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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: Development of heart failure in rabbits was monitored electrocardiographically and echocardiographically, after one or three months. Cardiac myocytes were also isolated. One group of animals were treated with cariporide (inhibitor of NHE-1) in the diet after one month. Cytoplasmic calcium, sodium and action potentials were measured with fluorescent markers and sarcoplasmic reticulum calcium content by rapid cooling. Calcium after-transients were elicited after rapid pacing. Sodium channel current (INa) was measured using patch-clamp techniques. KEY RESULTS: Hypertrophy and heart failure developed after one month and progressed during the next two months. After one month, dietary treatment with cariporide was initiated. Two months of treatment reduced hypertrophy and heart failure, duration of action potential QT-interval and QRS, and restored sodium and calcium handling and the incidence of calcium after-transients. In cardiac myocytes, parameters of INa were not changed by cariporide. CONCLUSION AND IMPLICATIONS: In rabbit hearts with hypertrophy and signs of heart failure one month after induction of pressure/volume overload, two months of dietary treatment with the NHE-1 inhibitor cariporide caused regression of hypertrophy, heart failure and ionic and electrophysiological remodelling.

Two distinct congenital arrhythmias evoked by a multidysfunctional Na(+) channel.

The congenital long-QT syndrome (LQT3) and the Brugada syndrome are distinct, life-threatening rhythm disorders linked to autosomal dominant mutations in SCN5A, the gene encoding the human cardiac Na(+) channel. It is believed that these two syndromes result from opposite molecular effects: LQT3 mutations induce a gain of function, whereas Brugada syndrome mutations reduce Na(+) channel function. Paradoxically, an inherited C-terminal SCN5A mutation causes affected individuals to manifest electrocardiographic features of both syndromes: QT-interval prolongation (LQT3) at slow heart rates and distinctive ST-segment elevations (Brugada syndrome) with exercise. In the present study, we show that the insertion of the amino acid 1795insD has opposite effects on two distinct kinetic components of Na(+) channel gating (fast and slow inactivation) that render unique, simultaneous effects on cardiac excitability. The mutation disrupts fast inactivation, causing sustained Na(+) current throughout the action potential plateau and prolonging cardiac repolarization at slow heart rates. At the same time, 1795insD augments slow inactivation, delaying recovery of Na(+) channel availability between stimuli and reducing the Na(+) current at rapid heart rates. Our findings reveal a novel molecular mechanism for the Brugada syndrome and identify a new dual mechanism whereby single SCN5A mutations may evoke multiple cardiac arrhythmia syndromes by influencing diverse components of Na(+) channel gating function. The full text of this article is available at http://www.circresaha.org.

Ionic mechanism of delayed afterdepolarizations in ventricular cells isolated from human end-stage failing hearts.

BACKGROUND: Animal studies have shown that the Ca(2+)-activated Cl(-) current (I(Cl(Ca))) and the Na(+)/Ca(2+) exchange current (I(Na/Ca)) contribute to the transient inward current (I(ti)). I(ti) is responsible for the proarrhythmic delayed afterdepolarizations (DADs). We investigated the ionic mechanism of I(ti) and DADs in human cardiac cells. METHODS AND RESULTS: Human ventricular cells were enzymatically isolated from explanted hearts of patients with end-stage heart failure and studied with patch-clamp methodology. I(ti)s were elicited in the presence of 1 micromol/L norepinephrine by trains of repetitive depolarizations from -80 to +50 mV. DADs were induced in the presence of 1 micromol/L norepinephrine at a stimulus frequency of 1 Hz. I(ti) currents were inwardly directed over the voltage range between -110 and + 50 mV. Neither the Cl(-) channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid nor changes in [Cl(-)](i) affected I(ti) or DAD amplitude. This excludes an important role for I(Cl(Ca)). Blockade of Na(+)/Ca(2+) exchange by substitution of all extracellular Na(+) by Li(+), conversely, completely inhibited I(ti). In rabbit, I(Cl(Ca)) density in ventricular cells isolated from control hearts did not differ significantly from that in ventricular cells isolated from failing hearts. CONCLUSIONS: In contrast to many animal species, I(ti) and DADs in human ventricular cells from failing hearts consist only of I(Na/Ca). In rabbits, heart failure per se does not alter I(Cl(Ca)) density, suggesting that I(Cl(Ca)) may also be absent during DADs in nonfailing human ventricular cells.

[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.

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.

Transsarcolemmal sodium-calcium exchange and myocardial oxygen consumption in isolated rat ventricular myocytes.

We studied oxygen consumption and energy metabolism in isolated rat ventricular myocytes which were subjected to an abrupt change in the cation composition of the extracellular medium ('transition'); extracellular [Na+] was decreased either alone or in combination with a change of [K+] or [Ca2+]. The magnitude of change of the cation concentration(s) was varied. The respiratory rate (vO2) of myocytes changed biphasically after such a transition. vO2 increases to a maximum after about 25 to 30 s and returns to almost control after 180 to 200 s. vO2-max depends on both the nature of the cation(s) of which the concentration(s) are varied and on the magnitude of these change(s); vO2-max can almost be as high as that induced by uncoupling of oxidative phosphorylation with DNP. The free energy of hydrolysis of cytoplasmic ATP hardly decreases after transition. Cell viability remains unaltered, although an increasingly larger fraction of rod-shaped cells transform to a hypercontracted state with increasing magnitude of the extracellular ion concentration change. Reversal of the ionic change or addition of EGTA at 30 s after transition accelerates the return of vO2 to the value prior to transition. In the presence of ouabain, vO2-max is higher and return to control is slower and incomplete. The total amount of oxygen consumption after transition, is linearly related to the initial change of the free energy of the Na+/Ca(2+)-exchanger caused by the cation concentration change(s); this relationship does not depend on the nature of the cation(s) changed. We conclude that the transient increase of vO2 after transition is regulated by intracellular free [Ca2+], which transiently increases. This transient increase is caused by change of the thermodynamic driving force on the Na+/Ca(2+)-exchanger after transition.

SR calcium depletion following reversal of the Na+/Ca2+-exchanger in rat ventricular myocytes.

We previously reported that cytosolic calcium transiently increases after reversal of the sarcolemmal Na+/Ca2+-exchanger. Calcium released from sarcoplasmic reticulum (SR) constituted the major part of this cytosolic transient. The aim of this study was to test whether reversal of the Na+/Ca2+-exchanger affects SR calcium content, and whether altered SR calcium content is associated with direct triggering of SR calcium release or calcium release secondary to SR calcium overload. To this purpose we studied the change of SR calcium content after reversal of the Na+/Ca2+-exchanger and the dependence on the magnitude of change of its free energy (delta Gexch) in isolated rat ventricular myocytes. The Na+/Ca2+-exchanger was reversed by abrupt reduction of extracellular sodium ([Na+]o). The magnitude of change of deltaGexch was varied with [Na+]o. Cytosolic free calcium ([Ca2+]i) was measured with indo-1 and SR calcium content was estimated from the increase of [Ca2+]i after rapid cooling (RC). SR function was manipulated either by blockade of the SR Ca2+-ATPase with thapsigargin or by blockade of SR calcium release channels with tetracaine. Reversal of the Na+/Ca2+-exchanger caused a transient increase of [Ca2+]i of about 180 s duration with a time to peak of about 30 s. During the first 30 s rapid small amplitude cytosolic calcium fluctuations were superimposed on this transient. The magnitude of the response of [Ca2+]i to RC, during the course of the cytosolic [Ca2+]i transient, also transiently increased from 174 in control myocytes to 480 nmol/l at the time of the peak value. After correction of [Ca2+]i data for the fraction of mitochondrially compartmentalized indo-1 and mitochondrial calcium, total calcium released from SR after RC was calculated with the use of literature data on cytosolic calcium buffer capacity. Contrary to the measured RC-dependent increase of measured [Ca2+]i, after reversal of the Na+/Ca2+-exchanger, calculated total calcium released from SR transiently decreased. The extent of SR calcium depletion after reversal of the Na+/Ca2+-exchanger increased with the magnitude of change of deltaGexch. Restitution of [Na+]o 30 s after reversal of the Na+/Ca2+-exchanger, greatly accelerated both recovery of [Ca2+]i and SR calcium content. Pretreatment of myocytes with thapsigargin caused almost entire depletion of SR and substantial reduction of the cytosolic transient of [Ca2+]i following reversal of the Na+/Ca2+-exchanger. Application of tetracaine hardly affected SR calcium content, but caused an increase of the SR calcium content following reversal of the Na+/Ca2+-exchanger, while the cytosolic transient increase of [Ca2+]i was substantially reduced. We conclude that reversal of the Na+/Ca2+-exchanger directly triggers SR calcium release and decreases SR calcium content in a deltaGexch dependent manner.

Small changes of cytosolic sodium in rat ventricular myocytes measured with SBFI in emission ratio mode.

The spectral properties of SBFI (sodium-binding benzofurzan isophthalate) were re-examined to arrive at a more specific and sensitive method to measure small changes of intracellular sodium ([Na+]i) particularly at low concentration. Relative to spectra of SBFI in protein- and cell-free solution, binding of SBFI to intracellular proteins caused a shift of excitation and emission spectra, and increased quantum efficiency. Excitation of SBFI at 340 nm caused an exclusively sodium-dependent fluorescence from 400-420 nm, and hardly any change of fluorescence above 530 nm upon replacing sodium by potassium. Due to these spectral and quantum efficiency changes, SBFI excitated at 340 nm can be used in a dual emission ratio mode to measure [Na+]i. In dual emission ratio mode (410 and 590 nm, respectively), the fluorescence ratio increased by a factor of 13 upon replacing sodium for potassium. The apparent equilibrium constant measured in single isolated rat ventricular myocytes was 22.5+/-0.3 mmol/l. Control [Na+]i was 9.6+/-0.4 mmol/l. After abrupt reduction of extracellular sodium from 156 to 29 or 11 mmol/l, [Na+]i decreased mono-exponentially to 2.5+/-0.3 and 1.9+/-0. 3 mmol/l, respectively, with a rate constant of about 0.02/s. We conclude that SBFI used in dual emission mode provides a more sensitive and more specific method to measure small changes of [Na+]i in single myocytes down to cytosolic sodium concentration as low as about 1 mmol/l.

Intracellular [Ca2+] and Vo2 after manipulation of the free-energy of the Na+/Ca(2+)-exchanger in isolated rat ventricular myocytes.

We have investigated whether the Na+/Ca(2+)-exchanger has a functional regulatory role in the control of oxidative metabolism in suspensions of isolated rat ventricular myocytes. Therefore we simultaneously measured intracellular [Ca2+] ([Ca2+]i) with Indo-1 and respiratory rate (Vo2) after abrupt manipulation of the free-energy of the Na+/Ca(2+)-exchanger (delta Gexch). The average fraction of viable myocytes was about 90% (82% rod-shaped plus 8% viable round cells). delta Gexch was manipulated either by an abrupt decrease of [Na+]o (in combination with an increase of [K+]o or [Ca2+]o) or by changing membrane potential and/or intracellular cation activities with the use of gramicidin or veratridine. A change of extracellular cation composition caused a transient increase of [Ca2+]i and Vo2, with peak values after 30 to 40 s and a new steady state near control values after 180 to 240 s. Peak values of the transients were associated with the magnitude of the thermodynamic disturbance. Inhibition of sodium-pump activity with ouabain greatly enhanced peak values and reduced the rate of return to a new steady state. Reversal of the initial disturbance of delta Gexch by restoring [Na+]o or reduction of [Ca2+]o during the time course of the transients greatly accelerated return to a new steady-state. An increase of sarcolemmal sodium permeability with the Na-channel ligand veratridine or manipulation of [Na+]i and [K+]i with the Na+/K(+)-exchanger gramicidin caused monophasic increase of both [Ca2+]i and Vo2. The relationship between VO2 and [Ca2+]i was the same, irrespective of the nature of the intervention (either extracellular or intracellular manipulation of delta Gexch). We conclude that cytoplasmic [Ca2+] (thermodynamically controlled by the Na+/Ca(2+)-exchanger) is a major regulator of the respiratory rate in (quiescent) myocytes.

Cytoplasmic sodium, calcium and free energy change of the Na+/Ca2+-exchanger in rat ventricular myocytes.

The relationship between changing driving force of the Na+/Ca2+-exchanger (deltaG(exch)) and associated cytosolic calcium fluxes was studied in rat ventricular myocytes. DeltaG(exch) was abruptly reversed by the reduction of extracellular sodium ([Na+]o) with or without sustained depolarization by the elevation of potassium ([K+]o). Cytosolic sodium ([Na+]i) and calcium ([Ca2+]i) were measured with SBFI and indo-1 respectively and the time course of recovery of deltaG(exch) was calculated. Following abrupt reversal of deltaG(exch) from +4.1 to -9.2 kJ/mol [Na+]i exponentially decreased from 9.6-2.5 mmol/l (t(1/2) about 30 s) and [Ca2+]i transiently increased to a peak value after about 30 s. Negative values of deltaG(exch) were associated with an increase and positive values with a decrease of [Ca2+]i. Equilibrium (deltaG(exch) = 0) was reached after about 30 s coinciding with the time to peak [Ca2+]i. After 180 s deltaG(exch) reached a new steady state at +3.5 kJ/mol. Inhibition of SR with ryanodine or thapsigargin reduced the amplitude of the [Ca2+]i transient and shifted its peak to 80 s, but did not affect the time course of [Na+]i changes. In the presence of ryanodine or thapsigargin the time required for deltaG(exch) to recover to equilibrium was also shifted to 80 s. When we changed the deltaG(exch) to the same extent by the reduction of [Na+]o in combination with a sustained depolarization, [Na+]i decreased less and the amplitude of [Ca2+]i transient was much enhanced. This increase of [Ca2+]i was completely abolished by verapamil. DeltaG(exch) only recovered to a little above equilibrium (+1 kJ/mol). Inhibition of the Na+/K+-ATPase with ouabain entirely prevented the decrease of [Na+]i and caused a much larger increase of [Ca2+]i, which remained elevated; deltaG(exch) recovered to equilibrium and never returned to positive values. The rate of change of total cytosolic calcium was related to deltaG(exch), despite the fact that the calcium flux associated with the exchanger itself contributed only about 10%; SR related flux contributed by about 90% to the rate of change of total cytosolic calcium. In summary, reduction of [Na+]o causes reversal of the Na+/Ca2+-exchanger and its driving force deltaG(exch), a transient increase of [Ca2+]i and a decrease of [Na+]i. The influx of calcium associated with reversed deltaG(exch) triggers the release of calcium from SR. Both the decrease of [Na+]i and the increase of [Ca2+]i contribute to the recovery of deltaG(exch) to equilibrium. The time at which deltaG(exch) reaches equilibrium always coincides with the time to peak of [Ca2+]i transient. Activation of the Na+/K+-ATPase is required to reduce [Na+]i and recover deltaG(exch) to positive values in order to reduce [Ca2+]i. We conclude that deltaG(exch) is a major regulator of cytosolic calcium by interaction with SR.

The cytoplasmic free energy of ATP hydrolysis in isolated rod-shaped rat ventricular myocytes.

The relationship between the percentage of rod-shaped rat heart myocytes and ATP, creatine phosphate, creatine and inorganic phosphate content was determined. With these values the free energy of ATP hydrolysis was calculated and found to be 59.2 kJ/mol, a much higher value than found for the perfused rat heart. When, during the isolation procedure, creatine was present in the perfusion medium during the low-calcium period, the total creatine content of the myocytes after isolation was comparable to that found in the perfused rat heart. However, when creatine was absent during this low-calcium perfusion period, total creatine content of the myocytes was significantly lower. This difference is caused by leakage of creatine from healthy cells. The free energy of ATP hydrolysis was not affected by the absence of creatine during the low-calcium perfusion period.

Osmotic changes and transsarcolemmal ion transport during total ischaemia of isolated rat ventricular myocytes.

Transsarcolemmal water and ion movement during 1, 7.5, 15, and 30 min of total ischaemia was studied in suspensions of isolated rat ventricular myocytes, with a control ratio of about 1 of intracellular volume (ICV) to extracellular volume (ECV). In this preparation, contrary to the intact heart: 1) There is no external exchange of matter, 2) the sum of ICV and ECV remains constant and 3) ECV is homogeneous; no separate interstitial and intravascular compartments are present and no extracellular metabolite or ion gradients develop as may occur in the intact heart. We demonstrate that: 1) It is possible to make an ischaemic preparation of isolated myocytes with a procedure which causes only minimal mechanical damage to intact myocytes. The preparation allows measurement of ECV with the non-cardiac enzyme alpha-amylase as a macromolecular extracellular marker. 2) The time course of change of metabolites relevant to energy metabolism (creatinephosphate (CrP), creatine (Cr), ATP, ADP, inorganic phosphate P(i) and lactate) is similar to that in the intact heart. 3) ECV has decreased and ICV increased by about 20% after 30 min of ischaemia. 4) Extracellular [Na+], [K+], [Cl-], and [P(i)] increase, but not in proportion to the decrease of ECV. There is net efflux of K+, P(i), H+, and lactate-; efflux of K+ and P(i) is quantitatively much less than influx of Na+ and Cl-. 5) Measured extracellular osmolality has increased with up to 70 mOsm/l after 30 min of ischaemia. The increase of extracellular [lactate-], [Na+], [K+], [Cl-], [P(i)] and the decrease of [glucose] account for the change of osmolality measured. 6) Summation of the electrical charges associated with measured increase of extracellular [lactate-], [Na+], [K+], [Cl-], [P(i)] shows a surplus of negative charge, which almost equals extracellular [lactate-], suggesting an equally large increase of osmotically inactive H+ as the compensatory ion. 7) Blockade of anaerobic metabolism with iodoacetic acid (IAA) reduces efflux of lactate and P(i) but greatly amplifies influx of sodium and chloride and efflux of potassium.

The origin of increased cytoplasmic calcium upon reversal of the Na+/Ca(2+)-exchanger in isolated rat ventricular myocytes.

Reversal of the driving force of the Na+/Ca(2+)-exchanger (delta Gexch) by a sufficiently large change of the transsarcolemmal electrochemical potential of sodium and calcium causes a transient increase of cytoplasmic calcium ([Ca2+]i). The objective of this study was to investigate the origin of this transient increase of calcium. In isolated quiescent rat ventricular myocytes delta Gexch was abruptly changed by reduction of extracellular sodium ([Na+]o), with or without a simultaneous increase of potassium ([K+]o) or calcium ([Ca2+]i). [Ca2+]i was measured with indo-1. A particular change of delta Gexch induced either by reduction of [Na+]o alone or in combination with increase of [Ca2+]o, produced a transient increase of [Ca2+]i of the same magnitude with a maximum after around 30s. The response of [Ca2+]i was insensitive to verapamil, but was greatly reduced by ryanodine, thapsigargin and caffeine, indicating a large contribution originating from the sarcoplasmic reticulum (SR). The magnitude of the response of [Ca2+]i and also the contribution from SR increased with increasing change of delta Gexch. A particular change of delta Gexch. Induced by a reduction of [Na+]o in combination with membrane depolarization (increase of [K+]o) increased the response of [Ca2+]i, compared that induced by reduction of [Na+]o alone at the same change of delta Gexch. This effect increased with the degree of depolarization, and was completely abolished by verapamil. Also in depolarized cells the response of [Ca2+]i was reduced by ryanodine. However, the contribution from SR to the response did not depend on the degree of depolarization, but only on the magnitude of the change of delta Gexch. Inhibition of the Na+/Ca(2+)-exchanger by Ni2+ almost completely abolished the response of [Ca2+]i to reduction of [Na+]o. Restitution of [Na+]o during the course of the calcium response greatly accelerated the rate of decay of [Ca2+]i. It is concluded that in quiescent rat ventricular myocytes, a large part of the transient increase of cytoplasmic calcium associated with reversal of the driving force of the Na+/Ca(2+)-exchanger originates from SR. Reversal of the exchanger combined with sustained depolarization increased the transient of [Ca2+]i, but the extra influx of calcium associated with depolarization did not affect the contribution from SR.

Energy-dependent transport of calcium to the extracellular space during acute ischemia of the rat heart.

OBJECTIVE: Acute ischemia is associated with rapidly decreasing contractility and Ca2+-transients. Diastolic intracellular Ca2+, however, only mildly increases until development of contracture. The purpose of this study was to investigate whether changes of cellular calcium handling during the early phase of ischemia are associated with active sarcolemmal calcium transport. METHODS: Changes of extracellular concentration of calcium ([Ca2+]o) and tetramethylammonium ([TMA+]o), to estimate extracellular space, were simultaneously measured with ion-specific electrodes in the globally ischemic rat heart. The magnitude and direction of sarcolemmal calcium transport were calculated from [Ca2]o corrected for changed extracellular water content. Energy dependence of sarcolemmal calcium transport was investigated by application of iodoaceticacid (IAA) to inhibit anaerobic glycolysis, and the involvement of the sarcoplasmic reticulum (SR) was studied by application of thapsigargin. The effect of anoxia and thapsigargin on cytosolic and SR calcium was studied in isolated myocytes with the fluorescent indicator indo-1. RESULTS: [Ca2+]o increased and extracellular space gradually decreased in the ischemic intact heart. During the first 7 min, the increase of [Ca2+]o was associated with net outward transport of calcium. Subsequently, net re-uptake occurred. IAA completely abolished outward transport and influx was accelerated and enhanced. Application of thapsigargin attenuated outward transport. In electrically-stimulated myocytes, anoxia caused little change of diastolic calcium and depletion of SR. Thapsigargin reduced both calcium transient amplitude and SR calcium without affecting diastolic calcium. During three successive short episodes of ischemia/reperfusion (preconditioning), outward transport of calcium progressively decreased. CONCLUSION: During the early phase of global ischemia, energy dependent transport of calcium to the extracellular space occurs. At least part of this calcium originates from SR. During the later stage of ischemia, re-uptake of calcium occurs, which is associated with development of contracture.

The change of the free energy of ATP hydrolysis during global ischemia and anoxia in the rat heart. Its possible role in the regulation of transsarcolemmal sodium and potassium gradients.

The timecourse of change of the cytoplasmic free energy of ATP hydrolysis during acute global ischemia and during anoxic perfusion was determined in the isolated rat heart. The timecourse of change of transsarcolemmal Na+ and K+ gradients during anoxia, and of extracellular K+ during ischemia were measured. The free energy of ATP hydrolysis was calculated from the equilibrium of the creatinekinase reaction, taking into account the pH-dependence of the equilibrium constant, and intracellular inorganic phosphate. In control aerobic hearts the mean free energy of ATP hydrolysis was 55.2 kJ/mol. Both during ischemia and anoxia it declines biphasically. The first rapid phase terminates within 4 min into a plateau of about 46 kJ/mol. The duration of this plateau is shorter during anoxia than during ischemia. The second phase of decrease starts after 6 to 8 min during anoxia and after 15 to 20 min during ischemia. After 30 min of anoxia the free energy of ATP hydrolysis has decreased to 31 kJ/mol and after 30 min of ischemia a value of 35.5 kJ/mol is reached. The timecourses of change of measured intracellular Na+ and K during anoxia and of extracellular K+ during ischemia were also biphasic. During anoxia the loss of intracellular K+ was almost equal to the gain of intracellular Na+ at any point. Based on the assumption that the sodium pump is in thermodynamic equilibrium or near-equilibrium during anoxia and ischemia, the time-course of change of Na+ and K+ gradients during anoxia and of extracellular K+ during ischemia were calculated from the respective timecourses of change of the free energy of ATP hydrolysis. Good agreement was observed between calculated and measured changes of Na+ and K+ gradients. It is concluded that the magnitude and direction of change of transsarcolemmal ion-gradients during anoxia and ischemia may be under direct thermodynamic control of myocardial energy metabolism.

Transmural inhomogeneity of energy metabolism during acute global ischemia in the isolated rat heart: dependence on environmental conditions.

Cardiac energy metabolism is one of the earliest metabolic activities affected when either anoxia or ischemia are induced, as evidenced by the rapid decline of the tissue high-energy phosphate content of creatinephosphate (CrP) and ATP. Several reports deal with the spatial inhomogeneity of these changes and it is generally found, that the subendocardium is more sensitive to ischemia than the subepicardium. The metabolic transmural gradients observed during in vivo ischemia were attributed to both variations in wall tension and collateral flow. Lowe et al. recently presented evidence that in addition to these variations the higher vulnerability of the subendocardium to ischemia could be secondary to an increased metabolic rate.

Norepinephrine induces action potential prolongation and early afterdepolarizations in ventricular myocytes isolated from human end-stage failing hearts.

AIMS: Congestive heart failure is characterized by high levels of norepinephrine which is considered to be arrhythmogenic. It is unclear whether increased norepinephrine is only a marker of the severity of heart failure or whether it directly triggers ventricular arrhythmias. METHODS AND RESULTS: Ventricular myocytes were isolated from eight explanted hearts of patients with end-stage heart failure (ischaemic or dilated cardiomyopathy). With the whole-cell configuration of the patch-clamp technique the effect of 1 micromol x l(-1)norepinephrine on action potentials and membrane currents was studied. The cells had a membrane capacitance of 256 +/- 25 pF (n = 26) and action potential duration (APD90) during control conditions was 620 +/- 45 ms at 1 Hz (n = 14). Norepinephrine induced action potential prolongation in all cells and early afterdepolarizations in 50% of them. Norepinephrine significantly increased the calcium current but had no effect on the delayed rectifier current, the inward rectifier current or the transient outward current. Norepinephrine also significantly increased the steady-state calcium window-current measured between -40 and 0 mV. CONCLUSIONS: In contrast to many animal species, norepinephrine induces action potential prolongation in ventricular myocytes from human failing hearts, as well as early afterdepolarization, by an increase in both the calcium peak current and window current. Thus norepinephrine seems to be an important arrhythmogenic factor in congestive heart failure.

Arrhythmogenesis in heart failure.

In a rabbit model of heart failure produced by combined pressure and volume overload, nonsustained ventricular tachycardias developed in 15 of 23 failing rabbits. Sinus rate was increased in rabbits dying suddenly, but was decreased in survivors. This also was true in isolated preparations. Microelectrode recordings from ventricular trabeculae both from patients with end-stage failure and from failing rabbits showed that in half of the preparations, delayed afterdepolarizations and triggered activity occurred, but only in the presence of norepinephrine and a lowered extracellular K+ concentration of 3 mM. This was due to spontaneous release of Ca2+ from the sarcoplasmic reticulum.