Guanabenz, an alpha2-selective adrenergic agonist, activates Ca2+-dependent chloride currents in cystic fibrosis human airway epithelial cells. Norez Caroline,Vandebrouck Clarisse,Antigny Fabrice,Dannhoffer Luc,Blondel Marc,Becq Frédéric European journal of pharmacology In cystic fibrosis respiratory epithelial cells, the absence or dysfunction of the chloride channel CFTR (Cystic Fibrosis Transmembrane conductance Regulator) results in reduced chloride ion transport. In contrast, Ca2+-stimulated Cl- secretion is intact in cystic fibrosis airway epithelia. One possible target for drug discovery aiming at treating cystic fibrosis is to correct the ionic imbalance through stimulation of alternative ionic pathways that may compensate the failure of epithelial Cl- conductance. Here, using a simple high-throughput screening assay to search for Cl- channels modulators in the cystic fibrosis nasal epithelial cell line JME-CF15, the compound guanabenz (Wytensin), an alpha2-selective adrenergic agonist was found positive. Using iodide effluxes and electrophysiological recordings, we showed that guanabenz-activated (EC50=831 nM) a DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) sensitive and Ca2+ dependent Cl- channel (CaCC). Guanabenz activated a linear Cl- channel with unitary single-channel conductance of 8 pS. Recording calcium signals in CF15 cells showed that guanabenz increased the intracellular Ca2+ concentration stimulating an influx of Ca2+. In the absence of extracellular Ca2+, the guanabenz effects on Ca2+ influx and activation of CaCC were both abolished. These data demonstrate that guanabenz activates Ca2+-dependent Cl- channels via a Ca2+ influx in human cystic fibrosis airway epithelial cells. 10.1016/j.ejphar.2008.06.103
    Increased passive stiffness promotes diastolic dysfunction despite improved Ca2+ handling during left ventricular concentric hypertrophy. Røe Åsmund T,Aronsen Jan Magnus,Skårdal Kristine,Hamdani Nazha,Linke Wolfgang A,Danielsen Håvard E,Sejersted Ole M,Sjaastad Ivar,Louch William E Cardiovascular research Aims:Concentric hypertrophy following pressure-overload is linked to preserved systolic function but impaired diastolic function, and is an important substrate for heart failure with preserved ejection fraction. While increased passive stiffness of the myocardium is a suggested mechanism underlying diastolic dysfunction in these hearts, the contribution of active diastolic Ca2+ cycling in cardiomyocytes remains unclear. In this study, we sought to dissect contributions of passive and active mechanisms to diastolic dysfunction in the concentrically hypertrophied heart following pressure-overload. Methods and results:Rats were subjected to aortic banding (AB), and experiments were performed 6 weeks after surgery using sham-operated rats as controls. In vivo ejection fraction and fractional shortening were normal, confirming preservation of systolic function. Left ventricular concentric hypertrophy and diastolic dysfunction following AB were indicated by thickening of the ventricular wall, reduced peak early diastolic tissue velocity, and higher E/e' values. Slowed relaxation was also observed in left ventricular muscle strips isolated from AB hearts, during both isometric and isotonic stimulation, and accompanied by increases in passive tension, viscosity, and extracellular collagen. An altered titin phosphorylation profile was observed with hypophosphorylation of the phosphosites S4080 and S3991 sites within the N2Bus, and S12884 within the PEVK region. Increased titin-based stiffness was confirmed by salt-extraction experiments. In contrast, isolated, unloaded cardiomyocytes exhibited accelerated relaxation in AB compared to sham, and less contracture at high pacing frequencies. Parallel enhancement of diastolic Ca2+ handling was observed, with augmented NCX and SERCA2 activity and lowered resting cytosolic [Ca2+]. Conclusion:In the hypertrophied heart with preserved systolic function, in vivo diastolic dysfunction develops as cardiac fibrosis and alterations in titin phosphorylation compromise left ventricular compliance, and despite compensatory changes in cardiomyocyte Ca2+ homeostasis. 10.1093/cvr/cvx087
    Mitochondrial Ca laMety directs fibrosis. Doynova Malina,Chatzieleftheriadis Konstantinos,Roderick H Llewelyn Cell calcium During development, disease or in response to changes in local environmental and/or nutrient supply, cellular metabolism is substantially remodeled. Reduced mitochondrial Ca uptake was recently reported to induce metabolic remodeling, which through stimulating alterations in the epigenome causes changes in gene expression associated with fibroblast to myofibroblast differentiation. 10.1016/j.ceca.2019.102155
    Mitochondrial Ca2+-dependent NLRP3 activation exacerbates the Pseudomonas aeruginosa-driven inflammatory response in cystic fibrosis. Rimessi Alessandro,Bezzerri Valentino,Patergnani Simone,Marchi Saverio,Cabrini Giulio,Pinton Paolo Nature communications The common pathological manifestation of cystic fibrosis (CF) is associated with an excessive lung inflammatory response characterized by interleukin-1β accumulation. CF airway epithelial cells show an exacerbated pro-inflammatory response to Pseudomonas aeruginosa; however, it is unclear whether this heightened inflammatory response is intrinsic to cells lacking CF transmembrane conductance regulator (CFTR). Here we demonstrate that the degree and quality of the inflammatory response in CF are supported by P. aeruginosa-dependent mitochondrial perturbation, in which flagellin is the inducer and mitochondrial Ca(2+) uniporter (MCU) is a signal-integrating organelle member for NLRP3 activation and IL-1β and IL-18 processing. Our work elucidates the regulation of the NLRP3 inflammasome by mitochondrial Ca(2+) in the P. aeruginosa-dependent inflammatory response and deepens our understanding of the significance of mitochondria in the Ca(2+)-dependent control of inflammation. 10.1038/ncomms7201
    CaMKII inhibition in type II pneumocytes protects from bleomycin-induced pulmonary fibrosis by preventing Ca2+-dependent apoptosis. Winters Christopher J,Koval Olha,Murthy Shubha,Allamargot Chantal,Sebag Sara C,Paschke John D,Jaffer Omar A,Carter A Brent,Grumbach Isabella M American journal of physiology. Lung cellular and molecular physiology The calcium and calmodulin-dependent kinase II (CaMKII) translates increases in intracellular Ca(2+) into downstream signaling events. Its function in pulmonary pathologies remains largely unknown. CaMKII is a well-known mediator of apoptosis and regulator of endoplasmic reticulum (ER) Ca(2+). ER stress and apoptosis of type II pneumocytes lead to aberrant tissue repair and progressive collagen deposition in pulmonary fibrosis. Thus we hypothesized that CaMKII inhibition alleviates fibrosis in response to bleomycin by attenuating apoptosis and ER stress of type II pneumocytes. We first established that CaMKII was strongly expressed in the distal respiratory epithelium, in particular in surfactant protein-C-positive type II pneumocytes, and activated after bleomycin instillation. We generated a novel transgenic model of inducible expression of the CaMKII inhibitor peptide AC3-I limited to type II pneumocytes (Tg SPC-AC3-I). Tg SPC-AC3-I mice were protected from development of pulmonary fibrosis after bleomycin exposure compared with wild-type mice. CaMKII inhibition also provided protection from apoptosis in type II pneumocytes in vitro and in vivo. Moreover, intracellular Ca(2+) levels and ER stress were increased by bleomycin and significantly blunted with CaMKII inhibition in vitro. These data demonstrate that CaMKII inhibition prevents type II pneumocyte apoptosis and development of pulmonary fibrosis in response to bleomycin. CaMKII inhibition may therefore be a promising approach to prevent or ameliorate the progression of pulmonary fibrosis. 10.1152/ajplung.00132.2015
    NAKα2 inhibits fibrosis formation and protects against cardiomyocyte hypertrophy by suppressing hypertrophy associated molecules and activating LTCC/NCX signaling pathway. Song S-S,Tang G,Tang L-X,Si L-Y,Xiong W European review for medical and pharmacological sciences OBJECTIVE:Cardiomyocyte hypertrophy is considered to be a compensatory process of heart suffering from pathological damages. This study aimed to evaluate effects of Na+/K+ APTaseα2 (NAKα2) on isoprenaline (ISO) induced cardiomyocyte hypertrophy. MATERIALS AND METHODS:Mouse atrial cardiomyocytes were cultured and treated with ISO to establish cardiomyocyte hypertrophy model. NAKα2 over-expression and small interfere RNA (siRNA) plasmids were constructed and transfected to cardiomyocytes. Influx Ca2+ ([Ca2+]i) was measured using flow cytometry method. Fibrosis formation was examined with Masson staining. Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining was used to examine apoptosis. Major histocompatibility complex β (β-MHC), atrial natriuretic peptides (ANP), B-type natriuretic peptides (BNP) were evaluated with quantitative Real-time PCR (qRT-PCR). Western blot was used to detect β-MHC, ANP, BNP, Na+/Ca2+ exchanger (NCX) and L-type calcium channel (LTCC). RESULTS:NAKα2 significantly inhibited NCX and LTCC expression compared to that in ISO-treated cardiomyocytes (p<0.05). NAKα2 significantly suppressed expression of β-MHC, ANP and BNP compared to that in ISO-treated cardiomyocytes (p<0.05). NAKα2 significantly alleviated fibrosis formation and inhibited apoptosis compared to that in ISO-treated cardiomyocytes (p<0.05). NAKα2 reduced intracellular calcineurin and activated phosphorylation of calcineurin-nuclear factor of activated T cells (NFAT) compared to ISO-treated cardiomyocytes (p<0.05). NAKα2 significantly strengthened effects of Klotho on ISO-induced up-regulation of hypertrophy associated molecules (p<0.05) by activating LTCC and NCX. Comparing to ISO-treated cardiomyocytes, NAKα2 combining Klotho treatment exhibited significantly better improvement of Ca2+ influx, alleviation of fibrosis and reduction of apoptosis by triggering LTCC/NCX signaling pathway. CONCLUSIONS:Over-expression of NKAα2 suppressed fibrosis formation and protected against cardiomyocyte hypertrophy by inhibiting hypertrophy associated molecules, alleviating apoptosis and activating LTCC/NCX signaling pathway. 10.26355/eurrev_201904_17584
    Evidence of a Role for Fibroblast Transient Receptor Potential Canonical 3 Ca2+ Channel in Renal Fibrosis. Saliba Youakim,Karam Ralph,Smayra Viviane,Aftimos Georges,Abramowitz Joel,Birnbaumer Lutz,Farès Nassim Journal of the American Society of Nephrology : JASN Transient receptor potential canonical (TRPC) Ca(2+)-permeant channels, especially TRPC3, are increasingly implicated in cardiorenal diseases. We studied the possible role of fibroblast TRPC3 in the development of renal fibrosis. In vitro, a macromolecular complex formed by TRPC1/TRPC3/TRPC6 existed in isolated cultured rat renal fibroblasts. However, specific blockade of TRPC3 with the pharmacologic inhibitor pyr3 was sufficient to inhibit both angiotensin II- and 1-oleoyl-2-acetyl-sn-glycerol-induced Ca(2+) entry in these cells, which was detected by fura-2 Ca(2+) imaging. TRPC3 blockade or Ca(2+) removal inhibited fibroblast proliferation and myofibroblast differentiation by suppressing the phosphorylation of extracellular signal-regulated kinase (ERK1/2). In addition, pyr3 inhibited fibrosis and inflammation-associated markers in a noncytotoxic manner. Furthermore, TRPC3 knockdown by siRNA confirmed these pharmacologic findings. In adult male Wistar rats or wild-type mice subjected to unilateral ureteral obstruction, TRPC3 expression increased in the fibroblasts of obstructed kidneys and was associated with increased Ca(2+) entry, ERK1/2 phosphorylation, and fibroblast proliferation. Both TRPC3 blockade in rats and TRPC3 knockout in mice inhibited ERK1/2 phosphorylation and fibroblast activation as well as myofibroblast differentiation and extracellular matrix remodeling in obstructed kidneys, thus ameliorating tubulointerstitial damage and renal fibrosis. In conclusion, TRPC3 channels are present in renal fibroblasts and control fibroblast proliferation, differentiation, and activation through Ca(2+)-mediated ERK signaling. TRPC3 channels might constitute important therapeutic targets for improving renal remodeling in kidney disease. 10.1681/ASN.2014010065
    Blockade of Orai1 Store-Operated Calcium Entry Protects against Renal Fibrosis. Mai Xiaoyi,Shang Jinyan,Liang Sijia,Yu Beixin,Yuan Jiani,Lin Yu,Luo Renfei,Zhang Feiran,Liu Yingying,Lv Xiaofei,Li Chunling,Liang Xinling,Wang Weidong,Zhou Jiaguo Journal of the American Society of Nephrology : JASN Evidence supports an important role of Ca release-activated Ca channel protein 1 (Orai1)-mediated Ca entry in the development of renal fibrosis, a common pathologic feature of CKDs that lead to ESRD, but the molecular mechanisms remain unclear. We determined the role of Orai1 calcium channel in renal fibrosis induced by high-fat diet and by unilateral ureteral obstruction. Mouse kidneys with fibrosis had higher levels of Orai1 protein expression than did kidneys without fibrosis. In vivo knockdown of Orai1 with adenovirus harboring Orai1-short hairpin RNA or inhibition of Orai1 with SKF96365 dramatically prevented renal fibrosis and significantly decreased protein expression of fibronectin, α‑smooth muscle actin, and TGF‑β1 in the kidney cortex of ApoE mice on a high-fat diet and in the obstructed kidneys of mice with unilateral ureteral obstruction. Compared with kidney biopsy specimens of patients with glomerular minimal change disease, those of patients with fibrotic nephropathy had higher expression levels of Orai1. In cultured human proximal tubule epithelial cells (HK2), knockdown of Orai1 Ca channel with adenovirus-Orai1-short hairpin RNA markedly inhibited TGF-β1-induced intracellular Ca influx and phosphorylation of smad2/3. Knockdown or blockade of the Orai1 Ca channel in HK2 cells also prevented epithelial-to-mesenchymal transition induced by TGF‑β1. In conclusion, blockade of the Orai1 Ca channel prevented progression of renal fibrosis in mice, likely by suppressing smad2/3 phosphorylation and TGF-β1-induced epithelial-to-mesenchymal transition. These results render the Orai1 Ca channel a potential therapeutic target against renal fibrosis. 10.1681/ASN.2015080889
    KCa3.1 ion channel: A novel therapeutic target for corneal fibrosis. Anumanthan Govindaraj,Gupta Suneel,Fink Michael K,Hesemann Nathan P,Bowles Douglas K,McDaniel Lindsey M,Muhammad Maaz,Mohan Rajiv R PloS one Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and α-smooth muscle actin (α-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGFβ-mediated transcription of pro-fibrotic collagen I mRNA and α-SMA mRNA and protein expression (p<0.001). No evidence of cytotoxicity was observed. Our study suggests that KCa3.1 regulates corneal wound healing and that blockade of KCa3.1 by TRAM-34 offers a potential therapeutic strategy for developing therapies to cure corneal fibrosis in vivo. 10.1371/journal.pone.0192145
    Ion channels as targets to treat cystic fibrosis lung disease. Martin S Lorraine,Saint-Criq Vinciane,Hwang Tzyh-Chang,Csanády László Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society Lung health relies on effective mucociliary clearance and innate immune defence mechanisms. In cystic fibrosis (CF), an imbalance in ion transport due to an absence of chloride ion secretion, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) and a concomitant sodium hyperabsorption, caused by dyregulation of the epithelial sodium channel (ENaC), results in mucus stasis which predisposes the lungs to cycles of chronic infection and inflammation leading to lung function decline. An increased understanding of CFTR structure and function has provided opportunity for the development of a number of novel modulators targeting mutant CFTR however, it is important to also consider other ion channels and transporters present in the airways as putative targets for drug development. In this review, we discuss recent advances in CFTR biology which will contribute to further drug discovery in the field. We also examine developments to inhibit the epithelial sodium channel (ENaC) and potentially activate alternative chloride channels and transporters as a multi-tracked strategy to hydrate CF airways and restore normal mucociliary clearance mechanisms in a manner independent of CFTR mutation. 10.1016/j.jcf.2017.10.006