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.

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

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.

A portable microprocessor-based triggering device for cardiac muscle electrical stimulation

We describe a microprocessor-based programmable triggering instrument designed to control the distribution in time of electrical stimuli delivered to a heart muscle preparation. Sequences of stimuli may be selected among those stored in the non-volatile memory of the instrument and new sequences may be programmed using a repertory of 25 commands. The instrument was used to study the inotropic effects of three irregular sequences of stimuli applied to the isolated rat left atrium. The mean peak tension developed by the tissue was unaltered by stimulus sequences, provided the mean stimulatory frequency (2 or 5 Hz) was maintained. The instrument may be useful to study the effect of different stimulatory patterns on cardiac inotropism, as well as for controlling the electrical simulation of tother biological preparation

Influence of the KCl concentration on rat sinus node recovery time in vitro

The influence of changes in the KCl concentration of the bath fluid ([KCl]o) on the corrected sinus node recovery time (CSNRT) was studied using the continuous pacing method (M1) and the method of stimulation with premature pulses (M2). M1 and M2 were compared in Krebs-Henseleit solution containing normal (4.6 mM), low (3.1mM, LoKCl) and hight (6.1 mM, HiKCl) [KCl]o. The results revealed that HiKCl increased CSNRT (P < 0.01) and changed the prematurit-CSNRT relationship (P<0.01), whereas LoKCL did not change CSNRT. M1 and M2 were different (P<0.01) regardless of [KCl]o, except for pacing intervals near the spontaneous cycle length

Calcium-osmolality interaction in the inotropic response of isolated rat atria to increased sodium concentration

The effects of osmolality (300 and 450 mOsm/l) and external calcium concentration ([Ca2 +]o: 0.87, 1.13, 1.47 and 1.92 mM) on the inotropic response of isolated rat left atria to an increase of 97.8 mM in extracellular sodium concentration ([Na + ]o) were studied. The evoked tensions developed during 30 min after the exposure to increased [Na + ]o increased with increasing [Ca2 +]o. In iso-osmotic solutions, tension was lower than in hyperosmotic solutions when [Ca2+]o was 0.87 and 1.13 mM, but not for higher [Ca2+]o, revealing a Ca2 -osmolality interaction in the determination of the inotropic response

Effect of hyperosmolality on the sensitivity of right and left atria of the rat to noradrenaline

This study analyzes the effects of hyperosmotic solutions on the sensitivity of the rat isolated right and left atria to the chronotropic and inotropic effects of noradrenaline (NA), respectively. Hyperosmotic NaCl solution caused subsensitivity to both effects of NA (pD2 values for NA, right atria: control 7.42 ñ 0.06, NaCl 6.83 ñ 0.18, P < 0.05; left atria: control 7.67 ñ 0.22, NaCl 6.61 ñ 0.18, P < 0.05). Hyperosmotic sucrose solution produced a similar effect in right atria (pD2NA: 6.96 ñ0.17, P < 0.05), whereas in left atria it depressed the maximum inotropic response to NA by about 80%. All chagnes, except that of maximun inotropic response, were abolished following combined pretreatment with 6-hydroxydopamine, phenoxybenzamine, atropine and imipramine. These data suggest that the noradrenergic subsensitivity induced in atrial tissue bu hyperosmolality is probablu not due to changes of beta-adrenoceptor function and/or coupling of these receptors to the effectors mechanisms

Influence of stimulatory parameters on simus node recovery time: an in vitro study

The effects of pacing frequency, overdrive duration and stimulus amplitude on the sinus node recovery time (SNRT) were studied in the isolated right atrium of the rat. a positive relationship between pacing frequency and the SNRT was observed, whereas overdrive duration ans stimulus amplitude did not affect SNRT. There was no significant interaction among the factors studied. The effect of frequency upon SNRT probably does not involve neurotransmitter release due to stimulation, since in vitro pretreatment with atropine plus propranolol does change the SNRT - frequency relation

Estimulador elétrico de corrente para estudos fisiológicos / Electric current stimulator for physiological studies

Com o objetivo de contribuir ao estudo da atividade elétrica e contrátil do músculo cardíaco foi desenvolvido um estimulador elétrico capaz de produzir pulsos mono e bipolares de corrente, na faixa de 1 micronA a 5 mA, com duraçäo variável de 50 microns a 500 ms e frequência estimulatória variável de 0,1 Hz a 2 KHz. Além disso, o instrumento permite o controle externo do intervalo entre pulsos. Uma entrada especial para acoplamento de um gerador de funçöes permite a estimulaçäo com qualquer forma de pulso de corrente. A calibraçäo do instrumento pode ser feita sem necessidade de acoplamento à preparaçäo biológica. A saída para estimulaçäo é isolada por acoplamento optico e alimentada por conversor DC/DC. Os testes efetuados em bancada revelaram que o instrumento possui resposta linear na faixa de -5 a 5 mA, tempo de subida ao degrau de 17 microns e sua curva de regulaçäo obedece as condiçöes impostas no projeto. Com base nestes resultados e na aplicaçäo a experimentos biológicos concluiu-se que o instrumento desenvolvido é perfeitamente adequado para a estimulaçäo do tecido cardíaco isolado