A deep learning-based approach for efficient detection and classification of local Ca²âº release events in Full-Frame confocal imaging.

The release of Ca2+ ions from intracellular stores plays a crucial role in many cellular processes, acting as a secondary messenger in various cell types, including cardiomyocytes, smooth muscle cells, hepatocytes, and many others. Detecting and classifying associated local Ca2+ release events is particularly important, as these events provide insight into the mechanisms, interplay, and interdependencies of local Ca2+release events underlying global intracellular Ca2+signaling. However, time-consuming and labor-intensive procedures often complicate analysis, especially with low signal-to-noise ratio imaging data. Here, we present an innovative deep learning-based approach for automatically detecting and classifying local Ca2+ release events. This approach is exemplified with rapid full-frame confocal imaging data recorded in isolated cardiomyocytes. To demonstrate the robustness and accuracy of our method, we first use conventional evaluation methods by comparing the intersection between manual annotations and the segmentation of Ca2+ release events provided by the deep learning method, as well as the annotated and recognized instances of individual events. In addition to these methods, we compare the performance of the proposed model with the annotation of six experts in the field. Our model can recognize more than 75 % of the annotated Ca2+ release events and correctly classify more than 75 %. A key result was that there were no significant differences between the annotations produced by human experts and the result of the proposed deep learning model. We conclude that the proposed approach is a robust and time-saving alternative to conventional full-frame confocal imaging analysis of local intracellular Ca2+ events.

Dual mode of action of IP3-dependent SR-Ca2+ release on local and global SR-Ca2+ release in ventricular cardiomyocytes.

In heart muscle, the physiological function of IP3-induced Ca2+ release (IP3ICR) from the sarcoplasmic reticulum (SR) is still the subject of intense study. A role of IP3ICR may reside in modulating Ca2+-dependent cardiac arrhythmogenicity. Here we observe the propensity of spontaneous intracellular Ca2+ waves (SCaW) driven by Ca2+-induced Ca2+ release (CICR) in ventricular myocytes as a correlate of arrhythmogenicity on the organ level. We observe a dual mode of action of IP3ICR on SCaW generation in an IP3R overexpression model. This model shows a mild cardiac phenotype and mimics pathophysiological conditions of increased IP3R activity. In this model, IP3ICR was able to increase or decrease the occurrence of SCaW depending on global Ca2+ activity. This IP3ICR-based regulatory mechanism can operate in two "modes" depending on the intracellular CICR activity and efficiency (e.g. SCaW and/or local Ryanodine Receptor (RyR) Ca2+ release events, respectively): a) in a mode that augments the CICR mechanism at the cellular level, resulting in improved excitation-contraction coupling (ECC) and ultimately better contraction of the myocardium, and b) in a protective mode in which the CICR activity is curtailed to prevent the occurrence of Ca2+ waves at the cellular level and thus reduce the probability of arrhythmogenicity at the organ level.

IP3 receptors: An "elementary" journey from structure to signals.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are large tetrameric channels which sit mostly in the membrane of the endoplasmic reticulum (ER) and mediate Ca2+ release from intracellular stores in response to extracellular stimuli in almost all cells. Dual regulation of IP3Rs by IP3 and Ca2+ itself, upstream "licensing", and the arrangement of IP3Rs into small clusters in the ER membrane, allow IP3Rs to generate spatially and temporally diverse Ca2+ signals. The characteristic biphasic regulation of IP3Rs by cytosolic Ca2+ concentration underpins regenerative Ca2+ signals by Ca2+-induced Ca2+-release, while also preventing uncontrolled explosive Ca2+ release. In this way, cells can harness a simple ion such as Ca2+ as a near-universal intracellular messenger to regulate diverse cellular functions, including those with conflicting outcomes such as cell survival and cell death. High-resolution structures of the IP3R bound to IP3 and Ca2+ in different combinations have together started to unravel the workings of this giant channel. Here we discuss, in the context of recently published structures, how the tight regulation of IP3Rs and their cellular geography lead to generation of "elementary" local Ca2+ signals known as Ca2+ "puffs", which form the fundamental bottleneck through which all IP3-mediated cytosolic Ca2+ signals must first pass.

Ligand sensitivity of type-1 inositol 1,4,5-trisphosphate receptor is enhanced by the D2594K mutation.

Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are homologous cation channels that mediate release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR) and thereby are involved in many physiological processes. In previous studies, we determined that when the D2594 residue, located at or near the gate of the IP3R type 1, was replaced by lysine (D2594K), a gain of function was obtained. This mutant phenotype was characterized by increased IP3 sensitivity. We hypothesized the IP3R1-D2594 determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states. To test this possibility, the relationship between the D2594 site and IP3R1 regulation by IP3, cytosolic, and luminal Ca2+ was determined at the cellular, subcellular, and single-channel levels using fluorescence Ca2+ imaging and single-channel reconstitution. We found that in cells, D2594K mutation enhances the IP3 ligand sensitivity. Single-channel IP3R1 studies revealed that the conductance of IP3R1-WT and -D2594K channels is similar. However, IP3R1-D2594K channels exhibit higher IP3 sensitivity, with substantially greater efficacy. In addition, like its wild type (WT) counterpart, IP3R1-D2594K showed a bell-shape cytosolic Ca2+-dependency, but D2594K had greater activity at each tested cytosolic free Ca2+ concentration. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. Taken together, our functional studies indicate that the substitution of a negatively charged residue by a positive one at the channels' pore cytosolic exit affects the channel's gating behavior thereby explaining the enhanced ligand-channel's sensitivity.

Termination of Ca2+ puffs during IP3-evoked global Ca2+ signals.

We previously described that cell-wide cytosolic Ca2+ transients evoked by inositol trisphosphate (IP3) are generated by two modes of Ca2+ liberation from the ER; 'punctate' release via an initial flurry of transient Ca2+ puffs from local clusters of IP3 receptors, succeeded by a spatially and temporally 'diffuse' Ca2+ liberation. Those findings were derived using statistical fluctuation analysis to monitor puff activity which is otherwise masked as global Ca2+ levels rise. Here, we devised imaging approaches to resolve individual puffs during global Ca2+ elevations to better investigate the mechanisms terminating the puff flurry. We find that puffs contribute about 40% (∼90 attomoles) of the total Ca2+ liberation, largely while the global Ca2+ signal rises halfway to its peak. The major factor terminating punctate Ca2+ release is an abrupt decline in puff frequency. Although the amplitudes of large puffs fall during the flurry, the amplitudes of more numerous small puffs remain steady, so overall puff amplitudes decline only modestly (∼30%). The Ca2+ flux through individual IP3 receptor/channels does not measurably decline during the flurry, or when puff activity is depressed by pharmacological lowering of Ca2+ levels in the ER lumen, indicating that the termination of punctate release is not a simple consequence of reduced driving force for Ca2+ liberation. We propose instead that the gating of IP3 receptors at puff sites is modulated such that their openings become suppressed as the bulk [Ca2+] in the ER lumen falls during global Ca2+ signals.

A KRAP(y) job: Defining the localization of active IP3R.

A recent publication documented an exciting role for KRAP in tethering/immobilizing IP3Rs to actin. This interaction "licenses" IP3R activity as disrupting the partnership markedly diminishes Ca2+ puffs and global signals. These findings highlight a unique mechanism for regulating IP3R activity.

CREB regulates the expression of type 1 inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a central role in regulating intracellular Ca2+ signals in response to a variety of internal and external cues. Dysregulation of IP3R signaling is the underlying cause for numerous pathological conditions. It is well established that the activities of IP3Rs are governed by several post-translational modifications, including phosphorylation by protein kinase A (PKA). However, the long-term effects of PKA activation on expression of IP3R subtypes remains largely unexplored. In this report, we investigate the effects of chronic stimulation and tonic activity of PKA on the expression of IP3R subtypes. We demonstrate that expression of the type 1 IP3R (IP3R1) is augmented upon prolonged activation of PKA or upon ectopic overexpression of cyclic AMP-response element-binding protein (CREB) without altering IP3R2 and IP3R3 abundance. By contrast, inhibition of PKA or blocking CREB diminished IP3R1 expression. We also demonstrate that agonist-induced Ca2+-release mediated by IP3R1 is significantly attenuated upon blocking of CREB. Moreover, CREB - by regulating the expression of KRAS-induced actin-interacting protein (KRAP) - ensures correct localization and licensing of IP3R1. Overall, we report a crucial role for CREB in governing both the expression and correct localization of IP3R1. This article has an associated First Person interview with the first author of the paper.

Noise analysis of cytosolic calcium image data.

Cellular Ca2+ signals are often constrained to cytosolic micro- or nano-domains where stochastic openings of Ca2+ channels cause large fluctuations in local Ca2+ concentration (Ca2+ 'noise'). With the advent of TIRF microscopy to image the fluorescence of Ca2+-sensitive probes from attoliter volumes it has become possible to directly monitor these signals, which closely track the gating of plasmalemmal and ER Ca2+-permeable channels. Nevertheless, it is likely that many physiologically important Ca2+ signals are too small to resolve as discrete events in fluorescence recordings. By analogy with noise analysis of electrophysiological data, we explore here the use of statistical approaches to detect and analyze such Ca2+ noise in images obtained using Ca2+-sensitive indicator dyes. We describe two techniques - power spectrum analysis and spatio-temporal correlation - and demonstrate that both effectively identify discrete, spatially localized calcium release events (Ca2+ puffs). Moreover, we show they are able to detect localized noise fluctuations in a case where discrete events cannot directly be resolved.

Spatial-temporal patterning of Ca2+ signals by the subcellular distribution of IP3 and IP3 receptors.

The patterning of cytosolic Ca2+ signals in space and time underlies their ubiquitous ability to specifically regulate numerous cellular processes. Signals mediated by liberation of Ca2+ sequestered in the endoplasmic reticulum (ER) through inositol trisphosphate receptor (IP3R) channels constitute a hierarchy of events; ranging from openings of individual IP3 channels, through the concerted openings of several clustered IP3Rs to generate local Ca2+ puffs, to global Ca2+ waves and oscillations that engulf the entire cell. Here, we review recent progress in elucidating how this hierarchy is shaped by an interplay between the functional gating properties of IP3Rs and their spatial distribution within the cell. We focus in particular on the subset of IP3Rs that are organized in stationary clusters and are endowed with the ability to preferentially liberate Ca2+.

Applications of FLIKA, a Python-based image processing and analysis platform, for studying local events of cellular calcium signaling.

The patterning of cytosolic Ca2+ signals underlies their ubiquitous ability to specifically regulate numerous cellular processes. Advances in fluorescence microscopy have made it possible to image these signals with unprecedented temporal and spatial resolution. However, this is a double-edged sword, as the resulting enormous data sets necessitate development of software to automate image processing and analysis. Here, we describe Flika, an open source, graphical user interface program written in the Python environment that contains a suite of built-in image processing tools to enable intuitive visualization of image data and analysis. We illustrate the utility and power of Flika by three applications for studying cellular Ca2+ signaling: a script for measuring single-cell global Ca2+ signals; a plugin for the detection, localization and analysis of subcellular Ca2+ puffs; and a script that implements a novel approach for fluctuation analysis of transient, local Ca2+ fluorescence signals. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Dynamic Ca2+ imaging with a simplified lattice light-sheet microscope: A sideways view of subcellular Ca2+ puffs.

We describe the construction of a simplified, inexpensive lattice light-sheet microscope, and illustrate its use for imaging subcellular Ca2+ puffs evoked by photoreleased i-IP3 in cultured SH-SY5Y neuroblastoma cells loaded with the Ca2+ probe Cal520. The microscope provides sub-micron spatial resolution and enables recording of local Ca2+ transients in single-slice mode with a signal-to-noise ratio and temporal resolution (2ms) at least as good as confocal or total internal reflection microscopy. Signals arising from openings of individual IP3R channels are clearly resolved, as are stepwise changes in fluorescence reflecting openings and closings of individual channels during puffs. Moreover, by stepping the specimen through the light-sheet, the entire volume of a cell can be scanned within a few hundred ms. The ability to directly visualize a sideways (axial) section through cells directly reveals that IP3-evoked Ca2+ puffs originate at sites in very close (≤a few hundred nm) to the plasma membrane, suggesting they play a specific role in signaling to the membrane.

Functional local crosstalk of inositol 1,4,5-trisphosphate receptor- and ryanodine receptor-dependent Ca2+ release in atrial cardiomyocytes.

AIMS: Enhanced inositol 1,4,5-trisphosphate receptor (InsP3R2) expression has been associated with a variety of proarrhythmogenic cardiac disorders. The functional interaction between the two major Ca2+ release mechanisms in cardiomyocytes, Ca2+ release mediated by ryanodine receptors (RyR2s) and InsP3-induced intracellular Ca2+ release (IP3ICR) remains enigmatic. We aimed at identifying characterizing local IP3ICR events, and elucidating functional local crosstalk mechanisms between cardiac InsP3R2s and RyR2s under conditions of enhanced cardiac specific InsP3R2 activity. METHODS AND RESULTS: Using confocal imaging and two-dimensional spark analysis, we demonstrate in atrial myocytes (mouse model cardiac specific overexpressing InsP3R2s) that local Ca2+ release through InsP3Rs (Ca2+ puff) directly activates RyRs and triggers elementary Ca2+ release events (Ca2+ sparks). In the presence of increased intracellular InsP3 concentrations IP3ICR can modulate RyRs openings and Ca2+ spark probability. We show as well that IP3ICR remains under local control of Ca2+ release through RyRs. CONCLUSIONS: Our results support the concept of bidirectional interaction between RyRs and InsP3Rs (i.e. Ca2+ sparks and Ca2+ puffs) in atrial myocytes. We conclude that highly efficient InsP3 dependent SR-Ca2+ flux constitute the main mechanism of functional crosstalk between InsP3Rs and RyRs resulting in more Ca2+ sensitized RyRs to trigger subsequent Ca2+-induced Ca2+ release activation. In this way, bidirectional local interaction of both SR-Ca2+ release channels may contribute to the shaping of global Ca2+ transients and thereby to contractility in cardiac myocytes.

Comparison of Ca2+ puffs evoked by extracellular agonists and photoreleased IP3.

The inositol trisphosphate (IP3) signaling pathway evokes local Ca2+ signals (Ca2+ puffs) that arise from the concerted openings of clustered IP3 receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP3 into the cytosol. Photorelease of IP3 from a caged precursor provides a convenient and widely employed means to study the final stage of IP3-mediated Ca2+ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP3. Here, we address whether Ca2+ puffs evoked by photoreleased IP3 fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP3 analog, i-IP3. The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1µm), suggesting that IP3 from both sources accesses the same, or closely co-localized clusters of IP3Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP3 uniformly throughout the cytoplasm, our results imply that IP3 generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP3Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain.

Ca(2+)-activation kinetics modulate successive puff/spark amplitude, duration and inter-event-interval correlations in a Langevin model of stochastic Ca(2+) release.

Through theoretical analysis of the statistics of stochastic calcium (Ca(2+)) release (i.e., the amplitude, duration and inter-event interval of simulated Ca(2+) puffs and sparks), we show that a Langevin description of the collective gating of Ca(2+) channels may be a good approximation to the corresponding Markov chain model when the number of Ca(2+) channels per Ca(2+) release unit (CaRU) is in the physiological range. The Langevin description of stochastic Ca(2+) release facilitates our investigation of correlations between successive puff/spark amplitudes, durations and inter-spark intervals, and how such puff/spark statistics depend on the number of channels per release site and the kinetics of Ca(2+)-mediated inactivation of open channels. When Ca(2+) inactivation/de-inactivation rates are intermediate-i.e., the termination of Ca(2+) puff/sparks is caused by an increase in the number of inactivated channels-the correlation between successive puff/spark amplitudes is negative, while the correlations between puff/spark amplitudes and the duration of the preceding or subsequent inter-spark interval are positive. These correlations are significantly reduced or change signs when inactivation/de-inactivation rates are extreme (slow or fast) and puff/sparks terminate via stochastic attrition.