Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophy.

Mechanical overloading (OVL) resulting from the ablation of muscle agonists, a supra-physiological model of resistance training, reduces skeletal muscle fragility, i.e. the immediate maximal force drop following lengthening contractions, and increases maximal force production, in mdx mice, a murine model of Duchene muscular dystrophy (DMD). Here, we further analyzed these beneficial effects of OVL by determining whether they were blocked by cyclosporin, an inhibitor of the calcineurin pathway, and whether there were also observed in the D2-mdx mice, a more severe murine DMD model. We found that cyclosporin did not block the beneficial effect of 1-month OVL on plantaris muscle fragility in mdx mice, nor did it limit the increases in maximal force and muscle weight (an index of hypertrophy). Fragility and maximal force were also ameliorated by OVL in the plantaris muscle of D2-mdx mice. In addition, OVL increased the expression of utrophin, cytoplamic γ-actin, MyoD, and p-Akt in the D2-mdx mice, proteins playing an important role in fragility, maximal force gain and muscle growth. In conclusion, OVL reduced fragility and increased maximal force in the more frequently used mild mdx model but also in D2-mdx mice, a severe model of DMD, closer to human physiopathology. Moreover, these beneficial effects of OVL did not seem to be related to the activation of the calcineurin pathway. Thus, this preclinical study suggests that resistance training could have a potential benefit in the improvement of the quality of life of DMD patients.

Nsp1 facilitates SARS-CoV-2 replication through calcineurin-NFAT signaling.

SARS-CoV-2, the causative agent of COVID-19, has been intensely studied in search of effective antiviral treatments. The immunosuppressant cyclosporine A (CsA) has been suggested to be a pan-coronavirus inhibitor, yet its underlying mechanism remained largely unknown. Here, we found that non-structural protein 1 (Nsp1) of SARS-CoV-2 usurped CsA-suppressed nuclear factor of activated T cells (NFAT) signaling to drive the expression of cellular DEAD-box helicase 5 (DDX5), which facilitates viral replication. Nsp1 interacted with calcineurin A (CnA) to displace the regulatory protein regulator of calcineurin 3 (RCAN3) of CnA for NFAT activation. The influence of NFAT activation on SARS-CoV-2 replication was also validated by using the Nsp1-deficient mutant virus. Calcineurin inhibitors, such as CsA and VIVIT, inhibited SARS-CoV-2 replication and exhibited synergistic antiviral effects when used in combination with nirmatrelvir. Our study delineated the molecular mechanism of CsA-mediated inhibition of SARS-CoV-2 replication and the anti-SARS-CoV-2 action of calcineurin inhibitors. IMPORTANCE: Cyclosporine A (CsA), commonly used to inhibit immune responses, is also known to have anti-SARS-CoV-2 activity, but its mode of action remains elusive. Here, we provide a model to explain how CsA antagonizes SARS-CoV-2 through three critical proteins: DDX5, NFAT1, and Nsp1. DDX5 is a cellular facilitator of SARS-CoV-2 replication, and NFAT1 controls the production of DDX5. Nsp1 is a viral protein absent from the mature viral particle and capable of activating the function of NFAT1 and DDX5. CsA and similar agents suppress Nsp1, NFAT1, and DDX5 to exert their anti-SARS-CoV-2 activity either alone or in combination with Paxlovid.

TRPV4 Channels Promote Pathological, but Not Physiological, Cardiac Remodeling through the Activation of Calcineurin/NFAT and TRPC6.

TRPV4 channels, which respond to mechanical activation by permeating Ca2+ into the cell, may play a pivotal role in cardiac remodeling during cardiac overload. Our study aimed to investigate TRPV4 involvement in pathological and physiological remodeling through Ca2+-dependent signaling. TRPV4 expression was assessed in heart failure (HF) models, induced by isoproterenol infusion or transverse aortic constriction, and in exercise-induced adaptive remodeling models. The impact of genetic TRPV4 inhibition on HF was studied by echocardiography, histology, gene and protein analysis, arrhythmia inducibility, Ca2+ dynamics, calcineurin (CN) activity, and NFAT nuclear translocation. TRPV4 expression exclusively increased in HF models, strongly correlating with fibrosis. Isoproterenol-administered transgenic TRPV4-/- mice did not exhibit HF features. Cardiac fibroblasts (CFb) from TRPV4+/+ animals, compared to TRPV4-/-, displayed significant TRPV4 overexpression, elevated Ca2+ influx, and enhanced CN/NFATc3 pathway activation. TRPC6 expression paralleled that of TRPV4 in all models, with no increase in TRPV4-/- mice. In cultured CFb, the activation of TRPV4 by GSK1016790A increased TRPC6 expression, which led to enhanced CN/NFATc3 activation through synergistic action of both channels. In conclusion, TRPV4 channels contribute to pathological remodeling by promoting fibrosis and inducing TRPC6 upregulation through the activation of Ca2+-dependent CN/NFATc3 signaling. These results pose TRPV4 as a primary mediator of the pathological response.

Activation of Ca2+ phosphatase Calcineurin regulates Parkin translocation to mitochondria and mitophagy in flies.

Selective removal of dysfunctional mitochondria via autophagy is crucial for the maintenance of cellular homeostasis. This event is initiated by the translocation of the E3 ubiquitin ligase Parkin to damaged mitochondria, and it requires the Serine/Threonine-protein kinase PINK1. In a coordinated set of events, PINK1 operates upstream of Parkin in a linear pathway that leads to the phosphorylation of Parkin, Ubiquitin, and Parkin mitochondrial substrates, to promote ubiquitination of outer mitochondrial membrane proteins. Ubiquitin-decorated mitochondria are selectively recruiting autophagy receptors, which are required to terminate the organelle via autophagy. In this work, we show a previously uncharacterized molecular pathway that correlates the activation of the Ca2+-dependent phosphatase Calcineurin to Parkin translocation and Parkin-dependent mitophagy. Calcineurin downregulation or genetic inhibition prevents Parkin translocation to CCCP-treated mitochondria and impairs stress-induced mitophagy, whereas Calcineurin activation promotes Parkin mitochondrial recruitment and basal mitophagy. Calcineurin interacts with Parkin, and promotes Parkin translocation in the absence of PINK1, but requires PINK1 expression to execute mitophagy in MEF cells. Genetic activation of Calcineurin in vivo boosts basal mitophagy in neurons and corrects locomotor dysfunction and mitochondrial respiratory defects of a Drosophila model of impaired mitochondrial functions. Our study identifies Calcineurin as a novel key player in the regulation of Parkin translocation and mitophagy.

Calcineurin contributes to RNAi-mediated transgene silencing and small interfering RNA production in the human fungal pathogen Cryptococcus neoformans.

Adaptation to external environmental challenges at the cellular level requires rapid responses and involves relay of information to the nucleus to drive key gene expression changes through downstream transcription factors. Here, we describe an alternative route of adaptation through a direct role for cellular signaling components in governing gene expression via RNA interference-mediated small RNA production. Calcium-calcineurin signaling is a highly conserved signaling cascade that plays central roles in stress adaptation and virulence of eukaryotic pathogens, including the human fungal pathogen Cryptococcus neoformans. Upon activation in C. neoformans, calcineurin localizes to P-bodies, membraneless organelles that are also the site for RNA processing. Here, we studied the role of calcineurin and its substrates in RNAi-mediated transgene silencing. Our results reveal that calcineurin regulates both the onset and the reversion of transgene silencing. We found that some calcineurin substrates that localize to P-bodies also regulate transgene silencing but in opposing directions. Small RNA sequencing in mutants lacking calcineurin or its targets revealed a role for calcineurin in small RNA production. Interestingly, the impact of calcineurin and its substrates was found to be different in genome-wide analysis, suggesting that calcineurin may regulate small RNA production in C. neoformans through additional pathways. Overall, these findings define a mechanism by which signaling machinery induced by external stimuli can directly alter gene expression to accelerate adaptative responses and contribute to genome defense.

Novel peptides from sea cucumber intestines hydrolyzed by neutral protease alleviate exercise-induced fatigue via upregulating the glutaminemediated Ca2+ /Calcineurin signaling pathway in mice.

Sea cucumber intestines are considered a valuable resource in the sea cucumber processing industry due to their balanced amino acid composition. Studies have reported that peptides rich in glutamate and branched-chain amino acids have anti-fatigue properties. However, the function of the sea cucumber intestine in reducing exercise-induced fatigue remains unclear. In this study, we enzymatically hydrolyzed low molecular weight peptides from sea cucumber intestines (SCIP) and administered SCIP orally to mice to examine its effects on exercise-induced fatigue using swimming and pole-climbing exhaustion experiments. The results revealed that supplementation with SCIP significantly prolonged the exhaustion time of swimming in mice, decreased blood lactate and urea nitrogen levels, and increased liver and muscle glycogen levels following a weight-loaded swimming test. Immunofluorescence analysis indicated a notable increase the proportion of slow-twitch muscle fiber and a significant decrease the proportion of fast-twitch muscle fiber following SCIP supplementation. Furthermore, SCIP upregulated mRNA expression levels of Ca2+ /Calcineurin upstream and downstream regulators, thereby contributing to the promotion of skeletal muscle fiber type conversion. This study presents the initial evidence establishing SCIP as a potential enhancer of skeletal muscle fatigue resistance, consequently providing a theoretical foundation for the valuable utilization of sea cucumber intestines.

Casein kinase 2 complex: a central regulator of multiple pathobiological signaling pathways in Cryptococcus neoformans.

The casein kinase 2 (CK2) complex has garnered extensive attention over the past decades as a potential therapeutic target for diverse human diseases, including cancer, diabetes, and obesity, due to its pivotal roles in eukaryotic growth, differentiation, and metabolic homeostasis. While CK2 is also considered a promising antifungal target, its role in fungal pathogens remains unexplored. In this study, we investigated the functions and regulatory mechanisms of the CK2 complex in Cryptococcus neoformans, a major cause of fungal meningitis. The cryptococcal CK2 complex consists of a single catalytic subunit, Cka1, and two regulatory subunits, Ckb1 and Ckb2. Our findings show that Cka1 plays a primary role as a protein kinase, while Ckb1 and Ckb2 have major and minor regulatory functions, respectively, in growth, cell cycle control, morphogenesis, stress response, antifungal drug resistance, and virulence factor production. Interestingly, triple mutants lacking all three subunits (cka1Δ ckb1Δ ckb2Δ) exhibited more severe phenotypic defects than the cka1Δ mutant alone, suggesting that Ckb1/2 may have Cka1-independent functions. In a murine model of systemic cryptococcosis, cka1Δ and cka1Δ ckb1Δ ckb2Δ mutants showed severely reduced virulence. Transcriptomic, proteomic, and phosphoproteomic analyses further revealed that the CK2 complex controls a wide array of effector proteins involved in transcriptional regulation, cell cycle control, nutrient metabolisms, and stress responses. Most notably, CK2 disruption led to dysregulation of key signaling cascades central to C. neoformans pathogenicity, including the Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin signaling pathways. In summary, our study provides novel insights into the multifaceted roles of the fungal CK2 complex and presents a compelling case for targeting it in the development of new antifungal drugs.IMPORTANCEThe casein kinase 2 (CK2) complex, crucial for eukaryotic growth, differentiation, and metabolic regulation, presents a promising therapeutic target for various human diseases, including cancer, diabetes, and obesity. Its potential as an antifungal target is further highlighted in this study, which explores CK2's functions in C. neoformans, a key fungal meningitis pathogen. The CK2 complex in C. neoformans, comprising the Cka1 catalytic subunit and Ckb1/2 regulatory subunits, is integral to processes like growth, cell cycle, morphogenesis, stress response, drug resistance, and virulence. Our findings of CK2's role in regulating critical signaling pathways, including Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin, underscore its importance in C. neoformans pathogenicity. This study provides valuable insights into the fungal CK2 complex, reinforcing its potential as a target for novel antifungal drug development and pointing out a promising direction for creating new antifungal agents.

Calcineurin inhibition, cardiovascular consequences, vascular resistance, and potential responses.

AIM: To place the consequences of calcineurin inhibition in a cardiovascular context. METHODS: Literature review coupled with personal encounters. RESULTS: Calcineurin is a calcium-binding and calmodulin-binding protein that is conserved across evolution from yeast to mammals. The enzyme functions as a calcium-dependent, calmodulin-stimulated protein phosphatase. Its role in regulating physiology has largely been elucidated by observing calcineurin inhibition. Calcineurin inhibition transformed organ transplantation from an experiment into a therapy and made much of general immunotherapy possible. The function of this phosphatase and how its inhibition leads to toxicity concern us to this date. Initial research from patients and animal models implicated a panoply of factors contributing to hypertension and vasculopathy. Subsequently, the role of calcineurin in regulating the effective fluid volume, sodium reabsorption, and potassium and hydrogen ion excretion was elucidated by investigating calcineurin inhibition. Understanding the regulatory effects of calcineurin on endothelial and vascular smooth muscle cell function has also made substantial progress. However, precisely how the increase in systemic vascular resistance arises requires further mechanistic research. CONCLUSION: Calcineurin inhibition continues to save lives; however, options to counteract the negative effects of calcineurin inhibition should be vigorously pursued.

The signalling pathways, calcineurin B-like protein 5 (CBL5)-CBL-interacting protein kinase 8 (CIPK8)/CIPK24-salt overly sensitive 1 (SOS1), transduce salt signals in seed germination in Arabidopsis.

For plant growth under salt stress, sensing and transducing salt signals are central to cellular Na+ homoeostasis. The calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CIPK) complexes play critical roles in transducing salt signals in plants. Here, we show that CBL5, an ortholog of CBL4 and CBL10 in Arabidopsis, interacts with and recruits CIPK8/CIPK24 to the plasma membrane. Yeast cells coexpressing CBL5, CIPK8/CIPK24 and SOS1 demonstrated lesser Na+ accumulation and a better growth phenotype than the untransformed or SOS1 transgenic yeast cells under salinity. Overexpression of CBL5 improved the growth of the cipk8 or cipk24 single mutant but not the cipk8 cipk24 double mutant under salt stress, suggesting that CIPK8 and CIPK24 were the downstream targets of CBL5. Interestingly, seed germination in cbl5 was severely inhibited by NaCl, which was recovered by the overexpression of CBL5. Furthermore, CBL5 was mainly expressed in the cotyledons and hypocotyls, which are essential to seed germination. Na+ efflux activity in the hypocotyls of cbl5 was reduced relative to the wild-type under salt stress, enhancing Na+ accumulation. These findings indicate that CBL5 functions in seed germination and protects seeds and germinating seedlings from salt stress through the CBL5-CIPK8/CIPK24-SOS1 pathways.

Shikonin reactivates TSGs GADD45B and PPP3CC to block NSCLC cell proliferation and migration through JNK/P38/MAPK signaling pathways.

BACKGROUND: Shikonin, a natural naphthoquinone compound extracted from the Chinese traditional herbal medicine "Lithospermum erythrorhizon", possesses antitumor activity against various cancer types. Tumor-suppressor genes (TSGs) negatively regulate cell growth, proliferation, and differentiation, thereby inhibiting tumor formation. However, the molecular mechanism of action of shikonin on TSGs in non-small-cell lung cancer (NSCLC) remains unclear. METHODS: The inhibitory effect of shikonin on the proliferation and migration abilities of lung cancer cells were measured by Cell Counting Kit 8 (CCK8) and wound healing assays. The alteration of genes by shikonin treatment was detected by mRNA high-throughput sequencing and further confirmed by qPCR and western blotting experiments. The dominant functions of the upregulated genes were analyzed by GO and KEGG profiling. RESULTS: Shikonin inhibited the proliferation and migration of A549 and H1299 NSCLC cells in a dose-dependent manner. mRNA high-throughput sequencing revealed a total of 1794 upregulated genes in shikonin-treated NSCLC cells. Moreover, bioinformatic analysis of GO and KEGG profiling revealed that the up-regulated genes were mostly involved in the JNK/P38/MAPK signaling pathway, among which the expression of GADD45B and PPP3CC was significantly enhanced. Finally, we confirmed that GADD45B and PPP3CC were indeed upregulated in JNK/P38/MAPK pathway. CONCLUSIONS: Taken together, these results suggested that shikonin might affect the expression of GADD45B and PPP3CC through the JNK/P38/MAPK pathway, therefore exerting an inhibitory effect on the proliferation and migration of cancer cells. To our knowledge, this is the first study reporting the role of shikonin in upregulating TSGs to activate the JNK/P38/MAPK signaling pathways in NSCLC.

Development of a monoclonal antibody specific for a calpain-generated ∆48 kDa calcineurin fragment, a marker of distressed astrocytes.

BACKGROUND: Calcineurin (CN) is a Ca2+/calmodulin-dependent protein phosphatase. In healthy tissue, CN exists mainly as a full-length (∼60 kDa) highly-regulated protein phosphatase involved in essential cellular functions. However, in diseased or injured tissue, CN is proteolytically converted to a constitutively active fragment that has been causatively-linked to numerous pathophysiologic processes. These calpain-cleaved CN fragments (∆CN) appear at high levels in human brain at early stages of cognitive decline associated with Alzheimer's disease (AD). NEW METHOD: We developed a monoclonal antibody to ∆CN, using an immunizing peptide corresponding to the C-terminal end of the ∆CN fragment. RESULTS: We obtained a mouse monoclonal antibody, designated 26A6, that selectively detects ∆CN in Western analysis of calpain-cleaved recombinant human CN. Using this antibody, we screened both pathological and normal human brain sections provided by the University of Kentucky's Alzheimer's Disease Research Center. 26A6 showed low reactivity towards normal brain tissue, but detected astrocytes both surrounding AD amyloid plaques and throughout AD brain tissue. In brain tissue with infarcts, there was considerable concentration of 26A6-positive astrocytes within/around infarcts, suggesting a link with anoxic/ischemia pathways. COMPARISON WITH EXISTING METHOD: The results obtained with the new monoclonal are similar to those obtained with a polyclonal we had previously developed. However, the monoclonal is an abundant tool available to the dementia research community. CONCLUSIONS: The new monoclonal 26A6 antibody is highly selective for the ∆CN proteolytic fragment and labels a subset of astrocytes, and could be a useful tool for marking insidious brain pathology and identifying novel astrocyte phenotypes.

Dephosphorylation of NFAT by Calcineurin inhibits Skp2-mediated degradation.

The transcription factor NFAT plays key roles in multiple biological activities, such as immune responses, tissue development and malignant transformation. NFAT is dephosphorylated by calcineurin, which is activated by intracellular calcium levels, and translocated into the nucleus, resulting in transcriptional activation. Calcineurin dephosphorylates various target proteins and regulates their functions. However, the regulation of NFAT degradation is largely unknown, and it is unclear whether calcineurin contributes to the stability of NFAT. We investigated the effect of calcineurin inhibition on NFAT protein stability and found that the dephosphorylation of NFAT by calcineurin promotes the NFAT stabilization, whereas calcineurin mutant that is defective in phosphatase activity was unable to stabilize NFAT. Increased intracellular calcium ion concentration, which is essential for calcineurin activation, also induced NFAT stability. In addition, we identified S-phase kinase associated protein 2 (Skp2), an F-box protein of the SCF ubiquitin ligase complex, as a factor mediating degradation of NFAT when calcineurin was depleted. In summary, these findings revealed that the dephosphorylation of NFAT by calcineurin protects NFAT from degradation by Skp2 and promotes its protein stability.

Generation of Bioactivity-Tailored FK506/FK520 Analogs by CRISPR Editing in Streptomyces tsukubaensis.

For a potential application of FK506 in the treatment of acute kidney failure only the FKBP12 binding capability of the compound is required, while the immunosuppressive activity via calcineurin binding is considered as a likely risk to the patients. The methoxy groups at C13 and C15 are thought to have significant influence on the immunosuppressive activity of the molecule. Consequently, FK506 analogs with different functionalities at C13 and C15 were generated by targeted CRISPR editing of the AT domains in module 7 and 8 of the biosynthetic assembly line in Streptomyces tsukubaensis. In addition, the corresponding FK520 (C21 ethyl derivative of FK506) analogs could be obtained by media adjustments. The compounds were tested for their bioactivity in regards to FKBP12 binding, BMP potentiation and calcineurin sparing. 15-desmethoxy FK506 was superior to the other tested analogs as it did not inhibit calcineurin but retained high potency towards FKBP12 binding and BMP potentiation.

LINC-PINT suppresses breast cancer cell proliferation and migration via MEIS2/PPP3CC/NF-κB pathway by sponging miR-576-5p.

BACKGROUND: Breast cancer (BCa) is the most frequent malignant tumor in women. Long non-coding RNAs (lncRNAs) have been acknowledged to exert critical regulating functions in various cancers. Long intergenic non-protein coding RNA, p53 induced transcript (LINC-PINT) has been reported to be a chemosensitizer and a tumor suppressor in BCa. However, its downstream molecular mechanism contributing to its tumor-suppressing role remains to be explored in BCa. METHODS: LINC-PINT expression in BCa tissues and cells was measured using quantitative real-time polymerase chain reaction (RT-qPCR). The proliferation of transfected BCa cells was examined by counting kit-8 (CCK-8) and EdU assay. The migrating ability of indicate BCa cells was assessed by wound healing assays. Bioinformatics analysis and mechanism experiments such as RNA immunoprecipitation (RIP), RNA pull down assay, and luciferase reporter assay, were applied to demonstrate the downstream targets of LINC-PINT. RESULTS: LINC-PINT was downregulated in BCa tissues and cell lines. Overexpression of LINC-PINT suppressed BCa cell proliferation and migration. LINC-PINT could interact with miR-576-5p to upregulate Meis homeobox 2 (MEIS2) that positively regulated protein phosphatase 3 catalytic subunit gamma (PPP3CC) by inactivating the nuclear factor-κB (NF-κB) pathway. CONCLUSIONS: These findings elucidated the anti-tumor role of LINC-PINT in BCa via the miR-576-5p/MEIS2/PPP3CC/NF-κB axis, which suggested that LINC-PINT might serve as a potential therapeutic target for BCa.

Dual action of macrophage miR-204 confines cyclosporine A-induced atherosclerosis.

BACKGROUND AND PURPOSE: Atherosclerosis induced by cyclosporine A (CsA), an inhibitor of the calcineurin/nuclear factor of activated T cells (NFAT) pathway, is a major concern after organ transplantation. However, the atherosclerotic mechanisms of CsA remain obscure. We previously demonstrated that calcineurin/NFAT signalling inhibition contributes to atherogenesis via suppressing microRNA-204 (miR-204) transcription. We therefore hypothesised that miR-204 is involved in the development of CsA-induced atherosclerosis. EXPERIMENTAL APPROACH: ApoE-/- mice with macrophage-miR-204 overexpression were generated to determine the effects of miR-204 on CsA-induced atherosclerosis. Luciferase reporter assays and chromatin immunoprecipitation sequencing were performed to explore the targets mediating miR-204 effects. KEY RESULTS: CsA alone did not significantly affect atherosclerotic lesions or serum lipid levels. However, it exacerbated high-fat diet-induced atherosclerosis and hyperlipidemia in C57BL/6J and ApoE-/- mice, respectively. miR-204 levels decreased in circulating monocytes and plaque lesions during CsA-induced atherosclerosis. The upregulation of miR-204 in macrophages inhibited CsA-induced atherosclerotic plaque formation but did not affect serum lipid levels. miR-204 limited the CsA-induced foam cell formation by reducing the expression of the scavenger receptors SR-BII and CD36. SR-BII was post-transcriptionally regulated by mature miR-204-5p via 3'-UTR targeting. Additionally, nuclear-localised miR-204-3p prevented the CsA-induced binding of Ago2 to the CD36 promoter, suppressing CD36 transcription. SR-BII or CD36 expression restoration dampened the beneficial effects of miR-204 on CsA-induced atherosclerosis. CONCLUSION AND IMPLICATIONS: Macrophage miR-204 ameliorates CsA-induced atherosclerosis, suggesting that miR-204 may be a potential target for the prevention and treatment of CsA-related atherosclerotic side effects.

Macromolecular Complex Including MLL3, Carabin and Calcineurin Regulates Cardiac Remodeling.

BACKGROUND: Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include extensive gene reprogramming, which is dependent on epigenetic regulation and chromatin remodeling. However, the chromatin remodelers and their regulatory functions involved in the pathogenesis of cardiac hypertrophy are not well characterized. METHODS: Protein interaction was determined by immunoprecipitation assay in primary cardiomyocytes and mouse cardiac samples subjected or not to transverse aortic constriction for 1 week. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) experiments were performed on the chromatin of adult mouse cardiomyocytes. RESULTS: We report that the calcium-activated protein phosphatase CaN (calcineurin), its endogenous inhibitory protein carabin, the STK24 (STE20-like protein kinase 3), and the histone monomethyltransferase, MLL3 (mixed lineage leukemia 3) form altogether a macromolecular complex at the chromatin of cardiomyocytes. Under basal conditions, carabin prevents CaN activation while the serine/threonine kinase STK24 maintains MLL3 inactive via phosphorylation. After 1 week of transverse aortic constriction, both carabin and STK24 are released from the CaN-MLL3 complex leading to the activation of CaN, dephosphorylation of MLL3, and in turn, histone H3 lysine 4 monomethylation. Selective cardiac MLL3 knockdown mitigates hypertrophy, and chromatin immunoprecipitation and DNA sequencing analysis demonstrates that MLL3 is de novo recruited at the transcriptional start site of genes implicated in cardiomyopathy in stress conditions. We also show that CaN and MLL3 colocalize at chromatin and that CaN activates MLL3 histone methyl transferase activity at distal intergenic regions under hypertrophic conditions. CONCLUSIONS: Our study reveals an unsuspected epigenetic mechanism of CaN that directly regulates MLL3 histone methyl transferase activity to promote cardiac remodeling.

Trpc6 knockout protects against renal fibrosis by restraining the CN­NFAT2 signaling pathway in T2DM mice.

Diabetic kidney disease (DKD), one of the common complications of type­2 diabetes mellitus (T2DM), has become the principal cause of end­stage kidney disease. Transient receptor potential channel 6 (TRPC6), one of non­selective cation channels with significant calcium­permeability, is associated with renal fibrosis. However, the mechanism of TRPC6 in T2DM­induced renal fibrosis is still not entirely understood. The present study explored the potential mechanism of Trpc6 knockout in T2DM­induced renal fibrosis in Trpc6­/­ mice. The results showed that Trpc6 knockout inhibited the loss of body weight and the increase of fasting blood glucose (FBG) and significantly improved renal dysfunction and glomerular fibrosis in T2DM mice. The present study also indicated that Trpc6 knockout significantly lowered the expression of phosphorylated (p­)SMAD2/3, TGF­ß, calcineurin (CN), nuclear factor of activated T­cell (NFAT)2 and Nod­like receptor (NLR) 3 inflammasome­associated proteins. Calcium imaging results revealed that Trpc6 knockdown could decrease the levels of [Ca2+]i and inhibited calcium homeostasis imbalance. Moreover, it was found that knockout of Trpc6 had no significant influence on lipid disposition and reactive oxygen species generation in the kidney cortex. The present study suggested that knockout of Trpc6 may alleviate glomerular fibrosis and delay DKD progression by reducing [Ca2+]i overload and inhibiting the CN­NFAT2 pathway in T2DM mice.

Oleanolic acid promotes skeletal muscle fiber type transformation by activating TGR5-mediated CaN signaling pathway.

In recent years, the impact of bile acids and their representative G protein-coupled bile acid receptor 1 Takeda-G-protein-receptor-5 (TGR5) signaling pathway on muscle function and metabolic health has gained considerable interest. Increasing the content of slow muscle fibers has been recognized as an effective strategy to improve metabolic health. Oleanolic acid (OA) is a naturally occurring triterpenoid compound derived from plants, which can activate TGR5. The aim of this study was to investigate the effect of OA and TGR5 on muscle fiber types and further explore the underlying TGR5-dependent mechanisms. In this study, mice were divided into three groups and dietary supplementation with 0, 50, or 100 mg/kg OA. In addition, C2C12 cells were treated with OA at concentrations of 0, 5, 10, and 20 µM. Our studies revealed that OA promoted the conversion of fast to slow muscle fibers. In addition, it was found that OA activated the TGR5-mediated calcineurin (CaN)/nuclear factor of activated T cells cytoplasmic 1 (NFATc1) signaling pathway. Further mechanistic investigations demonstrated that inhibiting TGR5 and CaN abolished the effects of OA on muscle fiber types transformation. In conclusion, this study found that OA promotes the transformation of fast muscle fibers to slow muscle fibers through the TGR5-mediated CaN/NFATc1 signaling pathway.

Synergistic effects of putative Ca2+-binding sites of calmodulin in fungal development, temperature stress and virulence of Aspergillus fumigatus.

In pathogenic fungi, calcium-calmodulin-dependent serine-threonine-specific phosphatase calcineurin is involved in morphogenesis and virulence. Therefore, calcineurin and its tightly related protein complexes are attractive antifungal drug targets. However, there is limited knowledge available on the relationship between in vivo Ca2+-binding sites of calmodulin (CaM) and its functions in regulating stress responses, morphogenesis, and pathogenesis. In the current study, we demonstrated that calmodulin is required for hyphal growth, conidiation, and virulence in the human fungal pathogen, Aspergillus fumigatus. Site-directed mutations of calmodulin revealed that a single Ca2+-binding site mutation had no significant effect on A. fumigatus hyphal development, but multiple Ca2+-binding site mutations exhibited synergistic effects, especially when cultured at 42 °C, indicating that calmodulin function in response to temperature stress depends on its Ca2+-binding sites. Western blotting implied that mutations in Ca2+-binding sites caused highly degraded calmodulin fragments, suggesting that the loss of Ca2+-binding sites results in reduced protein stability. Moreover, normal intracellular calcium homeostasis and the nuclear translocation of the transcriptional factor CrzA are dependent on Ca2+-binding sites of AfCaM, demonstrating that Ca2+-binding sites of calmodulin are required for calcium signalling and its major transcription factor CrzA. Importantly, in situ mutations for four Ca2+-binding sites of calmodulin resulted in an almost complete loss of virulence in the Galleria mellonella wax moth model. This study shed more light on the functional characterization of putative calcium-binding sites of calmodulin in the morphogenesis and virulence of A. fumigatus, which enhances our understanding of calmodulin biological functions in cells of opportunistic fungal pathogens.

Ezrin inhibition alleviates oxidative stress and pyroptosis via regulating TRPML1-calcineurin axis mediated enhancement of autophagy in spinal cord injury.

Spinal cord injury (SCI) presents profound ramifications for patients, leading to diminished motor and sensory capabilities distal to the lesion site. Once SCI occurs, it not only causes great physical and psychological problems for patients but also imposes a heavy economic burden. Ezrin is involved in various cellular processes, including signal transduction, cell death, inflammation, chemotherapy resistance and the stress response. However, whether Ezrin regulates functional repair after SCI and its underlying mechanism has not been elucidated. Here, our results showed that there is a marked augmentation of Ezrin levels within neurons and Ezrin inhibition markedly diminished glial scarring and bolstered functional recuperation after SCI. RNA sequencing indicated the potential involvement of pyroptosis, oxidative stress and autophagy in the enhancement of functional recovery upon reduced Ezrin expression. Moreover, the inhibition of Ezrin expression curtailed pyroptosis and oxidative stress by amplifying autophagy. Our studies further demonstrated that Ezrin inhibition promoted autophagy by increasing TFEB activity via the Akt-TRPML1-calcineurin pathway. Finally, we concluded that inhibiting Ezrin expression alleviates pyroptosis and oxidative stress by enhancing TFEB-driven autophagy, thereby promoting functional recovery after SCI, which may be a promising therapeutic target for SCI treatment.