Intracellular concentrations of redox-active molecules can significantly increase in the heart as a result of activation of specific signal transduction pathways or the development of certain pathophysiological conditions. Changes in the intracellular redox environment can affect many cellular processes, including the gating properties of ion channels and the activity of ion transporters. Because cardiac contraction is highly dependent on intracellular Ca(2+) levels ([Ca(2+)](i)) and [Ca(2+)](i) regulation, redox modification of Ca(2+) channels and transporters has a profound effect on cardiac function. The sarcoplasmic reticulum (SR) Ca(2+) release channel, or ryanodine receptor (RyR), is one of the well-characterized redox-sensitive ion channels in the heart. The redox modulation of RyR activity is mediated by the redox modification of sulfhydryl groups of cysteine residues. Other key components of cardiac excitation-contraction (e-c) coupling such as the SR Ca(2+) ATPase and L-type Ca(2+) channel are subject to redox modulation. Redox-mediated alteration of the activity of ion channels and pumps is directly involved in cardiac pathologies such as ischemia-reperfusion injury. Significant bursts of reactive oxygen species (ROS) generation occur during reperfusion of the ischemic heart, and changes in the activity of the major components of [Ca(2+)](i) regulation, such as RyR, Na(+)-Ca(2+) exchange and Ca(2+) ATPases, are likely to play an important role in ischemia-related Ca(2+) overload. This article summarizes recent findings on redox regulation of cardiac Ca(2+) transport systems and discusses contributions of this redox regulation to normal and pathological cardiac function.

Abstract-Recent studies have suggested that inositol-1,4,5-trisphosphate-receptor (IP 3 R)-mediated Ca 2ϩ 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). IP 3 R type-2 (IP 3 R2) is the predominant IP 3 R isoform expressed in atrial myocytes. To determine the role of IP 3 R2 in atrial arrhythmogenesis and ECC, we generated IP 3 R2-deficient mice. Our results revealed that endothelin-1 (ET-1) stimulation of wild-type (WT) atrial myocytes caused an increase in basal [Ca 2ϩ ] i , an enhancement of action potential (AP)-induced [Ca 2ϩ ] i transients, an improvement of the efficacy of ECC (increased fractional SR Ca 2ϩ release), and the occurrence of spontaneous arrhythmogenic Ca 2ϩ release events as the result of activation of IP 3 R-dependent Ca 2ϩ release. In contrast, ET-1 did not alter diastolic [Ca 2ϩ ] i or cause spontaneous Ca 2ϩ release events in IP 3 R2-deficient atrial myocytes. Under basal conditions the spatio-temporal properties (amplitude, rise-time, decay kinetics, and spatial spread) of [Ca 2ϩ ] i transients and fractional SR Ca 2ϩ release were not different in WT and IP 3 R2-deficient atrial myocytes. WT and IP 3 R2-deficient atrial myocytes also showed a significant and very similar increase in the amplitude of AP-dependent [Ca 2ϩ ] i transients and Ca 2ϩ spark frequency in response to isoproterenol stimulation, suggesting that both cell types maintained a strong inotropic reserve. No compensatory changes in Ca 2ϩ regulatory protein expression (IP 3 R1, IP 3 R3, RyR2, NCX, SERCA2) or morphology of the atria could be detected between WT and IP 3 R2-deficient mice. These results show that lack of IP 3 R2 abolishes the positive inotropic effect of neurohumoral stimulation with ET-1 and protects from its arrhythmogenic effects. (Circ Res. 2005;96:1274-1281.)Key Words: IP 3 receptor Ⅲ intracellular calcium Ⅲ atrial arrhythmias Ⅲ excitation-contraction coupling Ⅲ endothelin C hronic atrial fibrillation (AF) is the most common sustained form of cardiac arrhythmia. AF is characterized by an atrial activation rate of typically Ͼ400 beats per minute, and is associated with 2 major complications including cardiac dysfunction and thrombus formation, resulting in an increased risk of morbidity because of heart failure and stroke. 1,2 In recent years, considerable attention has focused on the cellular and molecular mechanisms involved in AF (for review see Nattel 3,4 activates the RyR which leads to massive Ca 2ϩ release from the SR by a mechanism known as Ca 2ϩ -induced Ca 2ϩ release (CICR 12 ), which is required for inducing contraction.Cardiac myocytes also contain IP 3 R channels, however their functional importance in the heart has remained controversial. IP 3 Rs release Ca 2ϩ from intracellular Ca 2ϩ stores when activated by IP 3 , a product generated by phospholipase C (PLC) metabolism of phosphoinositol-4,5-bisphosphate (PIP 2 ) in response to G-protein-c...

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). 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. 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. 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. 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. 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.

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. 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. 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. 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.

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.

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.

Sarcoplasmic reticulum (SR) Ca

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.