The extent of mitochondrial heterogeneity and the presence of mitochondrial archetypes in cancer remain unknown. Mitochondria play a central role in the metabolic reprogramming that occurs in cancer cells. This process adjusts the activity of metabolic pathways to support growth, proliferation, and survival of cancer cells. Using a panel of colorectal cancer (CRC) cell lines, we revealed extensive differences in their mitochondrial composition, suggesting functional specialisation of these organelles. We differentiated bioenergetic and mitochondrial phenotypes, which point to different strategies used by CRC cells to maintain their sustainability. Moreover, the efficacy of various treatments targeting metabolic pathways was dependent on the respiration and glycolysis levels of cancer cells. Furthermore, we identified metabolites associated with both bioenergetic profiles and cell responses to treatments. The levels of these molecules can be used to predict the therapeutic efficacy of anti-cancer drugs and identify metabolic vulnerabilities of CRC. Our study indicates that the efficacy of CRC therapies is closely linked to mitochondrial status and cellular bioenergetics.

During exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in phosphorylation of the cardiac ryanodine receptor (RyR2). Three RyR2 phosphorylation sites have taken prominence in excitation-contraction coupling: S2808 and S2030 are described as protein kinase A specific and S2814 as a Ca2+/calmodulin kinase type-2-specific site. To examine the contribution of these phosphosites to Ca2+ signalling, we generated double knock-in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2-S2808A/S2814A). These mice did not exhibit an overt phenotype. Heart morphology and haemodynamic parameters were not altered. However, they had a higher susceptibility to arrhythmias. We performed confocal Ca2+ imaging and electrophysiology experiments. Isoprenaline was used to stimulate β-ARs. Measurements of Ca2+ waves and latencies in myocytes revealed an increased propensity for spontaneous Ca2+ releases in DKI myocytes, both in control conditions and during β-AR stimulation. In DKI cells, waves were initiated from a lower threshold concentration of Ca2+ inside the sarcoplasmic reticulum, suggesting higher Ca2+ sensitivity of the RyRs. The refractoriness of Ca2+ spark triggering depends on the Ca2+ sensitivity of the RyR2. We found that RyR2-S2808A/S2814A channels were more Ca2+ sensitive in control conditions. Isoprenaline further shortened RyR refractoriness in DKI cardiomyocytes. Together, our results suggest that ablation of both the RyR2-Ser2808 and RyR2-S2814 sites increases the propensity for pro-arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs. Given that the DKI cells present a full response to isoprenaline, the data suggest that phosphorylation of Ser2030 might be sufficient for β-AR-mediated sensitization of RyRs. KEY POINTS: Phosphorylation of cardiac sarcoplasmic reticulum Ca2+-release channels (ryanodine receptors, RyRs) is involved in the regulation of cardiac function. Ablation of both the RyR2-Ser2808 and RyR2-Ser2814 sites increases the propensity for pro-arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs. The intra-sarcoplasmic reticulum Ca2+ threshold for spontaneous Ca2+ wave generation is lower in RyR2-double-knock-in cells. The RyR2 from double-knock-in cells exhibits increased Ca2+ sensitivity. Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the channel. Phosphorylation of Ser2030 might be sufficient for a β-adrenergic response.

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.

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.

Global Ca²⁺ increase in the cytosol of cardiomyocytes is crucial for the contraction of the heart. The malfunctioning of proteins involved in this process can trigger local events (e.g., sparks and puffs) and global events (e.g., waves). These are thought to be involved in the development of pathological conditions, such as arrhythmias. To understand the underlying mechanisms, it is important to detect and identify arrhythmogenic Ca²⁺ release events. We present a novel approach, based on a 3D U-Net architecture, to perform these tasks automatically. We employed data obtained with fast xyt confocal imaging of cardiomyocytes and provide a dataset of xyt image series where the subcellular Ca²⁺ events are manually segmented and labelled. We trained the neural network to infer comparable segmentation as outputs and processed them to obtain single event instances that are available for further analysis. We obtained promising results despite the relatively small amount of available annotated data and the challenges that it exhibits.

Cyclooxygenase inhibitors demonstrate effective antinociception in many clinical and experimental pain models. Acute uterine cervical distension (UCD) forms the basis for obstetric and some gynecologic pain, and acute UCD in rats results in nocifensor reflexes which are inhibited by morphine in animals lacking, but not in animals with circulating estrogen. We studied the antinociceptive effect of intravenous and intrathecal injection of the cyclooxygenase inhibitor, ketorolac in acute UCD rats and its dependency on estrogen. Virgin rats received estrogen or placebo treatment for 1 week following oviarectomy. An intrathecal catheter was inserted for drug administration. Rats were anesthetized, then the electromyographic response in the rectus abdominus muscle and mean arterial blood pressure change to UCD was recorded before and with cumulative dosing of intravenous or intrathecal ketorolac. Intravenous ketorolac produced dose dependent inhibition of the responses to UCD, but intrathecal ketorolac was ineffective at the maximum test dose (300 μg). Estrogen replacement did not affect the stimulus response or maximum efficacy of ketorolac. Unlike morphine, which reduces response to UCD by spinal and supraspinal mechanisms and whose action is blocked by estrogen, the cyclooxygenase inhibitor, ketorolac acts at an estrogen-independent, non spinal site.

Monitoring and manipulation of ionized intracellular calcium concentrations within intact, living cells using optical probes with organic chromophores is a core method for cell physiology. Since all these probes have multiple negative charges, they must be smuggled through the plasma membrane in a transiently neutral form, with intracellular esterases used to deprotect the masked anions. Here we explore the ability of the synthetically easily accessible n-butyl ester protecting group to deliver amphipathic cargoes to the cytosol. We show that the size of the caging chromophore conditions the ability of intracellular probe delivery and esterase charge unmasking.

Mutations of the RyR2 are channelopathies that can predispose to life threatening catecholaminergic polymorphic ventricular tachycardias (CPVTs) during exercise or stress. However, the cellular and molecular mechanisms that are causal for the arrhythmias downstream of the β-adrenergic receptor (β-AR) activation are not defined. They may be specific and different for each particular RyR2 mutation. Obvious possibilities are the phosphorylation of the mutated RyR2s or the stimulation of the SR Ca2+ pump (SERCA), which could increase SR Ca2+ loading. Potentially arrhythmogenic Ca2+ signals, such as Ca2+ waves, were recorded and analyzed from WT and RyR2R420Q+/− mouse cardiomyocytes with confocal microscopy after field stimulation at 1 Hz. In RyR2R420Q+/− cardiomyocytes we found a higher occurrence and frequency of Ca2+ waves, particularly upon β-AR stimulation with isoproterenol. This was accompanied by a shorter latency to the first spontaneous wave. Wave velocity from raw traces, as well as amplitude and decay time constant (τ) analyzed in de-skewed traces were comparable in both cell types. To obtain further insight into the role of the SERCA we selectively stimulated SERCA in permeabilized myocytes using Fab fragments of a PLB antibody (2D12). Surprisingly, SERCA stimulation alone resulted in considerably higher wave frequencies than when mimicking β-AR stimulation with cAMP, particularly in RyR2R420Q+/− cardiomyocytes. This may be a consequence of some protective SR Ca2+ unloading resulting from the SR Ca2+ leak via phosphorylated RyR2s in cAMP. Spark-to-spark recovery analysis suggested a remarkably higher Ca2+ release sensitivity in RyR2R420Q+/− cells, both in control and upon β-AR stimulation. Together these findings suggest that the fine balance between SR Ca2+ loading via SERCA and the Ca2+ leak via mutated and phosphorylated RyR2s is an important determinant for the overall cellular arrhythmogenicity prevailing in the RyR2R420Q+/− myocytes.

In the heart muscle, Ca2+-induced Ca2+ release (CICR) is the main mechanism for Ca2+ increase required for the excitation-contraction coupling. However, a second mechanism for Ca2+ release from the sarcoplasmic reticulum induced by inositol 1,4,5-trisphosphate (IP3), called inositol IP3-induced intracellular Ca2+ release (IP3ICR), has been verified in cardiomyocytes. Occurrence of global Ca2+ transients as well as spontaneous Ca2+ waves (SCaW) are driven by CICR. Under pathophysiological remodeling conditions in which an up-regulation of IP3 receptors (IP3Rs) was observed, a modulatory role of IP3ICR on CICR seems plausible.

Channelopathies of the cardiac ryanodine receptor (RyR2) can predispose to life threatening catecholaminergic polymorphic ventricular tachycardias (CPVTs) during exercise or stress, however the underlaying mechanism involving complex signaling pathways is not yet fully understood. Furthermore, the resulting arrhythmogenic events on the cellular level are most likely quite diverse, depending on the mutated residue on the RyR2. Obvious possibilities are the phosphorylation of the mutated RyR2s or the stimulation of the SR Ca2+ pump (SERCA), which could increase SR Ca2+ loading.

Key points In cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery. Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias. We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two‐photon photolysis of a new ultra‐effective caged calcium compound. The findings confirm the utility of the technique to define an elevated sensitivity of the calcium release mechanism in situ and to follow hastened recovery of spark trigger probabilities in a mouse model of an inherited cardiac arrhythmia, which was used for validation. Analogous methods are likely to be applicable to investigate other microscopic subcellular signalling systems in a variety of cell types. AbstractIn cardiac myocytes Ca2+‐induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca2+ release occurs via subcellular Ca2+ signalling events, Ca2+ sparks. Local recovery of Ca2+ release depends on both SR refilling and restoration of Ca2+ sensitivity of the RyRs. We used two‐photon (2P) photolysis of the ultra‐effective caged Ca2+ compound BIST‐2EGTA and laser‐scanning confocal Ca2+ imaging to probe refractoriness of local Ca2+ release in control conditions and in the presence of cAMP or low‐dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST‐2EGTA and rhod‐2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca2+ release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self‐learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyRR420Q mutation were used for further validation and revealed a higher Ca2+ sensitivity of CICR. This new 2P approach provides composite information of Ca2+ release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca2+ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias.

Augmented inositol 1,4,5-trisphosphate receptor (InsP3R2) expression and - function has been linked to a variety of cardiac pathologies including cardiac Ca2+-dependent arrhythmogenicity. However, a detailed understanding of its potential role in myocyte excitation-contraction coupling under pathophysiological conditions remains elusive. This is specially true for the functional local interaction between the two major Ca2+ release mechanisms Ca2+ release mediated by ryanodine receptors (RyR2s) and InsP3-induced SR-Ca2+ release (IP3ICR).

Analysis of Ca2+ signals obtained in various cell types (i.e., cardiomyocytes) is always a tradeoff between acquisition speed and signal/noise ratio of the fluorescence signal. This becomes especially apparent during fast two- or three-dimensional confocal imaging when local intracellular fluorescence signals originating from Ca2+ release from intracellular Ca2+ stores (e.g., sarcoplasmic reticulum) need to be examined. Mathematical methods have been developed to remedy a high noise level by fitting each pixel with a transient function to "denoise" the image. So far, current available analytical approaches have been impaired by a number of constraints (e.g., inability to fit local, concurrent, and consecutive events) and the limited ability to customize implementation. Here, we suggest a, to our knowledge, novel approach for detailed analysis of subcellular micro Ca2+ events based on pixel-by-pixel denoising of confocal frame- and line-scan images. The algorithm enables spatiotemporally overlapping events (e.g., a Ca2+ spark occurring during the decaying phase of a Ca2+ wave) to be extracted so that various types of Ca2+ events can be detected at a pixel time level of precision. The method allows a nonconstant baseline to be estimated for each pixel, foregoing the need to subtract fluorescence background or apply self-ratio methods before image analysis. Furthermore, by using a clustering algorithm, identified single-pixel events are grouped into "physiologically relevant" Ca2+ signaling events spanning multiple pixels (sparks, waves, puffs, transients, etc.), from which spatiotemporal event parameters (e.g., full duration at half maximal amplitude, full width at half maximal amplitude, amplitude, wave speed, rise, and decay times) can be easily extracted. The method was implemented with cross-platform open source software, providing a comprehensive and easy-to-use graphical user interface enabling rapid line-scan images and rapid frame-scan image sequences (up to 150 frames/s) to be analyzed and repetitive Ca2+ events (Ca2+ sparks and Ca2+ puffs) originating from clusters of Ca2+ release channels located in the sarcoplasmic reticulum membrane (ryanodine receptors and inositol 1,4,5-trisphosphate receptors) of isolated cardiomyocytes to be examined with a high level of precision.

In cardiac muscle, besides Ca2+-induced Ca2+ release (CICR), a second Ca2+ release mechanism activated by hormone binding to G-protein coupled receptors is present. This prompt intracellular production of the signaling molecule Inositol-1,4,5-trisphosphate (IP3) which then subsequently triggers sarcoplasmic reticulum (SR)-Ca2+ release through openings of IP3 receptors type 2 (IP3R2s). IP3-induced SR-Ca2+release (IP3ICR) may modulate Ryanodine receptor (RyR2s) function via locally regulated interactions and fine-tune excitation-contraction coupling in ventricular myocytes. A functional local interplay between IP3R2s and RyR2s may be significantly pronounced under several cardiac pathologies where IP3R2 has been found to be overexpressed. We examined IP3ICR and CICR in ventricular myocytes on a local scale in a cardiac specific IP3R2-overexpressing mouse model (IP3/tTA) with a phenotype of cardiac hypertrophy. Protein analysis confirmed a 12-fold increase in IP3R2 expression together with a reduction in RyR2 protein levels by 61.7%. IP3-pathway stimulation in wild-type permeabilized cells increased spontaneous Ca2+ events by 24.2%. Surprisingly, activation of IP3ICR in IP3/tTA myocytes induced a decrease in Ca2+ spark frequency by 25.2% together with a reduction of the SR-Ca2+ content by 14.4% that cannot be explained by RyR2 Ca2+ spark occurrence alone. We examined this phenomenon in more detail in intact myocytes by applying specific SR-Ca2+ leak/load protocols. We found that in IP3/tTA mice IP3ICR functions below the threshold for Ca2+ sparks, via efficient modulation of the SR-Ca2+ leak. Activation of this pathway acts as a protective mechanism against arrhythmogenic Ca2+ wave occurrence. We conclude that the overexpression of IP3R2 in ventricular myocytes may represent a new and so far not recognized anti-arrhythmogenic mechanism that can prevent SR-Ca2+ overload and reduce the propensity of aberrant SR- Ca2+ release preconditioned for arrhythmogenicity. This mechanism could be a potential target for precision medicine therapies treating ventricular tachycardia.

Key points Augmented inositol 1,4,5‐trisphosphate (IP 3 ) receptor (IP 3 R2) expression has been linked to a variety of cardiac pathologies. Although cardiac IP 3 R2 function has been in the focus of research for some time, a detailed understanding of its potential role in ventricular myocyte excitation–contraction coupling under pathophysiological conditions remains elusive. The present study focuses on mechanisms of IP 3 R2‐mediated sarcoplasmic reticulum (SR)‐Ca 2+ release in ventricular excitation–contraction coupling under IP 3 R2‐overexpressing conditions by studying intracellular Ca 2+ events. We report that, upon IP 3 R2 overexpression in ventricular myocytes, IP 3 ‐induced Ca 2+ release (IP 3 ICR) modulates the SR‐Ca 2+ content via “eventless” SR‐Ca 2+ release, affecting the global SR‐Ca 2+ leak. Thus, IP 3 R2 activation could act as a SR‐Ca 2+ gateway mechanism to escape ominous SR‐Ca 2+ overload. Our approach unmasks a so far unrecognized mechanism by which “eventless” IP 3 ICR plays a protective role against ventricular Ca 2+ ‐dependent arrhythmogenicity. Abstract Augmented inositol 1,4,5‐trisphosphate (IP 3 ) receptor (IP 3 R2) function has been linked to a variety of cardiac pathologies including cardiac arrhythmias. The functional role of IP 3 ‐induced Ca 2+ release (IP 3 ICR) within ventricular excitation–contraction coupling (ECC) remains elusive. As part of pathophysiological cellular remodelling, IP 3 R2s are overexpressed and have been repeatedly linked to enhanced Ca 2+ ‐dependent arrhythmogenicity. In this study we test the hypothesis that an opposite scenario might be plausible in which IP 3 ICR is part of an ECC protecting mechanism, resulting in a Ca 2+ ‐dependent anti‐arrhythmogenic response on the cellular scale. IP 3 R2 activation was triggered via endothelin‐1 or IP 3 ‐salt application in single ventricular myocytes from a cardiac‐specific IP 3 R type 2 overexpressing mouse model. Upon IP 3 R2 overexpression, IP 3 R activation reduced Ca 2+ ‐wave occurrence (46 vs . 21.72%; P < 0.001) while its block increased SR‐Ca 2+ content (∼29.4% 2‐aminoethoxydiphenyl borate, ∼16.4% xestospongin C; P < 0.001), suggesting an active role of IP 3 ICR in SR‐Ca 2+ content regulation and anti‐arrhythmogenic function. Pharmacological separation of ryanodine receptor RyR2 and IP 3 R2 functions and two‐dimensional Ca 2+ event analysis failed to identify local IP 3 ICR events (Ca 2+ puffs). SR‐Ca 2+ leak measurements revealed that under pathophysiological conditions, “eventless” SR‐Ca 2+ efflux via enhanced IP 3 ICR maintains the SR‐Ca 2+ content below Ca 2+ spark threshold, preventing aberrant SR‐Ca 2+ release and resulting in a protective mechanism against SR‐Ca 2+ overload and arrhythmias. Our results support a so far unrecognized modulatory mechanism in ventricular myocytes working in an anti‐arrhythmogenic fashion.

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.

Inositol-1,4,5-trisphosphate (IP3) is a second messenger produced upon agonist binding to a G-protein coupled receptor (GPCR) and subsequently triggers SR-Ca2+ release through openings of IP3 receptors (IP3Rs). In cardiac muscle, IP3R type 2 (IP3R2) is the predominant isoform expressed both in ventricle and atrial tissue. Several studies have focused on the functional interaction between ryanodine receptors (RyRs) and IP3Rs in atrial myocytes. However, it is still unclear how IP3-induced Ca2+ release (IP3ICR) may contribute to excitation-contraction coupling in ventricle. Evidence suggests that IP3ICR modulates the RyR function by affecting its local Ca2+ environment. Under pathophysiological cellular remodeling conditions (e.g. atrial fibrillation and heart failure) a functional interplay of IP3R and RyR Ca2+ events may be significantly pronounced. Our aim in this study was to examine this interaction in a cardiac-specific IP3R2 overexpressing (TG) mouse model. Our experimental approach includes: characterization of the IP3 signaling pathway by various pharmacological interventions; electrophysiology under whole-cell configuration of the patch clamp technique in combination with rapid confocal Ca2+ imaging and complemented with molecular biology approaches (RT-PCR, Western Blot, immunostaining). Western blot results show an increase in IP3R2 protein expression in the TG model both in atria and ventricular tissue compared to its wild-type littermate (FVB). However, a similar distribution on the junctional SR was found, where IP3Rs co-localize with RyRs. Supporting the protein data, preliminary results show an increase in basal spark frequency (SpF) in TG atrial and ventricular myocytes compared to FVB. Upon IP3 stimulation FVB atrial and ventricular cardiomyocytes presented an increase in SpF. Furthermore, a larger increment was seen in TG atrial myocytes. In order to examine the contribution of the SR-Ca2+ leak mediated by IP3R2 a SR-Ca2+ leak protocol was established. However, even under control conditions a significant contribution of IP3ICR on the SR-Ca2+ leak was found. We conclude that overexpression of IP3R2 in the TG model affects Ca2+ handling significantly by functional interplay with the RyR2.

Analysis of Ca2+ signaling in cardiac cells is often a trade-off between acquisition speed and signal-to-noise ratio. This becomes especially apparent in fast 2D scanning or when recording fluorescence signals from the sarcoplasmic reticulum, for example. Methods have been developed to remedy this via 'denoising' the image by fitting each pixel with a transient function. So far, adoption of such methods has been hindered by a number of limitations (e.g., inability to fit local, concurrent and consecutive events) and the limited availability of a customizable implementation. Here we present a novel method for performing per-pixel denoising of confocal frame- and linescans. Our algorithm permits the extraction of spatiotemporally overlapping events (e.g., a spark occurring during the decaying phase of a Ca2+ wave) and is able to detect various different types of events within a pixel time course. The method estimates a non-constant baseline for each pixel, negating the necessity of using background regions or self-ratio methods prior to performing the analysis. Furthermore, by applying a clustering algorithm, detected single-pixel events are grouped into physiologically relevant events spanning multiple pixels (sparks, waves, puffs,transients, etc.), from which traditional parameters such as FDHM, FWHM, amplitude, wave speed, rise and decay times, can be easily extracted. The method has been implemented as a cross-platform open source software with a comprehensive and easy to use graphical user interface. We have applied our method to analyzing linescans of repetitive sparks from individual RyR clusters; high-speed (150 frames/sec) framescans containing alterations in Ca2+ release events in atrial myocytes; and parallel analysis of Ca2+ release dynamics in the sarcoplasmic reticulum and cytosol. Supported by SNF and SciEx.

RATIONALE:In biomedical journals authors sometimes use the standard error of the mean (SEM) for data description, which has been called inappropriate or incorrect.OBJECTIVE:To assess the frequency of incorrect use of SEM in articles in three selected cardiovascular journals.METHODS AND RESULTS:All original journal articles published in 2012 in Cardiovascular Research, Circulation: Heart Failure and Circulation Research were assessed by two assessors for inappropriate use of SEM when providing descriptive information of empirical data. We also assessed whether the authors state in the methods section that the SEM will be used for data description. Of 441 articles included in this survey, 64% (282 articles) contained at least one instance of incorrect use of the SEM, with two journals having a prevalence above 70% and "Circulation: Heart Failure" having the lowest value (27%). In 81% of articles with incorrect use of SEM, the authors had explicitly stated that they use the SEM for data description and in 89% SEM bars were also used instead of 95% confidence intervals. Basic science studies had a 7.4-fold higher level of inappropriate SEM use (74%) than clinical studies (10%).LIMITATIONS:The selection of the three cardiovascular journals was based on a subjective initial impression of observing inappropriate SEM use. The observed results are not representative for all cardiovascular journals.CONCLUSION:In three selected cardiovascular journals we found a high level of inappropriate SEM use and explicit methods statements to use it for data description, especially in basic science studies. To improve on this situation, these and other journals should provide clear instructions to authors on how to report descriptive information of empirical data.