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Key Points: In atrial myocytes excitation-contraction coupling is strikingly different from ventricle because atrial myocytes lack a transverse tubule membrane system: Ca 2+ release starts in the cell periphery and propagates towards the cell centre by Ca 2+ -induced Ca 2+ release from the sarcoplasmic reticulum (SR) Ca 2+ store. The cytosolic Ca 2+ sensitivity of the ryanodine receptor (RyRs) Ca 2+ release channel is low and it is unclear how Ca 2+ release can be activated in the interior of atrial cells. Simultaneous confocal imaging of cytosolic and intra-SR calcium revealed a transient elevation of store Ca 2+ that we termed 'Ca 2+ sensitization signal'. We propose a novel paradigm of atrial ECC that is based on tandem activation of the RyRs by cytosolic and luminal Ca 2+ through a 'fire-diffuse-uptake-fire' (or FDUF) mechanism: Ca 2+ uptake by SR Ca 2+ pumps at the propagation front elevates Ca 2+ inside the SR locally, leading to luminal RyR sensitization and lowering of the cytosolic Ca 2+ activation threshold., Abstract: In atrial myocytes Ca 2+ release during excitation-contraction coupling (ECC) is strikingly different from ventricular myocytes. In many species atrial myocytes lack a transverse tubule system, dividing the sarcoplasmic reticulum (SR) Ca 2+ store into the peripheral subsarcolemmnal junctional (j-SR) and the much more abundant central non-junctional (nj-SR) SR. Action potential (AP)-induced Ca 2+ entry activates Ca 2+ -induced Ca 2+ release (CICR) from j-SR ryanodine receptor (RyR) Ca 2+ release channels. Peripheral elevation of [Ca 2+ ] i initiates CICR from nj-SR and sustains propagation of CICR to the cell centre. Simultaneous confocal measurements of cytosolic ([Ca 2+ ] i ; with the fluorescent Ca 2+ indicator rhod-2) and intra-SR ([Ca 2+ ] SR ; fluo-5N) Ca 2+ in rabbit atrial myocytes revealed that Ca 2+ release from j-SR resulted in a cytosolic Ca 2+ transient of higher amplitude compared to release from nj-SR; however, the degree of depletion of j-SR [Ca 2+ ] SR was smaller than nj-SR [Ca 2+ ] SR . Similarly, Ca 2+ signals from individual release sites of the j-SR showed a larger cytosolic amplitude (Ca 2+ sparks) but smaller depletion (Ca 2+ blinks) than release from nj-SR. During AP-induced Ca 2+ release the rise of [Ca 2+ ] i detected at individual release sites of the nj-SR preceded the depletion of [Ca 2+ ] SR , and during this latency period a transient elevation of [Ca 2+ ] SR occurred. We propose that Ca 2+ release from nj-SR is activated by cytosolic and luminal Ca 2+ (tandem RyR activation) via a novel 'fire-diffuse-uptake-fire' (FDUF) mechanism. This novel paradigm of atrial ECC predicts that Ca 2+ uptake by sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) at the propagation front elevates local [Ca 2+ ] SR , leading to luminal RyR sensitization and lowering of the activation threshold for cytosolic CICR., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Key Points: Impaired calcium (Ca(2+)) signalling is the main contributor to depressed ventricular contractile function and occurrence of arrhythmia in heart failure (HF). Here we report that in atrial cells of a rabbit HF model, Ca(2+) signalling is enhanced and we identified the underlying cellular mechanisms. Enhanced Ca(2+) transients (CaTs) are due to upregulation of inositol-1,4,5-trisphosphate receptor induced Ca(2+) release (IICR) and decreased mitochondrial Ca(2+) sequestration. Enhanced IICR, however, together with an increased activity of the sodium-calcium exchange mechanism, also facilitates spontaneous Ca(2+) release in form of arrhythmogenic Ca(2+) waves and spontaneous action potentials, thus enhancing the arrhythmogenic potential of atrial cells. Our data show that enhanced Ca(2+) signalling in HF provides atrial cells with a mechanism to improve ventricular filling and to maintain cardiac output, but also increases the susceptibility to develop atrial arrhythmias facilitated by spontaneous Ca(2+) release., Abstract: We studied excitation-contraction coupling (ECC) and inositol-1,4,5-triphosphate (IP3)-dependent Ca(2+) release in normal and heart failure (HF) rabbit atrial cells. Left ventricular HF was induced by combined volume and pressure overload. In HF atrial myocytes diastolic [Ca(2+)]i was increased, action potential (AP)-induced Ca(2+) transients (CaTs) were larger in amplitude, primarily due to enhanced Ca(2+) release from central non-junctional sarcoplasmic reticulum (SR) and centripetal propagation of activation was accelerated, whereas HF ventricular CaTs were depressed. The larger CaTs were due to enhanced IP3 receptor-induced Ca(2+) release (IICR) and reduced mitochondrial Ca(2+) buffering, consistent with a reduced mitochondrial density and Ca(2+) uptake capacity in HF. Elementary IP3 receptor-mediated Ca(2+) release events (Ca(2+) puffs) were more frequent in HF atrial myoctes and were detected more often in central regions of the non-junctional SR compared to normal cells. HF cells had an overall higher frequency of spontaneous Ca(2+) waves and a larger fraction of waves (termed arrhythmogenic Ca(2+) waves) triggered APs and global CaTs. The higher propensity of arrhythmogenic Ca(2+) waves resulted from the combined action of enhanced IICR and increased activity of sarcolemmal Na(+)-Ca(2+) exchange depolarizing the cell membrane. In conclusion, the data support the hypothesis that in atrial myocytes from hearts with left ventricular failure, enhanced CaTs during ECC exert positive inotropic effects on atrial contractility which facilitates ventricular filling and contributes to maintaining cardiac output. However, HF atrial cells were also more susceptible to developing arrhythmogenic Ca(2+) waves which might form the substrate for atrial rhythm disorders frequently encountered in HF., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

β-Adrenergic signalling induces positive inotropic effects on the heart that associate with pro-arrhythmic spontaneous Ca(2+) waves. A threshold level of sarcoplasmic reticulum (SR) Ca(2+) ([Ca(2+)](SR)) is necessary to trigger Ca(2+) waves, and whether the increased incidence of Ca(2+) waves during β-adrenergic stimulation is due to an alteration in this threshold remains controversial. Using the low-affinity Ca(2+) indicator fluo-5N entrapped within the SR of rabbit ventricular myocytes, we addressed this controversy by directly monitoring [Ca(2+)](SR) and Ca(2+) waves during β-adrenergic stimulation. Electrical pacing in elevated extracellular Ca(2+) ([Ca(2+)](o) = 7 mM) was used to increase [Ca(2+)](SR) to the threshold where Ca(2+) waves were consistently observed. The β-adrenergic agonist isoproterenol (ISO; 1 μM) increased [Ca(2+)](SR) well above the control threshold and consistently triggered Ca(2+) waves. However, when [Ca(2+)](SR) was subsequently lowered in the presence of ISO (by lowering [Ca(2+)](o) to 1 mM and partially inhibiting sarcoplasmic/endoplasmic reticulum calcium ATPase with cyclopiazonic acid or thapsigargin), Ca(2+) waves ceased to occur at a [Ca(2+)](SR) that was higher than the control threshold. Furthermore, for a set [Ca(2+)](SR) level the refractoriness of wave occurrence (Ca(2+) wave latency) was prolonged during β-adrenergic stimulation, and was highly dependent on the extent that [Ca](SR) exceeded the wave threshold. These data show that acute β-adrenergic stimulation increases the [Ca(2+)](SR) threshold for Ca(2+) waves, and therefore the primary cause of Ca(2+) waves is the robust increase in [Ca(2+)](SR) above this higher threshold level. Elevation of the [Ca(2+)](SR) wave threshold and prolongation of wave latency represent potentially protective mechanisms against pro-arrhythmogenic Ca(2+) release during β-adrenergic stimulation.

The widely accepted paradigm for cytosolic Ca(2+) wave propagation postulates a 'fire-diffuse-fire' mechanism where local Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca(2+) release channels diffuses towards and activates neighbouring release sites, resulting in a propagating Ca(2+) wave. A recent challenge to this paradigm proposed the requirement for an intra-SR 'sensitization' Ca(2+) wave that precedes the cytosolic Ca(2+) wave and primes RyRs from the luminal side to CICR. Here, we tested this hypothesis experimentally with direct simultaneous measurements of cytosolic ([Ca(2+)](i); rhod-2) and intra-SR ([Ca(2+)](SR); fluo-5N) calcium signals during wave propagation in rabbit ventricular myocytes, using high resolution fluorescence confocal imaging. The increase in [Ca(2+)](i) at the wave front preceded depletion of the SR at each point along the calcium wave front, while during this latency period a transient increase of [Ca(2+)](SR) was observed. This transient elevation of [Ca(2+)](SR) could be identified at individual release junctions and depended on the activity of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA). Increased SERCA activity (β-adrenergic stimulation with 1 μM isoproterenol (isoprenaline)) decreased the latency period and increased the amplitude of the transient elevation of [Ca(2+)](SR), whereas inhibition of SERCA (3 μM cyclopiazonic acid) had the opposite effect. In conclusion, the data provide experimental evidence that local Ca(2+) uptake by SERCA into the SR facilitates the propagation of cytosolic Ca(2+) waves via luminal sensitization of the RyR, and supports a novel paradigm of a 'fire-diffuse-uptake-fire' mechanism for Ca(2+) wave propagation in cardiac myocytes.

Photoactivated probes have gained interest as experimental tools to study intracellular signalling pathways all the way to the molecular level. However technical limitations of the means to activate such compounds have put constraints on their use in spatially highly restricted subcellular areas. The Mosaic digital illumination system uses a high-speed array of individually addressable, tiltable micromirrors to direct continuous-wave laser light onto a specimen with diffraction-limited precision. The system, integrated into a Nikon A1R confocal microscope, was used to uncage Ca²⁺ or IP3 and conduct photobleaching experiments from multiple geometrically complex subcellular regions while simultaneously measuring [Ca²⁺]i with high-speed confocal imaging.