Functional antagonism of ß-adrenoceptor subtypes in the catecholamine-induced automatism in rat myocardium.

BACKGROUND AND PURPOSE: Myocardial automatism and arrhythmias may ensue during strong sympathetic stimulation. We sought to investigate the relevant types of adrenoceptor, as well as the role of phosphodiesterase (PDE) activity, in the production of catecholaminergic automatism in atrial and ventricular rat myocardium. EXPERIMENTAL APPROACH: The effects of adrenoceptor agonists on the rate of spontaneous contractions (automatic response) and the amplitude of electrically evoked contractions (inotropic response) were determined in left atria and ventricular myocytes isolated from Wistar rats. KEY RESULTS: Catecholaminergic automatism was Ca(2+) -dependent, as it required a functional sarcoplasmic reticulum to be exhibited. Although both α- and ß-adrenoceptor activation caused inotropic stimulation, only ß(1) -adrenoceptors seemed to mediate the induction of spontaneous activity. Catecholaminergic automatism was enhanced and suppressed by ß(2) -adrenoceptor blockade and stimulation respectively. Inhibition of either PDE3 or PDE4 (by milrinone and rolipram, respectively) potentiated the automatic response of myocytes to catecholamines. However, only rolipram abolished the attenuation of automatism produced by ß(2) -adrenoceptor stimulation. CONCLUSIONS AND IMPLICATIONS: α- and ß(2) -adrenoceptors do not seem to be involved in the mediation of catecholaminergic stimulation of spontaneous activity in atrial and ventricular myocardium. However, a functional antagonism of ß(1) - and ß(2) -adrenoceptor activation was identified, the former mediating catecholaminergic myocardial automatism and the latter attenuating this effect. Results suggest that hydrolysis of cAMP by PDE4 is involved in the protective effect mediated by ß(2) -adrenoceptor stimulation.

Transient outward potassium current and Ca2+ homeostasis in the heart: beyond the action potential

The present review deals with Ca2+-independent, K+-carried transient outward current (Ito), an important determinant of the early repolarization phase of the myocardial action potential. The density of total Ito and of its fast and slow components (Ito,f and Ito,s, respectively), as well as the expression of their molecular correlates (pore-forming protein isoforms Kv4.3/4.2 and Kv1.4, respectively), vary during postnatal development and aging across species and regions of the heart. Changes in Ito may also occur in disease conditions, which may affect the profile of cardiac repolarization and vulnerability to arrhythmias, and also influence excitation-contraction coupling. Decreased Ito density, observed in immature and aging myocardium, as well as during several types of cardiomyopathy and heart failure, may be associated with action potential prolongation, which favors Ca2+ influx during membrane depolarization and limits voltage-dependent Ca2+ efflux via the Na+/Ca2+ exchanger. Both effects contribute to increasing sarcoplasmic reticulum (SR) Ca2+ content (the main source of contraction-activating Ca2+ in mammalian myocardium), which, in addition to the increased Ca2+ influx, should enhance the amount of Ca2+ released by the SR during systole. This change usually takes place under conditions in which SR function is depressed, and may be adaptive since it provides partial compensation for SR deficiency, although possibly at the cost of asynchronous SR Ca2+ release and greater propensity to triggered arrhythmias. Thus, Ito modulation appears to be an additional mechanism by which excitation-contraction coupling in myocardial cells is indirectly regulated.

Transient outward potassium current and Ca2+ homeostasis in the heart: beyond the action potential.

The present review deals with Ca2+-independent, K+-carried transient outward current (Ito), an important determinant of the early repolarization phase of the myocardial action potential. The density of total Ito and of its fast and slow components (I(to,f) and I(to,s), respectively), as well as the expression of their molecular correlates (pore-forming protein isoforms Kv4.3/4.2 and Kv1.4, respectively), vary during postnatal development and aging across species and regions of the heart. Changes in Ito may also occur in disease conditions, which may affect the profile of cardiac repolarization and vulnerability to arrhythmias, and also influence excitation-contraction coupling. Decreased Ito density, observed in immature and aging myocardium, as well as during several types of cardiomyopathy and heart failure, may be associated with action potential prolongation, which favors Ca2+ influx during membrane depolarization and limits voltage-dependent Ca2+ efflux via the Na+/Ca2+ exchanger. Both effects contribute to increasing sarcoplasmic reticulum (SR) Ca2+ content (the main source of contraction-activating Ca2+ in mammalian myocardium), which, in addition to the increased Ca2+ influx, should enhance the amount of Ca2+ released by the SR during systole. This change usually takes place under conditions in which SR function is depressed, and may be adaptive since it provides partial compensation for SR deficiency, although possibly at the cost of asynchronous SR Ca2+ release and greater propensity to triggered arrhythmias. Thus, Ito modulation appears to be an additional mechanism by which excitation-contraction coupling in myocardial cells is indirectly regulated.

Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation.

Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A), Na+-Ca2+ exchanger (NCX) and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake). To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT) and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW). Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 M), whereas SR-dependent flux was lower with TG-TW (77 vs 81 M). The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively) and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively) in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.

Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation

Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A), Na+-Ca2+ exchanger (NCX) and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake). To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT) and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW). Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 æM), whereas SR-dependent flux was lower with TG-TW (77 vs 81 æM). The relative participations of NCX (12.5 vs 8 percent with TG-TW and Cf-NT, respectively) and SR-A (85 vs 89.5 percent with TG-TW and Cf-NT, respectively) in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.

Electric field stimulation of cardiac myocytes during postnatal development.

Studies on cardiac cell response to electric field stimulation are important for understanding basic phenomena underlying cardiac defibrillation. In this work, we used a model of a prolate spheroidal cell in a uniform external field (Klee and Plonsey, 1976) to predict the threshold electric field (ET) for stimulation of isolated ventricular myocytes of rats at different ages. The model assumes that ET is primarily determined by cell shape and dimensions, which markedly change during postnatal development. Neonatal cells showed very high ET, which progressively decreased with maturation (experimental mean values were 29, 21, 13, and 5.9 and 6.3 V/cm for 3-6, 13-16, 20-21, 28-35, and 120-180 day-old rats, respectively, P < 0.001; theoretical values were 24, 18, 11, 9, and 6 V/cm, respectively). Estimated maximum membrane depolarization at threshold (deltaVT approximately equals 35 mV, under our experimental conditions) was reasonably constant during development, except for cells from 1-mo-old animals, in which deltaVT was lower than at other ages. We conclude that the model reasonably correlates ET with cell geometry and size in most cases. Our results might be relevant for the development of efficient procedures for defibrillation of pediatric patients.

Rest-dependence of twitch amplitude and sarcoplasmic reticulum calcium content in the developing rat myocardium.

Post-rest contractile response was studied in isolated ventricular muscle from rats aged 1 to 90 days. Amplitude of rapid cooling contractures (RCC) was taken as an index of the sarcoplasmic reticulum (SR) Ca2+ content. We observed that: (a) developed tension (per cross-section area) increased with age; (b) time to peak twitch force and relaxation half-time decreased from 87+/-6 to 56+/-2 ms and from 68+/-6 to 36+/-1 ms, respectively, from the neonatal period to adulthood; (c) post-rest twitch potentiation was observed at all ages, with greater relative potentiation in younger preparations, although relative potentiation of [Ca2+]i transient amplitude was similar in young and adult isolated ventricular myocytes; (d) rest did not significantly affect the amplitude of RCC in muscle or caffeine-evoked [Ca2+]i transients in myocytes at any studied age; (e) favoring Ca2+ efflux via Na+-Ca2+ exchange (NCX) during rest reversed twitch potentiation and caused a similar decrease in RCC amplitude ( approximately 40%) at all ages; (f) stimulation of Ca2+ influx via NCX during rest increased RCC amplitude ( approximately 40%) only in immature preparations. However, when this procedure was repeated after partial SR Ca2+ depletion, increase in RCC amplitude was not significantly age-dependent. We conclude that post-rest twitch potentiation is already present early after birth and does not require rest-dependent changes in SR Ca2+ content at any studied age. Our results suggest that NCX is close to equilibrium during rest in both adult and developing rat myocardium, and does not seem to mediate diastolic net Ca2+ fluxes which may affect the SR Ca2+ content.

Role of acetylcholine in electrical stimulation-induced arrhythmia in rat isolated atria.

In this study, we used the spontaneously beating, isolated rat right atrium as an in vitro model to study arrhythmogenic effects of electrical stimulation. A tetrapolar platinum electrode was used for stimulation and recording of atrial electrical activity at 36.5 degrees C (spontaneous rate, 4.9+/-0.3 Hz). A flutter-like pattern of arrhythmia was reproducibly induced by application of stimulus trains (250 pulses, 66.7 Hz). Arrhythmia was characterized by regular and very short cycle length (40-70 ms), each episode lasting from 3 s to >5 min. In control conditions, application of one to five pulse trains was sufficient to induce arrhythmia. However, atropine (but not propranolol) completely blocked arrhythmia induction (10-15 consecutive trains were ineffective). The ability of electrical stimulation to evoke arrhythmia was restored after atropine washout. A milder stimulation protocol (30 pulses, 50 Hz), which was unable to evoke arrhythmia in control conditions, was fully effective in the presence of 1 microM acetylcholine (ACh). Furthermore, a similar flutter-like pattern could be induced in isolated left atria in the presence of ACh. Our results point out an arrhythmogenic effect of neurally released ACh in the isolated right atrium on atrial electrical stimulation.

Effect of ryanodine on sinus node recovery time determined in vitro.

Evidence has indicated that the sarcoplasmic reticulum (SR) might be involved in the generation of spontaneous electrical activity in atrial pacemaker cells. We report the effect of disabling the SR with ryanodine (0.1 microM) on the sinus node recovery time (SNRT) measured in isolated right atria from 4-6-month-old male Wistar rats. Electrogram and isometric force were recorded at 36.5 degree C. Two methods for sinus node resetting were used: a) pulse: a single stimulus pulse interpolated at coupling intervals of 50, 65 or 80% of the regular spontaneous cycle length (RCL), and b) train: a 2-min train of pulses at intervals of 50, 65 or 80% of RCL. Corrected SNRT (cSNRT) was calculated as the difference between SNRT (first spontaneous cycle length after stimulation interruption) and RCL. Ryanodine only slightly increased RCL (<10%), but decreased developed force by 90%. When the pulse method was used, cSNRT ( approximately 40 ms), which represents intranodal/atrial conduction time, was independent of the coupling interval and unaffected by ryanodine. However, cSNRT obtained by the train method was significantly higher for shorter intervals between pulses, indicating the occurrence of overdrive suppression. In this case, ryanodine prolonged cSNRT in a rate-dependent fashion, with a greater effect at shorter intervals. These results indicate that: a) a functional SR, albeit important for force development, does not seem to play a major role in atrial automaticity in the rat; b) disruption of cell Ca2+ homeostasis by inhibition of SR function does not appear to affect conduction; however, it enhances overdrive-induced depression of sinusal automaticity.

Effect of ryanodine on sinus node recovery time determined in vitro

Evidence has indicated that the sarcoplasmic reticulum (SR) might be involved in the generation of spontaneous electrical activity in atrial pacemaker cells. We report the effect of disabling the SR with ryanodine (0.1 µM) on the sinus node recovery time (SNRT) measured in isolated right atria from 4-6-month-old male Wistar rats. Electrogram and isometric force were recorded at 36.5oC. Two methods for sinus node resetting were used: a) pulse: a single stimulus pulse interpolated at coupling intervals of 50, 65 or 80 percent of the regular spontaneous cycle length (RCL), and b) train: a 2-min train of pulses at intervals of 50, 65 or 80 percent of RCL. Corrected SNRT (cSNRT) was calculated as the difference between SNRT (first spontaneous cycle length after stimulation interruption) and RCL. Ryanodine only slightly increased RCL (<10 percent), but decreased developed force by 90 percent. When the pulse method was used, cSNRT (~40 ms), which represents intranodal/atrial conduction time, was independent of the coupling interval and unaffected by ryanodine. However, cSNRT obtained by the train method was significantly higher for shorter intervals between pulses, indicating the occurrence of overdrive suppression. In this case, ryanodine prolonged cSNRT in a rate-dependent fashion, with a greater effect at shorter intervals. These results indicate that: a) a functional SR, albeit important for force development, does not seem to play a major role in atrial automaticity in the rat; b) disruption of cell Ca2+ homeostasis by inhibition of SR function does not appear to affect conduction; however, it enhances overdrive-induced depression of sinusal automaticity

Changes in calcium uptake rate by rat cardiac mitochondria during postnatal development.

Ca2+ uptake, transmembrane electrical potential (Deltapsim) and oxygen consumption were measured in isolated ventricular mitochondria of rats from 3 days to 5 months of age. Estimated values of ruthenium red-sensitive, succinate-supported maximal rate of Ca2+ uptake (Vmax, expressed as nmol Ca2+/min/mg protein) were higher in neonates and gradually fell during postnatal development (from 435+/-24 at 3-6 days, to 156+/-10 in adults,P<0.001), whereas K0.5 values (approximately 10 microM were not significantly affected by age. Under similar conditions, mitochondria from adults (5 months old) and neonates (4-6 days old) showed comparable state 4 (succinate and alpha-ketoglutarate as substrates) and state 3ADP (alpha-ketoglutarate-supported) respiration rates, as well as Deltapsim values (approximately-150 mV). Respiration-independent Deltapsim and Ca2+ uptake, supported by valinomycin-induced K+ efflux were also investigated at these ages. A transient Deltapsim (approximately -30 mV) was evoked by valinomycin in both neonatal and adult mitochondria. Respiration-independent Ca2+ uptake was also transient, but its initial rate was significantly higher in neonates than in adults (49. 4+/-10.0v 28.0+/-5.7 mmol Ca2+/min/mg protein,P<0.01). These results indicate that Ca2+ uptake capacity of rat cardiac mitochondria is remarkably high just after birth and declines over the first weeks of postnatal life, without change in apparent affinity of the transporter. Increased mitochondrial Ca2+ uptake rate in neonates appears to be related to the uniporter itself, rather than to modification of the driving force of the transport.

Measuring [Ca2+] with fluorescent indicators: theoretical approach to the ratio method.

In this work we present a theoretical analysis of the ratio method, a widely used technique for measuring intracellular calcium concentration, [Ca2+]i, in isolated cells. From the ratio of fluorescence measured at two different excitation or emission wavelengths, [Ca2+]i may be estimated from the equation: [Ca2+]i = Kd.beta.(R-Rmin)/(Rmax-R). From this equation we determined the method sensitivity showing that its maximum is located at [Ca2+] = Kd.beta.(Rmin/Rmax)1/2, i.e. for [Ca2+] < Kd.beta. We also analyzed the error propagation due to inaccuracies in the calibration parameters. The fluorescence phenomenon was described, aiming at providing a basis for the microscopic interpretation of the method and giving physical meaning to the calibration parameters. In this sense beta, is shown to depend not only on the set-up, but also on the spectrum of the indicator for the particular sample studied. A new approach to estimate beta with higher accuracy is also proposed. Experimentally obtained beta values using this approach were not statistically different from those determined as Fmin2/Fmax2. A graphical interpretation of the method is presented to provide users of fluorescence systems with a simple technique to help understand equipment performance and design.

Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes.

Left ventricular hypertrophy (approximately 40%) was induced in rats by banding of the abdominal aorta. After 16 wk, ventricular homogenates were prepared for biochemical measurements and ventricular myocytes were isolated for functional studies. In myocytes, the effects of banding on intracellular Ca handling, contraction, and excitation-contraction (E-C) coupling were determined using indo 1 fluorescence and whole cell voltage clamp. After steady-state field or voltage-clamp stimulation to load the sarcoplasmic reticulum (SR), SR Ca content assessed by caffeine-induced Ca transients was the same in sham and banded groups. Despite this, cell shortening amplitudes were significantly depressed in the banded group, suggesting altered contractile properties. In banded rats, the SR Ca-adenosinetriphosphatase (Ca-ATPase) mRNA level was reduced, as was homogenate thapsigargin-sensitive SR Ca-ATPase, but cytosolic free Ca concentration ([Ca]i) decline attributed to SR Ca-ATPase activity in intact cells was not slowed. Banding also reduced Na/Ca exchange mRNA level but did not affect either Na-dependent sarcolemmal 45Ca transport in homogenate or the rate of [Ca]i decline in intact cells attributed to Na/Ca exchange (during caffeine-induced contractures). Banding also did not change the rate of [Ca]i decline mediated by the combined function of the mitochondrial Ca uptake and sarcolemmal Ca-ATPase in intact cells. Ca current (ICa) density and voltage dependence were the same in sham and banded groups. Ryanodine receptor mRNA, protein content, and ryanodine affinity were also unchanged in the banded group. At 1 mM extracellular Ca concentration ([Ca]o), banding did not affect E-C coupling efficacy in intact cells under voltage clamp (i.e., same contraction for given ICa and SR Ca load). However, when [Ca]o was reduced to 0.5 mM, the efficacy of E-C coupling was greatly depressed in the banded group (even though ICa and SR Ca content were matched). In summary, unloaded myocyte contraction was depressed in these hypertrophic hearts, but Ca transport was little altered, at 1 mM [Ca]o. However, reduction of [Ca]o to 0.5 mM appears to unmask a depressed fractional SR Ca release in response to a given ICa trigger and SR Ca load.

Passive Ca2+ binding in ventricular myocardium of neonatal and adult rats.

In this study, passive Ca2+ binding was determined in ventricular homogenates (VH) from neonatal (4-6 days) and adult rats, as well as in digitonin-permeabilized adult ventricular myocytes. Ca2+ binding sites, both endogenous and exogenous (Indo-1 and BAPTA) were titrated. Sarcoplasmic reticulum and mitochondrial Ca2+ uptake were blocked by thapsigargin and Ru360, respectively. Free [Ca2+] ([Ca2+]F) was measured with Indo-1 and bound Ca2+ ([Ca2+]B) was the difference between [Ca2+]F and total Ca2+. Apparent Ca2+ dissociation constants (Kd) for BAPTA and Indo-1 were increased by 10-20 mg VH protein/ml (from 0.35 to 0.92 microM for Indo-1 and from 0.20 to 0.76 microM for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl2 additions (2.5-10 nmoles) yielded delta[Ca2+]B/delta[Ca2+]F for the sum of [Ca2+]B at all three classes of binding sites. From this function, the apparent number of endogenous sites (Ben) and their Kd (Ken) were determined. Similar Ken values were obtained in neonatal and adult VH, as well as in adult myocytes (0.68 +/- 0.14 microM, 0.69 +/- 0.13 microM and 0.53 +/- 0.10 microM, respectively). However, Ben was significantly higher in adult myocytes than in adult VH (1.73 +/- 0.35 versus 0.70 +/- 0.12 nmol/mg protein, P < 0.01), which correspond to approximately 300 and 213 mumol/l cytosol. This indicates that binding sites are more concentrated in myocytes than in other ventricular components and that Ben determined in VH underestimates cellular Ben by 29%. Although Ben values in nmol/mg protein were similar in adult and neonatal VH (0.69 +/- 0.12), protein content was much higher in adult ventricle (125 +/- 7 versus 80 +/- 1 mg protein/g wet weight, P < 0.01). Expressing Ben per unit cell volume (accounting for fractional mitochondrial volume, and 29% dilution in homogenate), the passive Ca2+ binding capacity at high-affinity sites is approximately 300 and 176 mmol/l cytosol in adult and neonatal rat ventricular myocytes, respectively. Additional estimates suggest that passive Ca2+ buffering capacity in rat ventricle increases markedly during the first two weeks of life and that adult levels are attained by the end of the first month.

Diastolic SR Ca efflux in atrial pacemaker cells and Ca-overloaded myocytes.

Evidence has shown that the sarcoplasmic reticulum (SR) of cardiac cells releases Ca not only during excitation-contraction coupling but also during diastole, albeit at a much lower rate. This diastolic SR Ca release (leak) has also been implicated in the generation of spontaneous depolarization in latent atrial pacemaker cells of the cat right atrium. In the present work, we sought to measure Ca transients in pacemaker and nonpacemaker cells of the cat using the fluorescent Ca indicator indo 1. Atrial latent pacemaker cells develop a slow Ca transient when rested in the presence of both Na- and Ca-free solution and thapsigargin [used to inhibit Na/Ca exchange and SR Ca adenosinetriphosphatase (Ca-ATPase), respectively]. This increase in cytosolic Ca concentration ([Ca]i) is probably caused by the rate of SR Ca leak exceeding the capacity of the remaining Ca transport systems (e.g., sarcolemmal Ca-ATPase and mitochondrial Ca uptake). However, neither cat sinoatrial (SA) node cells nor myocytes from cat atrium or ventricle exhibited a similar increase in [Ca]i during the same protocol. This indicates that SR Ca leak in these cells occurred at a rate low enough to be within the capacity of the slow Ca transporters, as observed previously in rabbit ventricular myocytes. When atrial and ventricular myocytes were stimulated at higher frequencies, sufficient to markedly increase diastolic and systolic [Ca]i and approach Ca overload (and spontaneous activity), they responded to inhibition of SR Ca-ATPase and Na/Ca exchange with a slow Ca transient similar to that normally observed in atrial latent pacemaker cells. Furthermore, the SR Ca depletion by thapsigargin did not affect spontaneous activity of SA node cells, but it prevented or slowed pacemaker activity in the atrial latent pacemaker cells. These findings suggest that enhanced diastolic SR Ca efflux contributes significantly to the generation of spontaneous activity in atrial subsidiary pacemakers under normal conditions and in Ca-overloaded myocytes but not in SA node cells.

A method to estimate mitochondrial Ca2+ uptake in intact cardiac myocytes.

In the present paper we describe a method to estimate mitochondrial Ca2+ uptake during the declining phase of Ca2+ transients (cell relaxation) in intact isolated myocardial cells. This method is based on inhibition of sarcoplasmic reticulum (SR) Ca2+ accumulation by caffeine, blockade of Ca2+ transport via sarcolemmal Ca(2+)-ATPase by treatment with carboxyeosin and inhibition of sarcolemmal Na+/Ca2+ exchange by removal of extracellular Na+ and Ca2+.Ca2+ transients were evoked in rabbit ventricular myocytes by quick and sustained caffeine application (10 mM) after a 5-min period of electrical stimulation to load the SR with Ca2+. Mitochondrial Ca2+ transport was estimated using a model described by Sipido and Wier (Journal of Physiology (1991), 435: 605-630), which was originally proposed to describe Ca2+ fluxes during excitation-contraction coupling in cardiac cells. Our results indicate that, in intact rabbit myocytes, the Ca2+ flux due to net mitochondrial Ca2+ uptake may attain a value close to 1 microM/sec.

A method to estimate mitochondrial Ca2+ uptake in intact cardiac myocytes

In the present paper we describe a method to estimate mitochondrial Ca2+ uptake during the declining phase of Ca2+ transients (cell relaxation) in intact isolated myocardial cells. This method is based on inhibition of sarcoplasmic reticulum (SR) Ca2+ accumulation by caffeine, blockade of Ca2+ transport via sarcolemmal Ca2+ -ATPase by treatment with carboxyeosin and inhibition of sarcolemmal Na+/Ca+ exchange by removal of extracellular Na+ and Ca2+. Ca2+ transients were evoked in rabbit ventricular myocytes by quick and sustained caffeine application (10 mM) after a 5-min period of electrical stimulation to load the SR with Ca2+. Mitochondrial Ca2+ transport was estimated using a model described by Sipido and Wier (Journal of Physiology (1991), 435: 605-630), which was originally proposed to describe Ca2+ fluxes during excitation-contraction coupling in cardiac cells. Our results indicate that, in intact rabbit myocytes, the Ca2+ flux due to net mitochondrial CA2+ uptake may attain a value close to 1 muM/sec.

Temperature and relative contributions of Ca transport systems in cardiac myocyte relaxation.

The relative contributions of the different Ca transport systems involved in cardiac relaxation were evaluated at 25 and 35 degrees C in isolated rabbit, ferret, and cat ventricular myocytes during twitches, caffeine-induced contractures in normal Tyrode solution, and caffeine-induced contractures in Na- and Ca-free solution. The time course of intracellular [Ca] decline these contractions in rabbit ventricular myocytes allowed estimates of the relative contributions of the sarcoplasmic reticulum (SR) Ca pump, Na/Ca exchange, sarcolemmal Ca pump, and the mitochondrial calcium uniporter (with the latter two considered together as "slow mechanisms"). The percent contributions of the SR Ca pump, the Na/Ca exchange, and the slow mechanisms were 70, 27 and 3% at 25 degrees C and 74, 23, and 3% at 35 degrees C. Warming from 25 to 35 degrees C decreases twitch contractions in rabbit and ferret myocytes and caffeine-induced contractures in normal Tyrode solution and Na- and Ca-free solution in all species. In contrast, in cat myocytes warming increased twitches, possibly because of a stronger effect of temperature on Ca influx. We conclude that increased temperature accelerates all of the Ca transport systems involved in relaxation. Despite large changes in each Ca transport system with warming, the relative contributions during relaxation remain similar at physiological temperature.

Na-Ca exchange and Ca fluxes during contraction and relaxation in mammalian ventricular muscle.

There are four cellular Ca transport systems which compete to remove Ca from the myoplasm in mammalian ventricular myocytes. These are 1) the SR Ca-ATPase, 2) the sarcolemmal Na-Ca exchange, 3) the sarcolemmal Ca-ATPase and 4) the mitochondrial Ca uniporter. Using multiple experimental approaches we have evaluated the dynamic interaction of these systems during the normal cardiac contraction-relaxation cycle. The SR Ca-ATPase and Na-Ca exchange are clearly the most important, quantitatively; however, the relative roles vary in a species-dependent manner. In particular, the SR is much more strongly dominant in rat ventricular myocytes, where approximately 92% of Ca removal is via SR Ca-ATPase and only 7% via Na-Ca exchange during a twitch. In other species (rabbit, ferret, cat, and guinea pig) the balance is more in the range of 70% SR CA-ATPase and 25-30% Na-Ca exchange. Ferret ventricular myocytes also exhibit an unusually strong sarcolemmal Ca-ATPase. During the steady state the same amount of Ca must leave the cell as enters over a cardiac cycle. This implies that 25-30% of the Ca required to activate contraction must enter the cell, and experiments demonstrate that this amount of Ca may be supplied by the L-type Ca current.

Relaxation in ferret ventricular myocytes: role of the sarcolemmal Ca ATPase.

In ferret ventricular myocytes the rate of intracellular Ca concentration [Ca]i decline and relaxation is remarkably fast (compared with rabbit and rat) under conditions where both the sarcoplasmic reticulum Ca uptake and Na/Ca exchange are inhibited. Here we explore the possibility that this rapid [Ca]i decline in ferret cells is attributable to the sarcolemmal Ca ATPase by using carboxyeosin (a potent inhibitor of the sarcolemmal Ca-ATPase). We compare the effects of carboxyeosin with those of elevated extracellular [Ca] ([Ca]o) (a thermodynamic approach to limit Ca transport by the sarcolemmal Ca ATPase). In rabbit cells, carboxyeosin and high [Ca]o slowed [Ca]i decline similarly and both virtually abolished [Ca]i decline when mitochondrial Ca uptake was also inhibited. In ferret cells, carboxyeosin treatment produced these same effects on [Ca]i decline, but high [Ca]o did not mimic them. Moreover, only in carboxyeosin-treated ferret cells did additional inhibition of mitochondrial Ca uptake nearly abolish [Ca]i decline. We conclude that, carboxyeosin loading can inhibit the sarcolemmal Ca-ATPase in intact myocytes; that this pump seems likely to be responsible for the much faster relaxation observed in ferret cells after block of SR Ca accumulation and Na/Ca exchange transport and that the sarcolemmal Ca pump apparently has different characteristics in rabbit and ferret ventricular myocytes.