Inhibition of adenylyl cyclase 8 prevents the upregulation of Orai1 channel, which improves cardiac function after myocardial infarction.

The upregulation of Orai1 and subsequent store-operated Ca2+ entry (SOCE) has been associated with adverse cardiac remodeling and heart failure (HF). However, the mechanism underlying Orai1 upregulation and its role in myocardial infarction remains unclear. Our study investigated the role of Orai1 in activating adenylyl cyclase 8 (AC8) and cyclic AMP (cAMP) response element-binding protein (CREB), as well as its contribution to cardiac dysfunction induced by ischemia and reperfusion (I/R). We found that I/R evoked an increase in the expression of Orai1 and AC8 in rats' hearts, resulting in a substantial rise in diastolic Ca2+ concentration ([Ca2+]i), and reduced ventricular contractions. The expression of Orai1 and AC8 was also increased in ventricular biopsies of post-ischemic HF patients. Mechanistically, we demonstrate that I/R activation of Orai1 stimulated AC8, which produced cAMP and phosphorylated CREB. Subsequently, p-CREB activated the ORAI1 promoter, resulting in Orai1 upregulation and SOCE exacerbation. Intramyocardial administration of AAV9 carrying AC8 short hairpin RNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i and improved cardiac contraction. Therefore, our data suggests that the axis composed by Orai1/AC8/CREB plays a critical role in I/R-induced cardiac dysfunction, representing a potential new therapeutic target to limit the progression of the disease toward HF.

Orai1α and Orai1ß support calcium entry and mammosphere formation in breast cancer stem cells.

Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1ß, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1ß, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1ß support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1ß in spheroid forming efficiency and self-renewal in breast cancer stem cells.

The Ca2+ Sensor STIM in Human Diseases.

The STIM family of proteins plays a crucial role in a plethora of cellular functions through the regulation of store-operated Ca2+ entry (SOCE) and, thus, intracellular calcium homeostasis. The two members of the mammalian STIM family, STIM1 and STIM2, are transmembrane proteins that act as Ca2+ sensors in the endoplasmic reticulum (ER) and, upon Ca2+ store discharge, interact with and activate the Orai/CRACs in the plasma membrane. Dysregulation of Ca2+ signaling leads to the pathogenesis of a variety of human diseases, including neurodegenerative disorders, cardiovascular diseases, cancer, and immune disorders. Therefore, understanding the mechanisms underlying Ca2+ signaling pathways is crucial for developing therapeutic strategies targeting these diseases. This review focuses on several rare conditions associated with STIM1 mutations that lead to either gain- or loss-of-function, characterized by myopathy, hematological and immunological disorders, among others, and due to abnormal activation of CRACs. In addition, we summarize the current evidence concerning STIM2 allele duplication and deletion associated with language, intellectual, and developmental delay, recurrent pulmonary infections, microcephaly, facial dimorphism, limb anomalies, hypogonadism, and congenital heart defects.

New Insights into the Reparative Angiogenesis after Myocardial Infarction.

Myocardial infarction (MI) causes massive loss of cardiac myocytes and injury to the coronary microcirculation, overwhelming the limited capacity of cardiac regeneration. Cardiac repair after MI is finely organized by complex series of procedures involving a robust angiogenic response that begins in the peri-infarcted border area of the infarcted heart, concluding with fibroblast proliferation and scar formation. Efficient neovascularization after MI limits hypertrophied myocytes and scar extent by the reduction in collagen deposition and sustains the improvement in cardiac function. Compelling evidence from animal models and classical in vitro angiogenic approaches demonstrate that a plethora of well-orchestrated signaling pathways involving Notch, Wnt, PI3K, and the modulation of intracellular Ca2+ concentration through ion channels, regulate angiogenesis from existing endothelial cells (ECs) and endothelial progenitor cells (EPCs) in the infarcted heart. Moreover, cardiac repair after MI involves cell-to-cell communication by paracrine/autocrine signals, mainly through the delivery of extracellular vesicles hosting pro-angiogenic proteins and non-coding RNAs, as microRNAs (miRNAs). This review highlights some general insights into signaling pathways activated under MI, focusing on the role of Ca2+ influx, Notch activated pathway, and miRNAs in EC activation and angiogenesis after MI.

miRNA-132/212 Deficiency Disrupts Selective Corticosterone Modulation of Dorsal vs. Ventral Hippocampal Metaplasticity.

Cortisol is a potent human steroid hormone that plays key roles in the central nervous system, influencing processes such as brain neuronal synaptic plasticity and regulating the expression of emotional and behavioral responses. The relevance of cortisol stands out in the disease, as its dysregulation is associated with debilitating conditions such as Alzheimer's Disease, chronic stress, anxiety and depression. Among other brain regions, cortisol importantly influences the function of the hippocampus, a structure central for memory and emotional information processing. The mechanisms fine-tuning the different synaptic responses of the hippocampus to steroid hormone signaling remain, however, poorly understood. Using ex vivo electrophysiology and wild type (WT) and miR-132/miR-212 microRNAs knockout (miRNA-132/212-/-) mice, we examined the effects of corticosterone (the rodent's equivalent to cortisol in humans) on the synaptic properties of the dorsal and ventral hippocampus. In WT mice, corticosterone predominantly inhibited metaplasticity in the dorsal WT hippocampi, whereas it significantly dysregulated both synaptic transmission and metaplasticity at dorsal and ventral regions of miR-132/212-/- hippocampi. Western blotting further revealed significantly augmented levels of endogenous CREB and a significant CREB reduction in response to corticosterone only in miR-132/212-/- hippocampi. Sirt1 levels were also endogenously enhanced in the miR-132/212-/- hippocampi but unaltered by corticosterone, whereas the levels of phospo-MSK1 were only reduced by corticosterone in WT, not in miR-132/212-/- hippocampi. In behavioral studies using the elevated plus maze, miRNA-132/212-/- mice further showed reduced anxiety-like behavior. These observations propose miRNA-132/212 as potential region-selective regulators of the effects of steroid hormones on hippocampal functions, thus likely fine-tuning hippocampus-dependent memory and emotional processing.

Acute oxygen sensing by vascular smooth muscle cells.

An adequate supply of oxygen (O2) is essential for most life forms on earth, making the delivery of appropriate levels of O2 to tissues a fundamental physiological challenge. When O2 levels in the alveoli and/or blood are low, compensatory adaptive reflexes are produced that increase the uptake of O2 and its distribution to tissues within a few seconds. This paper analyzes the most important acute vasomotor responses to lack of O2 (hypoxia): hypoxic pulmonary vasoconstriction (HPV) and hypoxic vasodilation (HVD). HPV affects distal pulmonary (resistance) arteries, with its homeostatic role being to divert blood to well ventilated alveoli to thereby optimize the ventilation/perfusion ratio. HVD is produced in most systemic arteries, in particular in the skeletal muscle, coronary, and cerebral circulations, to increase blood supply to poorly oxygenated tissues. Although vasomotor responses to hypoxia are modulated by endothelial factors and autonomic innervation, it is well established that arterial smooth muscle cells contain an acute O2 sensing system capable of detecting changes in O2 tension and to signal membrane ion channels, which in turn regulate cytosolic Ca2+ levels and myocyte contraction. Here, we summarize current knowledge on the nature of O2 sensing and signaling systems underlying acute vasomotor responses to hypoxia. We also discuss similarities and differences existing in O2 sensors and effectors in the various arterial territories.

Correction: Sanchez-Collado et al. Orai2 Modulates Store-Operated Ca2+ Entry and Cell Cycle Progression in Breast Cancer Cells. Cancers 2021, 14, 114.

In the original publication [...].

Sulconazole inhibits PD-1 expression in immune cells and cancer cells malignant phenotype through NF-κB and calcium activity repression.

The overexpression of the immunoinhibitory receptor programmed death-1 (PD1) on T-cells is involved in immune evasion in cancer. The use of anti-PD-1/PDL-1 strategy has deeply changed the therapies of cancers and patient survival. However, their efficacy diverges greatly along with tumor type and patient populations. Thereby, novel treatments are needed to interfere with the anti-tumoral immune responses and propose an adjunct therapy. In the current study, we found that the antifungal drug Sulconazole (SCZ) inhibits PD-1 expression on activated PBMCs and T cells at the RNA and protein levels. SCZ repressed NF-κB and calcium signaling, both, involved in the induction of PD-1. Further analysis revealed cancer cells treatment with SCZ inhibited their proliferation, and migration and ability to mediate tumor growth in zebrafish embryos. SCZ found also to inhibit calcium mobilization in cancer cells. These results suggest the SCZ therapeutic potential used alone or as adjunct strategy to prevent T-cell exhaustion and promotes cancer cell malignant phenotype repression in order to improve tumor eradication.

Tissue engineered in-vitro vascular patch fabrication using hybrid 3D printing and electrospinning.

Three-dimensional (3D) engineered cardiovascular tissues have shown great promise to replace damaged structures. Specifically, tissue engineering vascular grafts (TEVG) have the potential to replace biological and synthetic grafts. We aimed to design an in-vitro patient-specific patch based on a hybrid 3D print combined with vascular smooth muscle cells (VSMC) differentiation. Based on the medical images of a 2 months-old girl with aortic arch hypoplasia and using computational modelling, we evaluated the most hemodynamically efficient aortic patch surgical repair. Using the designed 3D patch geometry, the scaffold was printed using a hybrid fused deposition modelling (FDM) and electrospinning techniques. The scaffold was seeded with multipotent mesenchymal stem cells (MSC) for later maturation to derived VSMC (dVSMC). The graft showed adequate resistance to physiological aortic pressure (burst pressure 101 â€‹± â€‹15 â€‹mmHg) and a porosity gradient ranging from 80 to 10 â€‹µm allowing cells to infiltrate through the entire thickness of the patch. The bio-scaffolds showed good cell viability at days 4 and 12 and adequate functional vasoactive response to endothelin-1. In summary, we have shown that our method of generating patient-specific patch shows adequate hemodynamic profile, mechanical properties, dVSMC infiltration, viability and functionality. This innovative 3D biotechnology has the potential for broad application in regenerative medicine and potentially in heart disease prevention.

Predicted Value of MicroRNAs, Vascular Endothelial Growth Factor, and Intermediate Monocytes in the Left Adverse Ventricular Remodeling in Revascularized ST-Segment Elevation Myocardial Infarction Patients.

Background: Primary percutaneous coronary intervention (PPCI) in patients with ST-segment elevation myocardial infarction (STEMI) improves the survival of patients; nevertheless, some patients develop left ventricular adverse remodeling (LVAR) a few months after the intervention. The main objective of this study was to characterize the role of pro-inflammatory cell populations, related cytokines, and microRNAs (miRNAs) released after PPCI as reliable prognostic biomarkers for LVAR in patients with STEMI. Methods: We evaluated the level of pro-inflammatory subsets, before and after revascularization, 1 and 6 months after PPCI, using flow cytometry. We also performed a miRNA microarray in isolated peripheral blood mononuclear cells (PBMCs) and examined the levels of 27 cytokines in patients' serum of patients by multiplex ELISA. Results: We observed that the levels of classical and intermediate monocytes increased 6 h after PPCI in patients who developed LVAR later. Multivariate regression analysis and ROC curves indicated that intermediate monocytes, after PPCI, were the best monocyte subset that correlated with LVAR. Within the 27 evaluated cytokines evaluated, we found that the increase in the level of vascular endothelial growth factor (VEGF) correlated with LVAR. Furthermore, the microarray analysis of PBMCs determined that up to 1,209 miRNAs were differentially expressed 6 h after PPCI in LVAR patients, compared with those who did not develop LVAR. Using RT-qPCR we confirmed a significant increase in miR-16, miR-21-5p, and miR-29a-3p, suggested to modulate the expression of different cytokines, 6 h post-PPCI in LVAR patients. Interestingly, we determined that the combined analysis of the levels of the intermediate monocyte subpopulation, VEGF, and miRNAs gave a better association with LVAR appearance. Similarly, combined ROC analysis provided high accurate specificity and sensibility to identify STEMI patients who will develop LVAR. Conclusion: Our data suggest that the combined analysis of intermediate monocytes, VEGF, and miRNAs predicts LVAR in STEMI patients.

Cardiac protection induced by urocortin-2 enables the regulation of apoptosis and fibrosis after ischemia and reperfusion involving miR-29a modulation.

Urocortin-2 (Ucn-2) has demonstrated cardioprotective actions against myocardial ischemia-reperfusion (I/R) injuries. Herein, we explored the protective role of Ucn-2 through microRNAs (miRNAs) post-transcriptional regulation of apoptotic and pro-fibrotic genes. We determined that the intravenous administration of Ucn-2 before heart reperfusion in a Wistar rat model of I/R recovered cardiac contractility and decreased fibrosis, lactate dehydrogenase release, and apoptosis. The infusion of Ucn-2 also inhibited the upregulation of 6 miRNAs in revascularized heart. The in silico analysis indicated that miR-29a and miR-451_1∗ are predicted to target many apoptotic and fibrotic genes. Accordingly, the transfection of neonatal rat ventricular myocytes with mimics overexpressing miR-29a, but not miR-451_1∗, prevented I/R-induced expression of pro- and anti-apoptotic genes such as Apaf-1, Hmox-1, and Cycs, as well as pro-fibrotic genes Col-I and Col-III. We also confirmed that Hmox-1, target of miR-29a, is highly expressed at the mRNA and protein levels in adult rat heart under I/R, whereas, Ucn-2 abolished I/R-induced mRNA and protein upregulation of HMOX-1. Interestingly, a significant upregulation of Hmox-1 was observed in the ventricle of ischemic patients with heart failure, correlating negatively with the left ventricle ejection fraction. Altogether, these data indicate that Ucn-2, through miR-29a regulation, provides long-lasting cardioprotection, involving the post-transcriptional regulation of apoptotic and fibrotic genes.

Orai1α, but not Orai1ß, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells.

The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1ß, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1ß differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1ß are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α-TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

Store-Operated Calcium Entry in Breast Cancer Cells Is Insensitive to Orai1 and STIM1 N-Linked Glycosylation.

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

Urocortin Role in Ischemia Cardioprotection and the Adverse Cardiac Remodeling.

Despite the considerable progress in strategies of myocardial protection, ischemic heart diseases (IHD) and consequent heart failure (HF) remain the main cause of mortality worldwide. Several procedures are used routinely to guarantee the prompt and successful reestablishment of blood flow to preserve the myocardial viability of infarcted hearts from ischemia injuries. However, ischemic heart reperfusion/revascularization triggers additional damages that occur when oxygen-rich blood re-enters the vulnerable myocardial tissue, which is a phenomenon known as ischemia and reperfusion (I/R) syndrome. Complications of I/R injuries provoke the adverse cardiac remodeling, involving inflammation, mishandling of Ca2+ homeostasis, apoptotic genes activation, cardiac myocytes loss, etc., which often progress toward HF. Therefore, there is an urgent need to develop new cardioprotective therapies for IHD and HF. Compelling evidence from animal studies and pilot clinical trials in HF patients suggest that urocortin (Ucn) isoforms, which are peptides associated with stress and belonging to the corticotropin releasing factor family, have promising potential to improve cardiovascular functions by targeting many signaling pathways at different molecular levels. This review highlights the current knowledge on the role of urocortin isoforms in cardioprotection, focusing on its acute and long-term effects.

SARAF and EFHB Modulate Store-Operated Ca2+ Entry and Are Required for Cell Proliferation, Migration and Viability in Breast Cancer Cells.

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Store-operated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells.

Corrigendum: SARAF and Orai1 Contribute to Endothelial Cell Activation and Angiogenesis.

[This corrects the article DOI: 10.3389/fcell.2021.639952.].

Ca2+ mishandling in heart failure: Potential targets.

Ca2+ mishandling is a common feature in several cardiovascular diseases such as heart failure (HF). In many cases, impairment of key players in intracellular Ca2+ homeostasis has been identified as the underlying mechanism of cardiac dysfunction and cardiac arrhythmias associated with HF. In this review, we summarize primary novel findings related to Ca2+ mishandling in HF progression. HF research has increasingly focused on the identification of new targets and the contribution of their role in Ca2+ handling to the progression of the disease. Recent research studies have identified potential targets in three major emerging areas implicated in regulation of Ca2+ handling: the innate immune system, bone metabolism factors and post-translational modification of key proteins involved in regulation of Ca2+ handling. Here, we describe their possible contributions to the progression of HF.

Furin Prodomain ppFurin Enhances Ca2+ Entry Through Orai and TRPC6 Channels' Activation in Breast Cancer Cells.

The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we showed that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced store-operated calcium entry (SOCE) and reduced the cell malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments revealed that the ppFurin-expressing cells are unable to adequately process the proprotein convertase (PC) substrates vascular endothelial growth factor C (proVEGF-C) and insulin-like growth factor-1 receptor (proIGF-1R). Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 and BT20 cells significantly enhanced SOCE and induced constitutive Ca2+ entry. The enhanced SOCE is impaired by inhibition of Orai channels while the constitutive Ca2+ entry is attenuated by silencing or inhibition of TRPC6 or inhibition of Orai channels. Analysis of TRPC6 activation revealed its upregulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. In addition, while ppFurin had no effect on MDA-MB-435 cell viability, in MDA-MB-231 cells ppFurin expression reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide and similar results were observed in BT20 cells. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca2+ mobilization, leading to breast cancer cells' malignant phenotype repression and reduction of their resistance to treatments.