Kay-Dietrich Wagner

Oxygen-regulated expression of the Wilms' tumor suppressor Wt1 involves hypoxia-inducible factor-1 (HIF-1).

The Wilms’ tumor gene Wt1 is unique among tumor suppressors because of its requirement for the development of certain organs. We recently described de novo expression of Wt1 in myocardial blood vessels of ischemic rat hearts. The purpose of this study was to analyze the mechanism(s) of hypoxic/ischemic induction of Wt1. We show here that Wt1 mRNA and protein is up‐regulated in the heart and kidneys of rats exposed to normobaric hypoxia (8% O2). Ectopic Wt1 immunoreactivity was detected in renal tubules of hypoxic rats, which also expressed the antiapoptotic protein Bcl‐2 and contained significantly fewer TUNEL‐positive cells than in normoxic kidneys. Wt1 expression was enhanced in the osteosarcoma line U‐2OS and in Reh lymphoblast cells that were grown either at 1% O2 or in the presence of CoCl2 and desferrioxamine, respectively. The promoter of the Wt1 gene was capable of mediating expression of a luciferase reporter in response to hypoxia. We identified a hypoxia‐responsive element in the Wt1 sequence that bound to hypoxia‐inducible factor‐1 (HIF‐1) and was required for activation of the Wt1 promoter by CoCl2 and HIF‐1. These findings demonstrate that Wt1 expression can be stimulated by hypoxia, which involves activation of the Wt1 promoter by HIF‐1.

Characterization of stretch-activated ion currents in isolated atrial myocytes from human hearts.

Abstract. To explore further the mechanisms that may underlie cardiac arrhythmia, we analysed stretch-activated ion currents in human atrial myocytes. Longitudinal stretch of freshly isolated atrial myocytes prolonged the duration of action potentials, depolarized the resting membrane potential and caused extra action potentials. Under voltage-clamp conditions, the amplitude of stretch-induced transmembrane currents increased reversibly with the intensity of stretch. Stretch-activated currents (ISAC) had a reversal potential of 0xa0mV and were insensitive to substitution of Cl– with aspartate ions in the extracellular fluid. ISAC was suppressed by 5xa0µM gadolinium (Gd3+). Furthermore, mechanical stretch decreased transmembrane ion fluxes through L-type calcium channels (ICa,L). This reduction of ICa,L was inhibited by dialysing the cells for 5xa0min with 5xa0mM BAPTA prior to application of stretch. In contrast, both BAPTA and removal of Ca2+ from the extracellular bathing solution had no significant effect on stretch activation of ISAC. These findings suggest that non-selective cation channels in human atrial myocytes are sensitive to mechanical stimulation. We propose that activation of transmembrane influx of cations, preferentially Na+, by local stretch may play a role in cardiac arrhythmia.

Cardiac fibroblasts and the mechano-electric feedback mechanism in healthy and diseased hearts.

Cardiac arrhythmia is a serious clinical condition, which is frequently associated with abnormalities of mechanical loading and changes in wall tension of the heart. Recent novel findings suggest that fibroblasts may function as mechano-electric transducers in healthy and diseased hearts. Cardiac fibroblasts are electrically non-excitable cells that respond to spontaneous contractions of the myocardium with rhythmical changes of their resting membrane potential. This phenomenon is referred to as mechanically induced potential (MIP) and has been implicated in the mechano-electric feedback mechanism of the heart. Mechano-electric feedback is thought to adjust the frequency of spontaneous myocardial contractions to changes in wall tension, which may result from variable filling pressure. Electrophysiological recordings of single atrial fibroblasts indicate that mechanical compression of the cells may activate a non-selective cation conductance leading to depolarisation of the membrane potential. Reduced amplitudes of MIPs due to pharmacological disruption of F-actin and tubulin suggest a role for the cytoskeleton in the mechano-electric signal transduction process. Enhanced sensitivity of the membrane potential of the fibroblasts to mechanical stretch after myocardial infarction correlates with depression of heart rates. It is assumed that altered electrical function of cardiac fibroblasts may contribute to the increased risk of post-infarct arrhythmia.

Mechanically Induced Potentials in Fibroblasts from Human Right Atrium

It has been shown that cardiac fibroblasts of the human heart are electrically non‐excitable and mechanosensitive. The resting membrane potential of these cells is ‐15.9 ± 2.1 mV and the membrane resistance is 4.1 ± 0.1 GΩ. Rhythmic contractions of the myocardium associated with stretch of the surrounding tissue produce reversible changes in the membrane potential of cardiac fibroblasts. These mechanically induced potentials (MIPs) follow the rhythm of myocardial contractions. Simultaneous recording of the action potential of cardiomyocytes and MIPs of cardiac fibroblasts demonstrates a delay of 40.0 ± 0.4 ms after the action potential before the appearance of the MIP. Contraction produces a MIP which is more positive or more negative than the reversal potential ‐ the membrane potential due to current injection at which the MIP reverses its direction. Regardless of the initial orientation of the MIP, intracellular polarization increases the amplitude towards the reversal potential if the background MIP had depolarized the membrane or away from the reversal potential if the initial background MIP had hyperpolarized the membrane. Artificial intracellular polarization changed the amplitude but not the frequency of the MIP. The pool of electrically non‐excitable mechanosensitive cells, which change their electrical activity during contraction and relaxation of the heart, may play a role in the mechano‐electrical feedback mechanism which has to be taken into account in the normal function of the heart as well as in pathological processes.

Mechanoelectric feedback after left ventricular infarction in rats

BACKGROUNDnMyocardial infarction can lead to electrical abnormalities and rhythm disturbances. However, there is limited data on the electrophysiological basis for these events. Since regional contraction abnormalities feature prominently in infarction, we investigated whether stretch of myocardium from the infarction borderzone can modulate the electrophysiological properties of cardiomyocytes via mechanoelectric feedback providing a mechanism for post-infarction arrhythmia.nnnMETHODSnFive weeks after experimental myocardial infarction (MI) in rats due to ligation of the left coronary artery (n = 26) or after sham operation (SO, n = 16), action potentials (AP) were measured in left ventricular preparations from the infarction borderzone. Sustained stretch was applied via a micrometer.nnnRESULTSnPreparations from MI generated spontaneous electrical and contractile activity. Cardiomyocytes from MI had a comparable AP amplitude, a more negative resting membrane potential, and a prolonged AP duration (APD) when compared to SO. In SO, stretch of 150 microns increased the APD90. This was associated with stretch activated depolarizations near APD90 (SAD-90). In MI, significantly lower stretch, of only 20 microns, elicited SAD-90s, or SADs near APD50 (SAD-50). Stretch-induced events were suppressed by gadolinium, at a concentration (40 microM) normally used to inhibit stretch-activated channels.nnnCONCLUSIONnAfter MI, SADs are generated in the infarction borderzone at lower degrees of stretch. Increased sensitivity of the membrane potential of cardiac myocytes to mechanical stimuli may contribute to the high risk of arrhythmia after infarction. These SADs may involve the opening of stretch-activated channels.

The Wilms' tumor suppressor Wt1 encodes a transcriptional activator of the class IV POU-domain factor Pou4f2 (Brn-3b).

The Wilms tumor gene Wt1 encodes a zinc finger protein, which is required for normal formation of the genitourinary system and mesothelial tissues. Our recent findings indicate that Wt1 also plays a critical role in the development of ganglion cells in the vertebrate retina. Here we show that the POU-domain factor Pou4f2 (formerly Brn-3b), which is necessary for retinal ganglion cell survival, is up-regulated in human embryonic kidney (HEK)293 cells with stable Wt1 expression. Consistent with our previous observations of increased Pou4f2 mRNA in stably Wt1-transfeced HEK293 cells [EMBO J. 21 (2002) 1398], endogenous Pou4f2 was also elevated at the protein level in the HEK293 transfectants as well as in U2OS osteosarcoma cells that expressed an inducible Wt1 isoform. Transient co-transfection of a Wt1 expression construct activated a Pou4f2 promoter-reporter construct approximately 4-fold. Stimulation of the Pou4f2 promoter required a Wt1 binding element that was similar to a degenerative consensus site previously identified in other Wt1 responsive genes. Double-immunofluorescent labeling revealed co-expression of Pou4f2 and Wt1 in glomerular podocytes of adult kidney and in developing retinal ganglion cells of mouse embryos. Pou4f2 immunoreactivity was absent from the retinas of Wt1(-/-) embryos. In conclusion, we identified Pou4f2 as a novel downstream target gene of Wt1. Co-localization of both proteins in glomerular podocytes of the kidney and in developing retinal ganglion cells suggests a role for Wt1-Pou4f2 interaction in these tissues.

Mechanically induced potentials in atrial fibroblasts from rat hearts are sensitive to hypoxia/reoxygenation

Membrane potential changes of atrial fibroblasts in response to mechanical stress have been considered to modulate the rhythmic electrical activity of healthy hearts. Our recent findings suggest that cardiac arrhythmia after infarction is related to enhanced susceptibility of the fibroblasts to physical stretch. In this study, we analysed the effect of hypoxia/reoxygenation, which are major components of tissue ischemia/reperfusion, on the membrane potential of atrial fibroblasts. Intracellular microelectrode recordings were performed together with isometric force measurements on isometrically contracting right atrial tissue preparations from adult rats. Lowering the oxygen tension in the perfusate from 80xa0kPa to 3.5xa0kPa reduced active force development and decreased the resting membrane potential of the cardiac fibroblasts from −23±5xa0mV to −5±2xa0mV (n=35). Application of gadolinium (40xa0μM) to inhibit non-selective cation channels prevented hypoxia-induced membrane depolarization of the fibroblasts. Reoxygenation of the myocardial tissue resulted in a transient increase of the resting membrane potential to maximally −60±8xa0mV. These findings indicate that transmembrane currents in atrial fibroblasts are sensitive to changes in tissue oxygenation. In conclusion, altered electro-mechanical function of the ischemic heart may possibly involve changes of the membrane potential of the cardiac fibroblasts.

Myocardial contractility after infarction and carnitine palmitoyltransferase I inhibition in rats.

Inhibition of carnitine palmitoyltransferase I with etomoxir increases sarcoplasmic reticulum Ca(2+)-transport and V(1) isomyosin expression. To test whether etomoxir attenuates contractile dysfunction after myocardial infarction, we compared the contractility of papillary muscles from etomoxir- and placebo-treated rats 6 weeks after infarction. Etomoxir induced cardiac hypertrophy in animals with small infarctions, and enhanced compensatory heart growth at large infarct size. Contractile function of papillary muscles from etomoxir-treated rats was improved particularly in animals with small infarctions. Thus, induction of mild cardiac hypertrophy by etomoxir in rats with small infarctions may be beneficial for myocardial performance.

Mechanically induced potentials in rat atrial fibroblasts depend on actin and tubulin polymerisation.

Abstract. When atrial tissue contracts, mechanically induced potentials (MIPs) are generated in fibroblasts, presumably by activation of a non-selective cation conductance Gns. Non-stimulated atrial fibroblasts had a mean (±SD) membrane potential (Em) of –22±2xa0mV and an input resistance of 510±10xa0MΩ. MIP amplitude (AMIP) was 38±4xa0mV when current injection had polarised Em to Vm=–50xa0mV. The slope of the function relating AMIP to Vm can be regarded as a mechanosensitive factor (Xms) that describes the relative increase in Gns during a MIP. Putative involvement of cytoskeletal fibres in activation of Gns was studied by delivering drugs from the intracellular recording microelectrode. Destabilisation of F-actin by 0.2xa0mM cytochalasin D reduced AMIP from 38 to 16xa0mV and Xms from 5 to 1.8. Destabilisation of tubulin with 0.2xa0mM colchicine reduced AMIP to 21xa0mV and Xms to 2.1. The combination colchicine plus cytochalasin D reduced AMIP to 9xa0mV and Xms to 1.4. Promoting F-actin stability with exogenous adenosine 5′-triphosphate (ATP) increased AMIP and Xms and attenuated the effects of cytochalasin D. Similarly, facilitation of tubulin stability with guanosine 5′-triphosphate (GTP) or taxol increased AMIP and Xms and attenuated the effects of colchicine. The results suggest that transfer of mechanical energy from the deformed fibroblast surface to the Gns channel protein depends on intact F-actin and tubulin fibres.

Combined Treatment with Ramipril and Metoprolol Prevents Changes in the Creatine Kinase Isoenzyme System and Improves Hemodynamic Function in Rat Hearts after Myocardial Infarction

Beneficial effects of monotherapy with ACE inhibitors or beta-blockers on hemodynamic function after myocardial infarction are well known. Until now, the effects of combined treatment on cardiac function and energy metabolism have been poorly described. This study examines the effects of combined ramipril and metoprolol treatment on the creatine kinase (CK) system and hemodynamic function in rats after infarction. Wistar rats with experimental infarction were randomized for treatment with ramipril (R), metoprolol (M), combined treatment (MR), or placebo (P). Sham-operated (SO) animals served as controls. After 6 weeks, we assayed for CK isoenzymes and performed hemodynamic measurements. In P versus SO, left ventricular systolic pressures (dp/dtmax and dp/dtmin) diminished, whereas left ventricular end-diastolic pressure (LVEDP) increased. Decreased total CK activity and mitochondrial CK isoenzyme, increased CK-MB, and increased CK-BB isoenzymes were measured in P versus SO. With infarct size ≤45%, mitochondrial CK increased in M and R versus P. Combined treatment had an additional enhancing effect on mitochondrial CK isoenzyme level versus M and R, decreased LVEDP versus P, as well as increased dp/dtmax and dp/dtmin versus R. These results provide evidence of an interaction between normalization of energy metabolism and improvement in cardiac function due to a combination of ACE inhibition and beta blockade after myocardial infarction.