Determination of the Upper Bound of Intracellular [Na+] by Electrophysiological Method: Probing the Subsarcolemmal [Na+] (original) (raw)
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Arrhythmogenic and antiarrhythmic actions of late sustained sodium current in the adult human heart
Scientific Reports, 2021
Late sodium current (late INa) inhibition has been proposed to suppress the incidence of arrhythmias generated by pathological states or induced by drugs. However, the role of late INa in the human heart is still poorly understood. We therefore investigated the role of this conductance in arrhythmias using adult primary cardiomyocytes and tissues from donor hearts. Potentiation of late INa with ATX-II (anemonia sulcata toxin II) and E-4031 (selective blocker of the hERG channel) slowed the kinetics of action potential repolarization, impaired Ca 2+ homeostasis, increased contractility, and increased the manifestation of arrhythmia markers. These effects could be reversed by late INa inhibitors, ranolazine and GS-967. We also report that atrial tissues from donor hearts affected by atrial fibrillation exhibit arrhythmia markers in the absence of drug treatment and inhibition of late INa with GS-967 leads to a significant reduction in arrhythmic behaviour. These findings reveal a critical role for the late INa in cardiac arrhythmias and suggest that inhibition of this conductance could provide an effective therapeutic strategy. Finally, this study highlights the utility of human ex-vivo heart models for advancing cardiac translational sciences. Development programs aiming to provide new treatments are frequently terminated prior to regulatory approval due to drug-induced cardiotoxicity or lack of adequate efficacy 1-5. Clinical attrition is in part a consequence of the inability of current preclinical models, including artificially engineered cell lines, stem cell-derived platforms and animal models, to generate data predictive of human clinical responses 6,7. The utilization of recently developed human ex vivo paradigms based on organ donor hearts provides novel opportunities for translational sciences 4,8 and may be instrumental for informing the development of a new generation of therapies by identifying critical ion channel conductances underlying pathological states 9-15. At present, limited information is available on the role of late sodium current (INa) in normal as well as pathological states in the human heart. An increase in late INa function has been proposed to play a pivotal role in rhythm disorders 16,17 , heart failure 18-22 , and ischemia and hypoxia 23,24. Consequently, a reduction of late INa could have therapeutic benefits, such as protection against drug-induced QT prolongation/pro-arrhythmia 25,26 , and may provide antiarrhythmic therapy for atrial fibrillation and ventricular tachycardia 27-31. Late INa contributes to the cardiac action potential morphology and is largely responsible for maintaining intracellular Na + homeostasis 17,20, 22,31. The current knowledge of the physiology and pathophysiology of late INa derives from studies conducted in rabbit, guinea pig, dog, and porcine hearts 32-43 , non-failing/failing human hearts or patients in sinus rhythm/atrial fibrillation 17,19, 44-48. In the context of arrhythmia genesis, role for late INa has been well studied in animal models 17. However, the specific contribution, if any, for late INa in the genesis or suppression of arrhythmias in human cardiomyocytes is still unknown. To address this knowledge gap, we performed a series of studies using adult human ex vivo preparations from normal and atrial fibrillation donor hearts to define the role of late INa in action potential morphology, intracellular Ca 2+ handling, contractility, arrhythmogenesis, and the potential of late INa as a target for antiarrhythmic treatment and protection against drug-induced pro-arrhythmia.
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2004
The mechanism underlying temperature-dependent shortening of action potential (AP) duration was examined in the fish (Carassius carassius L.) heart ventricle. Acute temperature change from ϩ5 to ϩ18°C (heat stress) shortened AP duration from 2.8 Ϯ 0.3 to 1.3 Ϯ 0.1 s in intact ventricles. In 56% (18 of 32) of enzymatically isolated myocytes, heat stress also induced reversible opening of ATP-sensitive K ϩ channels and increased their singlechannel conductance from 37 Ϯ 12 pS at ϩ8°C to 51 Ϯ 13 pS at ϩ18°C (Q10 ϭ 1.38) (P Ͻ 0.01; n ϭ 12). The ATP-sensitive K ϩ channels of the crucian carp ventricle were characterized by very low affinity to ATP both at ϩ8°C [concentration of Tris-ATP that produces half-maximal inhibition of the channel (K1/2)ϭ 1.35 mM] and ϩ18°C (K 1/2 ϭ 1.85 mM). Although acute heat stress induced ATP-sensitive K ϩ current (IK,ATP) in patch-clamped myocytes, similar heat stress did not cause any glibenclamide (10 M)-sensitive changes in AP duration in multicellular ventricular preparations. Examination of APs and K ϩ currents from the same myocytes by alternate recording under current-clamp and voltage-clamp modes revealed that changes in AP duration were closely correlated with temperature-specific changes in the voltage-dependent rectification of the background inward rectifier K ϩ current IK1. In ϳ15% of myocytes (4 out of 27), IK,ATP-dependent shortening of AP followed the IK1-induced AP shortening. Thus heat stress-induced shortening of AP duration in crucian carp ventricle is primarily dependent on IK1.
Effects of temperature on intracellular [Ca2+] in trout atrial myocytes
Journal of Experimental Biology, 2002
SUMMARYAcute temperature change can be cardioplegic to mammals, yet certain ectotherms maintain their cardiac scope over a wide temperature range. To better understand the acute effects of temperature on the ectothermic heart,we investigated the stimulus-induced change in intracellular Ca2+concentration ([Ca2+]i; cytosolic Ca2+transient) in isolated rainbow trout myocytes at 7°C, 14°C and 21°C. Myocytes were voltage-clamped and loaded with Fura-2 to measure the L-type Ca2+ channel current (ICa) and[Ca2+]i during physiological action potential (AP)pulses at frequencies that correspond to trout heart rates in vivo at 7°C, 14°C and 21°C. Additionally, [Ca2+]iand ICa were examined with square (SQ) pulses at slow (0.2 Hz) and physiologically relevant contraction frequencies. The amplitude of[Ca2+]i decreased with increasing temperature for both SQ and AP pulses, which may contribute to the well-known negative inotropic effect of warm temperature on contractile strength in trout hearts. W...
The effects of temperature upon calcium exchange in intact cultured cardiac myocytes
Cell Calcium, 1997
Cardiac myocyte Ca transport systems, such as sarcoplasmic reticulum Ca-ATPase and sarcolemmal Na/Ca exchange (Na/Ca), are critically dependent on temperature. The purpose of this work was to study the effect of temperature on cellular Ca compartmentation and its exchange characteristics in intact functional neonatal cultured myocytes. The Na/Ca mediated Ca exchange (CaNUca)-including its sarcoplasmic reticulum (SR) and sarcolemmal (SL) contributions, a slow exchange component related to mitochondrial Ca and the La displaceable Ca pool were studied combining isotopic and gas-dissection techniques for membrane isolation. The major findings of this study are: (i) The amount of Ca exchanged through CaNaica is clearly dependent on temperature (Q,,-1.6) in the range studied (17-37°C); (ii) the addition of 1 f.rM nifedipine does not modify the temperature dependence of CaNa/,..; (iii) the sarcolemmal bound fraction contributing to CaNtic= is not changed by temperature; (iv) the increase in CaNtica with temperature is explained by an increment in the contribution of SR-Ca to CaNtic.; (v) a fraction of SR which does not exchange via CaNaiCa at low temperatures can be released and mobilized by caffeine-this caffeine sensitive fraction is reduced as temperature is increased and is no longer measurable as a separate entity at 37°C; (vi) if we consider (iv) and (v) together, SR content would be temperature dependent with a Q,, of-1 S; (vii) a La displaceable pool, which represents over 66% of the total exchangeable Ca, increases in the range of 2233°C with a Q,, of 1.25 which is consistent with a pool distribution of 70% SL-bound and 30% W-derived [Post J.A., Langer G.A. Cellular origin of the rapidly exchangeable calcium pool in the cultured neonatal rat heart cell. Cell Calcium 1992; 13: 627-6341; and (viii) the rate constant for the mitochondrial Ca component increases by 60% from 22°C to 37"C, but Ca content in this organelle is not modified over this temperature range.
Temperature regulates the arrhythmogenic activity of pulmonary vein cardiomyocytes
Journal of Biomedical Science, 2003
Temperature plays an important role in the etectrophysiology of cardiomyocytes. Pulmonary veins (PVs) are known to initiate paroxysmal atrial fibrillation. The effects of temperature on the arrhythmogenic activity of rabbit single PV and atrial cardiomyocytes were assessed using the whole-cell clamp technique. PV cardiomyocytes had different beating rates at low (22-25°C), normal (38-39°C) and high (40-41 °C) temperatures (0.9 _+ 0.1, 3.2 ± 0.4, 6.4 +-0.6 Hz, respectively; p < 0.001). There were different action potential durations and incidences of delayed afterdepolarization in PV cardiomyocytes with pacemaker activity (31, 59, 63%; p < 0.05), PV cardiomyocytes without pacemaker activity (16, 47, 60%; p < 0.001), and atrial myocytes (0, 0, 21%; p < 0.05). However, oscillatory afterpotentials were only found in PV cardiomyocytes with pacemaker activity at normal (50%) or high (68%) temperatures, but not at low temperatures (p < 0.001). Both PV and atrial cardiomyocytes had larger transient inward currents and inward rectified currents at high temperatures. Additionally, PV cardiomyocytes with and without pacemaker activity had larger pacemaker currents at higher temperatures. This study dem-onstrated that PV cardiomyocytes have an increase in arrhythmogenic activity at high temperatures because of enhanced automaticity, induced triggered activity, or shortening of action potential duration.
Circulation Research, 1999
Ca 2ϩ influx via Ca 2ϩ current (I Ca ) during the action potential (AP) was determined at 25°C and 35°C in isolated rabbit ventricular myocytes using AP clamp. Contaminating currents through Na ϩ and K ϩ channels were eliminated by using Na ϩ -and K ϩ -free solutions, respectively. DIDS (0.2 mmol/L) was used to block Ca 2ϩ -activated chloride current (I Cl(Ca) ). When the sarcoplasmic reticulum (SR) was depleted of Ca 2ϩ by preexposure to 10 mmol/L caffeine, total Ca 2ϩ entry via I Ca during the AP was Ϸ12 mol/L cytosol (at both 25°C and 35°C). Similar Ca 2ϩ influx at 35°C and 25°C resulted from a combination of higher and faster peak I Ca , offset by more rapid I Ca inactivation at 35°C. During repeated AP clamps, the SR gradually fills with Ca 2ϩ , and consequent SR Ca 2ϩ release accelerates I Ca inactivation during the AP. During APs and contractions in steady state, total Ca 2ϩ influx via I Ca was reduced by Ϸ50% but was again unaltered by temperature (5.6Ϯ0.2 mol/L cytosol at 25°C, 6.0Ϯ0.2 mol/L cytosol at 35°C). Thus, SR Ca 2ϩ release is responsible for sufficient I Ca inactivation to cut total Ca 2ϩ influx in half. However, because of the kinetic differences in I Ca , the amount of Ca 2ϩ influx during the first 10 ms, which presumably triggers SR Ca 2ϩ release, is much greater at 35°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 2ϩ release and thus indicate the time course of local [Ca 2ϩ ] in the subsarcolemmal space near Ca 2ϩ channels produced by SR Ca 2ϩ release (eg, maximal at 20 ms after the AP activation at 35°C). Furthermore, the rate of change of this difference current may reflect the rate of SR Ca 2ϩ release as sensed by L-type Ca 2ϩ channels. These results suggest that peak SR Ca 2ϩ release occurs within 2.5 or 5 ms of AP upstroke at 35°C and 25°C, respectively. I Cl(Ca) might also indicate local [Ca 2ϩ ], and at 35°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 2ϩ release decreases the Ca 2ϩ influx during the AP by Ϸ50% (at both 25°C and 35°C) and that changes in I Ca (and I Cl(Ca) ), which depend on SR Ca 2ϩ release, provide information about local subsarcolemmal [Ca 2ϩ ]. The full text of this article is available at http://www.circresaha.org. (Circ Res. 1999;85:e7-e16.)
Cardiovascular Research, 1993
Objective: The aim was to investigate the effects of temperature on cycle length dependent changes of action potential duration and on restitution of action potential duration. Methods: Guinea pig papillary muscle action potentials were recorded using conventional microelectrode techniques. Action potential duration was measured at cycle lengths ranging from 500 to 2000 ms at both 27°C and 37°C. Restitution of action potential duration was determined by introducing an extra stimulus at progressively longer diastolic intervals from 40 to 9000 ms at pacing cycle lengths of 500, 1000, and 2000 ms. Results: At 37°C, action potential duration measured at 90% of repolarisation (APD 90 ) during continuous pacing and the maximum value of APD 90 achieved during restitution (APD 90 r 1 ) decreased by 18(SEM 6) ms (n=7) and 24(7) ms (n=6), respectively, when pacing cycle length was reduced from 2000 to 500 ms. At 27°C, the magnitude of the shortening of APD 90 and APD 90 r 1 observed when pacing cycle length was similarly reduced was greater than at 37°C, ie, 143(21) ms (n=6) and 115(11) ms (n=6), respectively. Thus the relation for restitution of action potential duration shifted downwards with reduction in pacing cycle length, and the magnitude of this shift was greater at 27°C than at 37°C. The difference between APD 90 at the shortest diastolic interval (40 ms) and at diastolic interval of 100 ms (range of premature action potential durations) was much greater at 27°C that at 37oC at all three pacing cycle lengths. Conclusions: Reduction in temperature magnifies the cycle length dependent changes in action potential duration both during abrupt changes in cycle length, as with an extra stimulus, and during changes of steady state cycle length. This may indicate a greater dispersion of premature action potential durations during hypothermia, and hence predispose to hypothermia induced arrhythmias. Cardiovascular Research 1993 ;27 :946-950
PLoS ONE, 2012
Heart failure constitutes a major public health problem worldwide. The electrophysiological remodeling of failing hearts sets the stage for malignant arrhythmias, in which the role of the late Na + current (I NaL) is relevant and is currently under investigation. In this study we examined the role of I NaL in the electrophysiological phenotype of ventricular myocytes, and its proarrhythmic effects in the failing heart. A model for cellular heart failure was proposed using a modified version of Grandi et al. model for human ventricular action potential that incorporates the formulation of I NaL. A sensitivity analysis of the model was performed and simulations of the pathological electrical activity of the cell were conducted. The proposed model for the human I NaL and the electrophysiological remodeling of myocytes from failing hearts accurately reproduce experimental observations. The sensitivity analysis of the modulation of electrophysiological parameters of myocytes from failing hearts due to ion channels remodeling, revealed a role for I NaL in the prolongation of action potential duration (APD), triangulation of the shape of the AP, and changes in Ca 2+ transient. A mechanistic investigation of intracellular Na + accumulation and APD shortening with increasing frequency of stimulation of failing myocytes revealed a role for the Na + / K + pump, the Na + /Ca 2+ exchanger and I NaL. The results of the simulations also showed that in failing myocytes, the enhancement of I NaL increased the reverse rate-dependent APD prolongation and the probability of initiating early afterdepolarizations. The electrophysiological remodeling of failing hearts and especially the enhancement of the I NaL prolong APD and alter Ca 2+ transient facilitating the development of early afterdepolarizations. An enhanced I NaL appears to be an important contributor to the electrophysiological phenotype and to the dysregulation of [Ca 2+ ] i homeostasis of failing myocytes.
Physiological and Biochemical Zoology, 2003
The purpose of this study was to investigate how the endogenous catecholamine adrenaline protects sarcolemmal Ca 2ϩ flux through the L-type Ca 2ϩ channel (I Ca ) during acute exposure to cold in the fish heart. We examined the response of I Ca to adrenergic stimulation at three temperatures (7Њ, 14Њ, and 21ЊC) in atrial myocytes isolated from rainbow trout acclimated to 14ЊC. We found that I Ca amplitude varied directly with test temperature and was increased by adrenergic stimulation (AD; 5 nM and 1 mM) at all temperatures. However, I Ca was significantly more sensitive to adrenergic stimulation at the coldest test temperature. In fact, at 7ЊC in the absence of AD, I Ca was extremely low. The addition of 1 mM AD increased peak I Ca 7.2-fold at 7ЊC, 2.6-fold at 14ЊC, and 1.6-fold at 21ЊC and ameliorated the temperature-dependent difference in Ca 2ϩ influx across the cell membrane. We suggest that this increased adrenergic sensitivity is a critical compensatory mechanism that allows the rainbow trout heart to maintain contractility during acute exposure to cold temperatures. In particular, the tonic level of adrenergic stimulation provided by circulating plasma catecholamines (i.e., in the nM concentration range) may be crucial for effective excitation-contraction coupling in the cold cardiomyocyte.