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.

Ca2+ spark latency and control of intrinsic Ca2+ release dyssynchrony in rat cardiac ventricular muscle cells.

Cardiac excitation-contraction coupling (ECC) depends on Ca2+ release from intracellular stores via ryanodine receptors (RyRs) triggered by L-type Ca2+ channels (LCCs). Uncertain numbers of RyRs and LCCs form 'couplons' whose activation produces Ca2+ sparks, which summate to form a cell-wide Ca2+ transient that switches on contraction. Voltage (Vm) changes during the action potential (AP) and stochasticity in channel gating should create variability in Ca2+ spark timing, but Ca2+ transient wavefronts have remarkable uniformity. To examine how this is achieved, we measured the Vm-dependence of evoked Ca2+ spark probability (Pspark) and latency over a wide voltage range in rat ventricular cells. With depolarising steps, Ca2+ spark latency showed a U-shaped Vm-dependence, while repolarising steps from 50 mV produced Ca2+ spark latencies that increased monotonically with Vm. A computer model based on reported channel gating and geometry reproduced our experimental data and revealed a likely RyR:LCC stoichiometry of âˆ¼ 5:1 for the Ca2+ spark initiating complex (IC). Using the experimental AP waveform, the model revealed a high coupling fidelity (Pcpl âˆ¼ 0.5) between each LCC opening and IC activation. The presence of âˆ¼ 4 ICs per couplon reduced Ca2+ spark latency and increased Pspark to match experimental data. Variability in AP release timing is less than that seen with voltage steps because the AP overshoot and later repolarization decrease Pspark due to effects on LCC flux and LCC deactivation respectively. This work provides a framework for explaining the Vm- and time-dependence of Pspark, and indicates how ion channel dispersion in disease can contribute to dyssynchrony in Ca2+ release.

Improved Workflow for Analysis of Vascular Myocyte Time-Series and Line-Scan Ca2+ Imaging Datasets.

Intracellular Ca2+ signals are key for the regulation of cellular processes ranging from myocyte contraction, hormonal secretion, neural transmission, cellular metabolism, transcriptional regulation, and cell proliferation. Measurement of cellular Ca2+ is routinely performed using fluorescence microscopy with biological indicators. Analysis of deterministic signals is reasonably straightforward as relevant data can be discriminated based on the timing of cellular responses. However, analysis of stochastic, slower oscillatory events, as well as rapid subcellular Ca2+ responses, takes considerable time and effort which often includes visual analysis by trained investigators, especially when studying signals arising from cells embedded in complex tissues. The purpose of the current study was to determine if full-frame time-series and line-scan image analysis workflow of Fluo-4 generated Ca2+ fluorescence data from vascular myocytes could be automated without introducing errors. This evaluation was addressed by re-analyzing a published "gold standard" full-frame time-series dataset through visual analysis of Ca2+ signals from recordings made in pulmonary arterial myocytes of en face arterial preparations. We applied a combination of data driven and statistical approaches with comparisons to our published data to assess the fidelity of the various approaches. Regions of interest with Ca2+ oscillations were detected automatically post hoc using the LCPro plug-in for ImageJ. Oscillatory signals were separated based on event durations between 4 and 40 s. These data were filtered based on cutoffs obtained from multiple methods and compared to the published manually curated "gold standard" dataset. Subcellular focal and rapid Ca2+ "spark" events from line-scan recordings were examined using SparkLab 5.8, which is a custom automated detection and analysis program. After filtering, the number of true positives, false positives, and false negatives were calculated through comparisons to visually derived "gold standard" datasets. Positive predictive value, sensitivity, and false discovery rates were calculated. There were very few significant differences between the automated and manually curated results with respect to quality of the oscillatory and Ca2+ spark events, and there were no systematic biases in the data curation or filtering techniques. The lack of statistical difference in event quality between manual data curation and statistically derived critical cutoff techniques leads us to believe that automated analysis techniques can be reliably used to analyze spatial and temporal aspects to Ca2+ imaging data, which will improve experiment workflow.

Cardiac Protein Kinase D1 ablation alters the myocytes ß-adrenergic response.

ß-adrenergic (ß-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute ß-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte ß-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal ß-adrenergic responses in Ca2+ handling.

The Role of Ca2+ Sparks in Force Frequency Relationships in Guinea Pig Ventricular Myocytes.

Calcium sparks are the elementary Ca2+ release events in excitation-contraction coupling that underlie the Ca2+ transient. The frequency-dependent contractile force generated by cardiac myocytes depends upon the characteristics of the Ca2+ transients. A stochastic computational local control model of a guinea pig ventricular cardiomyocyte was developed, to gain insight into mechanisms of force-frequency relationship (FFR). This required the creation of a new three-state RyR2 model that reproduced the adaptive behavior of RyR2, in which the RyR2 channels transition into a different state when exposed to prolonged elevated subspace [Ca2+]. The model simulations agree with previous experimental and modeling studies on interval-force relations. Unlike previous common pool models, this local control model displayed stable action potential trains at 7 Hz. The duration and the amplitude of the [Ca2+]myo transients increase in pacing rates consistent with the experiments. The [Ca2+]myo transient reaches its peak value at 4 Hz and decreases afterward, consistent with experimental force-frequency curves. The model predicts, in agreement with previous modeling studies of Jafri and co-workers, diastolic sarcoplasmic reticulum, [Ca2+]sr, and RyR2 adaptation increase with the increased stimulation frequency, producing rising, rather than falling, amplitude of the myoplasmic [Ca2+] transients. However, the local control model also suggests that the reduction of the L-type Ca2+ current, with an increase in pacing frequency due to Ca2+-dependent inactivation, also plays a role in the negative slope of the FFR. In the simulations, the peak Ca2+ transient in the FFR correlated with the highest numbers of SR Ca2+ sparks: the larger average amplitudes of those sparks, and the longer duration of the Ca2+ sparks.

A stochastic mathematical model for coupling the cytosolic and sarcoplasmic calcium movements in diseased cardiac myocytes.

Several computational studies have been undertaken to explore the Ca2+-induced Ca2+ release (CICR) events in cardiac myocytes and along with experimental studies it has given us invaluable insight into the mechanism of CICR from spark/blink initiation to termination and regulation, and their interplay under normal and pathological conditions. The computational modelling of this mechanism has mainly been investigated using coupled differential equations (DEs). However, there is a lack of computational investigation into (1) how the different formulation of coupled DEs capture the Ca2+ movement in the cytosol and sarcoplasmic reticulum (SR), (2) the buffer and dye inclusion in both compartments, and (3) the effect of buffer and dye properties on the calcium behaviour. This work is set out to explore (1) the effect of different coupled formulation of DEs on spark/blink occurrence, (2) the inclusion of improved sarcoplasmic buffering properties, and (3) the effects of cytosolic and sarcoplasmic dye and buffer properties on Ca2+ movement. The simulation results show large discrepancies between different formulations of the governing equations. Additionally, extension of the model to include sarcoplasmic buffering properties show normalised fluorescent dye profiles to be in good agreement with experimental and amongst its one- and two-dimensional representations.

MicroRNA-210-mediated mtROS confer hypoxia-induced suppression of STOCs in ovine uterine arteries.

BACKGROUND AND PURPOSE: Hypoxia during pregnancy is associated with increased uterine vascular resistance and elevated blood pressure both in women and female sheep. A previous study demonstrated a causal role of microRNA-210 (miR-210) in gestational hypoxia-induced suppression of Ca2+ sparks/spontaneous transient outward currents (STOCs) in ovine uterine arteries, but the underlying mechanisms remain undetermined. We tested the hypothesis that miR-210 perturbs mitochondrial metabolism and increases mitochondrial reactive oxygen species (mtROS) that confer hypoxia-induced suppression of STOCs in uterine arteries. EXPERIMENTAL APPROACH: Resistance-sized uterine arteries were isolated from near-term pregnant sheep and were treated ex vivo in normoxia and hypoxia (10.5% O2 ) for 48 h. KEY RESULTS: Hypoxia increased mtROS and suppressed mitochondrial respiration in uterine arteries, which were also produced by miR-210 mimic to normoxic arteries and blocked by antagomir miR-210-LNA in hypoxic arteries. Hypoxia or miR-210 mimic inhibited Ca2+ sparks/STOCs and increased uterine arterial myogenic tone, which were inhibited by the mitochondria-targeted antioxidant MitoQ. Hypoxia and miR-210 down-regulated iron-sulfur cluster scaffold protein (ISCU) in uterine arteries and knockdown of ISCU via siRNAs suppressed mitochondrial respiration, increased mtROS, and inhibited STOCs. In addition, blockade of mitochondrial electron transport chain with antimycin and rotenone inhibited large-conductance Ca2+ -activated K+ channels, decreased STOCs and increased uterine arterial myogenic tone. CONCLUSION AND IMPLICATIONS: This study demonstrates a novel mechanistic role for the miR-210-ISCU-mtROS axis in inhibiting Ca2+ sparks/STOCs in the maladaptation of uterine arteries and provides new insights into the understanding of mitochondrial perturbations in the pathogenesis of pregnancy complications resulted from hypoxia.

Calcium Spark Detection and Event-Based Classification of Single Cardiomyocyte Using Deep Learning.

Ca2+ sparks are the elementary Ca2+ release events in cardiomyocytes, altered properties of which lead to impaired Ca2+ handling and finally contribute to cardiac pathology under various diseases. Despite increasing use of machine-learning algorithms in deciphering the content of biological and medical data, Ca2+ spark images and data are yet to be deeply learnt and analyzed. In the present study, we developed a deep residual convolutional neural network method to detect Ca2+ sparks. Compared to traditional detection methods with arbitrarily defined thresholds to distinguish signals from noises, our new method detected more Ca2+ sparks with lower amplitudes but similar spatiotemporal distributions, thereby indicating that our new algorithm detected many very weak events that are usually omitted when using traditional detection methods. Furthermore, we proposed an event-based logistic regression and binary classification model to classify single cardiomyocytes using Ca2+ spark characteristics, which to date have generally been used only for simple statistical analyses and comparison between normal and diseased groups. Using this new detection algorithm and classification model, we succeeded in distinguishing wild type (WT) vs RyR2-R2474S± cardiomyocytes with 100% accuracy, and vehicle vs isoprenaline-insulted WT cardiomyocytes with 95.6% accuracy. The model can be extended to judge whether a small number of cardiomyocytes (and so the whole heart) are under a specific cardiac disease. Thus, this study provides a novel and powerful approach for the research and application of calcium signaling in cardiac diseases.

Modulated Calcium Homeostasis and Release Events Under Atrial Fibrillation and Its Risk Factors: A Meta-Analysis.

Background: Atrial fibrillation (AF) is associated with calcium (Ca2+) handling remodeling and increased spontaneous calcium release events (SCaEs). Nevertheless, its exact mechanism remains unclear, resulting in suboptimal primary and secondary preventative strategies. Methods: We searched the PubMed database for studies that investigated the relationship between SCaEs and AF and/or its risk factors. Meta-analysis was used to examine the Ca2+ mechanisms involved in the primary and secondary AF preventative groups. Results: We included a total of 74 studies, out of the identified 446 publications from inception (1982) until March 31, 2020. Forty-five were primary and 29 were secondary prevention studies for AF. The main Ca2+ release events, calcium transient (standardized mean difference (SMD) = 0.49; I 2 = 35%; confidence interval (CI) = 0.33-0.66; p < 0.0001), and spark amplitude (SMD = 0.48; I 2 = 0%; CI = -0.98-1.93; p = 0.054) were enhanced in the primary diseased group, while calcium transient frequency was increased in the secondary group. Calcium spark frequency was elevated in both the primary diseased and secondary AF groups. One of the key cardiac currents, the L-type calcium current (ICaL) was significantly downregulated in primary diseased (SMD = -1.07; I 2 = 88%; CI = -1.94 to -0.20; p < 0.0001) and secondary AF groups (SMD = -1.28; I 2 = 91%; CI = -2.04 to -0.52; p < 0.0001). Furthermore, the sodium-calcium exchanger (INCX) and NCX1 protein expression were significantly enhanced in the primary diseased group, while only NCX1 protein expression was shown to increase in the secondary AF studies. The phosphorylation of the ryanodine receptor at S2808 (pRyR-S2808) was significantly elevated in both the primary and secondary groups. It was increased in the primary diseased and proarrhythmic subgroups (SMD = 0.95; I 2 = 64%; CI = 0.12-1.79; p = 0.074) and secondary AF group (SMD = 0.66; I 2 = 63%; CI = 0.01-1.31; p < 0.0001). Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression was elevated in the primary diseased and proarrhythmic drug subgroups but substantially reduced in the secondary paroxysmal AF subgroup. Conclusions: Our study identified that ICaL is reduced in both the primary and secondary diseased groups. Furthermore, pRyR-S2808 and NCX1 protein expression are enhanced. The remodeling leads to elevated Ca2+ functional activities, such as increased frequencies or amplitude of Ca2+ spark and Ca2+ transient. The main difference identified between the primary and secondary diseased groups is SERCA expression, which is elevated in the primary diseased group and substantially reduced in the secondary paroxysmal AF subgroup. We believe our study will add new evidence to AF mechanisms and treatment targets.

Ca2+ release via InsP3Rs enhances RyR recruitment during Ca2+ transients by increasing dyadic [Ca2+] in cardiomyocytes.

Excitation-contraction coupling (ECC) relies on temporally synchronized sarcoplasmic reticulum (SR) Ca2+ release via ryanodine receptors (RyRs) at dyadic membrane compartments. Neurohormones, such as endothelin-1 (ET-1), that act via Gαq-associated G protein-coupled receptors (GPCRs) modulate Ca2+ dynamics during ECC and induce SR Ca2+ release events involving Ca2+ release via inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs). How the relatively modest Ca2+ release via InsP3Rs elicits this action is not resolved. Here, we investigated whether the actions of InsP3Rs on Ca2+ handling during ECC were mediated by a direct influence on dyadic Ca2+ levels and whether this mechanism contributes to the effects of ET-1. Using a dyad-targeted genetically encoded Ca2+ reporter, we found that InsP3R activation augmented dyadic Ca2+ fluxes during Ca2+ transients and increased Ca2+ sparks. RyRs were required for these effects. These data provide the first direct demonstration of GPCR and InsP3 effects on dyadic Ca2+, and support the notion that Ca2+ release via InsP3Rs influences Ca2+ transients during ECC by facilitating the activation and recruitment of proximal RyRs. We propose that this mechanism contributes to neurohormonal modulation of cardiac function. This article has an associated First Person interview with the first author of the paper.

Calcium signaling consequences of RyR2 mutations associated with CPVT1 introduced via CRISPR/Cas9 gene editing in human-induced pluripotent stem cell-derived cardiomyocytes: Comparison of RyR2-R420Q, F2483I, and Q4201R.

BACKGROUND: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) created from patients with catecholaminergic polymorphic ventricular tachycardia 1 (CPVT1) have been used to study CPVT1 arrhythmia. OBJECTIVE: The purpose of this study was to evaluate the Ca2+ signaling aberrancies and pharmacological sensitivities of 3 CRISPR/Cas9-introduced CPVT1 mutations located in different molecular domains of ryanodine receptor 2 (RyR2). METHODS: CRISPR/Cas9-engineered hiPSC-CMs carrying RyR2 mutations-R420Q, Q4201R, and F2483I-were voltage clamped, and their electrophysiology, pharmacology, and Ca2+ signaling phenotypes measured using total internal reflection fluorescence microscopy. RESULTS: R420Q and Q4201R mutant hiPSC-CMs exhibit irregular, long-lasting, spatially wandering Ca2+ sparks and aberrant Ca2+ releases similar to F2483I unlike the wild-type myocytes. Large sarcoplasmic reticulum (SR) Ca2+ leaks and smaller SR Ca2+ contents were detected in cells expressing Q4201R and F2483I, but not R420Q. Fractional Ca2+ release and calcium-induced calcium release gain were higher in Q4201R than in R420Q and F2483I hiPSC-CMs. JTV519 was equally effective in suppressing Ca2+ sparks, waves, and SR Ca2+ leaks in hiPSC-CMs derived from all 3 mutant lines. Flecainide and dantrolene similarly suppressed SR Ca2+ leaks, but were less effective in decreasing spark frequency and durations. CONCLUSION: CRISPR/Cas9 gene editing of hiPSCs provides a novel approach in studying CPVT1-associated RyR2 mutations and suggests that Ca2+-signaling aberrancies and drug sensitivities may vary depending on the mutation site.

Ryanodine receptor subtypes regulate Ca2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy.

AIMS: Our recent study demonstrated that increased Ca2+ sparks and spontaneous transient outward currents (STOCs) played an important role in uterine vascular tone and haemodynamic adaptation to pregnancy. The present study examined the role of ryanodine receptor (RyR) subtypes in regulating Ca2+ sparks/STOCs and myogenic tone in uterine arterial adaptation to pregnancy. METHODS AND RESULTS: Uterine arteries isolated from non-pregnant and near-term pregnant sheep were used in the present study. Pregnancy increased the association of α and ß1 subunits of large-conductance Ca2+-activated K+ (BKCa) channels and enhanced the co-localization of RyR1 and RyR2 with the ß1 subunit in the uterine artery. In contrast, RyR3 was not co-localized with BKCa ß1 subunit. Knockdown of RyR1 or RyR2 in uterine arteries of pregnant sheep downregulated the ß1 but not α subunit of the BKCa channel and decreased the association of α and ß1 subunits. Unlike RyR1 and RyR2, knockdown of RyR3 had no significant effect on either expression or association of BKCa subunits. In addition, knockdown of RyR1 or RyR2 significantly decreased Ca2+ spark frequency, suppressed STOCs frequency and amplitude, and increased pressure-dependent myogenic tone in uterine arteries of pregnant animals. RyR3 knockdown did not affect Ca2+ sparks/STOCs and myogenic tone in the uterine artery. CONCLUSION: Together, the present study demonstrates a novel mechanistic paradigm of RyR subtypes in the regulation of Ca2+ sparks/STOCs and uterine vascular tone, providing new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.

Arrhythmogenic late Ca2+ sparks in failing heart cells and their control by action potential configuration.

Sudden death in heart failure patients is a major clinical problem worldwide, but it is unclear how arrhythmogenic early afterdepolarizations (EADs) are triggered in failing heart cells. To examine EAD initiation, high-sensitivity intracellular Ca2+ measurements were combined with action potential voltage clamp techniques in a physiologically relevant heart failure model. In failing cells, the loss of Ca2+ release synchrony at the start of the action potential leads to an increase in number of microscopic intracellular Ca2+ release events ("late" Ca2+ sparks) during phase 2-3 of the action potential. These late Ca2+ sparks prolong the Ca2+ transient that activates contraction and can trigger propagating microscopic Ca2+ ripples, larger macroscopic Ca2+ waves, and EADs. Modification of the action potential to include steps to different potentials revealed the amount of current generated by these late Ca2+ sparks and their (subsequent) spatiotemporal summation into Ca2+ ripples/waves. Comparison of this current to the net current that causes action potential repolarization shows that late Ca2+ sparks provide a mechanism for EAD initiation. Computer simulations confirmed that this forms the basis of a strong oscillatory positive feedback system that can act in parallel with other purely voltage-dependent ionic mechanisms for EAD initiation. In failing heart cells, restoration of the action potential to a nonfailing phase 1 configuration improved the synchrony of excitation-contraction coupling, increased Ca2+ transient amplitude, and suppressed late Ca2+ sparks. Therapeutic control of late Ca2+ spark activity may provide an additional approach for treating heart failure and reduce the risk for sudden cardiac death.

Elementary calcium signaling in arterial smooth muscle.

Vascular smooth muscle cells (VSMCs) of small peripheral arteries contribute to blood pressure control by adapting their contractile state. These adaptations depend on the VSMC cytosolic Ca2+ concentration, regulated by complex local elementary Ca2+ signaling pathways. Ca2+ sparks represent local, transient, rapid calcium release events from a cluster of ryanodine receptors (RyRs) in the sarcoplasmic reticulum. In arterial SMCs, Ca2+ sparks activate nearby calcium-dependent potassium channels, cause membrane hyperpolarization and thus decrease the global intracellular [Ca2+] to oppose vasoconstriction. Arterial SMC Cav1.2 L-type channels regulate intracellular calcium stores content, which in turn modulates calcium efflux through RyRs. Cav3.2 T-type channels contribute to a minor extend to Ca2+ spark generation in certain types of arteries. Their localization within cell membrane caveolae is essential. We summarize present data on local elementary calcium signaling (Ca2+ sparks) in arterial SMCs with focus on RyR isoforms, large-conductance calcium-dependent potassium (BKCa) channels, and cell membrane-bound calcium channels (Cav1.2 and Cav3.2), particularly in caveolar microdomains.

Calcium as a Key Player in Arrhythmogenic Cardiomyopathy: Adhesion Disorder or Intracellular Alteration?

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca2+ handling machinery, focusing on the available data supporting this hypothesis. The Ca2+ toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation‒contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca2+, thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca2+ signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease.

Calcium overload-induced arrhythmia is suppressed by farnesol in rat heart.

Cardiac arrhythmias are among the most important pathologies that lead to sudden death. The discovery of new therapeutic options against arrhythmias with low adverse effects is of paramount importance. Farnesol is found in essential oils with antioxidant, anti-inflammatory and cardioprotective properties. The aim of this work was to investigate the effects of farnesol on the contractile and electrophysiological properties in rat heart and evaluate its antiarrhythmic action. It was evaluated farnesol effects on the left ventricular developed pressure, ECG, potassium (Ik) and L-type Ca2+ currents (ICa,L), action potential, intracellular Ca2+ transient, Ca2+ sparks and waves and reactive oxygen species production. Antiarrhythmic activity of farnesol was determined in vivo and ex vivo. The results showed that 50 µM farnesol did not alter left ventricular developed pressure, heart rate, ECG parameters and intracellular Ca2+ transient but reduced ICa,L. Farnesol reduced action potential duration at 90% repolarization. Notably, farnesol improved arrhythmia score and the incidence of the most severe arrhythmias. Farnesol attenuated the generation of reactive oxygen species, Ca2+ sparks and waves in isolated cardiomyocytes submitted to Ca2+ overload. In conclusion, farnesol has antiarrhythmic effect mediated by reducing of ICa,L and IK along with a decrease of reactive oxygen species production and normalized Ca2+ sparks and waves.

Role of Ryanodine Type 2 Receptors in Elementary Ca2+ Signaling in Arteries and Vascular Adaptive Responses.

Background Hypertension is the major risk factor for cardiovascular disease, the most common cause of death worldwide. Resistance arteries are capable of adapting their diameter independently in response to pressure and flow-associated shear stress. Ryanodine receptors (RyRs) are major Ca2+-release channels in the sarcoplasmic reticulum membrane of myocytes that contribute to the regulation of contractility. Vascular smooth muscle cells exhibit 3 different RyR isoforms (RyR1, RyR2, and RyR3), but the impact of individual RyR isoforms on adaptive vascular responses is largely unknown. Herein, we generated tamoxifen-inducible smooth muscle cell-specific RyR2-deficient mice and tested the hypothesis that vascular smooth muscle cell RyR2s play a specific role in elementary Ca2+ signaling and adaptive vascular responses to vascular pressure and/or flow. Methods and Results Targeted deletion of the Ryr2 gene resulted in a complete loss of sarcoplasmic reticulum-mediated Ca2+-release events and associated Ca2+-activated, large-conductance K+ channel currents in peripheral arteries, leading to increased myogenic tone and systemic blood pressure. In the absence of RyR2, the pulmonary artery pressure response to sustained hypoxia was enhanced, but flow-dependent effects, including blood flow recovery in ischemic hind limbs, were unaffected. Conclusions Our results establish that RyR2-mediated Ca2+-release events in VSCM s specifically regulate myogenic tone (systemic circulation) and arterial adaptation in response to changes in pressure (hypoxic lung model), but not flow. They further suggest that vascular smooth muscle cell-expressed RyR2 deserves scrutiny as a therapeutic target for the treatment of vascular responses in hypertension and chronic vascular diseases.

Estimation of stochastic behaviour in cardiac myocytes: I. Ca2+ movements inside the cytosol and sarcoplasmic reticulum on curvilinear domains.

Since the discovery of Ca2+ sparks and their stochastic behaviour in cardiac myocytes, models have focused on the inclusion of stochasticity in their studies. While most models pay much attention to the stochastic modelling of cytosolic Ca2+ concentration the coupling of Ca2+ sparks and blinks in a stochastic model has not been explored fully. The cell morphology in in silico studies in the past is assumed to be Cartesian, spherical or cylindrical. The application on curvilinear grids can easily address certain restrictions posed by such grid set up and provide more realistic cell morphology. In this paper, we present a stochastic reaction-diffusion model that couples Ca2+ sparks and blinks in realistic shapes of cells in curvilinear domains. Methodology: Transformation of the model was performed to the curvilinear coordinate system. The set of equations is used to produce Ca2+ waves initiated from sparks and blinks. A non-buffered and non-dyed version as well as a buffered and dyed version of these equations were studied in light of observing the dynamics on the two different systems. For comparison, results for both the Cartesian and curvilinear grids are provided. Results and conclusions: A successful demonstration of the application of curvilinear grids serving as basis for future developments.