Lothar A. Blatter

Surface:volume relationship in cardiac myocytes studied with confocal microscopy and membrane capacitance measurements: species-dependence and developmental effects.

The quantitative analysis of the contribution of ion fluxes through membrane channels to changes of intracellular ion concentrations would benefit from the exact knowledge of the cell volume. It would allow direct correlation of ionic current measurements with simultaneous measurements of ion concentrations in individual cells. Because of various limitations of conventional light microscopy a simple method for accurate cell volume determination is lacking. We have combined the optical sectioning capabilities of fluorescence laser scanning confocal microscopy and the whole-cell patch-clamp technique to study the correlation between cell volume and membrane capacitance. Single cardiac myocytes loaded with the fluorescent dye calcein were optically sectioned to produce a series of confocal images. The volume of cardiac myocytes of three different mammalian species was determined by three-dimensional volume rendering of the confocal images. The calculated cell volumes were 30.4 +/- 7.3 pl (mean +/- SD) in rabbits (n = 28), 30.9 +/- 9.0 pl in ferrets (n = 23), and 34.4 +/- 7.0 pl in rats (n = 21), respectively. There was a positive linear correlation between membrane capacitance and cell volume in each animal species. The capacitance-volume ratios were significantly different among species (4.58 +/- 0.45 pF/pl in rabbit, 5.39 +/- 0.57 pF/pl in ferret, and 8.44 +/- 1.35 pF/pl in rat). Furthermore, the capacitance-volume ratio was dependent on the developmental stage (8.88 +/- 1.14 pF/pl in 6-month-old rats versus 6.76 +/- 0.62 pF/pl in 3-month-old rats). The data suggest that the ratio of surface area:volume of cardiac myocytes undergoes significant developmental changes and differs among mammalian species. We further established that the easily measurable parameters of cell membrane capacitance or the product of cell length and width provide reliable but species-dependent estimates for the volume of individual cells.

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.

Endothelin-1–Induced Arrhythmogenic Ca2+ Signaling Is Abolished in Atrial Myocytes of Inositol-1,4,5-Trisphosphate(IP3)–Receptor Type 2–Deficient Mice

Recent studies have suggested that inositol-1,4,5-trisphosphate-receptor (IP3R)–mediated Ca2+ release plays an important role in the modulation of excitation–contraction coupling (ECC) in atrial tissue and the generation of arrhythmias, specifically chronic atrial fibrillation (AF). IP3R type-2 (IP3R2) is the predominant IP3R isoform expressed in atrial myocytes. To determine the role of IP3R2 in atrial arrhythmogenesis and ECC, we generated IP3R2-deficient mice. Our results revealed that endothelin-1 (ET-1) stimulation of wild-type (WT) atrial myocytes caused an increase in basal [Ca2+]i, an enhancement of action potential (AP)-induced [Ca2+]i transients, an improvement of the efficacy of ECC (increased fractional SR Ca2+ release), and the occurrence of spontaneous arrhythmogenic Ca2+ release events as the result of activation of IP3R-dependent Ca2+ release. In contrast, ET-1 did not alter diastolic [Ca2+]i or cause spontaneous Ca2+ release events in IP3R2-deficient atrial myocytes. Under basal conditions the spatio-temporal properties (amplitude, rise-time, decay kinetics, and spatial spread) of [Ca2+]i transients and fractional SR Ca2+ release were not different in WT and IP3R2-deficient atrial myocytes. WT and IP3R2-deficient atrial myocytes also showed a significant and very similar increase in the amplitude of AP-dependent [Ca2+]i transients and Ca2+ spark frequency in response to isoproterenol stimulation, suggesting that both cell types maintained a strong inotropic reserve. No compensatory changes in Ca2+ regulatory protein expression (IP3R1, IP3R3, RyR2, NCX, SERCA2) or morphology of the atria could be detected between WT and IP3R2-deficient mice. These results show that lack of IP3R2 abolishes the positive inotropic effect of neurohumoral stimulation with ET-1 and protects from its arrhythmogenic effects.

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.

Inositol‐1,4,5‐trisphosphate‐dependent Ca2+ signalling in cat atrial excitation–contraction coupling and arrhythmias

Inositol‐1,4,5‐trisphosphate (IP3)‐dependent Ca2+ release represents the major Ca2+ mobilizing pathway responsible for diverse functions in non‐excitable cells. In the heart, however, its role is largely unknown or controversial. In intact cat atrial myocytes, endothelin (ET‐1) increased basal [Ca2+]i levels, enhanced action potential‐evoked [Ca2+]i transients, caused [Ca2+]i transients with alternating amplitudes (Ca2+ alternans), and facilitated spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) in the form of Ca2+ sparks and arrhythmogenic Ca2+ waves. These effects were prevented by the IP3 receptor (IP3R) blocker aminoethoxydiphenyl borate (2‐APB), suggesting the involvement of IP3‐dependent SR Ca2+ release. In saponin‐permeabilized myocytes IP3 and the more potent IP3R agonist adenophostin increased basal [Ca2+]i and the frequency of spontaneous Ca2+ sparks. In the presence of tetracaine to eliminate Ca2+ release from ryanodine receptor (RyR) SR Ca2+ release channels, IP3 and adenophostin triggered unique elementary, non‐propagating IP3R‐dependent Ca2+ release events with amplitudes and kinetics that were distinctly different from classical RyR‐dependent Ca2+ sparks. The effects of IP3 and adenophostin were prevented by heparin and 2‐APB. The data suggest that IP3‐dependent Ca2+ release increases [Ca2+]i in the vicinity of RyRs and thus facilitates Ca2+‐induced Ca2+ release during excitation–contraction coupling. It is concluded that in the adult mammalian atrium IP3‐dependent Ca2+ release enhances atrial Ca2+ signalling and exerts a positive inotropic effect. In addition, by facilitating Ca2+ release, IP3 may also be an important component in the development of Ca2+‐mediated atrial arrhythmias.

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.

Cardiac Alternans Do Not Rely on Diastolic Sarcoplasmic Reticulum Calcium Content Fluctuations

Cardiac alternans are thought to be a precursor to life-threatening arrhythmias. Previous studies suggested that alterations in sarcoplasmic reticulum (SR) Ca2+ content are either causative or not associated with myocyte Ca2+ alternans. However, those studies used indirect measures of SR Ca2+. Here we used direct continuous measurement of intra-SR free [Ca2+] ([Ca2+]SR) (using Fluo5N) during frequency-dependent Ca2+ alternans in rabbit ventricular myocytes. We tested the hypothesis that alternating [Ca2+]SR is required for Ca2+ alternans. Amplitudes of [Ca2+]SR depletions alternated in phase with cytosolic Ca2+ transients and contractions. Some cells showed clear alternation in diastolic [Ca2+]SR during alternans, with higher [Ca2+]SR before the larger SR Ca2+ releases. However, the extent of SR Ca2+ release during the small beats was smaller than expected for the modest decrease in [Ca2+]SR. In other cells, clear Ca2+ alternans was observed without alternations in diastolic [Ca2+]SR. Additionally, alternating cells were observed, in which diastolic [Ca2+]SR fluctuations occurred interspersed by depletions in which the amplitude was unrelated to the preceding diastolic [Ca2+]SR. In all forms of alternans, the SR Ca2+ release rate was higher during large depletions than during small depletions. Although [Ca2+]SR exerts major influence on SR Ca2+ release, alternations in [Ca2+]SR are not required for Ca2+ alternans to occur. Rather, it seems likely that some other factor, such as ryanodine receptor availability after a prior beat (eg, recovery from inactivation), is of greater importance in initiating frequency-induced Ca2+ alternans. However, once such a weak SR Ca2+ release occurs, it can result in increased [Ca2+]SR and further enhance SR Ca2+ release at the next beat. In this way, diastolic [Ca2+]SR alternans can enhance frequency-induced Ca2+ alternans, even if they initiate by other means.

Nitric oxide decreases [Ca2+]i in vascular smooth muscle by inhibition of the calcium current

Endothelium derived relaxing factor (nitric oxide, or NO) activates cytoplasmic guanylate cyclase in vascular smooth muscle and decreases vascular tone through cGMP-dependent mechanisms that are not yet understood fully. In cultured vascular smooth muscle cells (A7r5 cell line) sodium nitroprusside (NP), a vasodilator that decomposes into nitric oxide, lowered [Ca2+]i in cells in which [Ca2+]i was elevated after depolarization. NP decreased current through voltage-gated calcium channels, but did not affect release of calcium from intracellular stores. Hemoglobin, a scavenger of NO, reversed the effect of NP on [Ca2+]i and 8-Br-cGMP, a membrane permeant form of cGMP, mimicked the effect of NP on [Ca2+]i and on calcium currents. Thus, the signal transduction mechanism of endothelium dependent relaxation of vascular smooth muscle involves a decrease in [Ca2+]i by inhibition of Ca2+ entry. Relaxation or vasodilation would then result from decreased activity of myosin light chain kinase, in addition to myosin light chain dephosphorylation.

Emerging roles of inositol 1,4,5-trisphosphate signaling in cardiac myocytes

Inositol 1,4,5-trisphosphate (IP(3)) is a ubiquitous intracellular messenger regulating diverse functions in almost all mammalian cell types. It is generated by membrane receptors that couple to phospholipase C (PLC), an enzyme which liberates IP(3) from phosphatidylinositol 4,5-bisphosphate (PIP(2)). The major action of IP(3), which is hydrophilic and thus translocates from the membrane into the cytoplasm, is to induce Ca(2+) release from endogenous stores through IP(3) receptors (IP(3)Rs). Cardiac excitation-contraction coupling relies largely on ryanodine receptor (RyR)-induced Ca(2+) release from the sarcoplasmic reticulum. Myocytes express a significantly larger number of RyRs compared to IP(3)Rs (~100:1), and furthermore they experience substantial fluxes of Ca(2+) with each heartbeat. Therefore, the role of IP(3) and IP(3)-mediated Ca(2+) signaling in cardiac myocytes has long been enigmatic. Recent evidence, however, indicates that despite their paucity cardiac IP(3)Rs may play crucial roles in regulating diverse cardiac functions. Strategic localization of IP(3)Rs in cytoplasmic compartments and the nucleus enables them to participate in subsarcolemmal, bulk cytoplasmic and nuclear Ca(2+) signaling in embryonic stem cell-derived and neonatal cardiomyocytes, and in adult cardiac myocytes from the atria and ventricles. Intriguingly, expression of both IP(3)Rs and membrane receptors that couple to PLC/IP(3) signaling is altered in cardiac disease such as atrial fibrillation or heart failure, suggesting the involvement of IP(3) signaling in the pathology of these diseases. Thus, IP(3) exerts important physiological and pathological functions in the heart, ranging from the regulation of pacemaking, excitation-contraction and excitation-transcription coupling to the initiation and/or progression of arrhythmias, hypertrophy and heart failure.