Katherine A. Sheehan

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.

Regulation of junctional and non‐junctional sarcoplasmic reticulum calcium release in excitation‐contraction coupling in cat atrial myocytes

We have characterized the dependence on membrane potential (Vm) and calcium current (ICa) of calcium‐induced calcium release (CICR) from the junctional‐SR (j‐SR, in the subsarcolemmal (SS) space) and non‐junctional‐SR (nj‐SR, in the central (CT) region of the cell) of cat atrial myocytes using whole‐cell voltage‐clamp together with spatially resolved laser‐scanning confocal microscopy. Subsarcolemmal and central [Ca2+]i transient amplitudes and ICa had a bell‐shaped dependence on Vm, but [Ca2+]i reached a maximum at more negative Vm (‐10 to 0 mV) than ICa (+10 mV). Termination of ICa after a brief depolarization (2.5 to 22.5 ms) immediately interrupted only the SS [Ca2+]i transient, leaving the development of the CT [Ca2+]i transient unaffected. Block of SR function with 20 μm ryanodine and 2 μm thapsigargin, revealed that > 90 % of the control [Ca2+]i transient amplitude was attributable to active SR Ca2+ release through ryanodine receptors (RyRs). The gain of SR Ca2+ release was highest in the SS space at negative test potentials and was less pronounced in the CT region. Inhibition of Na+‐Ca2+ exchange resulted in prolonged and higher amplitude [Ca2+]i transients, elevated resting [Ca2+]i, accelerated propagation of CICR, decreased extrusion of Ca2+ and an increase in j‐SR Ca2+ load. Increasing the cytosolic Ca2+ buffer capacity by internal perfusion with 1 mm EGTA limited SR Ca2+ release to the SS region, indicating that Ca2+ release from nj‐SR is initiated by diffusion of Ca2+ from the cell periphery and propagating CICR. Junctional‐SR Ca2+ release occurred at discrete sites whose order of activation and amplitude of release varied from beat to beat. In conclusion, during normal excitation‐contraction coupling in cat atrial myocytes, only Ca2+ release from the j‐SR is directly activated by Ca2+ entering via ICa. Elevation of SS [Ca2+]i is required to provide the cytosolic Ca2+ gradient needed to initiate regenerative and propagating CICR from nj‐SR.

Regional differences in spontaneous Ca2+ spark activity and regulation in cat atrial myocytes

Calcium sparks result from the concerted opening of a small number of Ca2+ release channels (ryanodine receptors, RyRs) organized in clusters in the membrane of the sarcoplasmic reticulum (SR). Calcium sparks represent the elementary events of SR Ca2+ release in cardiac myocytes, and their spatial and temporal summation results in whole‐cell [Ca2+]i transients observed during excitation–contraction coupling (ECC). Atrial myocytes generally lack transverse tubules; however, during ECC Ca2+ release is initiated from junctional SR (j‐SR) in the cell periphery from where activation propagates inwardly through Ca2+‐induced Ca2+ release (CICR) from non‐junctional SR (nj‐SR). Despite the structural differences in the microdomains of RyRs of j‐SR and nj‐SR, spontaneous Ca2+ sparks are observed from both types of SR, albeit at different frequencies. In cells that showed spontaneous Ca2+ sparks from j‐SR and nj‐SR, subsarcolemmal (SS) Ca2+ sparks from the j‐SR were 3–4 times more frequent than central (CTR) Ca2+ sparks occurring from nj‐SR. Subsarcolemmal Ca2+ sparks had a slightly higher amplitude, but were essentially identical in their spatial spread and duration when compared to CTR Ca2+ sparks. Sensitization of RyRs with a low concentration (0.1 mm) of caffeine led to a 107% increase in the frequency of CTR Ca2+ sparks, whereas the SS Ca2+ spark frequency increased by only 58%, suggesting that the nj‐SR is capable of much higher Ca2+ spark activity than observed normally in unstimulated cells. The L‐type Ca2+ channel blocker verapamil reduced SS Ca2+ spark frequency to 38% of control values, whereas Ca2+ spark activity from nj‐SR was reduced by only 19%, suggesting that SS Ca2+ sparks are under the control of Ca2+ influx from the extracellular space. Removal of extracellular Ca2+ eliminated SS Ca2+ sparks completely, whereas Ca2+ sparks from the nj‐SR continued, albeit at a lower frequency. In membrane‐permeabilized (saponin‐treated) atrial myocytes, where [Ca2+] can be experimentally controlled throughout the entire myocyte, j‐SR and nj‐SR Ca2+ spark frequencies were identical, and Ca2+ sparks could be observed spaced at sarcomeric distances throughout the entire cell, suggesting that all release sites of the nj‐SR can become active. Measurement of SR Ca2+ load (10 mm caffeine) revealed no difference between j‐SR and nj‐SR. The data suggest that in atrial myocytes, which lack a t‐tubular system, the nj‐SR is fully equipped with a three‐dimensional array of functional SR Ca2+ release sites; however, in intact cells under resting conditions, peripheral RyR clusters have a higher probability of activation owing to their association with surface membrane Ca2+ channels, leading to higher spontaneous Ca2+ spark activity. In conclusion, Ca2+ sparks originating from both j‐SR and nj‐SR are rather stereotypical and show little differences in their spatiotemporal properties. In intact cells, however, the higher frequency of spontaneous SS Ca2+ sparks arises from the structural arrangement of sarcolemma and j‐SR membrane and thus from the difference in the trigger mechanism.

P21-Activated Kinase-1 and Fty720 Alter Atrial Excitation-Contraction Coupling

We previously reported that Pak1 participates in the integrated regulation of excitation-contraction coupling (e-cc) in adult rat ventricular myocytes via activation of protein phosphatase 2A (PP2A). Pak1 inhibited the ability of isoproterenol (ISO) to increase the peak of the Ca2+ transient in a single myocyte without altering the increase in shortening. Here we test the hypothesis that Pak1 signaling regulates atrial e-cc and potentiates arrhythmicity. Under normal conditions single adult rat atrial myocytes expressing constitutively active Pak1 (CA-Pak1) and paced at 0.5 Hz have a slower time constant of [Ca2+ ]i-transient decay compared to uninfected controls as assessed by confocal microscopy and fluo-4 fluorescence. Increase of the pacing frequency to 2.0 Hz resulted in a reduced peak of the [Ca2+ ]i-transient, an increase in diastolic [Ca2+ ]I, and more rapid [Ca2+ ]i decay compared to 0.5 Hz for both CA-Pak1 expressing myocytes and controls. Return to 0.5 Hz pacing restored these values to near controls, with some extra-systolic Ca2+ release. Stimulation with ISO (100 nM) of CA-Pak1-expressing myocytes paced at 3.0 Hz caused 20% missed beats that were subsequently restored on washout. Uninfected myocytes paced at 2.0 Hz in the presence of ISO had 1:1 fidelity of beating to stimulation, however treatment with fingolimod (FTY720, 25 nM) in addition to ISO caused 50% missed beats that were restored on washout of the drug. Cultured monolayers of rat neonatal myocytes expressing CA-Pak1 had a more rapid rate of spontaneous beating than controls that was resistant to further increase with ISO, as assessed by microelectrode array (MEA). We conclude that Pak1 and FTY720 act through the same pathways in atrial myocytes and induce arrhythmic activity under conditions of stress.