Connexin-43 is a promising target for pulmonary hypertension due to hypoxaemic lung disease.

The mechanisms underlying pulmonary hypertension (PH) are complex and multifactorial, and involve different cell types that are interconnected through gap junctional channels. Although connexin (Cx)-43 is the most abundant gap junction protein in the heart and lungs, and critically governs intercellular signalling communication, its contribution to PH remains unknown. The focus of the present study is thus to evaluate Cx43 as a potential new target in PH.Expressions of Cx37, Cx40 and Cx43 were studied in lung specimens from patients with idiopathic pulmonary arterial hypertension (IPAH) or PH associated with chronic hypoxaemic lung diseases (chronic hypoxia-induced pulmonary hypertension (CH-PH)). Heterozygous Cx43 knockdown CD1 (Cx43+/-) and wild-type littermate (Cx43+/+) mice at 12 weeks of age were randomly divided into two groups, one of which was maintained in room air and the other exposed to hypoxia (10% oxygen) for 3 weeks. We evaluated pulmonary haemodynamics, remodelling processes in cardiac tissues and pulmonary arteries (PAs), lung inflammation and PA vasoreactivity.Cx43 levels were increased in PAs from CH-PH patients and decreased in PAs from IPAH patients; however, no difference in Cx37 or Cx40 levels was noted. Upon hypoxia treatment, the Cx43+/- mice were partially protected against CH-PH when compared to Cx43+/+ mice, with reduced pulmonary arterial muscularisation and inflammatory infiltration. Interestingly, the adaptive changes in cardiac remodelling in Cx43+/- mice were not affected. PA contraction due to endothelin-1 (ET-1) was increased in Cx43+/- mice under normoxic and hypoxic conditions.Taken together, these results indicate that targeting Cx43 may have beneficial therapeutic effects in PH without affecting compensatory cardiac hypertrophy.

Effect of hypoxia on TRPV1 and TRPV4 channels in rat pulmonary arterial smooth muscle cells.

Transient receptor potential (TRP) channels of the vanilloid subfamily, mainly TRPV1 and TRPV4, are expressed in pulmonary artery smooth muscle cells (PASMC) and implicated in the remodeling of pulmonary artery, a landmark of pulmonary hypertension (PH). Among a variety of PH subtypes, PH of group 3 are mostly related to a prolonged hypoxia exposure occurring in a variety of chronic lung diseases. In the present study, we thus investigated the role of hypoxia on TRPV1 and TRPV4 channels independently of the increased pulmonary arterial pressure that occurs during PH. We isolated PASMC from normoxic rat and cultured these cells under in vitro hypoxia. Using microspectrofluorimetry and the patch-clamp technique, we showed that hypoxia (1 % O2 for 48 h) significantly increased stretch- and TRPV4-induced calcium responses. qRT-PCR, Western blotting, and immunostaining experiments revealed that the expression of TRPV1 and TRPV4 was not enhanced under hypoxic conditions, but we observed a membrane translocation of TRPV1. Furthermore, hypoxia induced a reorganization of the F-actin cytoskeleton, the tubulin, and intermediate filament networks (immunostaining experiments), associated with an enhanced TRPV1- and TRPV4-induced migratory response (wound-healing assay). Finally, as assessed by immunostaining, exposure to in vitro hypoxia elicited a significant increase in NFATc4 nuclear localization. Cyclosporin A and BAPTA-AM inhibited NFATc4 translocation, indicating the activation of the Ca(2+)/calcineurin/NFAT pathway. In conclusion, these data point out the effect of hypoxia on TRPV1 and TRPV4 channels in rat PASMC, suggesting that these channels can act as direct signal transducers in the pathophysiology of PH.

10th European Calcium Society symposium: The Ca2+-signaling toolkit in cell function, health and disease.

The 10th European Calcium Society symposium, organized in Leuven, Belgium on November 15-17, 2023, focused on the role of Ca2+ signaling in cell function, health and disease. The symposium featured six scientific sessions, 16 invited speakers - of whom two were postdoctoral researchers - and 14 short talks. The talks covered various aspects of intracellular Ca2+ signaling and its implications in pathology. Each session was opened by one or more invited speakers, followed by a series of presentations from speakers selected from submitted abstracts. Through short talks, poster presentations, awards, and sustainable travel fellowships, the symposium also fostered opportunities for the active participation of early-career researchers. At least half of the short talks were allocated to early-career researchers, thereby offering a platform for the presentation of ongoing work and unpublished results. Presentations were also broadcast in real-time for online attendees. In this Meeting Review, we aim to capture the spirit of the meeting and discuss the main take-home messages that emerged during the symposium.

Piezo1 Is Required for Myoblast Migration and Involves Polarized Clustering in Association with Cholesterol and GM1 Ganglioside.

A specific plasma membrane distribution of the mechanosensitive ion channel Piezo1 is required for cell migration, but the mechanism remains elusive. Here, we addressed this question using WT and Piezo1-silenced C2C12 mouse myoblasts and WT and Piezo1-KO human kidney HEK293T cells. We showed that cell migration in a cell-free area and through a porous membrane decreased upon Piezo1 silencing or deletion, but increased upon Piezo1 activation by Yoda1, whereas migration towards a chemoattractant gradient was reduced by Yoda1. Piezo1 organized into clusters, which were preferentially enriched at the front. This polarization was stimulated by Yoda1, accompanied by Ca2+ polarization, and abrogated by partial cholesterol depletion. Piezo1 clusters partially colocalized with cholesterol- and GM1 ganglioside-enriched domains, the proportion of which was increased by Yoda1. Mechanistically, Piezo1 activation induced a differential mobile fraction of GM1 associated with domains and the bulk membrane. Conversely, cholesterol depletion abrogated the differential mobile fraction of Piezo1 associated with clusters and the bulk membrane. In conclusion, we revealed, for the first time, the differential implication of Piezo1 depending on the migration mode and the interplay between GM1/cholesterol-enriched domains at the front during migration in a cell-free area. These domains could provide the optimal biophysical properties for Piezo1 activity and/or spatial dissociation from the PMCA calcium efflux pump.

Characterization of the thoracodorsal artery: morphology and reactivity.

OBJECTIVES: In this paper, we describe the histological and contractile properties of the thoracodorsal artery (TDA), which indirectly feeds the spinotrapezius muscle. METHODS: We used immunolabelling techniques to histologically characterize the TDA while the contractile properties were assessed using pressure arteriography. RESULTS: Our results demonstrate that the TDA is composed of approximately one to two layers of smooth muscle cells, is highly innervated with adrenergic nerves, and develops spontaneous tone at intraluminal pressures above 80 mmHg. The reactivity of the TDA in response to various contractile agonists such as phenylephrine, noradrenaline, angiotensin II, serotonin, endothelin 1, and ATP, as well as vasodilators, shows that the TDA exhibits a remarkably comparable reactivity to what has been observed in mesenteric arteries. We further studied the different components of the TDA response to acetylcholine, and found that the TDA was sensitive to TRAM 34, a blocker of the intermediate conductance potassium channel, which is highly suggestive of an endothelium-dependent hyperpolarization. CONCLUSIONS: We conclude that the TDA exhibits comparable characteristics to other current vascular models, with the additional advantage of being easily manipulated for molecular and ex vivo vasoreactivity studies.

PIEZO channels and newcomers in the mammalian mechanosensitive ion channel family.

Many ion channels have been described as mechanosensitive according to various criteria. Most broadly defined, an ion channel is called mechanosensitive if its activity is controlled by application of a physical force. The last decade has witnessed a revolution in mechanosensory physiology at the molecular, cellular, and system levels, both in health and in diseases. Since the discovery of the PIEZO proteins as prototypical mechanosensitive channel, many proteins have been proposed to transduce mechanosensory information in mammals. However, few of these newly identified candidates have all the attributes of bona fide, pore-forming mechanosensitive ion channels. In this perspective, we will cover and discuss new data that have advanced our understanding of mechanosensation at the molecular level.

Piezo1-Pannexin1 complex couples force detection to ATP secretion in cholangiocytes.

Cholangiocytes actively contribute to the final composition of secreted bile. These cells are exposed to abnormal mechanical stimuli during obstructive cholestasis, which has a deep impact on their function. However, the effects of mechanical insults on cholangiocyte function are not understood. Combining gene silencing and pharmacological assays with live calcium imaging, we probed molecular candidates essential for coupling mechanical force to ATP secretion in mouse cholangiocytes. We show that Piezo1 and Pannexin1 are necessary for eliciting the downstream effects of mechanical stress. By mediating a rise in intracellular Ca2+, Piezo1 acts as a mechanosensor responsible for translating cell swelling into activation of Panx1, which triggers ATP release and subsequent signal amplification through P2X4R. Co-immunoprecipitation and pull-down assays indicated physical interaction between Piezo1 and Panx1, which leads to stable plasma membrane complexes. Piezo1-Panx1-P2X4R ATP release pathway could be reconstituted in HEK Piezo1 KO cells. Thus, our data suggest that Piezo1 and Panx1 can form a functional signaling complex that controls force-induced ATP secretion in cholangiocytes. These findings may foster the development of novel therapeutic strategies for biliary diseases.

Patch-seq of mouse DRG neurons reveals candidate genes for specific mechanosensory functions.

A variety of mechanosensory neurons are involved in touch, proprioception, and pain. Many molecular components of the mechanotransduction machinery subserving these sensory modalities remain to be discovered. Here, we combine recordings of mechanosensitive (MS) currents in mechanosensory neurons with single-cell RNA sequencing. Transcriptional profiles are mapped onto previously identified sensory neuron types to identify cell-type correlates between datasets. Correlation of current signatures with single-cell transcriptomes provides a one-to-one correspondence between mechanoelectric properties and transcriptomically defined neuronal populations. Moreover, a gene-expression differential comparison provides a set of candidate genes for mechanotransduction complexes. Piezo2 is expectedly found to be enriched in rapidly adapting MS current-expressing neurons, whereas Tmem120a and Tmem150c, thought to mediate slow-type MS currents, are uniformly expressed in all mechanosensory neuron subtypes. Further knockdown experiments disqualify them as mediating MS currents in sensory neurons. This dataset constitutes an open resource to explore further the cell-type-specific determinants of mechanosensory properties.

Unravelling the genetic causes of multiple malformation syndromes: A whole exome sequencing study of the Cypriot population.

Multiple malformation syndromes (MMS) belong to a group of genetic disorders characterised by neurodevelopmental anomalies and congenital malformations. Here we explore for the first time the genetic aetiology of MMS using whole-exome sequencing (WES) in undiagnosed patients from the Greek-Cypriot population after prior extensive diagnostics workup including karyotype and array-CGH. A total of 100 individuals (37 affected), from 32 families were recruited and family-based WES was applied to detect causative single-nucleotide variants (SNVs) and indels. A genetic diagnosis was reported for 16 MMS patients (43.2%), with 10/17 (58.8%) of the findings being novel. All autosomal dominant findings occurred de novo. Functional studies were also performed to elucidate the molecular mechanism relevant to the abnormal phenotypes, in cases where the clinical significance of the findings was unclear. The 17 variants identified in our cohort were located in 14 genes (PCNT, UBE3A, KAT6A, SPR, POMGNT1, PIEZO2, PXDN, KDM6A, PHIP, HECW2, TFAP2A, CNOT3, AGTPBP1 and GAMT). This study has highlighted the efficacy of WES through the high detection rate (43.2%) achieved for a challenging category of undiagnosed patients with MMS compared to other conventional diagnostic testing methods (10-20% for array-CGH and ~3% for G-banding karyotype analysis). As a result, family-based WES could potentially be considered as a first-tier cost effective diagnostic test for patients with MMS that facilitates better patient management, prognosis and offer accurate recurrence risks to the families.

Piezo channels.

Parpaite and Coste introduce Piezo channels and their role in mechanotransduction.

Calcium signalling induced by in vitro exposure to silicium dioxide nanoparticles in rat pulmonary artery smooth muscle cells.

The development and use of nanomaterials, especially engineered nanoparticles (NP), is expected to provide many benefits. But at the same time the development of such materials is also feared because of their potential human health risks. Indeed, NP display some characteristics similar to ultrafine environmental particles which are known to exert deleterious cardiovascular effects including pro-hypertensive ones. In this context, the effect of NP on calcium signalling, whose deregulation is often involved in hypertensive diseases, remain poorly described. We thus assessed the effect of SiO2 NP on calcium signalling by fluorescence imaging and on the proliferation response in rat pulmonary artery smooth muscle cells (PASMC). In PASMC, acute exposure to SiO2 NP, from 1 to 500µg/mL, produced an increase of the [Ca2+]i. In addition, when PASMC were exposed to NP at 200µg/mL, a proliferative response was observed. This calcium increase was even greater in PASMC isolated from rats suffering from pulmonary hypertension. The absence of extracellular calcium, addition of diltiazem or nicardipine (L-type voltage-operated calcium channel inhibitors both used at 10µM), and addition of capsazepine or HC067047 (TRPV1 and TRPV4 inhibitors used at 10µM and 5µM, respectively) significantly reduced this response. Moreover, this response was also inhibited by thapsigargin (SERCA inhibitor, 1µM), ryanodine (100µM) and dantrolene (ryanodine receptor antagonists, 10µM) but not by xestospongin C (IP3 receptor antagonist, 10µM). Thus, NP induce an intracellular calcium rise in rat PASMC originating from both extracellular and intracellular calcium sources. This study also provides evidence for the implication of TRPV channels in NP induced calcium rise that may highlight the role of these channels in the deleterious cardiovascular effects of NP.

A molecular signature in the pannexin1 intracellular loop confers channel activation by the α1 adrenoreceptor in smooth muscle cells.

Both purinergic signaling through nucleotides such as ATP (adenosine 5'-triphosphate) and noradrenergic signaling through molecules such as norepinephrine regulate vascular tone and blood pressure. Pannexin1 (Panx1), which forms large-pore, ATP-releasing channels, is present in vascular smooth muscle cells in peripheral blood vessels and participates in noradrenergic responses. Using pharmacological approaches and mice conditionally lacking Panx1 in smooth muscle cells, we found that Panx1 contributed to vasoconstriction mediated by the α1 adrenoreceptor (α1AR), whereas vasoconstriction in response to serotonin or endothelin-1 was independent of Panx1. Analysis of the Panx1-deficient mice showed that Panx1 contributed to blood pressure regulation especially during the night cycle when sympathetic nervous activity is highest. Using mimetic peptides and site-directed mutagenesis, we identified a specific amino acid sequence in the Panx1 intracellular loop that is essential for activation by α1AR signaling. Collectively, these data describe a specific link between noradrenergic and purinergic signaling in blood pressure homeostasis.