Activity recognition in live-cell imaging is labor-intensive and requires significant human effort. Existing automated analysis tools are largely limited in versatility. We present the Intelligent Vesicle Exocytosis Analysis (IVEA) platform, an ImageJ plugin for automated, reliable analysis of fluorescence-labeled vesicle fusion events and other burst-like activity. IVEA includes three specialized modules for detecting: (1) synaptic transmission in neurons, (2) single-vesicle exocytosis in any cell type, and (3) nano-sensor-detected exocytosis. Each module uses distinct techniques, including deep learning, allowing the detection of rare events often missed by humans at a speed estimated to be approximately 60 times faster than manual analysis. IVEA’s versatility can be expanded by refining or training new models via an integrated interface. With its impressive speed and remarkable accuracy, IVEA represents a seminal advancement in exocytosis image analysis and other burst-like fluorescence fluctuations applicable to a wide range of microscope types and fluorescent dyes.

Microendoscopy, a crucial technology for minimally invasive investigations of organs, facilitates studies within confined cavities. However, conventional microendoscopy is often limited by probe size and the constraint of using a single excitation wavelength. In response to these constraints, a multichannel microendoscope with a slender profile of only 360 µm is engineered. Functional signals both in situ and in vivo are successfully captured from individual single cells, employing a specially developed software suite for image processing, and exhibiting an effective resolution of 4.6 µm, allowing for the resolution of subcellular neuronal structures. This system enabled the first examination of calcium dynamics in vivo in murine tracheal tuft cells (formerly named brush cells) and in situ in kidney podocytes. Additionally, it recorded ratiometric redox reactions in various biological settings, including intact explanted organs and pancreatic islet cultures. The flexibility and streamlined operation of the microendoscopic technique open new avenues for conducting in vivo research, allowing for studies of tissue and organ function at cellular resolution.

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

Pseudomonas aeruginosa is a critical priority pathogen and causes life-threatening acute and biofilm-associated chronic infections. The choice of suitable treatment for complicated infections requires lengthy culturing for species identification from swabs or an invasive biopsy. To date, no fast, pathogen-specific diagnostic tools for P. aeruginosa infections are available. Here, we present the noninvasive pathogen-specific detection of P. aeruginosa using novel fluorescent probes that target the bacterial biofilm-associated lectins LecA and LecB. Several glycomimetic probes were developed to target these extracellular lectins and demonstrated to stain P. aeruginosa biofilms in vitro. Importantly, for the targeting of LecA an activity boost to low-nanomolar affinity could be achieved, which is essential for in vivo application. In vitro, the nanomolar divalent LecA-targeted imaging probe accumulated effectively in biofilms under flow conditions, independent of the fluorophore identity. Investigation of these glycomimetic imaging probes in a murine lung infection model and fluorescence imaging revealed accumulation at the infection site. These findings demonstrate the use of LecA- and LecB-targeting probes for the imaging of P. aeruginosa infections and suggest their potential as pathogen-specific diagnostics to accelerate the start of the appropriate treatment.

Microendoscopy enables minimally invasive investigations of organs even within small cavities. Conventional microendoscopy is limited by probe size and often restricted to a single excitation wavelength. We developed and characterized a multichannel microendoscope as thin as 360 µm and recorded functional cellular signals in-situ using custom written software for image processing. The endoscope had an effective resolution of 4.64 µm and resolved subcellular structures of neurons. The system enabled analysis of in-situ calcium responses in murine tracheal brush cells and kidney podocytes. Additionally, ratiometric redox responses were recorded in whole, explanted organs and pancreatic islet culture. The flexibility and simplicity of our approach for imaging a variety of tissues and organs paves the way for in-vivo, longitudinal studies with cellular resolution.### Competing Interest StatementThe authors have declared no competing interest.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is caused by genetic variants that often increase sarcomeric Ca2+ sensitivity. While Ca2+ sensitization explains diastolic dysfunction, the genesis of ventricular arrhythmias remains unresolved. Here, we show that HCM mutations or pharmacological interventions that increase myofilament Ca2+ sensitivity generate bioenergetic mismatch and oxidative stress during β-adrenergic stimulation, which provide a trigger and a substrate for arrhythmias. For any given sarcomere shortening that produces work and consumes ATP, less Ca2+ stimulates the Krebs cycle to maintain mitochondrial NADH. This reverses the mitochondrial transhydrogenase to regenerate NADH from NADPH, supporting ATP production at the cost of NADPH-dependent antioxidative capacity. The ensuing overflow of reactive oxygen species (ROS) from mitochondria and glutathione oxidation induce spontaneous Ca2+ release from the sarcoplasmic reticulum and Ca2+ waves, well-defined triggers of arrhythmias. Furthermore, transhydrogenase-dependent ROS formation slows electrical conduction during β-adrenergic stimulation in vivo, providing a substrate for arrhythmias. Chronic treatment with a mitochondrially-targeted ROS scavenger abolishes the arrhythmic burden during β-adrenergic stimulation in HCM mice in vivo, while inducing mitochondrial ROS with a redox cycler is sufficient to induce arrhythmias in wild-type animals. These findings may lead to new strategies to prevent sudden cardiac death in patients with HCM. ### Competing Interest Statement C.M. received honoraria for speaking from Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, Novartis, Novo Nordisk, Servier; and has participated in advisory boards for Amgen, AstraZeneca, Boehringer Ingelheim, Servier, and Pharmacosmos. V.S. received research funding from Bristol Myers Squibb. M.B. has received honoraria for speaking from Abbott, Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, Cytokinetics, Medtronic, Novartis, Servier, and Vifor; and has participated in advisory boards for Amgen, Bayer, Boehringer Ingelheim, Cytokinetics, Medtronic, Novartis, Pfizer, ReCor, Servier, and Vifor.

Gonadotropes in the anterior pituitary gland are essential for fertility and provide a functional link between the brain and the gonads. To trigger ovulation, gonadotrope cells release massive amounts of luteinizing hormone (LH). The mechanism underlying this remains unclear. Here, we utilize a mouse model expressing a genetically encoded Ca2+ indicator exclusively in gonadotropes to dissect this mechanism in intact pituitaries. We demonstrate that female gonadotropes exclusively exhibit a state of hyperexcitability during the LH surge, resulting in spontaneous [Ca2+]i transients in these cells, which persist in the absence of any in vivo hormonal signals. L-type Ca2+ channels and transient receptor potential channel A1 (TRPA1) together with intracellular reactive oxygen species (ROS) levels ensure this state of hyperexcitability. Consistent with this, virus-assisted triple knockout of Trpa1 and L-type Ca2+ subunits in gonadotropes leads to vaginal closure in cycling females. Our data provide insight into molecular mechanisms required for ovulation and reproductive success in mammals.

Inter-organ communication is a major hallmark of health and is often orchestrated by hormones released by the anterior pituitary gland. Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to regulate gonadal function and control fertility. Whether FSH and LH also act on organs other than the gonads is debated. Here, we find that gonadotrope depletion in adult female mice triggers profound hypogonadism, obesity, glucose intolerance, fatty liver, and bone loss. The absence of sex steroids precipitates these phenotypes, with the notable exception of fatty liver, which results from ovary-independent actions of FSH. We uncover paracrine FSH action on pituitary corticotropes as a mechanism to restrain the production of corticosterone and prevent hepatic steatosis. Our data demonstrate that functional communication of two distinct hormone-secreting cell populations in the pituitary regulates hepatic lipid metabolism.

The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels. We then use epicardioids to investigate the functional cross-talk between cardiac cell types, gaining new insights into the role of IGF2/IGF1R and NRP2 signaling in human cardiogenesis. Finally, we show that epicardioids mimic the multicellular pathogenesis of congenital or stress-induced hypertrophy and fibrotic remodeling. As such, epicardioids offer a unique testing ground of epicardial activity in heart development, disease and regeneration.

Both particular myocardial locations in the human heart and the canonical transient receptor potential 6 (TRPC6) cation channel have been linked with cardiac pathophysiologies. Thus, the present study mapped TRPC6-protein distribution in select anatomic locations associated with cardiac disease in the context of an orienting pathological assessment. Specimens were obtained from 5 body donors (4 formalin fixation, 1 nitrite pickling salt-ethanol-polyethylene glycol (NEP) fixation; median age 81 years; 2 females) and procured for basic histological stains and TRPC6-immunohistochemistry. The latter was analyzed descriptively regarding distribution and intensity of positive signals. The percentage of positively labelled myocardium was also determined (optical threshold method). Exclusively exploratory statistical analyses were performed. TRPC6-protein was distributed widespread and homogenously within each analyzed sample. TRPC6-immunoreactive myocardial area was comparable regarding the different anatomic regions and sex. A significantly larger area of TRPC6-immunoreactive myocardium was found in the NEP-fixed donor compared to the formalin fixed donors. Two donors with more severe heart disease showed smaller areas of myocardial TRPC6-immunoreactivity overall compared to the other 3 donors. In summary, in the elderly, TRPC6-protein is widely and homogenously distributed, and severe cardiac disease might be associated with less TRPC6-immunoreactive myocardial area. The tissue fixation method represents a potential confounder.

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first -line defense mechanisms against bacterial infections of the lung.

TRPM4 is a Ca2+-activated channel mediating the transport of monovalent cations across the cell membrane. Mutations in the TRPM4 gene have been associated with cardiac arrhythmias in humans. Using CRISPR/Cas9 gene editing technology, we established two TRPM4 knockout human iPSC lines - one heterozygous (MRli003-A-3) and one homozygous (MRli003-A-4) - by inserting a frameshift mutation in exon 2 of the TRPM4 gene. Both lines maintained pluripotency, a normal karyotype, parental cell morphology, and the ability to differentiate into the three germ layers.

Assessment of the electrophysiological properties of cardiomyocytes is necessary for phenotyping cardiac disorders and for drug screening. Optical action potential imaging using a genetically encoded voltage-sensing fluorescent protein (VSFP) allows for high-throughput functional characterization of cardiomyocytes, which offers an advantage over the traditional patch-clamp technique. Here, we knocked VSFP into the AAVS1 safe harbor locus of human iPSCs, generating two stable voltage indicator lines - one heterozygous (MRIi003-A-5) and the other homozygous (MRI003-A-6). Both lines can be used for optical membrane potential recordings and provide a powerful platform for a wide range of applications in cardiovascular biomedicine.

There was an error published in Fig. 6 in J. Cell Sci. (2002) 115, 2497-2504 (doi:10.1242/jcs.115.12.2497).There is a duplication of a segment of signal in Fig. 6A. According to the authors, this was due to an error in the process of assembling the trace. They made every effort to find the original data, but, as the work dates back more than twenty years, they were unable to recover the original files.The authors state that this error does not significantly impact the interpretation of the article, the data reliability or the reader's understanding of the paper and that the scholarly integrity of the article remains intact. “Data reported in Fig. 6A and B aimed to illustrate variability of spontaneous Ca2+ signals in two RyR3-expressing cells; spontaneous Ca2+ release events are also reported in Fig. 5 and duplication of signal in Fig. 6Ab does not impact on the main interpretation of the article which was that HEK293 cells expressing RyR3 display spontaneous Ca2+ release events and those expressing RyR1 do not. This suggested that, at least in these cells, isoform-specific functional properties may contribute to generation of intracellular Ca2+ signals.”Without the original data, it is difficult to determine how the issues arose, so the journal is publishing this note to alert readers to our concerns, with the agreement of the authors.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) represent an excellent in vitro model in cardiovascular research. Changes in their action potential (AP) dynamics convey information that is essential for disease modeling, drug screening and toxicity evaluation. High-throughput optical AP recordings utilizing intramolecular Förster resonance energy transfer (FRET) of the voltage-sensitive fluorescent protein (VSFP) have emerged as a substitute or complement to the resource-intensive patch clamp technique. Here, we functionally validated our recently generated voltage indicator hiPSC lines stably expressing CAG-promoter-driven VSFP in the AAVS1 safe harbor locus. By combining subtype-specific cardiomyocyte differentiation protocols, we established optical AP recordings in ventricular, atrial, and nodal CMs in 2D monolayers using fluorescence microscopy. Moreover, we achieved high-throughput optical AP measurements in single hiPSC-derived CMs in a 3D context. Overall, this system greatly expands the spectrum of possibilities for high-throughput, non-invasive and long-term AP analyses in cardiovascular research and drug discovery.

Aims: After summarizing current concepts for the role of TRPC cation channels in cardiac cells and in processes triggered by mechanical stimuli arising e.g. during pressure overload, we analysed the role of TRPC1 and TRPC4 for background Ca2+ entry (BGCE) and for cardiac pressure overload induced transcriptional remodelling. Methods and results: Mn2+-quench analysis in cardiomyocytes from several Trpc-deficient mice revealed that both TRPC1 and TRPC4 are required for BGCE. Electrically-evoked cell shortening of cardiomyocytes from TRPC1/C4-DKO mice was reduced, whereas parameters of cardiac contractility and relaxation assessed in vivo were unaltered. As pathological cardiac remodelling in mice depends on their genetic background, and the development of cardiac remodelling was found to be reduced in TRPC1/C4-DKO mice on a mixed genetic background, we studied TRPC1/C4-DKO mice on a C57BL6/N genetic background. Cardiac hypertrophy was reduced in those mice after chronic isoproterenol infusion (-51.4%) or after one week of transverse aortic constriction (TAC; -73.0%). This last manoeuvre was preceded by changes in the pressure overload induced transcriptional program as analysed by RNA sequencing. Genes encoding specific collagens, the Mef2 target myomaxin and the gene encoding the mechanosensitive channel Piezo2 were up-regulated after TAC in wild type but not in TRPC1/C4-DKO hearts. Conclusions: Deletion of the TRPC1 and TRPC4 channel proteins protects against development of pathological cardiac hypertrophy independently of the genetic background. To determine if the TRPC1/C4-dependent changes in the pressure overload induced alterations in the transcriptional program causally contribute to cardio-protection needs to be elaborated in future studies. (c) 2020 Elsevier Ltd. All rights reserved.

Background: Cardiovascular diseases and chronic kidney disease (CKD) are highly prevalent, aggravate each other, and account for substantial mortality. Both conditions are characterized by activation of the innate immune system. The alarmin interleukin-1α (IL-1α) is expressed in a variety of cell types promoting (sterile) systemic inflammation. The aim of the present study was to examine the role of IL-1α in mediating inflammation in the setting of acute myocardial infarction (AMI) and CKD. Methods: We assessed the expression of IL-1α on the surface of monocytes from patients with AMI and patients with CKD and determined its association with atherosclerotic cardiovascular disease events during follow-up in an explorative clinical study. Furthermore, we assessed the inflammatory effects of IL-1α in several organ injury models in Il1a −/ − and Il1b −/ − mice and investigated the underlying mechanisms in vitro in monocytes and endothelial cells. Results: IL-1α is strongly expressed on the surface of monocytes from patients with AMI and CKD compared with healthy controls. Higher IL-1α surface expression on monocytes from patients with AMI and CKD was associated with a higher risk for atherosclerotic cardiovascular disease events, which underlines the clinical relevance of IL-1α. In mice, IL-1α, but not IL-1β, mediates leukocyte-endothelial adhesion as determined by intravital microscopy. IL-1α promotes accumulation of macrophages and neutrophils in inflamed tissue in vivo. Furthermore, IL-1α on monocytes stimulates their homing at sites of vascular injury. A variety of stimuli such as free fatty acids or oxalate crystals induce IL-1α surface expression and release by monocytes, which then mediates their adhesion to the endothelium via IL-1 receptor-1. IL-1α also promotes expression of the VCAM-1 (vascular cell adhesion molecule-1) on endothelial cells, thereby fostering the adhesion of circulating leukocytes. IL-1α induces inflammatory injury after experimental AMI, and abrogation of IL-1α prevents the development of CKD in oxalate or adenine-fed mice. Conclusions: IL-1α represents a key mediator of leukocyte-endothelial adhesion and inflammation in AMI and CKD. Inhibition of IL-1α may serve as a novel anti-inflammatory treatment strategy.

Angiogenesis crucially contributes to various diseases, such as cancer and diabetic retinopathy. Hence, anti-angiogenic therapy is considered as a powerful strategy against these diseases. Previous studies reported that the acyclic monoterpene linalool exhibits anticancer, anti-inflammatory and anti-oxidative activity. However, the effects of linalool on angiogenesis still remain elusive. Therefore, we investigated the action of (3 R )-(−)-linalool, a main enantiomer of linalool, on the angiogenic activity of human dermal microvascular endothelial cells (HDMECs) by a panel of angiogenesis assays. Non-cytotoxic doses of linalool significantly inhibited HDMEC proliferation, migration, tube formation and spheroid sprouting. Linalool also suppressed the vascular sprouting from rat aortic rings. In addition, Matrigel plugs containing linalool exhibited a significantly reduced microvessel density 7 days after implantation into BALB/c mice. Mechanistic analyses revealed that linalool promotes the phosphorylation of extracellular signal-regulated kinase (ERK), downregulates the intracellular level of adenosine triphosphate (ATP) and activates the transient receptor potential cation channel subfamily M (melastatin) member (TRPM)8 in HDMECs. Inhibition of ERK signaling, supplementation of ATP and blockade of TRPM8 significantly counteracted linalool-suppressed HDMEC spheroid sprouting. Moreover, ATP supplementation completely reversed linalool-induced ERK phosphorylation. In addition, linalool-induced ERK phosphorylation inhibited the expression of bone morphogenetic protein (BMP)-2 and linalool-induced TRPM8 activation caused the inhibition of β1 integrin/focal adhesion kinase (FAK) signaling. These findings indicate an anti-angiogenic effect of linalool, which is mediated by downregulating intracellular ATP levels and activating TRPM8.