Dirk von Lewinski

The slow force response to stretch in atrial and ventricular myocardium from human heart - functional relevance and subcellular mechanisms

Mechanical load is an important regulator of cardiac force. Stretching human atrial and ventricular trabeculae elicited a biphasic force increase: an immediate increase (Frank-Starling mechanism) followed by a further slow increase (slow force response, SFR). In ventricle, the SFR was unaffected by AT- and ET-receptor antagonism, by inhibition of protein-kinase-C, PI-3-kinase, and NO-synthase, but attenuated by inhibition of Na+/H+- (NHE) and Na+/Ca2+ exchange (NCX). In atrium, however, neither NHE- nor NCX-inhibition affected the SFR. Stretch elicited a large NHE-dependent [Na+]i increase in ventricle but only a small, NHE-independent [Na+]i increase in atrium. Stretch-activated non-selective cation channels contributed to basal force development in atrium but not ventricle and were not involved in the SFR in either tissue. Interestingly, inhibition of AT receptors or pre-application of angiotensin II or endothelin-1 reduced the atrial SFR. Furthermore, stretch increased phosphorylation of atrial myosin light chain 2 (MLC2) and inhibition of myosin light chain kinase (MLCK) attenuated the SFR in atrium and ventricle. Thus, in human heart both atrial and ventricular myocardium exhibit a stretch-dependent SFR that might serve to adjust cardiac output to increased workload. In ventricle, there is a robust NHE-dependent (but angiotensin II- and endothelin-1-independent) [Na+]i increase that is translated into a [Ca2+]i and force increase via NCX. In atrium, on the other hand, there is an angiotensin II- and endothelin-dependent (but NHE- and NCX-independent) force increase. Increased myofilament Ca2+ sensitivity through MLCK-induced phosphorylation of MLC2 is a novel mechanism contributing to the SFR in both atrium and ventricle.

Glucose-transporter-mediated positive inotropic effects in human myocardium of diabetic and nondiabetic patients.

Insulin causes inotropic effects via Ca(2+)-dependent and Ca(2+)-independent pathways. The latter one is potentially glucose dependent. We examined inotropic responses and signal transduction of insulin in human atrial myocardium of diabetic and nondiabetic patients to test for the role of glucose transporters. Experiments were performed in isolated atrial myocardium of 88 patients undergoing cardiac surgery and 28 ventricular muscle samples of explanted hearts. Influence of insulin (0.02 micromol/L) on isometric twitch force was examined with and without blocking glucose transporter (GLUT) 4 translocation (latrunculin), sodium-coupled glucose transporter (SGLT) 1 (phlorizin, T-1095A), or PI3-kinase (wortmannin). Experiments were performed in Tyrode solution containing glucose or pyruvate as energetic substrate. Messenger RNA expression of glucose transporters (GLUT1, GLUT4, SGLT1, SGLT2) was analyzed in atrial and ventricular myocardium of both diabetic and nondiabetic patients. Developed force increases after insulin (to 117.8% +/- 2.4% and 115.8% +/- 1.9%) in trabeculae from patients with and without diabetes. Inotropic effect was reduced after displacing glucose with pyruvate as well as after PI3-kinase inhibition (to 103% +/- 2%) or inhibition of glucose transporters GLUT4 (to 105% +/- 2%) and SGLT1 (phlorizin to 106% +/- 2%, T-1095A to 105% +/- 2%), without differences between the 2 groups. In glucose-free pyruvate-containing solution, only inhibition of PI3-kinase but not blocking glucose transporters resulted in further inhibitory effects. Messenger RNA expression did not show significant differences between patients with or without diabetes. Insulin exerts positive inotropic effects in human atrial myocardium. These effects are mediated via a PI3-kinase-sensitive and a glucose-transport-sensitive pathway. Differences in functional effects or messenger RNA expression of glucose transporters were not detectable between patients with and without diabetes.

Bile acids induce arrhythmias in human atrial myocardium—implications for altered serum bile acid composition in patients with atrial fibrillation

Objective High bile acid serum concentrations have been implicated in cardiac disease, particularly in arrhythmias. Most data originate from in vitro studies and animal models. We tested the hypotheses that (1) high bile acid concentrations are arrhythmogenic in adult human myocardium, (2) serum bile acid concentrations and composition are altered in patients with atrial fibrillation (AF) and (3) the therapeutically used ursodeoxycholic acid has different effects than other potentially toxic bile acids. Methods and Results Multicellular human atrial preparations (‘trabeculae’) were exposed to primary bile acids and the incidence of arrhythmic events was assessed. Bile acid concentrations were measured in serum samples from 250 patients and their association with AF and ECG parameters analysed. Additionally, we conducted electrophysiological studies in murine myocytes. Taurocholic acid (TCA) concentration-dependently induced arrhythmias in atrial trabeculae (14/28 at 300 µM TCA, p<0.01) while ursodeoxycholic acid did not. Patients with AF had significantly decreased serum levels of ursodeoxycholic acid conjugates and increased levels of non-ursodeoxycholic bile acids. In isolated myocytes, TCA depolarised the resting membrane potential, enhanced Na+/Ca2+ exchanger (NCX) tail current density and induced afterdepolarisations. Inhibition of NCX prevented arrhythmias in atrial trabeculae. Conclusions High TCA concentrations induce arrhythmias in adult human atria while ursodeoxycholic acid does not. AF is associated with higher serum levels of non-ursodeoxycholic bile acid conjugates and low levels of ursodeoxycholic acid conjugates. These data suggest that higher levels of toxic (arrhythmogenic) and low levels of protective bile acids create a milieu with a decreased arrhythmic threshold and thus may facilitate arrhythmic events.

Direct pro‐arrhythmogenic effects of angiotensin II can be suppressed by AT1 receptor blockade in human atrial myocardium

Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. Indirect evidence from clinical trials demonstrates that chronic inhibition of the renin–angiotensin‐system (RAS) significantly reduces the incidence of AF. Since mechanisms of this protective effect of RAS‐blockade are poorly understood, we directly tested proarrhythmic effects of angiotensin II (Ang II) in human atrial myocardium.

Exenatide exerts a PKA-dependent positive inotropic effect in human atrial myocardium: GLP-1R mediated effects in human myocardium

Glucagon-like peptide-1 receptor (GLP-1R) agonists are a rapidly growing class of drugs developed for treating type-2 diabetes mellitus. Patients with diabetes carry an up to 5-fold greater mortality risk compared to non-diabetic patients, mainly as a result of cardiovascular diseases. Although beneficial cardiovascular effects have been reported, exact mechanisms of GLP-1R-agonist action in the heart, especially in human myocardium, are poorly understood. The effects of GLP-1R-agonists (exenatide, GLP-1(7-36)NH2, PF-06446009, PF-06446667) on cardiac contractility were tested in non-failing atrial and ventricular trabeculae from 72 patients. The GLP-1(7-36)NH2 metabolite, GLP-1(9-36)NH2, was also examined. In electrically stimulated trabeculae, the effects of compounds on isometric force were measured in the absence and presence of pharmacological inhibitors of signal transduction pathways. The role of β-arrestin signaling was examined using a β-arrestin partial agonist, PF-06446667. Expression levels were tested by immunoblots. Translocation of GLP-1R downstream molecular targets, Epac2, GLUT-1 and GLUT-4, were assessed by fluorescence microscopy. All tested GLP-1R-agonists significantly increased developed force in human atrial trabeculae, whereas GLP-1(9-36)NH2 had no effect. Exendin(9-39)NH2, a GLP-1R-antagonist, and H-89 blunted the inotropic effect of exenatide. In addition, exenatide increased PKA-dependent phosphorylation of phospholamban (PLB), GLUT-1 and Epac2 translocation, but not GLUT-4 translocation. Exenatide failed to enhance contractility in ventricular myocardium. Quantitative real-time PCR (qRT-PCR) revealed a significant higher GLP-1R expression in the atrium compared to ventricle. Exenatide increased contractility in a dose-dependent manner via GLP-1R/cAMP/PKA pathway and induced GLUT-1 and Epac2 translocation in human atrial myocardium, but had no effect in ventricular myocardium. Therapeutic use of GLP-1R-agonists may therefore impart beneficial effects on myocardial function and remodelling.

AMPK - Activated Protein Kinase and its Role in Energy Metabolism of the Heart

Adenosine monophosphate – activated kinase (AMPK) plays a key role in the coordination of the heart’s anabolic and catabolic pathways. It induces a cellular cascade at the center of maintaining energy homeostasis in the cardiomyocytes.. The activated AMPK is a heterotrimeric protein, separated into a catalytic α - subunit (63kDa), a regulating β - subunit (38kDa) and a γ - subunit (38kDa), which is allosterically adjusted by adenosine triphosphate (ATP) and adenosine monophosphate (AMP). The actual binding of AMP to the γ – subunit is the step which activates AMPK. AMPK serves also as a protein kinase in several metabolic pathways of the heart, including cellular energy sensoring or cardiovascular protection. The AMPK cascade represents a sensitive system, activated by cellular stresses that deplete ATP and acts as an indicator of intracellular ATP/AMP. In the context of cellular stressors (i.e. hypoxia, pressure overload, hypertrophy or ATP deficiency) the increasing levels of AMP promote allosteric activation and phosphorylation of AMPK. As the concentration of AMP begins to increase, ATP competitively inhibits further phosphorylation of AMPK. The increase of AMP may also be induced either from an iatrogenic emboli, percutaneous coronary intervention, or from atherosclerotic plaque rupture leading to an ischemia in the microcirculation. To modulate energy metabolism by phosphorylation and dephosphorylation is vital in terms of ATP usage, maintaining transmembrane transporters and preserving membrane potential. In this article, we review AMPK and its role as an important regulatory enzyme during periods of myocardial stress, regulating energy metabolism, protein synthesis and cardiovascular protection.

Functional effects of glucose transporters in human ventricular myocardium

Insulin‐dependent positive inotropic effects (PIE) are partially Ca2+ independent. This mechanism is potentially glucose dependent. In contrast to most animal species, human myocardium expresses high levels of sodium‐glucose‐transporter‐1 (SGLT‐1) mRNA besides the common glucose‐transporters‐1 and ‐4 (GLUT1, GLUT4).

Efficacy and Safety of Glucose Control with Space GlucoseControl in the Medical Intensive Care Unit—An Open Clinical Investigation

BACKGROUND We aimed to investigate the performance of the Space GlucoseControl system (SGC) (B. Braun, Melsungen, Germany) in medical critically ill patients. The SGC is a nurse-driven, computer-assisted device for glycemic control combining infusion pumps with the enhanced Model Predictive Control algorithm. SUBJECTS AND METHODS The trial was designed as a single-center, open clinical investigation in a nine-bed medical intensive care unit in a tertiary center in Graz, Austria. Efficacy was assessed by percentage of time within the target range (4.4-8.3 mmol/L; primary end point), mean blood glucose, and sampling interval. Safety was assessed by the number of hypoglycemic episodes (≤2.2 mmol/L). RESULTS Twenty mechanically ventilated patients (age, 63±16 years; body mass index, 31.0±10.7 kg/m(2); Acute Physiology and Chronic Health Evaluation II score, 25.4±6.3; 14 males; six with diabetes) were included for a period of 7.0±3.6 days. Time within target range was 83.4±8.9% (mean±SD), and mean arterial blood glucose was 6.8±0.4 mmol/L. No severe hypoglycemic episodes (<2.2 mmol/L) occurred, and the percentage of time within 2.2 and 3.3 mmol/L was low (0.03±0.07%). The sampling interval was 2.0±0.4 h. The mean insulin dose was 93.5±80.1 IU/day, and the adherence to the given insulin dose advice was high (98.3%). A total of 11 unintended therapy interruptions (0.08 events/treatment day) caused by software problems occurred in four patients. CONCLUSIONS SGC is a safe and efficient method to control blood glucose in critically ill patients in the medical intensive care unit.

Angiotensin II and myosin light-chain phosphorylation contribute to the stretch-induced slow force response in human atrial myocardium

AIMS Stretch is an important regulator of atrial function. The functional effects of stretch on human atrium, however, are poorly understood. Thus, we characterized the stretch-induced force response in human atrium and evaluated the underlying cellular mechanisms. METHODS AND RESULTS Isometric twitch force of human atrial trabeculae (n = 252) was recorded (37 degrees C, 1 Hz stimulation) following stretch from 88 (L88) to 98% (L98) of optimal length. [Na(+)](i) and pH(i) were measured using SBFI and BCECF epifluorescence, respectively. Stretch induced a biphasic force increase: an immediate increase [first-phase, Frank-Starling mechanism (FSM)] to approximately 190% of force at L88 followed by an additional slower increase [5-10 min; slow force response (SFR)] to approximately 120% of the FSM. FSM and SFR were unaffected by gender, age, ejection fraction, and pre-medication with major cardiovascular drugs. There was a positive correlation between the amplitude of the FSM and the SFR. [Na(+)](i) rose by approximately 1 mmol/L and pH(i) remained unchanged during the SFR. Inhibition of Na(+)/H(+)-exchange (3 microM HOE642), Na(+)/Ca(2+)-exchange (5 microM KB-R7943), or stretch-activated channels (0.5 microM GsMtx-4 and 80 microM streptomycin) did not reduce the SFR. Inhibition of angiotensin-II (AngII) receptors (5 microM saralasin and 0.5 microM PD123319) or pre-application of 0.5 microM AngII, however, reduced the SFR by approximately 40-60%. Moreover, stretch increased phosphorylation of myosin light chain 2 (MLC2a) and inhibition of MLC kinase (10 microM ML-7 and 5 microM wortmannin) decreased the SFR by approximately 40-85%. CONCLUSION Stretch elicits a SFR in human atrium. The atrial SFR is mediated by stretch-induced release and autocrine/paracrine actions of AngII and increased myofilament Ca(2+) responsiveness via phosphorylation of MLC2a by MLC kinase.

Branched-Chain Amino Acids as New Biomarkers of Major Depression - A Novel Neurobiology of Mood Disorder.

Background The proteinogenic branched-chain amino acids (BCAAs) valine, leucine and isoleucine might play an unrecognised crucial role in the development of depression through their activation of the mammalian target of rapamycin (mTor) pathway. The aim of this research project is to evaluate whether BCAAs are altered in patients with major depression and might thus be appropriate biomarkers for major depression. Methods The concentrations of valine, leucine and isoleucine were determined in 71 in-patients with major depression and 48 healthy controls by high-pressure liquid chromatography. Psychiatric and laboratory assessments were obtained at the time of in-patient admittance. Results The BCAAs are significantly decreased in patients with major depression in comparison with healthy subjects (valine: Mann-Whitney-U: 968.0; p <0.0001, leucine: Mann-Whitney-U: 1246.5; p = 0.013, isoleucine: Mann-Whitney-U: 1252.5; p = 0.014). Furthermore, as shown by Spearmans rank correlation coefficients, there is a significant negative correlation between valine, leucine and isoleucine concentrations and the Hamilton Depression Rating Scale (HAMD-17) as well as Beck Depression Inventory (BDI-II) scores. Conclusions Our study results are strong evidence that in patients with major depression, BCAAs might be appropriate biomarkers for depression. Reduced activation of the mammalian target of rapamycin (mTor) due to a reduction of BCAAs might play a crucial unrecognised factor in the etiology of depression and may evoke depressive symptomatology and lower energy metabolism in patients with major depression. In the future, mTor and its up- and downstream signalling partners might be important targets for the development of novel antidepressants.