Enhancing mitochondrial Ca2+ uptake in myocytes from failing hearts restores energy supply and demand matching

Mitochondrial ATP production is continually adjusted to energy demand through coordinated increases in oxidative phosphorylation and NADH production mediated by mitochondrial Ca2+([Ca2+]m). Elevated cytosolic Na+ impairs [Ca2+]m accumulation during rapid pacing of myocytes, resulting in a decrease in NADH/NAD+ redox potential. Here, we determined 1) if accentuating [Ca2+]m accumulation prevents the impaired NADH response at high [Na+]i; 2) if [Ca2+]m handling and NADH/NAD+ balance during stimulation is impaired with heart failure (induced by aortic constriction); and 3) if inhibiting [Ca2+]m efflux improves NADH/NAD+ balance in heart failure. [Ca2+]m and NADH were recorded in cells at rest and during voltage clamp stimulation (4Hz) with either 5 or 15 mmol/L [Na+]i. Fast [Ca2+]m transients and a rise in diastolic [Ca2+]m were observed during electric stimulation. [Ca2+]m accumulation was [Na+]i-dependent; less [Ca2+]m accumulated in cells with 15 Na+ versus 5 mmol/L Na+ and NADH oxidation was evident at 15 mmol/L Na+, but not at 5 mmol/L Na+. Treatment with either the mitochondrial Na+/Ca2+ exchange inhibitor CGP-37157 (1 micromol/L) or raising cytosolic Pi (2 mmol/L) enhanced [Ca2+]m accumulation and prevented the NADH oxidation at 15 mmol/L [Na+]i. In heart failure myocytes, resting [Na+]i increased from 5.2+/-1.4 to 16.8+/-3.1mmol/L and net NADH oxidation was observed during pacing, whereas NADH was well matched in controls. Treatment with CGP-37157 or lowering [Na+]i prevented the impaired NADH response in heart failure. We conclude that high [Na+]i (at levels observed in heart failure) has detrimental effects on mitochondrial bioenergetics, and this impairment can be prevented by inhibiting the mitochondrial Na+/Ca2+ exchanger.     

Dominant role of mitochondria in calcium homeostasis of single rat pituitary corticotropes

The rise in cytosolic free Ca2+ concentration ([Ca2+]i) is the major trigger for secretion of ACTH from pituitary corticotropes. To better understand the shaping of the Ca2+ signal in corticotropes, we investigated the mechanisms regulating the depolarization-triggered Ca2+ signal using patch-clamp techniques and indo-1 fluorometry. The rate of cytosolic Ca2+ clearance was unaffected by inhibitors of Na+/Ca2+ exchanger or plasma membrane Ca2+-ATPase (PMCA), slightly slowed by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, but dramatically slowed by mitochondrial uncouplers or inhibitor of mitochondrial uniporter. Measurements with rhod-2 revealed that depolarization-triggered increase in mitochondrial Ca2+ concentration. Thus, mitochondria have a dominant role in cytosolic Ca2+ clearance. Using the Mn2+ quench technique, we found the presence of a continuous basal Ca2+ influx in corticotropes. This basal Ca2+ influx was balanced by the combined actions of mitochondrial uniporter and PMCA and SERCA pumps. Inhibition of the mitochondrial uniporter or PMCA or SERCA pumps elevated basal [Ca2+]i. Using membrane capacitance measurement, we found that the change in the shape of the depolarization-triggered Ca2+ signal after mitochondrial inhibition was associated with enhancement of the exocytotic response. Thus, mitochondria have a dominant role in the regulation of Ca2+ signal and exocytosis in corticotropes.     

Transient receptor potential channel 6-mediated, localized cytosolic [Na+] transients drive Na+/Ca2+ exchanger-mediated Ca2+ entry in purinergically stimulated aorta smooth muscle cells

The Na+/Ca2+ exchanger (NCX) is increasingly recognized as a physiological mediator of Ca2+ influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca2+ influx by the NCX (1) is the primary mechanism of Ca2+ entry in purinergically stimulated rat aorta smooth muscle cells and (2) requires functional coupling with transient receptor potential channel 6 nonselective cation channels. Using the Na+ indicator CoroNa Green, we now directly observed and characterized the localized cytosolic [Na+] ([Na+]i) elevations that have long been hypothesized to underlie physiological NCX reversal but that have never been directly shown. Stimulation of rat aorta smooth muscle cells caused both global and monotonic [Na+]i elevations and localized [Na+]i transients (LNats) at the cell periphery. Inhibition of nonselective cation channels with SKF-96365 (50 micromol/L) and 2-amino-4-phosphonobutyrate (75 micromol/L) reduced both global and localized [Na+]i elevations in response to ATP (1 mmol/L). This effect was mimicked by expression of a dominant negative construct of transient receptor potential channel 6. Selective inhibition of NCX-mediated Ca2+ entry with KB-R7943 (10 micromol/L) enhanced the LNats, whereas the global cytosolic [Na+] signal was unaffected. Inhibition of mitochondrial Na+ uptake with CGP-37157 (10 micromol/L) increased both LNats and global cytosolic [Na+] elevations. These findings directly demonstrate NCX regulation by LNats, which are restricted to subsarcolemmal, cytoplasmic microdomains. Analysis of the LNats, which facilitate Ca2+ entry via NCX, suggests that mitochondria limit the cytosolic diffusion of LNats generated by agonist-mediated activation of transient receptor potential channel 6-containing channels.     

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.     

Cardiac submembrane [Na+] transients sensed by Na+-Ca2+ exchange current

Na+ influx via INa during cardiac action potentials can raise bulk [Na+]i by 10 to 15 micromol/L. However, larger rises in submembrane [Na+] ([Na+]sm) local to Na+-Ca2+ exchangers (NCX) could enhance Ca2+ influx via NCX (and Ca2+-induced Ca2+ release). We tested whether INa could increase [Na+]sm, using NCX current (INCX) as a biosensor in rabbit ventricular myocytes (with [Ca2+]i buffered, [Na+]i=10 mmol/L, and other currents blocked). We measured INCX as early as 5 ms after INa. Prior INa activation did not affect INCX at physiological membrane potentials (Em=-100 to +50 mV), but for Em >+50 mV (where INCX is especially sensitive to [Na+]i), INCX shifted outward. At 5 ms and +100 mV, INa shifted INCX outward by 0.23 A/F (corresponding to Delta[Na+]sm=0.24 mmol/L). The effect of INa dissipated with a time constant of approximately 15 ms. Thus, the impact of INa on NCX is almost undetectable at physiological Em and short lived. This suggests that INa effects on excitation-contraction coupling (via outward INCX) are minimal and limited to early during the action potential. However, local Delta[Na+]sm during INa may be 60 times higher than bulk Delta[Na+]i.     

On the mechanism of the failure of mitochondrial function in isolated guinea-pig myocytes subjected to a Ca2+ overload

OBJECTIVE: The influence of agents that inhibit the movement of Ca2+ across the mitochondrial membrane or Ca2+ dependent changes to this membrane upon the response of isolated ventricular myocytes to a Ca2+ overload has been investigated. METHODS: The changes of intracellular Ca2+ and Mg2+ ([Ca2+]i, [Mg2+]i) (as reflected by cellular ATP), mitochondrial membrane potential (psi m) and NADH was measured upon the response of isolated ventricular myocytes to a Ca2+ overload. RESULTS: A slow depolarization of psi m during Ca2+ depletion and its prompt recovery on Ca2+ repletion were unaffected by ruthenium red, clonazepam, CGP-37157 which is a high potent inhibitor of the mitochondrial Na+/Ca2+ antiport or cyclosporin A but a large delayed sustained depolarization was inhibited. The slow small fall in [Mg2+]i on Ca2+ depletion and a rapid recovery on Ca2+ repletion were unaffected by ruthenium red, clonazepam, CGP-37157 or cyclosporin A. A delayed sustained larger rise in [Mg2+]i was inhibited. The marked sustained fall in NADH autofluorescence that occurs on Ca2+ overload was attenuated and transient in the presence of ruthenium red, CGP-37157 and cyclosporin A. CONCLUSION: These results are consistent with an increase in Ca2+ cycling across the mitochondrial membrane provoked by the combined Na+ and Ca2+ overload of cardiac myocytes, causing a depolarization sufficient to uncouple respiration and lead to the depletion of cellular ATP.     

Stimulation of muscarinic receptors raises free intracellular Ca2+ concentration in rat ventricular myocytes

The effect of carbachol on free intracellular calcium concentration, ([Ca2+]i) and on intracellular hydrogen concentration (pHi) was determined from fluorescence signals obtained from rat ventricular myocytes. Application of carbachol (300 mumol/l) to quin2-loaded myocytes bathed in 2 mmol/l Ca2+-containing solution caused [Ca2+]i to increase within 7-10 minutes from 182 +/- 9 to 212 +/- 11 nmol/l (n = 4). Carbachol acted via stimulation of muscarinic receptors because atropine (1 mumol/l) either prevented or abolished the increase in [Ca2+]i. Carbachol also produced a positive inotropic effect in rat papillary muscles contracting isometrically at a frequency of 0.5 Hz and enhanced contracture in resting preparations in the presence of high extracellular Ca2+ concentration ([Ca2+]o) (20 mmol/l). The effect of carbachol on [Ca2+]i was dependent on [Ca2+]o. In the presence of 10 mmol/l [Ca2+]o, the increase in [Ca2+]i was about two times that elicited by carbachol when bath [Ca2+]o was 2 mmol/l. Reduction of [Ca2+]o to 50 mumol/l abolished the carbachol effect but did not prevent caffeine-induced Ca2+ release. The carbachol-induced rise in [Ca2+]i remained unchanged in the presence of either 10 mmol/l caffeine or 1 mumol/l ryanodine. In the absence of extracellular Na+ concentration [( Na+]o), carbachol no longer produced an increase in [Ca2+]i of cardiomyocytes and failed to enhance Na+-withdrawal contracture of the rat papillary muscle. In contrast to the effect on [Ca2+]i, carbachol did not produce any change in pHi as determined from fluorescence signals obtained from rat ventricular myocytes loaded with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adverse bioenergetic consequences of Na+-Ca2+ exchanger-mediated Ca2+ influx in cardiac myocytes

BACKGROUND: In heart failure, the Na+-Ca2+ exchanger (NCX) is upregulated and mediates Ca2+ influx (instead of efflux) during the cardiac action potential. Although this partly compensates for impaired sarcoplasmic reticulum Ca2+ release and supports inotropy, the energetic consequences have never been considered. Because NCX-mediated Ca2+ influx is rather slow and mitochondrial Ca2+ uptake (which stimulates NADH production by the Krebs cycle) is thought to be facilitated by high Ca2+ gradients in a "mitochondrial Ca2+ microdomain," we speculated that NCX-mediated Ca2+ influx negatively affects the bioenergetic feedback response. Methods and Results- With the use of a patch-clamp-based approach in guinea-pig myocytes, cytosolic and mitochondrial Ca2+ ([Ca2+](c) and [Ca2+](m), respectively) was determined within the same cell after varying Ca2+ influx via L-type Ca2+ channels (I(Ca,L)) or the NCX. The efficiency of mitochondrial Ca2+ uptake, indexed by the slope of plotting [Ca2+](m) against [Ca2+](c) during each Ca2+ transient, was maximal during I(Ca,L)-triggered sarcoplasmic reticulum Ca2+ release. Depletion of sarcoplasmic reticulum Ca2+ load and increased contribution of the NCX to cytosolic Ca2+ influx independently reduced the efficiency of mitochondrial Ca2+ uptake. The upstroke velocity of cytosolic Ca2+ transients closely correlated with the efficiency of mitochondrial Ca2+ uptake. Despite comparable [Ca2+](c), sarcoplasmic reticulum Ca2+ release, but not NCX-mediated Ca2+ influx, led to stimulation of Ca2+-sensitive dehydrogenases of the Krebs cycle. Conclusions- Increased contribution of the NCX to cytosolic Ca2+ transients, which occurs in cardiac myocytes from failing hearts, impairs mitochondrial Ca2+ uptake and the bioenergetic feedback response. This mechanism could contribute to energy starvation of failing hearts.     

Transgenic CaMKIIdeltaC overexpression uniquely alters cardiac myocyte Ca2+ handling: reduced SR Ca2+ load and activated SR Ca2+ release

Ca2+/calmodulin-dependent protein kinase II (CaMKII) delta is the predominant cardiac isoform, and the deltaC splice variant is cytoplasmic. We overexpressed CaMKIIdeltaC in mouse heart and observed dilated heart failure and altered myocyte Ca2+ regulation in 3-month-old CaMKIIdeltaC transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca2+]i transient amplitude, and diastolic [Ca2+]i were all reduced by approximately 50% in TG versus WT. This is explained by >50% reduction in SR Ca2+ content in TG versus WT. Peak Ca2+ current (ICa) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca2+ load and diastolic [Ca2+]i, fractional SR Ca2+ release was increased and resting spontaneous SR Ca2+ release events (Ca2+ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca2+ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca2+ spark frequency and ICa, consistent with direct CaMKII activation of ryanodine receptors (and ICa) in TG. The rate of [Ca2+]i decline during caffeine exposure was faster in TG, indicating enhanced Na+-Ca2+ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca2+ leak (via sparks), reduced SR Ca2+-ATPase expression, and increased Na+-Ca2+ exchanger explain the reduced diastolic [Ca2+]i and SR Ca2+ content in TG. We conclude that CaMKIIdeltaC overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure.     

Intracellular Na+ regulation in cardiac myocytes

Intracellular [Na+] ([Na+]i) is regulated in cardiac myocytes by a balance of Na+ influx and efflux mechanisms. In the normal cell there is a large steady state electrochemical gradient favoring Na+ influx. This potential energy is used by numerous transport mechanisms, including Na+ channels and transporters which couple Na+ influx to either co- or counter-transport of other ions and solutes. Six sarcolemmal Na+ influx pathways are discussed in relatively quantitative terms: Na+ channels, Na+/Ca2+ exchange, Na+/H+ exchange, Na+/Mg2+ exchange, Na+/HCO3- cotransport and Na+/K+/2Cl- cotransport. Under normal conditions Na+/Ca2+ exchange and Na+ channels are the dominant Na+ influx pathways, but other transporters may become increasingly important during altered conditions (e.g. acidosis or cell volume stress). Mitochondria also exhibit Na+/Ca2+ antiporter and Na+/H+ exchange activity that are important in mitochondrial function. These coupled fluxes of Na+ with Ca2+, H+ and HCO3- make the detailed understanding of [Na+]i regulation pivotal to the understanding of both cardiac excitation-contraction coupling and pH regulation. The Na+/K+-ATPase is the main route for Na+ extrusion from cells and [Na+]i is a primary regulator under physiological conditions. [Na+]i is higher in rat than rabbit ventricular myocytes and the reason appears to be higher Na+ influx in rat with a consequent rise in Na+/K+-ATPase activity (rather than lower Na+/K+-ATPase function in rat). This has direct functional consequences. There may also be subcellular [Na+]i gradients locally in ventricular myocytes and this may also have important functional implications. Thus, the balance of Na+ fluxes in heart cells may be complex, but myocyte Na+ regulation is functionally important and merits focused attention as in this issue.     

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.     

Na+-Ca2+ exchange in cultured vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) contract as intracellular free calcium ([Ca2+]i) rises. While Na+-Ca2+ exchange has been proposed to contribute to transmembrane Ca2+ flux, its role in cultured VSMC is unknown. Accordingly, we have investigated the role of Na+-Ca2+ exchange in unidirectional and net transmembrane Ca2+ fluxes in cultured rat aortic VSMC under basal conditions and following agonist-mediated stimulation. Transmembrane Ca2+ uptake was significantly increased in response to a low external Na+ concentration ([Na+]o) compared with 140 mM [Na+]o. Na+-dependent Ca2+ uptake in response to low [Na+]o was further increased by intracellular Na+ loading by preincubation of the VSMC with 1 mM ouabain. Under steady-state conditions, Ca2+ content varied inversely with [Na+]o, increasing from 1.0 nmol Ca2+/mg protein at 140 mM [Na+]o to 4.0 nmol Ca2+/mg protein at 20 mM [Na+]o. Increasing [K+]o to 55 mM also enhanced Na+-dependent Ca2+ influx. Augmentation of Ca2+ uptake with K+ depolarization was not significantly inhibited by the calcium channel antagonist verapamil. Transmembrane Ca2+ efflux was increased in response to 130 mM [Na+]o compared with zero [Na+]o (iso-osmotic substitution with choline+), and was further stimulated by the vasoconstrictor angiotensin II, which is known to elevate [Ca2+]i. These changes in [Ca2+]i were studied directly using fura-2 fluorescence measurements. Elevated [Ca2+]i levels returned to baseline more rapidly in the presence of normal (130 mM) [Na+]o compared with zero [Na+]o (iso-osmotic substitution with choline+). These findings suggest that a bidirectional Na+-Ca2+ exchange mechanism is present in cultured rat aortic VSMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation of mitochondrial and cytosolic free calcium to cardiac myocyte recovery after exposure to anoxia.

Mitochondrial calcium overload has been suggested as a marker for irreversible injury in the ischemic heart. A new technique is used to measure dynamic changes in mitochondrial free calcium concentration ([Ca2+]m) in electrically stimulated (0.2 Hz) adult rat cardiac myocytes during exposure to anoxia and reoxygenation. Cells were incubated with indo-1 AM, which distributes in both the cytosol and mitochondria. After Mn2+ quenching of the cytosolic signal, cells were exposed to anoxia, and the residual fluorescence was monitored. [Ca2+]m averaged 94 +/- 3 nM (n = 16) at baseline, less than the baseline diastolic cytosolic free calcium concentration ([Ca2+]c, 124 +/- 4 nM, n = 12), which was measured in cells loaded with the pentapotassium salt of indo-1. [Ca2+]m and [Ca2+]c rose steadily only after the onset of ATP-depletion rigor contracture. At reoxygenation 35 minutes later, [Ca2+]c fell rapidly to preanoxic levels and then often showed a transient further rise. In contrast, [Ca2+]m showed only a slight transient fall and a secondary rise at reoxygenation. At reoxygenation, cells immediately either recovered, demonstrating partial relengthening and retaining their rectangular shape and response to stimulation, or they hypercontracted to rounded dysfunctional forms. Recovery occurred only in cells in which [Ca2+]m or [Ca2+]c remained below 250 nM before reoxygenation. Early during reoxygenation, [Ca2+]m remained higher in cells that hypercontracted (305 +/- 36 nM) than in cells that recovered (138 +/- 9 nM, p less than 0.05), whereas [Ca2+]c did not differ between the two groups (156 +/- 10 versus 128 +/- 10 nM, respectively; p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+/Ca2+ exchange current (I(Na/Ca)) and sarcoplasmic reticulum Ca2+ release in catecholamine-induced cardiac hypertrophy

OBJECTIVE: Catecholamines that accompany acute physiological stress are also involved in mediating the development of hypertrophy and failure. However, the cellular mechanisms involved in catecholamine-induced cardiac hypertrophy, particularly Ca2+ handling, are largely unknown. We therefore investigated the effects of cardiac hypertrophy, produced by isoprenaline, on I(Na/Ca) and sarcoplasmic reticulum (SR) function in isolated myocytes. METHODS: I(Na/Ca) was studied in myocytes from Wistar rats, using descending (+80 to -110 mV) voltage ramps under steady state conditions. Myocytes were also loaded with fura-2 and either field stimulated or voltage clamped to assess [Ca2+]i and SR Ca2+ content. RESULTS: Ca2+-dependent, steady state I(Na/Ca) density was increased in hypertrophied myocytes (P<0.05). Ca2+ release from the SR was also increased, whereas resting [Ca2+]i and the rate of decline of [Ca2+]i to control levels were unchanged. SR Ca2+ content, estimated by using 10.0 mmol/l caffeine, was also significantly increased in hypertrophied myocytes, but only when myocytes were held and stimulated from their normal resting potential (-80 mV) but not from -40 mV. However, the rate of decline of caffeine-induced Ca2+ transients or I(Na/Ca) was not significantly different between control and hypertrophied myocytes. Ca2+-dependence of I(Na/Ca), examined by comparing the slope of the descending phase of the hysteresis plots of I(Na/Ca) vs. [Ca2+]i, was also similar in the two groups of cells. CONCLUSION: Data show that SR Ca2+ release and SR Ca2+ content were increased in hypertrophied myocytes, despite an increase in the steady state I(Na/Ca) density. The observation that increased SR function occurred only when myocytes were stimulated from -80 mV suggests that Na+ influx may play a role in altering Ca2+ homeostasis in hypertrophied cardiac muscle, possibly through increased reverse Na+/Ca2+ exchange, particularly at low stimulation frequencies.     

The loading of fura-2 into mitochondria in the intact perfused rat heart and its use to estimate matrix Ca2+ under various conditions.

When rat hearts were perfused with a medium containing 10 microM fura-2/AM for 1 hr at 37 degrees C a significant amount of the derived fura-2 could be detected in subsequently isolated mitochondria. This procedure allowed the measurement of matrix free Ca2+ concentration ([Ca2+]m) of mitochondria rapidly isolated from whole hearts by a method which avoids artefactual redistribution of Ca2+. [Ca2+]m in mitochondria prepared from control hearts and incubated with respiratory substrates and EGTA was found to be 172 +/- 23 nM (mean +/- S.E.M.). When hearts were subjected to either increased mechanical work or treatment with 1 microM L-epinephrine (for 2 mins) [Ca2+]m increased to 916 +/- 138 nM and 727 +/- 65 nM respectively. The presence of ruthenium red (2.5 microM) in the perfusion medium prior to and during inotropic intervention diminished these increases in [Ca2+]m(to 316 +/- 28 nM and 218 +/- 18 nM respectively) but did not affect control values. Addition of Na+ ions to incubated mitochondria to enhance mitochondrial Ca2+ egress diminished these increases in [Ca2+]m due to pre-treatment with positive inotropes (compared to controls). These changes in [Ca2+]m were broadly parallelled by changes in the active non-phosphorylated form of pyruvate dehydrogenase (PDH) under all circumstances. These results provide further evidence that the activation of PDH by positive inotropes is accomplished by, and at least in part due to, raised mitochondrial matrix free [Ca2+] and that such increases can be maintained in isolated and suitably incubated mitochondria.     

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.     

Inhibition by KB-r7943 of the reverse mode of the Na+/Ca2+ exchanger reduces Ca2+ overload in ischemic-reperfused rat hearts.

Ca2+ influx via the Na+/Ca2+ exchanger (NCX) may lead to Ca2+ overload and myocardial injury in ischemia-reperfusion. Direct evidence that increased cytoplasmic Ca2+ concentration ([Ca2+]i) is mediated by the reverse mode of the NCX is limited, so in the present study the [Ca2+]i dynamics and left ventricular pressure were monitored in perfused beating hearts. The effects of KB-R7943 (KBR), a selective inhibitor of the NCX in the reverse mode, were analyzed during low-Na+ exposure and ischemia-reperfusion. Hearts from Sprague-Dawley rats were retrogradely perfused and loaded with 4 micromol/L fura-2 to measure the fluorescence ratio as an index of [Ca2+]i. To evaluate KBR effects on the reverse mode exchanger, the increase in [Ca2+]i induced by low-Na+ exposure (Na+: 30 mmol/L, 10 mmol/L caffeine pre-treatment) was measured with and without 10 micromol/L KBR (n=5). In another series, the hearts were subjected to 10 min of low-flow ischemia with pacing, followed by reperfusion in the absence (n=6) or in the presence of 10 micromol/L KBR (n=6). Background autofluorescence was subtracted to estimate the ratio in the ischemia-reperfusion protocol. KBR significantly suppressed the increase in [Ca2+]i induced by low-Na+ (40.2 +/- 11.2% of control condition, p=0.014), as well as on increase in diastolic [Ca2+]i during ischemia (% increase from pre-ischemia in [Ca2+]i at 10 min: KBR, 17.9 +/- 6.4%; no KBR, 44.4 +/- 7.7%; p=0.024). After reperfusion, diastolic [Ca2+]i normalized more rapidly in KBR-treated hearts (% increase at 1 min: KBR, 4.5 +/- 7.0%; no KBR, 39.8 +/- 12.2%; p=0.03). Treatment with KBR also accelerated recovery of the rate-pressure product on reperfusion (1 min: KBR, 8,944 +/- 1,554 min(-1) mmHg; no KBR, 4,970 +/- 1,325; p<0.05). Thus, inhibition of the reverse mode exchanger by KBR reduced ischemic Ca2+ overload and possibly improved functional myocardial recovery during reperfusion in a whole heart model.     

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.     

Effects of intracellular acidosis on [Ca2+]i transients, transsarcolemmal Ca2+ fluxes, and contraction in ventricular myocytes

We examined the effects of intracellular acidosis produced by washout of NH4Cl on [Ca2+]i transients (indo-1 fluorescence), cell contraction (video motion detector), and 45Ca and 24Na fluxes in cultured chick embryo ventricular myocytes. Exposure of cells to 10 mM NH4Cl produced intracellular alkalosis (pH 7.6), and subsequent washout resulted in a transient acidosis (pH 6.5). Exposure to 10 mM NH4Cl slightly decreased [Ca2+]i transients but increased the amplitude of cell contraction. Subsequent washout of NH4Cl initially increased diastolic [Ca2+]i and decreased the peak positive and negative d[Ca2+]i/dt, while the amplitude of cell contraction was markedly decreased. Subsequently, peak systolic [Ca2+]i increased with partial recovery of contraction. A similar increase in [Ca2+]i and decrease in contraction after washout of NH4Cl was observed in single paced adult guinea pig ventricular cells. Acidosis decreased 45Ca uptake by sarcoplasmic reticulum vesicles isolated from chick embryo ventricle. However, the [Ca2+]i increase caused by intracellular acidosis was also observed in the presence of 10 mM caffeine, suggesting that altered sarcoplasmic reticulum handling of calcium is not the only mechanism involved. Intracellular acidosis only slightly increased total 24Na uptake under these conditions, an effect resulting from the combination of a stimulation of amiloride-sensitive sodium influx (Na(+)-H+ exchange) and inhibition of sodium influx via Na(+)-Ca2+ exchange, manifested by a significant decrease in 45Ca efflux. Further support for a lack of involvement of an increased [Na+]i in the observed increase in [Ca2+]i during acidosis was low-sodium, nominal 0-calcium extracellular solution, an experimental condition that minimizes the possible effects of Na(+)-H+ exchange and Na(+)-Ca2+ exchange. We conclude that the [Ca2+]i increase caused by intracellular acidosis in cultured ventricular cells is primarily due to changes in [Ca2+]i buffering and [Ca2+]i extrusion, rather than to an increase in transsarcolemmal calcium influx. Intracellular acidosis also markedly decreases the sensitivity of the contractile elements to [Ca2+]i in cultured chick embryonic and adult guinea pig ventricular myocytes.     

Quantitative assessment of the SR Ca2+ leak-load relationship

Increased diastolic SR Ca2+ leak (J(leak)) could depress contractility in heart failure, but there are conflicting reports regarding the J(leak) magnitude even in normal, intact myocytes. We have developed a novel approach to measure SR Ca2+ leak in intact, isolated ventricular myocytes. After stimulation, myocytes were exposed to 0 Na+, 0 Ca2+ solution +/-1 mmol/L tetracaine (to block resting leak). Total cell [Ca2+] does not change under these conditions with Na+-Ca2+ exchange inhibited. Resting [Ca2+]i declined 25% after tetracaine addition (126+/-6 versus 94+/-6 nmol/L; P<0.05). At the same time, SR [Ca2+] ([Ca2+](SRT)) increased 20% (93+/-8 versus 108+/-6 micromol/L). From this Ca2+ shift, we calculate J(leak) to be 12 micromol/L per second or 30% of the SR diastolic efflux. The remaining 70% is SR pump unidirectional reverse flux (backflux). The sum of these Ca2+ effluxes is counterbalanced by unidirectional forward Ca2+ pump flux. J(leak) also increased nonlinearly with [Ca2+](SRT) with a steeper increase at higher load. We conclude that J(leak) is 4 to 15 micromol/L cytosol per second at physiological [Ca2+](SRT). The data suggest that the leak is steeply [Ca2+](SRT)-dependent, perhaps because of increased [Ca2+]i sensitivity of the ryanodine receptor at higher [Ca2+](SRT). Key factors that determine [Ca2+](SRT) in intact ventricular myocytes include (1) the thermodynamically limited Ca2+ gradient that the SR can develop (which depends on forward flux and backflux through the SR Ca2+ ATPase) and (2) diastolic SR Ca2+ leak (ryanodine receptor mediated).