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Pulmonary veins (PV) are the main source of ectopy, triggering atrial fibrillation. This study investigated the roles of G protein-coupled inwardly rectifying potassium (GIRK) channels in the PV and the left atrium (LA) of the rat. Simultaneous intracellular microelectrode recording from the LA and the PV of the rat found that in the presence or absence of acetylcholine, the GIRK channel blocker tertiapin-Q induced AP duration elongation in the LA and the loss of over-shooting AP in the PV, suggesting the presence of constitutively active GIRK channels in these tissues. Patch-clamp recordings from isolated myocytes showed that tertiapin-Q inhibited a basal inwardly rectified background current in PV cells with little effect in LA cells. Experiments with ROMK1 and KCa1.1 channel blockers ruled out the possibility of an off-target effect. Western blot showed that GIRK4 subunit expression was greater in PV cardiomyocytes, which may explain the differences observed between PV and LA in response to tertiapin-Q. In conclusion, GIRK channels blockade abolishes AP only in the PV, providing a molecular target to induce electrical disconnection of the PV from the LA.

Cardiac excitation-contraction coupling can be different between regions of the heart. Little is known at the atria level, specifically in different regions of the left atrium. This is important given the role of cardiac myocytes from the pulmonary vein sleeves, which are responsible for ectopic activity during atrial fibrillation. In this study, we present a new method to isolate atrial cardiac myocytes from four different regions of the left atrium of a large animal model, sheep, highly relevant to humans. Using collagenase/protease we obtained calcium-tolerant atrial cardiac myocytes from the epicardium, endocardium, free wall and pulmonary vein regions. Calcium transients were slower (time to peak and time to decay) in free wall and pulmonary vein myocytes compared to the epicardium and endocardium. This is associated with lower t-tubule density. Overall, these results suggest regional differences in calcium transient and t-tubule density across left atria, which may play a major role in the genesis of atrial fibrillation.

In the heart, cardiac function is regulated by the autonomic nervous system (ANS) that extends through the myocardium and establishes junctions at the sinus node and ventricular levels. Thus, an increase or decrease in neuronal activity acutely affects myocardial function and chronically affects its structure through remodeling processes. The neuro-cardiac junction (NCJ), which is the major structure of this system, is poorly understood and only a few cell models allow us to study it. Here, we present an innovant neuro-cardiac organ-on-chip model to study this structure to better understand the mechanisms involved in the establishment of NCJ. To create such a system, we used microfluidic devices composed of two separate cell culture compartments interconnected by asymmetric microchannels. Rat PC12 cells were differentiated to recapitulate the characteristics of sympathetic neurons, and cultivated with cardiomyocytes derived from human induced pluripotent stem cells (hiPSC). We confirmed the presence of a specialized structure between the two cell types that allows neuromodulation and observed that the neuronal stimulation impacts the excitation-contraction coupling properties including the intracellular calcium handling. Finally, we also co-cultivated human neurons (hiPSC-NRs) with human cardiomyocytes (hiPSC-CMs), both obtained from the same hiPSC line. Hence, we have developed a neuro-cardiac compartmentalized in vitro model system that allows us to recapitulate the structural and functional properties of the neuro-cardiac junction and that can also be used to better understand the interaction between the heart and brain in humans, as well as to evaluate the impact of drugs on a reconstructed human neuro-cardiac system.

Background: The intrinsic cardiac nervous system (ICNS) refers to clusters of neurons, located within the heart, that participate in the neuronal regulation of cardiac functions and that are involved in the initiation of cardiac arrhythmias. Therefore, deciphering its role in cardiac physiology and physiopathology is mandatory., Objective: The aim of this study was to provide a phenotypic, electrophysiological, and pharmacological characterization of the mouse ICNS, which is still poorly characterized., Methods: Global cardiac innervation and phenotypic diversity were investigated using immunohistochemistry on cleared murine hearts and on tissue sections. The patch clamp technique was used for the electrophysiological and pharmacological characterization of isolated mouse intracardiac neurons., Results: We have identified the expression of 7 distinct neuronal markers within the mouse ICNS, thus proving the neurochemical diversity of this network. Of note, it was the first time that the existence of neurons expressing the calcium-binding protein calbindin, neuropeptide Y, and cocaine and amphetamine regulated transcript peptide was described in the mouse. Electrophysiology studies also revealed the existence of 4 different neuronal populations on the basis of their electrical behavior. Finally, we showed that these neurons can be modulated by several neuromodulators., Conclusion: This study showed that the mouse ICNS presents a molecular and functional complexity similar to other species and is therefore a suitable model to decipher the role of individual neuronal subtypes regarding the modulation of cardiac function and the initiation of cardiac arrhythmias., (Copyright © 2022 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Introduction: Gabapentinoids are ligands of the α2-δ subunit of voltage-gated calcium channels (Cav) that have been associated with a risk of peripheral edema and acute heart failure in connection with a potentially dual mechanism, vascular and cardiac., Objectives & Methods: All cases of peripheral edema or heart failure involving gabapentin or pregabalin reported to the French Pharmacovigilance Centers between January 1, 1994 and April 30, 2020 were included to describe their onset patterns (e.g., time to onset). Based on these data, we investigated the impact of gabapentinoids on the myogenic tone of rat third-order mesenteric arteries and on the electrophysiological properties of rat ventricular cardiomyocytes., Results: A total of 58 reports were included (gabapentin n = 5, pregabalin n = 53). The female-to-male ratio was 4:1 and the median age was 77 years (IQR 57-85, range 32-95). The median time to onset were 23 days (IQR 10-54) and 17 days (IQR 3-30) for non-cardiogenic edema and acute heart failure, respectively. Cardiogenic and non-cardiogenic peripheral edema occurred frequently after a dose escalation (27/45, 60%), and the course was rapidly favorable after discontinuation of gabapentinoid (median 7 days, IQR 5-13). On rat mesenteric arteries, gabapentinoids significantly decreased the myogenic tone to the same extent as verapamil and nifedipine. Acute application of gabapentinoids had no significant effect on Ca v 1.2 currents of ventricular cardiomyocytes., Conclusion: Gabapentinoids can cause concentration-dependent peripheral edema of early onset. The primary mechanism of non-cardiogenic peripheral edema is vasodilatory edema secondary to altered myogenic tone, independent of Ca v 1.2 blockade under the experimental conditions tested., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Introduction and Objective: Nowadays, investigations of heart physiology and pathophysiology rely more and more upon image analysis, whether for the detection and characterization of events in single cells or for the mapping of events and their characteristics across an entire tissue. These investigations require extensive skills in image analysis and/or expensive software, and their reproducibility may be a concern. Our objective was to build a robust, reliable and open-source software tool to quantify excitation-contraction related experimental data at multiple scales, from single isolated cells to the whole heart., Methods and Results: A free and open-source ImageJ plugin, Spiky, was developed to detect and analyze peaks in experimental data streams. It allows rapid and easy analysis of action potentials, intracellular calcium transient and contraction data from cardiac research experiments. As shown in the provided examples, both classical bi-dimensional data (XT signals) and video data obtained from confocal microscopy and optical mapping experiments (XYT signals) can be analyzed. Spiky was written in ImageJ Macro Language and JAVA, and works under Windows, Mac and Linux operating systems., Conclusion: Spiky provides a complete working interface to process and analyze cardiac physiology research data.

Ectopic activity in the pulmonary vein cardiac muscle sleeves can both induce and maintain human atrial fibrillation. A central issue in any study of the pulmonary veins is their difference from the left atrial cardiac muscle. Here, we attempt to summarize the physiological phenomena underlying the occurrence of ectopic electrical activity in animal pulmonary veins. We emphasize that the activation of multiple signaling pathways influencing not only myocyte electrophysiology but also the means of excitation-contraction coupling may be required for the initiation of triggered or automatic activity. We also gather information regarding not only the large-scale structure of cardiac muscle sleeves but also recent studies suggesting that cellular heterogeneity may contribute to the generation of arrythmogenic phenomena and to the distinction between pulmonary vein and left atrial heart muscle.

The modulation of SK3 ion channels can be efficiently and selectively achieved by using the amphiphilic compound Ohmline (a glyco-glycero-ether-lipid). We report herein a series of Ohmline analogues featuring the replacement of one ether function by a thioether function located at the same position or shifted close to its initial position. The variation of the lipid chain length and the preparation of two analogues featuring either one sulfoxide or one sulfone moiety complete this series. Patch clamp measurements indicate that the presence of the thioether function (compounds 7 and 17a) produces strong activators of SK3 channels, whereas the introduction of a sulfoxide or a sulfone function at the same place produces amphiphiles devoid of an effect on SK3 channels. Compounds 7 and 17a are the first amphiphilic compounds featuring strong activation of SK3 channels (close to 200% activation). The cytosolic calcium concentration determined from fluorescence at 3 different times for compound 7b (13 min, 1 h, 24 h) revealed that the effect is different suggesting that the compound could be metabolized over time. This compound could be used as a strong SK3 activator for a short time. The capacity of 7b to activate SK3 was then used to induce vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. For the first time, we report that an amphiphilic compound can affect the endothelium dependent vasorelaxation.

Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV). It is still under discussion, in which cells viral integration and MCC development occurs. Recently, we demonstrated that a virus-positive MCC derived from a trichoblastoma, an epithelial neoplasia bearing Merkel cell (MC) differentiation potential. Accordingly, we hypothesized that MC progenitors may represent an origin of MCPyV-positive MCC. To sustain this hypothesis, phenotypic comparison of trichoblastomas and physiologic human MC progenitors was conducted revealing GLI family zinc finger 1 (GLI1), Keratin 17 (KRT 17), and SRY-box transcription factor 9 (SOX9) expressions in both subsets. Furthermore, GLI1 expression in keratinocytes induced transcription of the MC marker SOX2 supporting a role of GLI1 in human MC differentiation. To assess a possible contribution of the MCPyV T antigens (TA) to the development of an MC-like phenotype, human keratinocytes were transduced with TA. While this led only to induction of KRT8, an early MC marker, combined GLI1 and TA expression gave rise to a more advanced MC phenotype with SOX2, KRT8, and KRT20 expression. Finally, we demonstrated MCPyV-large T antigens' capacity to inhibit the degradation of the MC master regulator Atonal bHLH transcription factor 1 (ATOH1). In conclusion, our report suggests that MCPyV TA contribute to the acquisition of an MC-like phenotype in epithelial cells.

Background/objectives: Hibiscus sabdariffa L. ( H. sabdariffa (HS)) extract has a vascular relaxant effect on isolated rat thoracic aorta, but data on small resistance arteries, which play an important role on the development of hypertension, are still missing. The purposes of this study were (1) to assess the effect on isolated mesenteric arteries (MA) from normotensive (Wistar and Wistar-Kyoto (WKY)) and spontaneous hypertensive rats (SHR); (2) to elucidate the mechanism(s) of action underling the relaxant effect in light of bioactive components., Methods: Vascular effects of HS aqueous fraction (AF) on isolated MA rings, as well as its mechanisms of action, were assessed using the contractility and intracellular microelectrode technique. The patch clamp technique was used to evaluate the effect of HS AF on the L-type calcium current. Extraction and enrichment of AF were carried out using liquid-liquid extraction, and the yield was analyzed using HPLC., Results: The HS AF induced a concentration-dependent relaxant effect on MA rings of SHR (EC 50 = 0.83 ± 0.08 mg/mL), WKY (EC 50 = 0.46 ± 0.04 mg/mL), and Wistar rats (EC 50 = 0.44 ± 0.08 mg/mL) pre-contracted with phenylephrine (10 µM). In Wistar rats, the HS AF maximum relaxant effect was not modified after endothelium removal or when a guanylate cyclase inhibitor (ODQ, 10 µM) and a selective β2-adrenergic receptor antagonist (ICI-118551, 1 µM) were incubated with the preparation. Otherwise, it was reduced by 34.57 ± 10.66% when vascular rings were pre-contracted with an 80 mM [K+] solution ( p < 0.001), which suggests an effect on ionic channels. HS AF 2 mg/mL significantly decreased the peak of the L-type calcium current observed in cardiac myocytes by 24.4%. Moreover, though the vasorelaxant effect of HS, AF was reduced by 27% when the nonselective potassium channels blocker (tetraethylammonium (TEA) 20 mM) was added to the bath ( p < 0.01). The extract did not induce a membrane hyperpolarization of smooth muscle cells, which might suggest an absence of a direct effect on background potassium current., Conclusion: These results highlight that the antihypertensive effect of HS probably involves a vasorelaxant effect on small resistance arteries, which is endothelium independent. L-type calcium current reduction contributes to this effect. The results could also provide a link between the vasorelaxant effect and the bioactive compounds, especially anthocyanins., Competing Interests: The authors declare that they have no conflict of interest.

Pulmonary veins (PV) are involved in the pathophysiology of paroxysmal atrial fibrillation. In the rat, left atrium (LA) and PV cardiomyocytes have different reactions to α1-adrenergic receptor activation. In freely beating atria-PV preparations, we found that electrical field potential (EFP) originated from the sino-atrial node propagated through the LA and the PV. The α1-adrenergic receptor agonist cirazoline induced a progressive loss of EFP conduction in the PV whereas it was maintained in the LA. This could be reproduced in preparations electrically paced at 5 Hz in LA. During pacing at 10 Hz in the PV where high firing rate ectopic foci can occur, cirazoline stopped EFP conduction from the PV to the LA, which allowed the sino-atrial node to resume its pace-making function. Loss of conduction in the PV was associated with depolarization of the diastolic membrane potential of PV cardiomyocytes. Adenosine, which reversed the cirazoline-induced depolarization of the diastolic membrane potential of PV cardiomyocytes, restored full over-shooting action potentials and EFP conduction in the PV. In conclusion, selective activation of α1-adrenergic receptors results in the abolition of electrical conduction within the PV. These results highlight a potentially novel pharmacological approach to treat paroxysmal atrial fibrillation by targeting directly the PV myocardium.

The composition of periprostatic adipose tissue (PPAT) has been shown to play a role in prostate cancer (PCa) progression. We recently reported an inverse association between PCa aggressiveness and elevated PPAT linoleic acid (LA) and eicosapentaenoic acid (EPA) content. In the present study, we identified a new signaling pathway with a positive feedback loop between the epithelial-to-mesenchymal transition (EMT) transcription factor Zeb1 and the Ca 2+ -activated K + channel SK3, which leads to an amplification of Ca 2+ entry and cellular migration. Using in vitro experiments and ex vivo cultures of human PCa slices, we demonstrated that LA and EPA exert anticancer effects, by modulating Ca 2+ entry, which was involved in Zeb1 regulation and cancer cellular migration. This functional approach using human prostate tumors highlights the clinical relevance of our observations, and may allow us to consider the possibility of targeting cancer spread by altering the lipid microenvironment., Competing Interests: The authors declare no conflicts of interest.

Vascular aging is characterized by increase in arterial stiffness and remodeling of the arterial wall with a loss of elastic properties. Silicon is an essential trace element highly present in arteries. It is involved in the constitution and stabilization of elastin fibers. The nutritional supply and bioavailability of silicon are often inadequate. Spirulina (Sp), micro algae have recognized nutritional properties and are able to incorporate minerals in a bioavailable form. We evaluated the effects of nutritional supplementation with silicon-enriched spirulina (SpSi) on arterial system structure and function in hypertension. Experiments were performed on hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats supplemented with SpSi or Sp over a period of three months. Arterial pressure, vascular function and morphometric parameters of thoracic aorta were analyzed. SpSi supplementation lowered arterial pressure in SHR and minimized morphometric alterations induced by hypertension. Aortic wall thickness and elastic fibers fragmentation were partially reversed. Collagen and elastin levels were increased in association with extracellular matrix degradation decrease. Vascular reactivity was improved with better contractile and vasorelaxant responses to various agonists. No changes were observed in SHR supplemented with Sp. The beneficial effects of SpSi supplementation evidenced here, may be attributable to Si enrichment and offer interesting opportunities to prevent cardiovascular risks.

In prostate cancer research, there is a lack of valuable preclinical models. Tumor cell heterogeneity and sensitivity to microenvironment signals, such as hypoxia or extracellular calcium concentration, are difficult to reproduce. Here, we developed and characterized an ex vivo tissue culture model preserving these properties. Prostate tissue slices from 26 patients were maintained ex vivo under optimized culture conditions. The expression of markers associated with proliferation, androgen-receptor signaling, and hypoxia was assessed by immunostaining. A macroscope was used to achieve real-time calcium fluorescence optical imaging. Tissue morphology was maintained successfully without necrosis for 5 days. Compared with native tumors and tissue cultured with androgens, androgen deprivation in the medium led to decreased expression of both androgen receptor and its target gene products, prostate specific antigen (PSA) and ETS-related gene (ERG). Ex vivo cultured slices also were sensitive to hypoxia because carbonic anhydrase IX and zinc finger E-box binding homeobox 1 (Zeb1) protein levels increased in 1% oxygen. Exposure of slices to supraphysiological extracellular Ca 2+ concentration induced a robust and rapid Ca 2+ entry, with a greater response in tumor compared with nontumor tissue. This ex vivo model reproduces the morphologic and functional characteristics of human prostate cancer, including sensitivity to androgen deprivation and induced response to hypoxia and extracellular Ca 2+ . It therefore could become an attractive tool for drug response prediction studies., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Sepsis is a life-threatening condition often associated with a high incidence of multiple organs injury. Several papers suggested the immune response by itself, with the production of humoral inflammatory mediators, is crucial in determining organ injury. However, little is known of how sepsis directly induces organ injury at the cellular levels. To assess this point, we set up an in vitro study to investigate the response of renal tubular cells (RTCs), monocytes (U937) and hepatocytes (HepG2) after 24 h-incubation with septic patients' plasma., Methods: We enrolled 26 septic patients ("test" group). We evaluated cell viability, apoptosis and necrosis by flow cytometer. Caspase-3,-8,-9 and cytochrome-c concentrations have been analyzed using the Human enzyme-linked immunosorbent assay kit., Results: We found that a decrease of cell viability in all cell lines tested was associated to the increase of apoptosis in RTCs and U937 (p < 0.0001) and increase of necrosis in HepG2 (p < 0.5). The increase of apoptosis in RTCs and U937 cells was confirmed by higher levels of caspase-3 (p < 0.0001). We showed that apoptosis in both RTCs and U937 was triggered by the activation of the intrinsic pathway, as caspase-9 and cytochrome-c levels significantly increased (p < 0.0001), while caspase-8 did not change. This assumption was strengthened by the significant correlation of caspase-9 with both cytochrome-c (r = 0.73 for RTCs and r = 0.69 for U937) and caspase-3 (r = 0.69 for RTCs and r = 0.63 for U937)., Conclusion: Humoral mediators in septic patients' plasma induce apoptosis. This fact suggests that apoptosis inhibitors should be investigated as future strategy to reduce sepsis-induced organ damages., (© 2018 S. Karger AG, Basel.)

Summary: Simultaneous recordings of myocytes contractility and their cytoplasmic calcium concentration allow powerful studies, particularly on heart failure and other cardiac dysfunctions. Such studies require dedicated and expensive experimental devices that are difficult to use. Thus we propose SarConfoCal, the first and only software to simultaneously analyse both cytoplasmic calcium variations (from fluorescence signal) and myocytes contractility (from sarcomere length measurement) on laser scanning confocal microscopy images. SarConfoCal is easy to set up and use, especially by people without programming skills., Availability and Implementation: The software is freely distributed under the GNU General Public License. Download and setup instructions are available at http://pccv.univ-tours.fr/ImageJ/SarConfoCal . It is provided as a toolset for ImageJ (the open-source program for image analysis provided by the National Institutes of Health). SarConfoCal has been tested under Windows, Mac and Linux operating systems., Contact: come.pasqualin@univ-tours.fr., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Accurate measurement of cardiomyocyte contraction is a critical issue for scientists working on cardiac physiology and physiopathology of diseases implying contraction impairment. Cardiomyocytes contraction can be quantified by measuring sarcomere length, but few tools are available for this, and none is freely distributed. We developed a plug-in (SarcOptiM) for the ImageJ/Fiji image analysis platform developed by the National Institutes of Health. SarcOptiM computes sarcomere length via fast Fourier transform analysis of video frames captured or displayed in ImageJ and thus is not tied to a dedicated video camera. It can work in real time or offline, the latter overcoming rotating motion or displacement-related artifacts. SarcOptiM includes a simulator and video generator of cardiomyocyte contraction. Acquisition parameters, such as pixel size and camera frame rate, were tested with both experimental recordings of rat ventricular cardiomyocytes and synthetic videos. It is freely distributed, and its source code is available. It works under Windows, Mac, or Linux operating systems. The camera speed is the limiting factor, since the algorithm can compute online sarcomere shortening at frame rates >10 kHz. In conclusion, SarcOptiM is a free and validated user-friendly tool for studying cardiomyocyte contraction in all species, including human., (Copyright © 2016 the American Physiological Society.)

The transverse tubule system in mammalian striated muscle is highly organized and contributes to optimal and homogeneous contraction. Diverse pathologies such as heart failure and atrial fibrillation include disorganization of t-tubules and contractile dysfunction. Few tools are available for the quantification of the organization of the t-tubule system. We developed a plugin for the ImageJ/Fiji image analysis platform developed by the National Institutes of Health. This plugin (TTorg) analyzes raw confocal microscopy images. Analysis options include the whole image, specific regions of the image (cropping), and z-axis analysis of the same image. Batch analysis of a series of images with identical criteria is also one of the options. There is no need to either reorientate any specimen to the horizontal or to do a thresholding of the image to perform analysis. TTorg includes a synthetic "myocyte-like" image generator to test the plugin's efficiency in the user's own experimental conditions. This plugin was validated on synthetic images for different simulated cell characteristics and acquisition parameters. TTorg was able to detect significant differences between the organization of the t-tubule systems in experimental data of mouse ventricular myocytes isolated from wild-type and dystrophin-deficient mice. TTorg is freely distributed, and its source code is available. It provides a reliable, easy-to-use, automatic, and unbiased measurement of t-tubule organization in a wide variety of experimental conditions., (Copyright © 2015 the American Physiological Society.)

Background. Cardiorenal Syndrome Type 5 (CRS Type 5) reflects concomitant cardiac and renal dysfunctions in the setting of a wide spectrum of systemic disorders. Our aim was to study in vitro effects of CRS Type 5 plasma on renal tubular cells (RTCs), in terms of cellular death and the characterization of inflammatory plasma profile in these patients. Material and Methods. We enrolled 11 CRS Type 5 patients from ICU and 16 healthy controls. Plasma from patients and controls was incubated with renal tubular cells (RTCs) and cell death was evaluated. Plasma cytokines were detected. Results. RTCs incubated with CRS Type 5 plasma showed significantly higher apoptosis and necrosis with respect to controls. Plasma cytokine profile of CRS Type 5 patients was significantly different from controls: we observed the production of pro- and anti-inflammatory mediators in these patients. Caspase-3, caspase-8, and caspase-9 were activated in cells treated with CRS Type 5 plasma compared to controls. Conclusions. Our results underline the cytotoxic effect of CRS Type 5 mediators on RTC viability, probably due to the activation of both intrinsic and extrinsic pathways of apoptosis and to the deregulation of cytokine release. The consequence may be the damage of distant organs which lead to the worsening of condition of patients.