Factores asociados a recidiva de hernia de disco lumbar luego de una microdiscectomía / Factors associated with lumbar disc hernia recurrence after microdiscectomy

Introducción. Las hernias de disco lumbares son una causa frecuente de cirugía lumbar. La recurrencia herniaria es una complicación prevalente. Objetivo. Analizar los factores de riesgo asociados a recurrencia herniaria en pacientes intervenidos por hernia discal lumbar en nuestra institución. Materiales y métodos. Se analizaron en forma retrospectiva una serie de 177 microdiscectomías lumbares entre 2010 y 2014; se excluyeron pacientes con cirugías previas, hernias foraminales y extraforaminales. Los pacientes con recurrencia herniaria constituyeron el grupo de casos y los que no presentaron recurrencia fueron el grupo control; se analizó: sexo, edad, índice de masa corporal, nivel, degeneración facetaria, altura discal y protrusión discal. Resultados. Hubo 177 pacientes con microdiscectomía lumbar, de los cuales 30 (16%) presentaron recurrencia herniaria; de ellos, 27 fueron reoperados. Entre los factores de riesgo asociados a recurrencia observamos mayor índice de altura discal, mayor porcentaje de canal ocupado por la hernia y mayor presencia de cambios degenerativos facetarios; no observamos diferencias en cuanto al sexo, índice de masa corporal y edad. Discusión. Estudios previos muestran el aumento de altura discal y pacientes jóvenes como posibles factores asociados a recidiva. Conclusión. En nuestra serie encontramos que el mayor índice de altura discal, el porcentaje de canal ocupado por la hernia y los cambios degenerativos facetarios se asociaron a recurrencia herniaria (AU)

Introduction. Lumbar disc hernias are a common cause of spinal surgery. Hernia recurrence is a prevalent complication. Objective. To analyse the risk factors associated with hernia recurrence in patients undergoing surgery in our institution. Materials and methods. Lumbar microdiscectomies between 2010 and 2014 were analysed, patients with previous surgeries, extraforaminales and foraminal hernias were excluded. Patients with recurrent hernia were the case group and those who showed no recurrence were the control group. Results. 177 patients with lumbar microdiscectomy, of whom 30 experienced recurrence (16%), and of these 27 were reoperated. Among the risk factors associated with recurrence, we observed a higher rate of disc height, higher percentage of spinal canal occupied by the hernia and presence of degenerative facet joint changes; we observed no differences in sex, body mass index or age. Discussion. Previous studies show increased disc height and young patients as possible factors associated with recurrence. Conclusion. In our series we found that the higher rate of disc height, the percentage of spinal canal occupied by the hernia and degenerative facet joint changes were associated with hernia recurrence (AU)

Factors associated with lumbar disc hernia recurrence after microdiscectomy. / Factores asociados a recidiva de hernia de disco lumbar luego de una microdiscectomía.

INTRODUCTION: Lumbar disc hernias are a common cause of spinal surgery. Hernia recurrence is a prevalent complication. OBJECTIVE: To analyse the risk factors associated with hernia recurrence in patients undergoing surgery in our institution. MATERIALS AND METHODS: Lumbar microdiscectomies between 2010 and 2014 were analysed, patients with previous surgeries, extraforaminales and foraminal hernias were excluded. Patients with recurrent hernia were the case group and those who showed no recurrence were the control group. RESULTS: 177 patients with lumbar microdiscectomy, of whom 30 experienced recurrence (16%), and of these 27 were reoperated. Among the risk factors associated with recurrence, we observed a higher rate of disc height, higher percentage of spinal canal occupied by the hernia and presence of degenerative facet joint changes; we observed no differences in sex, body mass index or age. DISCUSSION: Previous studies show increased disc height and young patients as possible factors associated with recurrence. CONCLUSION: In our series we found that the higher rate of disc height, the percentage of spinal canal occupied by the hernia and degenerative facet joint changes were associated with hernia recurrence.

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.

The negative inotropic action of canrenone is mediated by L-type calcium current blockade and reduced intracellular calcium transients.

BACKGROUND AND PURPOSE: Adding spironolactone to standard therapy in heart failure reduces morbidity and mortality, but the underlying mechanisms are not fully understood. We analysed the effect of canrenone, the major active metabolite of spironolactone, on myocardial contractility and intracellular calcium homeostasis. EXPERIMENTAL APPROACH: Left ventricular papillary muscles and cardiomyocytes were isolated from male Wistar rats. Contractility of papillary muscles was assessed with force transducers, Ca(2+) transients by fluorescence and Ca(2+) fluxes by electrophysiological techniques. KEY RESULTS: Canrenone (300-600 micromol L(-1)) reduced developed tension, maximum rate of tension increase and maximum rate of tension decay of papillary muscles. In cardiomyocytes, canrenone (50 micromol L(-1)) reduced cell shortening and L-type Ca(2+) channel current, whereas steady-state activation and inactivation, and reactivation curves were unchanged. Canrenone also decreased the Ca(2+) content of the sarcoplasmic reticulum, intracellular Ca(2+) transient amplitude and intracellular diastolic Ca(2+) concentration. However, the time course of [Ca(2+)](i) decline during transients evoked by caffeine was not affected by canrenone. CONCLUSION AND IMPLICATIONS: Canrenone reduced L-type Ca(2+) channel current, amplitude of intracellular Ca(2+) transients and Ca(2+) content of sarcoplasmic reticulum in cardiomyocytes. These changes are likely to underlie the negative inotropic effect of canrenone.

Cost management of medical equipment maintenance.

In the present study we combine Activity Based Costing (ABC) with a microprocess-based custom-made management system used to control of the medical equipment maintenance service performed by a clinical engineering group in a public health institution in Brazil. Results show the cost of service orders calculated through the allocation of the expenditure per cost center to activities performed during the year 2003. As this model can estimate how the activities affect profitability, managers can use ABC information to interpret possible strategies needed to investigate the viability of cost minimization.

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.

Functional imaging of the retinal layers by laser scattering: an approach for the study of Leão's spreading depression in intact tissue.

This paper presents a novel optical approach for the study of spreading depression in isolated retina. The method makes it possible to register the laser light scattered from each layer of the tissue, yielding a functional image of the retina during spreading depression. The tissue is kept intact, since histological cuts are not necessary. Measurements of other variables, such as extracellular potential, are also allowed by the described method. This is done simultaneously with the functional image in a high spatial resolution, with the positioning of the microelectrode tip being easily monitored. The information about temporal and spatial evolution of light was compacted in a single image. The image-processing technique used here enables the visualization of the light scattered by the inner plexiform layer (IPL), which is the most prominent scatter layer during spreading depression. The wavefront velocity and its increase as two wavefronts approach each other can then be determined, and it is also possible to observe the thickness variation of the tissue during the wave travel. The relationship between two peaks of light-scattering sequence during the phenomenon was studied at two wavelengths (632.8 and 543.5 nm). This relationship is shown to be dependent on the wavelength.

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.

Ca(2+) influx through Ca(2+) channels in rabbit ventricular myocytes during action potential clamp: influence of temperature.

Ca(2+) influx via Ca(2+) current (I(Ca)) during the action potential (AP) was determined at 25 degrees C and 35 degrees C in isolated rabbit ventricular myocytes using AP clamp. Contaminating currents through Na(+) and K(+) channels were eliminated by using Na(+)- and K(+)-free solutions, respectively. DIDS (0.2 mmol/L) was used to block Ca(2+)-activated chloride current (I(Cl(Ca))). When the sarcoplasmic reticulum (SR) was depleted of Ca(2+) by preexposure to 10 mmol/L caffeine, total Ca(2+) entry via I(Ca) during the AP was approximately 12 micromol/L cytosol (at both 25 degrees C and 35 degrees C). Similar Ca(2+) influx at 35 degrees C and 25 degrees C resulted from a combination of higher and faster peak I(Ca), offset by more rapid I(Ca) inactivation at 35 degrees C. During repeated AP clamps, the SR gradually fills with Ca(2+), and consequent SR Ca(2+) release accelerates I(Ca) inactivation during the AP. During APs and contractions in steady state, total Ca(2+) influx via I(Ca) was reduced by approximately 50% but was again unaltered by temperature (5.6+/-0.2 micromol/L cytosol at 25 degrees C, 6.0+/-0.2 micromol/L cytosol at 35 degrees C). Thus, SR Ca(2+) release is responsible for sufficient I(Ca) inactivation to cut total Ca(2+) influx in half. However, because of the kinetic differences in I(Ca), the amount of Ca(2+) influx during the first 10 ms, which presumably triggers SR Ca(2+) release, is much greater at 35 degrees C. I(Ca) during a first pulse, given just after the SR was emptied with caffeine, was subtracted from I(Ca) during each of 9 subsequent pulses, which loaded the SR. These difference currents reflect I(Ca) inactivation due to SR Ca(2+) release and thus indicate the time course of local [Ca(2+)] in the subsarcolemmal space near Ca(2+) channels produced by SR Ca(2+) release (eg, maximal at 20 ms after the AP activation at 35 degrees C). Furthermore, the rate of change of this difference current may reflect the rate of SR Ca(2+) release as sensed by L-type Ca(2+) channels. These results suggest that peak SR Ca(2+) release occurs within 2.5 or 5 ms of AP upstroke at 35 degrees C and 25 degrees C, respectively. I(Cl(Ca)) might also indicate local [Ca(2+)], and at 35 degrees C in the absence of DIDS (when I(Cl(Ca)) is prominent), peak I(Cl(Ca)) also occurred at a time comparable to the peak I(Ca) difference current. We conclude that SR Ca(2+) release decreases the Ca(2+) influx during the AP by approximately 50% (at both 25 degrees C and 35 degrees C) and that changes in I(Ca) (and I(Cl(Ca))), which depend on SR Ca(2+) release, provide information about local subsarcolemmal [Ca(2+)].

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.

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.

Cardiac myocyte volume, Ca2+ fluxes, and sarcoplasmic reticulum loading in pressure-overload hypertrophy.

Alterations in cellular Ca2+ transport and excitation-contraction coupling may contribute to dysfunction in cardiac hypertrophy. Left ventricular myocytes were isolated from rat hearts after 15-18 wk of suprarenal abdominal aortic banding to evaluate the hypothesis that hypertrophy alters the relationship between Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ load during steady-state voltage-clamp depolarization. Mean arterial pressure (MAP) and heart weight-to-body weight ratio of banded (B) animals were significantly higher than in control or sham-operated animals (C). Isolated myocyte dimensions and volume increased in parallel with whole heart hypertrophy and elevation in MAP. However, the relationship between membrane surface area (measured by capacitance) and cell volume (measured by laser scanning confocal microscopy) was unaltered (C: 8.9 +/- 0.3; B: 8.5 +/- 0.4 pF/pl). No differences in the voltage dependence of ICa activation, steady-state inactivation, or recovery from inactivation were detected between C and B myocytes. Maximal ICa density for the two groups was also not different either under basal conditions (C: 4.28 +/- 0.98; B: 4.57 +/- 0.60 pA/pF) or in the presence of 1 microM isoproterenol (C: 16.6 +/- 2.3; B: 16.5 +/- 2.3 pA/pF). The fraction of Ca2+ released from the SR by a single twitch was 55.4 +/- 9.4% in C and 37.1 +/- 6.9% in B (not significantly different). Steady-state Ca2+ influx during a twitch was calculated in units of micromoles per liter of nonmitochondrial volume from the integral of ICa (C: 13.4 +/- 0.7 microM; B: 13.3 +/- 0.8 microM). The SR Ca2+ load was similarly calculated by integration of Na+/Ca2+ exchange current induced by rapid caffeine application (C: 140 +/- 9 microM; B: 169 +/- 18 microM). We conclude that significant cellular hypertrophy is associated with proportional increases in sarcolemmal ICa influx, SR Ca2+ loading, and the amount of SR Ca2+ released in this model of pressure overload.

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.