Jan W.T. Fiolet

Potassium accumulation in the globally ischemic mammalian heart. A role for the ATP-sensitive potassium channel.

We investigated the contribution of opening of the ATP-sensitive K+ channel to extracellular accumulation of K+ during ischemia with the use of glibenclamide, a specific blocker of this K+ channel. To characterize the electrophysiological effects of glibenclamide during metabolic inhibition (by either application of dinitrophenol or hypoxia) we performed patch-clamp studies in isolated membrane patches of guinea pig myocytes and in intact guinea pig myocytes and studied action potential parameters in isolated superfused guinea pig papillary muscle. We studied the effect of glibenclamide on extracellular accumulation of K+ and H+ in isolated retrogradely perfused globally ischemic hearts of rat, guinea pig, and rabbit. Experimental evidence is presented that supports the conclusions that glibenclamide 1) effectively blocks open K+ATP channels, 2) reverses the dinitrophenol-induced increase of the outward current and prevents the hypoxia-induced shortening of the action potential, 3) decreases the rate of K+ accumulation during the first minutes of ischemia in stimulated hearts, an effect which was entirely absent in quiescent hearts, and 4) does not influence the rate and extent of ischemia-induced extracellular acidification.

Intracellular Ca2+, Intercellular Electrical Coupling, and Mechanical Activity in Ischemic Rabbit Papillary Muscle: Effects of Preconditioning and Metabolic Blockade

During myocardial ischemia, electrical uncoupling and contracture herald irreversible damage. In the present study, we tested the hypothesis that an increase of intracellular Ca2+ is an important factor initiating these events. Therefore, we simultaneously determined tissue resistance, mechanical activity, pH(0), and intracellular Ca2+ (with the fluorescent indicator indo 1, Molecular Probes, Inc) in arterially perfused rabbit papillary muscles. Sustained ischemia was induced in three experimental groups: (1) control, (2) preparations preconditioned with two 5-minute periods of ischemia followed by reperfusion, and (3) preparations pretreated with 1 mmol/L iodoacetate to block anaerobic metabolism and minimize acidification during ischemia. In a fourth experimental group, intracellular Ca2+ was increased under nonischemic conditions by perfusing with 0.1 mmol/L ionomycin and 0.1 mumol/L gramicidin. Ca2+ transients and contractions rapidly disappeared after the induction of ischemia. In the control group, diastolic Ca2+ began to rise after 12.6 +/- 1.3 minutes of ischemia; uncoupling, after 14.5 +/- 1.2 minutes of ischemia; and contracture, after 12.6 +/- 1.5 minutes of ischemia (mean +/- SEM). Preconditioning significantly postponed Ca2+ rise, uncoupling, and contracture (21.5 +/- 4.0, 24.0 +/- 4.1, and 23.0 +/- 5.3 minutes of ischemia, respectively). Pretreatment with iodoacetate significantly advanced these events (1.9 +/- 0.7, 3.6 +/- 0.9, and 1.9 +/- 0.2 minutes of ischemia, respectively). In all groups, the onset of uncoupling always followed the start of Ca2+ rise, whereas the start of contracture was not different from the rise in Ca2+. Perfusion with ionomycin and gramicidin permitted estimation of a threshold [Ca2+] for electrical uncoupling of 685 +/- 85 nmol/L. In conclusion, the rise in intracellular Ca2+ is the main trigger for cellular uncoupling during ischemia. Contracture is closely associated with the increase of intracellular Ca2+ during ischemia.

Increased Na+/H+-exchange activity is the cause of increased [Na+]i and underlies disturbed calcium handling in the rabbit pressure and volume overload heart failure model

OBJECTIVE Cytosolic sodium ([Na+]i) is increased in heart failure (HF). We hypothesize that up-regulation of Na+/H+-exchanger (NHE) in heart failure is causal to the increase of [Na+]i and underlies disturbance of cytosolic calcium ([Ca2+]i) handling. METHODS Heart failure was induced in rabbits by combined volume and pressure overload. Age-matched animals served as control. [Na+]i, cytosolic calcium [Ca2+]i and cytosolic pH (pH(i)) were measured in isolated left ventricular midmural myocytes with SBFI, indo-1 and SNARF. SR calcium content was measured as the response of [Ca2+]i to rapid cooling (RC). Calcium after-transients were elicited by cessation of rapid stimulation (3 Hz) in the presence of 100 nmol/l noradrenalin. NHE and Na+/K+-ATPase activity were inhibited with 10 micromol/l cariporide and 100 micromol/l ouabain, respectively. RESULTS At all stimulation rates (0-3 Hz) [Na+]i and diastolic [Ca2+]i were significantly higher in HF than in control. With increasing frequency [Na+]i and diastolic [Ca2+]i progressively increased in HF and control, and the calcium transient amplitude (measured as total calcium released from SR) decreased in HF and increased in control. In HF (at 2 Hz), SR calcium content was reduced by 40% and the calcium gradient across the SR membrane by 60%. Fractional systolic SR calcium release was 90% in HF and 60% in control. In HF the rate of pH(i) recovery following acid loading was much faster at all pH(i) and NHE dependent sodium influx was almost twice as high as in control. In HF cariporide (10 micromol/l, 5 min) reduced [Na+]i and end diastolic [Ca2+]i to almost control values, and reversed the relation between calcium transient amplitude and stimulation rate from negative to positive. It increased SR calcium content and SR membrane gradient and decreased fractional systolic SR depletion to 60%. Cariporide greatly reduced the susceptibility to develop calcium after-transients. In control animals, cariporide had only minor effects on all these parameters. Increase of [Na+]i with ouabain in control myocytes induced abnormal calcium handling as found in HF. CONCLUSIONS In HF up-regulation of NHE activity is causal to increased [Na+]i and secondarily to disturbed diastolic, systolic and SR calcium handling. Specific inhibition of NHE partly normalized [Na+]i, end diastolic [Ca2+]i, and SR calcium handling and reduced the incidence of calcium after-transients. Chronic treatment with specific NHE inhibitors may provide a useful future therapeutic option in treatment of developing hypertrophy and heart failure.

The fluorescent properties of acridines in the presence of chloroplasts or liposomes. On the quantitative relationship between the fluorescence quenching and the transmembrane proton gradient

Abstract The fluorescent properties of 9-aminoacridine were studied in chloroplasts and phospholipid liposomes. In energized chloroplasts it was found that the percentage of fluorescence quenching was dependent on both the 9-aminoacridine concentration and the chlorophyll concentration. On the other hand, it was independent of the osmolarity of the medium. In phospholipid liposomes the dependence of the fluorescence quenching on the concentration of 9-aminoacridine was similar to that in chloroplasts. Moreover, the fluorescence quenching depended on the presence of charged compounds in the membrane being larger in negatively charged than in positively charged liposomes. The fluorescence of both the monoamine 9-amino-6-chloro-2-methoxyacridine and the diamine atebrin is quenched more extensively than that of 9-aminoacridine. Although the percentage of fluorescence quenching of both atebrin and 9-aminoacridine is dependent on the outside pH, the relationship between the fluorescence quenching of the two probes under similar conditions is not pH-dependent. It is concluded that calculation of ΔpH from the percentage of fluorescence quenching of fluorescent amines is not meaningful, that the osmotic volume of chloroplasts is not involved in the quenching process and, consequently, that the interaction between the acridines and energized membranes is more likely to occur at the level of the membrane proper.

Acute administration of fish oil inhibits triggered activity in isolated myocytes from rabbits and patients with heart failure

Background— Fish oil reduces sudden death in patients with prior myocardial infarction. Sudden death in heart failure may be due to triggered activity based on disturbed calcium handling. We hypothesized that superfusion with &ohgr;3-polyunsaturated fatty acids (&ohgr;3-PUFAs) from fish inhibits triggered activity in heart failure. Methods and Results— Ventricular myocytes were isolated from explanted hearts of rabbits with volume- and pressure-overload–induced heart failure and of patients with end-stage heart failure. Membrane potentials (patch-clamp technique) and intracellular calcium (indo-1 fluorescence) were recorded after 5 minutes of superfusion with Tyrode’s solution (control), &ohgr;-9 monounsaturated fatty acid oleic acid (20 &mgr;mol/L), or &ohgr;3-PUFAs (docosahexaenoic acid or eicosapentaenoic acid 20 &mgr;mol/L). &ohgr;3-PUFAs shortened the action potential at low stimulation frequencies and caused an ≈25% decrease in diastolic and systolic calcium (all P<0.05). Subsequently, noradrenalin and rapid pacing were used to evoke triggered activity, delayed afterdepolarizations, and calcium aftertransients. &ohgr;3-PUFAs abolished triggered activity and reduced the number of delayed afterdepolarizations and calcium aftertransients compared with control and oleic acid. &ohgr;3-PUFAs reduced action potential shortening and intracellular calcium elevation in response to noradrenalin. Results from human myocytes were in accordance with the findings obtained in rabbit myocytes. Conclusion— Superfusion with &ohgr;3-PUFAs from fish inhibits triggered arrhythmias in myocytes from rabbits and patients with heart failure by lowering intracellular calcium and reducing the response to noradrenalin.

Reperfusion arrhythmias in isolated perfused pig hearts. Inhomogeneities in extracellular potassium, ST and TQ potentials, and transmembrane action potentials.

We recorded direct current electrograms and local [K+]o at multiple sites and transmembrane potentials at selected sites during reperfusion after 5 minutes and 10 minutes of regional ischemia in isolated perfused pig hearts. After 10 minutes of ischemia, the incidence of ventricular fibrillation (VF) was 38%. At 80-90 seconds after reperfusion, [K+]o was 0.8 mM less than in normal tissue in half of the reperfused tissue, especially in the border zone. This was associated with TQ elevation of +4.5 mV and large peaked T waves. The latter was caused by an abrupt decrease of action potential duration in reperfused tissue, leading to a difference of up to 165 msec with normal tissue. Reperfusion VF started with a closely coupled ventricular premature beat. Activation block between reperfused and normal tissue permitted reentrant activation, leading to VF. Pretreatment with ryanodine (10(-6) M) and reperfusion with elevated [K+] (both of which prevent delayed afterdepolarizations) did not prevent closely coupled ventricular premature beats or VF. Five minutes of ischemia never caused VF. K+ depletion and TQ elevation in the reperfused zone was less frequent and smaller (-0.4 mM and 1.8 mV, respectively). Peaked T waves did not occur, and shortening of the action potential duration was less. We conclude that extracellular K+ depletion and marked action potential duration shortening in the reperfused tissue play a role in the genesis of reperfusion VF, which is caused by reentry. The closely coupled ventricular premature beat that initiates reentry is not caused by delayed afterdepolarizations but most likely by intramural reentry.

[Na+](i) and the driving force of the Na+/Ca2+-exchanger in heart failure

OBJECTIVE Diastolic calcium is increased in myocytes from failing hearts despite up-regulation of the principal calcium extruding mechanism the Na+/Ca2+-exchanger (NCX). We hypothesize that increased diastolic calcium ([Ca2+]i) is secondary to increased cytosolic sodium ([Na+]i) and decreased driving force of NCX (DeltaG(exch)). METHODS The stimulation rate dependence of simultaneously measured cytosolic sodium ([Na+]i), calcium transients ([Ca2+]i) and action potentials were determined with SBFI, indo-1 and the perforated patch technique in midmural left ventricular myocytes isolated from rabbits with pressure and volume overload induced heart failure (HF) and in age matched controls. Dynamic changes of DeltaG(exch) were calculated. RESULTS With increasing stimulation frequency, 0.2-3 Hz (all data HF versus control): [Na+]i increased (6.4 to 10.8 versus 3.8 to 6.4 mmol/l), diastolic [Ca2+]i increased (142 to 219 versus 47 to 98 nmol/l), calcium transient amplitude decreased in HF (300 to 250 nmol/l) but increased in control (201 to 479 nmol/l), action potential duration (APD90) decreased (380 to 260 versus 325 to 205 ms) and time averaged DeltaG(exch) decreased (6.8 to 2.8 versus 8.7 to 6.4 kJ/mol. With increasing stimulation rate the forward mode time integral of DeltaG(exch) decreased in HF by about 30%, the reversed mode time integral increased about ninefold and the duration of reversed mode operation more than sixfold relative to control. CONCLUSIONS [Na+]i is increased in HF and the driving force of NCX is decreased. NCX exerts thermodynamic control over diastolic calcium. Disturbed diastolic calcium handling in HF is due to decreased forward mode DeltaG(exch) secondary to increased [Na+]i and prolongation of the action potential. Enhanced reversed mode DeltaG(exch) may account for increased contribution of NCX to e-c coupling in HF.

Chronic inhibition of the Na+/H+‐ exchanger causes regression of hypertrophy, heart failure, and ionic and electrophysiological remodelling

Increased activity of the Na+/H+‐exchanger (NHE‐1) in heart failure underlies raised [Na+]i causing disturbances of calcium handling. Inhibition of NHE‐1, initiated at the onset of pressure/volume overload, prevents development of hypertrophy, heart failure and remodelling. We hypothesized that chronic inhibition of NHE‐1, initiated at a later stage, would induce regression of hypertrophy, heart failure, and ionic and electrophysiological remodelling.

Relevance of Na+-Ca2+ exchange in heart failure.

Time for primary review 21 days. Sarcolemmal Na+–Ca2+ exchange plays a pivotal role in ion transport of the myocardium which is crucial for cardiac contractile performance. The driving force of the exchanger molecule depends on sodium and calcium concentrations at either side of the plasma membrane and on the membrane potential. Section 2 of this article aims to review structural and thermodynamic aspects of the Na+–Ca2+ exchanger and its function. Recently, it has been recognized that Ca2+ as well as Na+ homeostasis is impaired in the failing myocardium. Thus, it has been postulated by numerous authors that Na+–Ca2+ exchanger may be altered with respect to expression and function. However, the literature is controversial. Section 3 comments upon a number of recent publications that have been published on this topic. Section 4 summarizes functional consequences of altered expression and function of the exchanger with respect to excitation–contraction coupling which have to be considered with the concomitant changes of other important Ca2+ cycling proteins such as sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and ryanodine receptor. Finally, besides contractile dysfunction in hypertrophy and heart failure, cytoplasmic Ca2+ overload can induce spontaneous SR Ca2+ release. This Ca2+ is partly removed from the cytoplasm by Na+–Ca2+ exchange generating transient inward currents, which were blamed for provoking arrhythmogenic, delayed afterdepolarizations. We have dedicated section 5 to recent literature and clinical studies that may elucidate the role of Na+–Ca2+ exchange in arrhythmogenesis. ### 2.1 Isoforms, expression, structure and regulation Besides the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA), the sarcolemmal Na+–Ca2+ exchanger (NCX) is the most important Ca2+ transport protein responsible for maintaining the Ca2+ balance of the myocyte. Molecular aspects of its function have been subject to … * Corresponding author. Tel.: +49-551-39-6351; fax: +49-551-39-6389. hasenfus{at}med.uni-goettingen.de

Transmural inhomogeneity of extracellular [K+] and pH and myocardial energy metabolism in the isolated rat heart during acute global ischemia; dependence on gaseous environment

SummaryWe investigated in the isolated rat heart the influence of the gas surrounding the globally ischemic heart on transmural inhomogeneity of energy metabolism, extracellular K+ accumulation, and change of extracellular pH. Hearts were made ischemic in 100% N2 (N2-ischemia), 100% O2 (O2-ischemia) or 100% CO2 (CO2-ischemia). We measured: 1) Midmural, subepicardial, and epicardial changes of extracellular [K+] and pH during successive 6-min periods of global ischemia, and 2) content of creatinephosphate (CrP) in consecutive tissue sections of 100 μm, from the subepicardium after 10 min of ischemia.A)During O2-ischemia both extracellular [K+] and change of pH in the subepicardium are significantly less than in the midmyocardium. During N2-ischemia only minor differences exist in [K+] and pH between the subepicardium and the midmyocardium. During CO2-ischemia midmural and subepicardial [K+] are similar to those during N2-ischemia. The midmural change of pH resembles that during N2-ischemia; subepicardial change of pH, however, was slightly larger. Midmural changes in [K+] and pH were not influenced by the nature of the surrounding gas.B)After 10 min of O2-ischemia a gradient of tissue content of CrP extends from the epicardium (CrP about 30 μmoles/g dry weight) to a distance of about 1000 μm (CrP 1 μmoles/g dry weight). In N2-and CO2-ischemia a CrP gradient is absent; CrP is appreciably less than 1 μmoles/g dry weight at any distances from the epicardium.C)We conclude that diffusion of O2 into the myocardium and of CO2 from the myocardium affects transmural gradients of [K+], pH, and energy metabolism during ischemia. Local availability of O2 increases the capacity of the ischemic tissue to generate high energy phosphates and mitigates ischemia-induced changes of transsarcolemmal ion gradients.