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.

On the stimulation of the light-induced proton uptake by uncoupling aminoacridine derivatives in spinach chloroplasts.

1. Light-induced proton uptake by spinach chloroplasts is enhanced several-fold by 9-(4-diethylamino-1-methylbutylamino)-6-chloro-2-methoxyacridine (atebrin). This stimulation does not depend on the chlorophyll concentration. The amount of extra protons taken up in the presence of atebrin is determined by the pKa values of atebrin and the pH of the incubation medium. 2. Both the stimulation of the proton uptake and the maximal binding capacity for atebrin is sensitive to uncouplers. However, the ratio of bound to free atebrin does not depend on the presence of uncoupler up to the saturating atebrin concentration. 3. From simultanious kinetic measurements of atebrin fluorescence and proton movement it seems that after binding of the completely protonated atebrin the dye and the protons can move separately. This can also be inferred from the spectral behaviour of atebrin in illuminated chloroplasts. 4. The stimulation of the proton uptake by atebrin does not depend on the presence of salts in the incubation medium. However, the 'saturating' atebrin concentration increases strongly with increasing salt concentration in the medium. 5. It is concluded that the interaction of atebrin and other acridines with energized chloroplasts most likely occurs at the level of the membrane proper. 6. It is proposed that uncoupling by atebrin is a consequence of the creation of a high proton activity at the periphery of the thylakoid membrane, which opposes a proton gradient across the membrane. The uncoupling by atebrin is not of the protonophoric type according to this mechanism.

[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.

Determination of creatine kinase isoenzyme MB (CK-MB): comparison of methods and clinical evaluation.

An ion-exchange chromatography method based on the method of Mercer is advocated for the routine determination of serum CK-MB. This method has some prominent advantages over other methods with which it is compared, and which include electrophoresis and an immunological technique. This method proves to be reliable and highly reproducible, while it allows a rather large number of samples to be analyzed within a relatively short period of time. Some parameters of the release pattern of CK-MB after acute myocardial infarction are characterized: normal values, time of first rise, time of peak value and rate-constant of inactivation. The clinical significance of serum CK-MB determination is evaluated.

beta-Blockade and acute myocardial infarction.

Results from experimental and clinical studies suggest that beta-blockade may have beneficial effects in acute myocardial infarction. These effects relate to decrease of cardiac work and improvement of metabolism without deleterious effects on perfusion of the ischemic myocardium. Several well designed randomized controlled studies with beta blockers have been carried out in the acute phase of myocardial infarction. Although beneficial effects could be demonstrated in some subsets, most studies however included small numbers of patients, thus indicating that more large scale studies are necessary to assess the efficacy of beta blockers in reducing infarct size and hospital mortality.

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.

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.

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.

Boehringer immunoinhibition procedure for creatine kinase-MB evaluated and compared with column ion-exchange chromatography.

In determination of creatine kinase isoenzyme MB (CK-MB), the Boehringer immunoinhibition method gives a high and variable blank activity as compared with column-chromatography. Thus a correction must be applied. Furthermore, a second correction of 1% of total creatine kinase activity is necessary to compensate for nonspecific creatine phosphate-dependent activity. As a consequence, two immunoinhibition determinations--one for CK-MB and one for blank activity--and a determination of total creatine kinase are required. Use of the manufacturer's diagnostic criteria, on the basis of which suspected myocardial infarction is confirmed or eliminated, leads to a high frequency of false-negative conclusions.

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.

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.

Evaluation of a radioenzymic kit for determination of plasma catecholamines.

We evaluated a commercially available reagent test-kit (Upjohn) for simultaneously determining norepinephrine, epinephrine, and dopamine in 50-microL of plasma. The three catecholamines are enzymically converted into the radioactive O-methyl derivatives and separated by thin-layer chromatography. Day-to-day precision (CV) was 11, 10, and 14% for norepinephrine, epinephrine, and dopamine, respectively. The relationship between concentration of catecholamine and radioactivity (net dpm) was linear to at least 8 ng (corresponding to 1 mumol/L in plasma). Sensitivity was approximately 2 pg for dopamine, 1 pg for each of the other two catecholamines. Under our conditions, epinephrine was not quite completely resolved from the other two fractions. Catecholamine values determined in normal humans, after 30 min supine and after normal laboratory activity, agreed well with those found by other investigators. Correlation was good between kit results and those obtained in another laboratory that used self-prepared reagents and "high-performance" liquid chromatography for the separation.