Jörg Hüser

Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells

1 The cellular mechanisms governing cardiac atrial pacemaker activity are not clear. In the present study we used perforated patch voltage clamp and confocal fluorescence microscopy to study the contribution of intracellular Ca2+ release to automaticity of pacemaker cells isolated from cat right atrium. 2 In spontaneously beating pacemaker cells, an increase in subsarcolemmal intracellular Ca2+ concentration occurred concomitantly with the last third of diastolic depolarization due to local release of Ca2+ from the sarcoplasmic reticulum (SR), i.e. Ca2+ sparks. Nickel (Ni2+; 25–50 μM), a blocker of low voltage‐activated T‐type Ca2+ current ((ICa,T), decreased diastolic depolarization, prolonged pacemaker cycle length and suppressed diastolic Ca2+ release. 3 Voltage clamp analysis indicated that the diastolic Ca2+ release was voltage dependent and triggered at about ‐60 mV. Ni2+ suppressed low voltage‐activated Ca2+ release. Moreover, low voltage‐activated Ca2+ release was paralleled by a slow inward current presumably due to stimulation of Na+‐Ca2+ exchange (INa‐Ca). Low voltage‐activated Ca2+ release was found in both sino‐atrial node and latent atrial pacemaker cells but not in working atrial myocytes. 4 These findings suggest that low voltage‐activated ICa,T triggers subsarcolemmal Ca2+ sparks, which in turn stimulate INa‐Ca to depolarize the pacemaker potential to threshold. This novel mechanism indicates a pivotal role for ICa,T and subsarcolemmal intracellular Ca2+ release in normal atrial pacemaker activity and may contribute to the development of ectopic atrial arrhythmias.

CALCIUM GRADIENTS DURING EXCITATION-CONTRACTION COUPLING IN CAT ATRIAL MYOCYTES

1. Confocal microscopy in combination with the calcium‐sensitive fluorescent probe fluo‐3 was used to study spatial aspects of intracellular Ca2+ signals during excitation‐contraction coupling in isolated atrial myocytes from cat heart. 2. Imaging of [Ca2+]i transients evoked by electrical stimulation revealed that Ca2+ release started at the periphery and subsequently spread towards the centre of the myocyte. 3. Blocking sarcoplasmic reticulum (SR) Ca2+ release with 50 microM ryanodine unmasked spatial inhomogeneities in the [Ca2+]i was higher in the periphery than in central regions of the myocyte. 4. Positive (or negative) staircase or postrest potentiation of the ‘whole‐cell’ [Ca2+] transients were paralleled by characteristic changes in the spatial profile of the [Ca2+]i signal. With low SR Ca2+ load [Ca2+]i transients in the subsarcolemmal space were small and no Ca2+ release in the centre of the cell was observed. Loading of the SR increased subsarcolemmal [Ca2+]i transient amplitude and subsequently triggered further release in more central regions of the cell. 5. Spontaneous Ca2+ release from functional SR units, i.e. Ca2+ sparks, occurred at higher frequency in the subsarcolemmal space than in more central regions of the myocyte. 6. Visualization of the surface membrane using the membrane‐selective dye Di‐8‐ANEPPS demonstrated that transverse tubules (t‐tubules) were absent in atrial cells. 7. It is concluded that in atrial myocytes voltage‐dependent Ca2+ entry triggers Ca2+ release from peripheral coupling SR that subsequently induces further Ca2+ release from stores in more central regions of the myocyte. Spreading of Ca2+ release from the cell periphery to the centre accounts for [Ca2+]i gradients underlying the whole‐cell [Ca2+]i transient. The finding that cat atrial myocytes lack t‐tubules demonstrates the functional importance of Ca2+ release from extended junctional (corbular) SR in these cells.

Functional coupling between glycolysis and excitation-contraction coupling underlies alternans in cat heart cells.

1 Electromechanical alternans was characterized in single cat atrial and ventricular myocytes by simultaneous measurements of action potentials, membrane current, cell shortening and changes in intracellular Ca2+ concentration ([Ca2+]i). 2 Using laser scanning confocal fluorescence microscopy, alternans of electrically evoked [Ca2+]i transients revealed marked differences between atrial and ventricular myocytes. In ventricular myocytes, electrically evoked [Ca2+]i transients during alternans were spatially homogeneous. In atrial cells Ca2+ release started at subsarcolemmal peripheral regions and subsequently spread toward the centre of the myocyte. In contrast to ventricular myocytes, in atrial cells propagation of Ca2+ release from the sarcoplasmic reticulum (SR) during the small‐amplitude [Ca2+]i transient was incomplete, leading to failures of excitation‐contraction (EC) coupling in central regions of the cell. 3 The mechanism underlying alternans was explored by evaluating the trigger signal for SR Ca2+ release (voltage‐gated L‐type Ca2+ current, ICa, L) and SR Ca2+ load during alternans. Voltage‐clamp experiments revealed that peak ICa, L was not affected during alternans when measured simultaneously with changes of cell shortening. The SR Ca2+ content, evaluated by application of caffeine pulses, was identical following the small‐amplitude and the large‐amplitude [Ca2+]i transient. These results suggest that the primary mechanism responsible for cardiac alternans does not reside in the trigger signal for Ca2+ release and SR Ca2+ load. 4 β‐Adrenergic stimulation with isoproterenol (isoprenaline) reversed electromechanical alternans, suggesting that under conditions of positive cardiac inotropy and enhanced efficiency of EC coupling alternans is less likely to occur. 5 The occurrence of electromechanical alternans could be elicited by impairment of glycolysis. Inhibition of glycolytic flux by application of pyruvate, iodoacetate or β‐hydroxybutyrate induced electromechanical and [Ca2+]i transient alternans in both atrial and ventricular myocytes. 6 The data support the conclusion that in cardiac myocytes alternans is the result of periodic alterations in the gain of EC coupling, i.e. the efficacy of a given trigger signal to release Ca2+ from the SR. It is suggested that the efficiency of EC coupling is locally controlled in the microenvironment of the SR Ca2+ release sites by mechanisms utilizing ATP, produced by glycolytic enzymes closely associated with the release channel.

Local calcium gradients during excitation–contraction coupling and alternans in atrial myocytes

Subcellular Ca2+ signalling during normal excitation‐contraction (E‐C) coupling and during Ca2+ alternans was studied in atrial myocytes using fast confocal microscopy and measurement of Ca2+ currents (ICa). Ca2+ alternans, a beat‐to‐beat alternation in the amplitude of the [Ca2+]i transient, causes electromechanical alternans, which has been implicated in the generation of cardiac fibrillation and sudden cardiac death. Cat atrial myocytes lack transverse tubules and contain sarcoplasmic reticulum (SR) of the junctional (j‐SR) and non‐junctional (nj‐SR) types, both of which have ryanodine‐receptor calcium release channels. During E‐C coupling, Ca2+ entering through voltage‐gated membrane Ca2+ channels (ICa) triggers Ca2+ release at discrete peripheral j‐SR release sites. The discrete Ca2+ spark‐like increases of [Ca2+]i then fuse into a peripheral ‘ring’ of elevated [Ca2+]i, followed by propagation (via calcium‐induced Ca2+ release, CICR) to the cell centre, resulting in contraction. Interrupting ICa instantaneously terminates j‐SR Ca2+ release, whereas nj‐SR Ca2+ release continues. Increasing the stimulation frequency or inhibition of glycolysis elicits Ca2+ alternans. The spatiotemporal [Ca2+]i pattern during alternans shows marked subcellular heterogeneities including longitudinal and transverse gradients of [Ca2+]i and neighbouring subcellular regions alternating out of phase. Moreover, focal inhibition of glycolysis causes spatially restricted Ca2+ alternans, further emphasising the local character of this phenomenon. When two adjacent regions within a myocyte alternate out of phase, delayed propagating Ca2+ waves develop at their border. In conclusion, the results demonstrate that (1) during normal E‐C coupling the atrial [Ca2+]i transient is the result of the spatiotemporal summation of Ca2+ release from individual release sites of the peripheral j‐SR and the central nj‐SR, activated in a centripetal fashion by CICR via ICa and Ca2+ release from j‐SR, respectively, (2) Ca2+ alternans is caused by subcellular alterations of SR Ca2+ release mediated, at least in part, by local inhibition of energy metabolism, and (3) the generation of arrhythmogenic Ca2+ waves resulting from heterogeneities in subcellular Ca2+ alternans may constitute a novel mechanism for the development of cardiac dysrhythmias.

Mitochondrial calcium in heart cells: beat-to-beat oscillations or slow integration of cytosolic transients?

Mitochondria have been implicated in intracellular Ca2+ signaling in many cell types. Theinner mitochondrial membrane contains Ca2+-transporting proteins, which catalyze Ca2+ uptakeand extrusion. Intramitochondrial (matrix) Ca2+, in turn, regulates the activity of Krebs cycledehydrogenases and, ultimately, the rate of ATP synthesis. In the myocardium, controversyremains whether the fast cytosolic Ca2+ transients underlying excitation-contraction couplingin beating cells are rapidly transmitted into the matrix compartment or slowly integratedby the mitochondrial Ca2+ transporters. This mini-review critically summarizes the recentexperimental work in this field.

Elementary events of agonist-induced Ca2+ release in vascular endothelial cells

The subcellular spatial and temporal organization of agonist-induced Ca2+ signals was investigated in single cultured vascular endothelial cells. Extracellular application of ATP initiated a rapid increase of intracellular Ca2+ concentration ([Ca2+]i) in peripheral cytoplasmic processes from where activation propagated as a [Ca2+]iwave toward the central regions of the cell. The average propagation velocity of the [Ca2+]iwave in the peripheral processes was 20-60 μm/s, whereas in the central region the wave propagated at <10 μm/s. The time course of the recovery of [Ca2+]idepended on the cell geometry. In the peripheral processes (i.e., regions with a high surface-to-volume ratio) [Ca2+]ideclined monotonically, whereas in the central region [Ca2+]idecreased in an oscillatory fashion. Propagating [Ca2+]iwaves were preceded by small, highly localized [Ca2+]itransients originating from 1- to 3-μm-wide regions. The average amplitude of these elementary events of Ca2+ release was 23 nM, and the underlying flux of Ca2+ amounted to ∼1-2 × 10-18mol/s or ∼0.3 pA, consistent with a Ca2+ flux through a single or small number of endoplasmic reticulum Ca2+-release channels.

Capacitative Ca2+ entry is graded with degree of intracellular Ca2+ store depletion in bovine vascular endothelial cells

1 In endothelial cells, release of Ca2+ from endoplasmic reticulum (ER) Ca2+ stores activates Ca2+ influx via the capacitative Ca2+ entry (CCE) pathway. In cultured bovine pulmonary artery endothelial cells, we investigated the relationship between intracellular Ca2+ store load and CCE activity, as well as the kinetics of CCE activation and deactivation, by simultaneously measuring changes in [Ca2+]i and unidirectional manganese (Mn2+) entry through the CCE pathway. 2 Submaximal concentrations of ATP caused quantal release of Ca2+ from the ER, resulting in a dose‐dependent depletion of Ca2+ stores and acceleration of Mn2+ entry. Mn2+ entry rate, as a measure of CCE activity, was graded with the amount of released Ca2+. Maximal activation of CCE did not require complete store depletion. 3 Slow depletion of the ER by exposure to the ER Ca2+ pump inhibitor cyclopiazonic acid resulted in a delayed activation of CCE, revealing a temporal dissociation between release of Ca2+ from intracellular stores and activation of CCE. 4 During [Ca2+]i oscillations, at frequencies higher than 0·5 spikes min−1, each Ca2+ spike resulted in a progressive acceleration of CCE without leading to oscillations of Ca2+ entry. In contrast, low frequency [Ca2+]i oscillations were paralleled by transient CCE that was activated and deactivated with each Ca2+ spike, resulting in an oscillatory pattern of Ca2+ entry. 5 It is concluded that CCE is a rapidly activating process which is graded with store depletion and becomes fully activated before complete depletion. The duration of CCE activation correlates with the degree of store depletion and the time that is required to refill depleted stores. Overall, a mechanism of graded CCE prevents exhaustion of intracellular Ca2+ reserves and provides an efficient way to respond to variable degrees of intracellular store depletion.

Subcellular properties of [Ca2+]i transients in phospholamban-deficient mouse ventricular cells

The regulatory protein phospholamban exerts a physiological inhibitory effect on the sarcoplasmic reticulum (SR) Ca2+ pump that is relieved with phosphorylation. We have studied the subcellular properties of intracellular Ca2+([Ca2+]i) transients in ventricular myocytes isolated from wild-type (WT) and phospholamban-deficient (PLB-KO) mice. In PLB-KO myocytes, steady-state twitch [Ca2+]itransients revealed an accelerated relaxation and the occurrence of highly localized failures of Ca2+release. The acceleration of SR Ca2+ uptake caused an increase in SR Ca2+ load with the frequent occurrence of spontaneous [Ca2+]iwaves and Ca2+ sparks. [Ca2+]iwaves in PLB-KO cells showed a marked decrease in spatial width and more frequently appeared to abort. Local Ca2+ release events (Ca2+ sparks) were larger and more variable in amplitude and [Ca2+]ideclined faster in PLB-KO myocytes. Increased local buffering and reduction in the refractoriness of SR Ca2+ release caused by the increased SR pump rate led to an overall enhancement of local [Ca2+]igradients and inhomogeneities in the [Ca2+]idistribution during spontaneous Ca2+ release, [Ca2+]iwaves, and excitation-contraction coupling.The regulatory protein phospholamban exerts a physiological inhibitory effect on the sarcoplasmic reticulum (SR) Ca2+ pump that is relieved with phosphorylation. We have studied the subcellular properties of intracellular Ca2+ ([Ca2+]i) transients in ventricular myocytes isolated from wild-type (WT) and phospholamban-deficient (PLB-KO) mice. In PLB-KO myocytes, steady-state twitch [Ca2+]i transients revealed an accelerated relaxation and the occurrence of highly localized failures of Ca2+ release. The acceleration of SR Ca2+ uptake caused an increase in SR Ca2+ load with the frequent occurrence of spontaneous [Ca2+]i waves and Ca2+ sparks. [Ca2+]i waves in PLB-KO cells showed a marked decrease in spatial width and more frequently appeared to abort. Local Ca2+ release events (Ca2+ sparks) were larger and more variable in amplitude and [Ca2+]i declined faster in PLB-KO myocytes. Increased local buffering and reduction in the refractoriness of SR Ca2+ release caused by the increased SR pump rate led to an overall enhancement of local [Ca2+]i gradients and inhomogeneities in the [Ca2+]i distribution during spontaneous Ca2+ release, [Ca2+]i waves, and excitation-contraction coupling.

Withdrawal of Acetylcholine Elicits Ca2+-Induced Delayed Afterdepolarizations in Cat Atrial Myocytes

BACKGROUND Recent experiments in atrial myocytes indicate that withdrawal of cholinergic agonist can directly increase Ca2+ influx via L-type Ca2+ current and stimulate Ca2+ uptake into the sarcoplasmic reticulum (SR), thereby increasing intracellular Ca2+. Overload of cellular Ca2+ within the SR can initiate various types of atrial dysrhythmias. The present study was designed to determine whether withdrawal of acetylcholine (ACh) can elicit Ca2+-induced delayed afterdepolarizations (DADs) in atrial myocytes. METHODS AND RESULTS A nystatin perforated-patch whole-cell method and fluorescence microscopy (indo 1) were used to measure electrical activities and intracellular free Ca2+ ([Ca2+]i), respectively. Withdrawal of ACh (1 micromol/L) increased action potential duration, shifted plateau voltage toward positive, and generated DADs that initiated spontaneous action potentials. Voltage-clamp analysis revealed that withdrawal of ACh elicited a rebound stimulation of L-type Ca2+ current (I(Ca,L)) (+45%) and Na/Ca exchange current (I(NaCa)) (+16%) and the appearance of transient inward current (I(ti)) and spontaneous [Ca2+]i transients. Each of these changes induced by withdrawal of ACh was abolished by Rp-cAMPs (50 to 100 micromol/L) or H-89 (2 micromol/L), inhibitors of cAMP-dependent protein kinase A. Ryanodine (1 micromol/L) abolished I(NaCa) and the appearance of I(ti) without decreasing the rebound stimulation of I(Ca,L) elicited by withdrawal of ACh. CONCLUSIONS Withdrawal of ACh can elicit cAMP-mediated stimulation of Ca2+ influx via I(Ca,L) and uptake of SR Ca2+. As a result, cellular Ca2+ overload causes enhanced SR Ca2+ release and the initiation of DADs. These mechanisms may generate triggered and/or spontaneous atrial depolarizations elicited by withdrawal of vagal nerve activity.

Focal agonist stimulation results in spatially restricted Ca2+ release and capacitative Ca2+ entry in bovine vascular endothelial cells

1 Intracellular Ca2+ ([Ca2+]i) signals were studied with spatial resolution in bovine vascular endothelial cells using the fluorescent Ca2+ indicator fluo‐3 and confocal laser scanning microscopy. Single cells were stimulated with the purinergic receptor agonist ATP resulting in an increase of [Ca2+]i due to intracellular Ca2+ release from inositol 1,4,5‐trisphosphate (IP3)‐sensitive stores. ATP‐induced Ca2+ release was quantal, i.e. submaximal concentrations mobilized only a fraction of the intracellularly stored Ca2+. 2 Focal receptor stimulation in Ca2+‐free solution by pressure application of agonist‐containing solution through a fine glass micropipette resulted in a spatially restricted increase in [Ca2+]i. Ca2+ release was initiated at the site of stimulation and frequently propagated some tens of micrometres into non‐stimulated regions. 3 Local Ca2+ release caused activation of capacitative Ca2+ entry (CCE). CCE was initially colocalized with Ca2+ release. Following repetitive focal stimulation, however, CCE became detectable at remote sites where no Ca2+ release had been observed. In addition, the rate of Ca2+ store depletion with repetitive local activation of release in Ca2+‐free solution was markedly slower than that elicited by ATP stimulation of the entire cell. 4 From these experiments it is concluded that both intracellular IP3‐dependent Ca2+ release and activation of CCE are controlled locally at the subcellular level. Moreover, redistribution of intracellular Ca2+ stored within the endoplasmic reticulum efficiently counteracts local store depletion and accounts for the spatial spread of CCE activation.