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Morphological characterization of cardiomyocyte apoptosis in rat tissues. (a) H&E staining in rat tissues (400 ×). (b) Ultrastructural changes of cardiomyocyte apoptosis. (A) Control, (B) CsA, (C) CsA + GdCl 3 , and (D) GdCl 3. Magnification is 10,000 × (A,D), 5,000 × (B), and 8,000 × (C). (c) Representative illustration of TUNEL staining. Nuclei with brown staining indicate TUNEL-positive cells (400 ×). (d) Quantitative results of the TUNEL staining for each group. Values represent the group means ± SEM. * p < 0.05 vs. control, ** p < 0.05 vs. CsA
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In this study, we sought to determine whether the calcium-sensing receptor (CaSR) is involved in Cyclosporin A (CsA)-induced cardiomyocyte apoptosis and identify its signal transduction pathway. Forty Wistar rats were randomly divided into four groups: the control group, the CsA group (CsA 15 mg/kg/day intraperitoneally, i.p.), the GdCl3 group (GdC...
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... microscopic examination revealed the effect of CsA and GdCl 3 on myocardial cells in rats (Fig. 1a). No significant changes were observed in the control group. In the CsA group, myocardial cells shrank and were arranged in a disordered man- ner, with increased amounts of collagen fibers between the cells. In the CsA + GdCl 3 group, myocardial cells shrank and were arranged in a disordered manner. Fibrosis emerged in the myocardial ...
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... ultrastructural changes of cardiomyocyte apoptosis were observed by transmission electron microscopy (Fig. 1b). There were no significant ultrastructural changes in the control groups. In the CsA group, many apoptotic bodies formed surrounding the myocardial cells, the density of the mitochondrial matrix increased, and some of the myofilaments were destroyed. In the CsA + GdCl 3 groups, multiple apoptotic bodies formed surrounding the ...
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... cardiomyocyte apoptosis. We detected a low level of TUNEL- positive cells in the control group (8.19 ± 1.0 %). The number of apoptotic cells in the CsA group was 27.5 ± 1.3 % (p < 0.05 vs. control), 38.4 ± 2.1 % in the CsA + GdCl 3 group (p < 0.05 vs. control, p < 0.05 vs. CsA), and 16.5 ± 0.7 % in the GdCl 3 group (p < 0.05 vs. control) (Fig. 1c, d). These results demonstrated that CsA could induce cardiomyocyte apoptosis in rats and that GdCl 3 further enhanced cyclosporine A-induced cardiomyocyte ...
Citations
... Some studies have indicated that CsA can ameliorate heart failure (Sharov et al., 2007) and cardiac hypertrophy (Schreiner et al., 2004) and protect the heart from ischemia-reperfusion injury (Bes et al., 2005). However, our previous report and other studies have found that CsA results in myocardial structural damage and increased cardiomyocyte apoptosis Zhao et al., 2011) and myocardial fibrosis in rats (Rezzani et al., 2003). Therefore, CsA-induced cardiotoxicity cannot be ignored. ...
... Selcoki et al. (2007) showed that CsA increased infiltrated inflammatory cells, disorganization of the myocardial fibers, and myocardial fibrosis in rats. Our previous studies found that CsA induced cardiomyocyte apoptosis in vivo and in vitro Zhao et al., 2011). CsA-induced cardiotoxicity cannot be neglected. ...
... Several experimental studies have shown that CsA-mediated cardiotoxicity may be due to mitochondrial damage (Jurado et al., 1998), oxidative stress (Rezzani, 2006;Sagiroglu et al., 2014), intracellular calcium overload (Florio et al., 2003;Tang et al., 2011;Zhao et al., 2011), and cell apoptosis (Rezzani et al., 2009;Tang et al., 2011). The molecular mechanism of CsA-induced cardiotoxicity still needs to be fully investigated. ...
Cyclosporin A (CsA) is a well-known and effective drug that is commonly used in autoimmune diseases and allotransplantation. However, kidney toxicity and cardiotoxicity limit its use. Circular RNAs (circRNAs) play a crucial role in disease, especially cardiovascular disease. We aimed to explore the circRNA expression profiles and potential mechanisms during CsA-induced cardiotoxicity. Sixty male adult Wistar rats were randomly divided into two groups. The CsA group was injected with CsA (15 mg/kg/day body weight) intraperitoneally (ip) for 2 weeks, whereas the control group was injected ip with the same volume of olive oil. We assessed CsA-induced cardiotoxicity by light microscopy, transferase-mediated dUTP nick-end labeling (TUNEL) staining, and electron microscopy. Microarray analysis was used to detect the expression profiles of circRNAs deregulated in the heart during CsA-induced cardiotoxicity. We confirmed the changes in circRNAs by quantitative PCR. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the microarray data were performed. A conventional dose of CsA induced cardiotoxicity in rats. We identified 67 upregulated and 37 downregulated circRNAs compared with those in the control group. Six of 12 circRNAs were successfully verified by quantitative real-time polymerase chain reaction (qRT-PCR). GO analyses of the differentially expressed circRNAs indicated that these molecules might play important roles in CsA-induced cardiotoxicity. KEGG pathway analyses showed that the differentially expressed circRNAs in CsA-induced cardiotoxicity may be related to autophagy or the Hippo signaling pathway. We identified differential circRNA expression patterns and provided more insight into the mechanism of CsA-induced cardiotoxicity. CircRNAs may serve as potential biomarkers or therapeutic targets of CsA-mediated cardiotoxicity in the future.
... Ciclosporin A (CsA) was described to induce cell apoptosis and to increase CaSR expression both in NRVMs [97], Wistar rats [98] and H9c2 cardiomyoblasts [99]. Gd 3+ was found to exacerbate CsA-induced effects, while NPS2390 appeared somehow protective. ...
The Calcium Sensing Receptor (CaSR), a G-protein-coupled receptor mainly known for its role in the homeostatic regulation of Ca²⁺ levels in the extracellular fluid, is also expressed in a multiplicity of tissues where it regulates a variety of physiological and pathological processes.
The main features of CaSR are its capacity to activate multiple downstream signaling pathways and its ability to itself be activated by a variety of ligands.
Recent data have demonstrated that these features are actually connected by the concept of biased signaling.
The recent availability of crystal structures of CaSR extracellular domain, and the functional characterization of clinically-relevant mutations, have catalyzed a great step forward in the field of CaSR signaling.
In the past two years, CaSR signaling characteristics have been shown to be even more complicated than expected: heterodimerization, phosphate sensing, and compartment bias are only a fraction of the exciting developments.
This review will focus on some of these topics, and on the debated case of CaSR signaling in cardiomyocytes.
... The effects of known proapoptotic substances were also investigated. Ciclosporin A (CsA) was described to exert its adverse effects (also) by increasing CaR expression both in vitro [207] and in vivo [208]. ...
The current interest of the scientific community for research in the field of calcium sensing in general and on the calcium-sensing Receptor (CaR) in particular is demonstrated by the still increasing number of papers published on this topic. The extracellular calcium-sensing receptor is the best-known G-protein-coupled receptor (GPCR) able to sense external Ca2+changes. Widely recognized as a fundamental player in systemic Ca2+homeostasis, the CaR is ubiquitously expressed in the human body where it activates multiple signalling pathways. In this review, old and new notions regarding the mechanisms by which extracellular Ca2+microdomains are created and the tools available to measure them are analyzed. After a survey of the main signalling pathways triggered by the CaR, a special attention is reserved for the emerging concepts regarding CaR function in the heart, CaR trafficking and pharmacology. Finally, an overview on other Ca2+sensors is provided.
Ischemia-reperfusion (I/R) injury is a multifactorial process triggered when an organ is subjected to transiently reduced blood supply. The result is a cascade of pathological complications and organ damage due to the production of reactive oxygen species following reperfusion. The present study aims to evaluate the role of activated calcium-sensing receptor (CaR)-cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway in I/R injury. Firstly, an I/R rat model with CSE knockout was constructed. Transthoracic echocardiography, TTC and HE staining were performed to determine the cardiac function of rats following I/R Injury, followed by TUNEL staining observation on apoptosis. Besides, with the attempt to better elucidate how CaR-CSE/H2S affects I/R, in-vitro culture of human coronary artery endothelial cells (HCAECs) was conducted with gadolinium chloride (GdCl3, a CaR agonist), H2O2, siRNA against CSE (siCSE), or W7 (a CaM inhibitor). The interaction between CSE and CaM was subsequently detected. Plasma oxidative stress indexes, H2S and CSE, and apoptosis-related proteins were all analyzed following cell apoptosis. We found that H2S elevation led to the improvement whereas CSE knockdown decreased cardiac function in rats with I/R injury. Moreover, oxidative stress injury in I/R rats with CSE knockout was aggravated, while the increased expression of H2S and CSE in the aortic tissues resulted in alleviated the oxidative stress injury. Moreover, increased H2S and CSE levels were found to inhibit cell apoptotic ability in the aortic tissues after I/R injury, thus attenuating oxidative stress injury, accompanied by inhibited expression of apoptosis-related proteins. In HCAECs following oxidative stress treatment, siCSE and CaM inhibitor were observed to reverse the protection of CaR agonist. Coimmunoprecipitation assay revealed the interaction between CSE and CaM. Taken together, all above-mentioned data provides evidence that activation of the CaR-CSE/H2S pathway may confer a potent protective effect in cardiac I/R injury.
Activation of calcium sensing receptor (CaSR) contributes to cardiac injury, but the underlying mechanism has not yet been examined. Astragaloside IV (AsIV) was previously reported to exhibit protective effects against various myocardial injuries. The aim of the present study was to investigate the underlying mechanism of CaSR in cardiac hypertrophy and apoptosis and to evaluate whether the protective effect of AsIV against myocardial injury is associated with CaSR and its related signaling pathway. In vivo and in vitro myocardial injury was induced by isoproterenol (Iso) or GdCl3 (a CaSR agonist) in rats and heart H9C2 cells. Cardiac cell hypertrophy, apoptosis, function, Mitochondrial Membrane Potential (MMP), mitochondrial ultrastructure, and [Ca²⁺]i, as well as the protein expression of CaSR, calcium/calmodulin-dependent protein kinase II (CaMKII), calcineurin (CaN), sarcoplasmic reticulum Ca²⁺-ATPase2a (SERCA2a), and the inositol 1,4,5-trisphosphate receptor (IP3R), were measured in vivo and/or in vitro. The results showed that AsIV attenuated cardiac hypertrophy and apoptosis and attenuated impairments in cardiac function, mitochondrial structure, and MMP induced by Iso or GdCl3 in rat myocardial tissue and H9C2 cells. Importantly, AsIV treatment inhibited the enhancement of [Ca²⁺]i and CaSR expression induced by Iso or GdCl3, an effect similar to that of the CaSR antagonist NPS2143. In addition, AsIV treatment repressed CaSR, CaMKII, and CaN activation and inhibited NFAT-3 nuclear translocation. Mechanistic analysis using lentivirus infection showed that CaSR overexpression activated the CaMKII and CaN signaling pathways and that this response was enhanced by Iso. The results suggested that CaSR-mediated changes in [Ca²⁺]i and CaMKII and CaN signaling pathways contribute to cardiac hypertrophy and apoptosis and are involved in the protective effect of astragaloside IV against cardiac injury.
Cardiac cell replacement therapy by using human embryonic stem cell derivatives remains a potential approach to regenerate myocardium. The major hurdles to clinical application of this technology are immunogenicity and post-transplantation cell death. Here we examined the effects of calcineurin-targeting immunosuppressants cyclosporine A (CsA), and FK506, as well as rapamycin (RAP) and a selective inhibitor of calcineurin-binding downstream NFAT transcription factor VIVIT on the proliferative activity, function and survival of hESC-derived cardiomyocytes (hESC-CM) and endothelial cells (hESC-EC) in culture. As shown by automated microscopy, treatments with CsA, FK506 and RAP all decreased proliferation, reducing the percentage of hESC-CM and hESC-EC cells with the mitotic marker Ki67+ by as much as 60% and 74%, respectively. Administration of the cell permeable analogue 11R-VIVIT protein did not modulate their proliferative activity. All immunosuppressants reversed the pro-apoptotic effect of chelerythrine in hESC-CM demonstrating an inhibitory role of calcineurin/NFAT and mTOR pathways in hESC-CM survival (using apoptotic marker caspase-3) whereas the protection was less obvious in hESC-EC exposed to H2O2. Immunosuppressants did not affect cell viability in hESC-EC. Our results show that immunosuppressants reduce proliferation while offsetting cell loss to a smaller extent by reduction in apoptosis of hESC-CM. Immunosuppressants therapy would be compatible with stem cell transplantation but the resulting reduction in graft expansion capabilities would potentially necessitate implantation of increased cell numbers when immunosuppressants are given. The effects of NFAT-binding immunosuppressant molecules, which do not affect hESC-CM proliferation, may point the way forward for new classes of compounds better suited to cell implantation.
Cyclosporine (CsA) has become a mainstay for immune suppression of organ transplants. It is known that patients receiving CsA manifest increased growth of aggressive cardiotoxicity. We have demonstrated that CsA induces myocardium cell apoptosis in vivo and vitro. Recently, dishevelled-1 (Dvl-1) protein, which is a cytoplasmic mediator of Wnt/β-catenin signaling, was explored in cardiac diseases. However, whether Dvl-1 is involved in CsA-induced apoptosis remains to be determined. The aim of this study was to explore the role of Dvl-1 in CsA-induced apoptosis in H9c2 cardiomyoblast cells and to investigate the role of the Wnt/β-catenin signaling cascade in this progress. H9c2 cells were treated with CsA in dose and time-dependent manners. We found that the appropriate concentrations and time-points of CsA-induced the expression of Dvl-1 and subsequent up-regulation of β-catenin and c-Myc, which is consistent with previously demonstrated concentrations and time-points when H9c2 cells apoptosis occurred. Then, cells were transfected with small interfering RNA (siRNA) against Dvl-1 and stimulated with previously demonstrated concentration of CsA. Dvl-1 down-regulation decreased the apoptotic rate, caspase-3 activity, and the Bax/Bcl-2 ratio in H9c2 cells treated with CsA. Furthermore, knocking down the expression of Dvl-1 partially suppressed the activity of the Wnt/β-catenin pathway. Moreover, we further deleted the downstream member β-catenin by specific siRNA, and found that CsA-induced the Bax/Bcl-2 ratio and the expression of c-Myc, which were attenuated. Our results are the first to unveil this novel aspect of Dvl-1 signaling. In addition, these data provide insight into the pathogenesis and the therapeutic strategies of CsA-induced myocardial injury.
