den Berufsbildenden Akademien – neun öffentliche Pädagogische Hochschulen und darüber hinaus fünf private Pädagogische Hochschulen entstanden. Letztere befinden sich meist in kirchlicher Trägerschaft

A549 in-silico 1.0: A first computational model to simulate cell cycle dependent ion current modulation in the human lung adenocarcinoma.

Lung cancer is still a leading cause of death worldwide. In recent years, knowledge has been obtained of the mechanisms modulating ion channel kinetics and thus of cell bioelectric properties, which is promising for oncological biomarkers and targets. The complex interplay of channel expression and its consequences on malignant processes, however, is still insufficiently understood. We here introduce the first approach of an in-silico whole-cell ion current model of a cancer cell, in particular of the A549 human lung adenocarcinoma, including the main functionally expressed ion channels in the plasma membrane as so far known. This hidden Markov-based model represents the electrophysiology behind proliferation of the A549 cell, describing its rhythmic oscillation of the membrane potential able to trigger the transition between cell cycle phases, and it predicts membrane potential changes over the cell cycle provoked by targeted ion channel modulation. This first A549 in-silico cell model opens up a deeper insight and understanding of possible ion channel interactions in tumor development and progression, and is a valuable tool for simulating altered ion channel function in lung cancer electrophysiology

Hypochlorite-Modified LDL Induces Arrhythmia and Contractile Dysfunction in Cardiomyocytes

Neutrophil-derived myeloperoxidase (MPO) and its potent oxidant, hypochlorous acid (HOCl), gained attention as important oxidative mediators in cardiac damage and dysfunction. As cardiomyocytes generate low-density lipoprotein (LDL)-like particles, we aimed to identify the footprints of proatherogenic HOCl-LDL, which adversely affects cellular signalling cascades in various cell types, in the human infarcted myocardium. We performed immunohistochemistry for MPO and HOCl-LDL in human myocardial tissue, investigated the impact of HOCl-LDL on electrophysiology and contractility in primary cardiomyocytes, and explored underlying mechanisms in HL-1 cardiomyocytes and human atrial appendages using immunoblot analysis, qPCR, and silencing experiments. HOCl-LDL reduced ICa,L and IK1, and increased INaL, leading to altered action potential characteristics and arrhythmic events including early- and delayed-afterdepolarizations. HOCl-LDL altered the expression and function of CaV1.2, RyR2, NCX1, and SERCA2a, resulting in impaired contractility and Ca2+ homeostasis. Elevated superoxide anion levels and oxidation of CaMKII were mediated via LOX-1 signaling in HL-1 cardiomyocytes. Furthermore, HOCl-LDL-mediated alterations of cardiac contractility and electrophysiology, including arrhythmic events, were ameliorated by the CaMKII inhibitor KN93 and the INaL blocker, ranolazine. This study provides an explanatory framework for the detrimental effects of HOCl-LDL compared to native LDL and cardiac remodeling in patients with high MPO levels during the progression of cardiovascular disease

NADH supplementation decreases pinacidil-primed I(K(ATP)) in ventricular cardiomyocytes by increasing intracellular ATP

1. The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current I(K(ATP)) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. 2. Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36–37°C. Adenine nucleotides were determined by HPLC. 3. Under physiological conditions (4.3 mM ATP in the pipette solution, ATP(i)) I(K(ATP)) did not contribute to basal electrical activity. 4. The ATP-dependent potassium (K((ATP))) channel opener pinacidil activated I(K(ATP)) dependent on [ATP](i) showing a significantly more pronounced activation at lower (1 mM) [ATP](i). 5. Supplementation of cardiomyocytes with 300 μg ml(−1) NADH (4–6 h) resulted in a significantly reduced I(K(ATP)) activation by pinacidil compared to control cells. The current density was 13.8±3.78 (n=6) versus 28.9±3.38 pA pF(−1) (n=19; P<0.05). 6. Equimolar amounts of the related compounds nicotinamide and NAD(+) did not achieve a similar effect like NADH. 7. Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6±1.88 nmol mg(−1) protein versus control: 35.4±2.57 nmol mg(−1) protein, P<0.000005). 8. These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of I(K(ATP)) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. 9. Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP

Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of CaV1.2 calcium channels in hippocampal neurons

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes

The sulphonylurea glibenclamide inhibits voltage dependent potassium currents in human atrial and ventricular myocytes

1. It was the aim of our study to investigate the effects of the sulphonylurea glibenclamide on voltage dependent potassium currents in human atrial myocytes. 2. The drug blocked a fraction of the quasi steady state current (ramp response) which was activated positive to −20 mV, was sensitive to 4-aminopyridine (500 μM) and was different from the ATP dependent potassium current I(K(ATP)). 3. Glibenclamide dose dependently inhibited both, the peak as well as the late current elicited by step depolarization positive to −20 mV. The IC(50) for reduction in charge area of total outward current was 76 μM. 4. The double-exponential inactivation time-course of the total outward current was accelerated in the presence of glibenclamide with a τ(fast) of 12.7±1.5 ms and a τ(slow) of 213±25 ms in control and 5.8±1.9 ms (P<0.001) and 101±20 ms (P<0.05) under glibenclamide (100 μM). 5. Our data suggest, that both repolarizing currents in human atrial myocytes, the transient outward current (I(to1)) and the ultrarapid delayed rectifier current (I(Kur)) were inhibited by glibenclamide. 6. In human ventricular myocytes glibenclamide inhibited I(to1) without affecting the late current. 7. Our data suggest that glibenclamide inhibits human voltage dependent cardiac potassium currents at concentrations above 10 μM