miR-1 as a Key Epigenetic Regulator in Early Differentiation of Cardiac Sinoatrial Region.

A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. In this study, miR-1 potential role in early cardiac chamber differentiation was analyzed through specific signaling pathways. For this, we performed in chick embryos functional experiments by means of miR-1 microinjections into the posterior cardiac precursors-of both primitive endocardial tubes-committed to sinoatrial region fates. Subsequently, embryos were subjected to whole mount in situ hybridization, immunohistochemistry and RT-qPCR analysis. As a relevant novelty, our results revealed that miR-1 increased Amhc1, Tbx5 and Gata4, while this microRNA diminished Mef2c and Cripto expressions during early differentiation of the cardiac sinoatrial region. Furthermore, we observed in this developmental context that miR-1 upregulated CrabpII and Rarß and downregulated CrabpI, which are three crucial factors in the retinoic acid signaling pathway. Interestingly, we also noticed that miR-1 directly interacted with Hdac4 and Calm1/Calmodulin, as well as with Erk2/Mapk1, which are three key factors actively involved in Mef2c regulation. Our study shows, for the first time, a key role of miR-1 as an epigenetic regulator in the early differentiation of the cardiac sinoatrial region through orchestrating opposite actions between retinoic acid and Mef2c, fundamental to properly assign cardiac cells to their respective heart chambers. A better understanding of those molecular mechanisms modulated by miR-1 will definitely help in fields applied to therapy and cardiac regeneration and repair.

Associations of CALM1 and DRD1 polymorphisms, and their expression levels, with Taihang chicken egg-production traits.

Egg production by hens is an important reproductive performance index in the poultry industry. To investigate the effects of the CALM1 and DRD1 genes on egg production in chicken, their mRNA expression and single nucleotide polymorphisms (SNP) levels were investigated, and bioinformatics and egg-production association analyses were performed. Three SNPs (g.44069941G > A and g.44069889A > G in CALM1 and g.10742639C > T in DRD1) were detected in the exons and introns of CALM1 and DRD1 in 400 Taihang chickens. Among them, g.44069941G > A was significantly associated with Taihang chicken egg production on the 500th day (p < 0.05), whereas g.10742639C > T was significantly associated with the 300th day (p < 0.05). The expression levels of CALM1 and DRD1 in ovarian tissues of a high-yielding Taihang group were greater than in a low-yielding group (p < 0.05). The bioinformatics analysis revealed that the mutations influenced the mRNA secondary structures of CALM1 and DRD1. This study provides new insights into the potential effects of CALM1 and DRD1 polymorphisms on chicken egg production. The two SNPs g.44069941G > A and g.10742639C > T are potential molecular markers for improving the reproductive traits of Taihang chicken.

CALM1 and DRD1 were two important genes for reproduction. In this study, the entire coding regions of both genes were sequenced and mutations were detected in Taihang chickens. The results showed that two single nucleotide polymorphisms (SNPs), g.44069941G > A in the CALM1 gene and g.10742639C > T in the DRD1 gene, were associated with egg-laying traits. g.10742639C > T is a synonymous mutation predicted to affect the secondary structure of mRNA. Therefore, these two mutations might be potential molecular markers for improving reproductive traits in Taihang chickens.

Navigating the intricacies of cellular machinery.

Voltage-gated sodium channels (NaVs) underlie the initiation of action potentials in various excitable cell types and are regulated by channel-interacting proteins, including the cellular calcium sensor calmodulin and fibroblast growth factor homologous factors. Both of these are known to bind the NaV cytosolic C-terminal domain and modulate the channel's electrophysiology, but it was unknown whether they had any allosteric interactions with each other. A recent rigorous study provides insights into the molecular interactions of these ion channels and their partners that crucially take the cellular landscape into consideration.

CALM1 promotes progression and dampens chemosensitivity to EGFR inhibitor in esophageal squamous cell carcinoma.

BACKGROUND: Calmodulin1 (CALM1) has been identified as one of the overexpression genes in a variety of cancers and EGFR inhibitor have been widely used in clinical treatment but it is unknown whether CALM1 and epidermal growth factor receptor (EGFR) have a synergistic effect in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to explore the synergistic effects of knock-out CALM1 combined with EGFR inhibitor (Afatinib) and to elucidate the role of CALM1 in sensitizing the resistance to Afatinib in ESCC. METHOD: Immunohistochemistry (IHC) and qRT-PCR were used to examine the expression of CALM1 and EGFR in ESCC tissues. Kaplan-Meier survival analysis was used to analyze the clinical and prognostic significance of CALM1 and EGFR expression in ESCC. Furthermore, to evaluate the biological function of CALM1 in ESCC, the latest gene editing technique CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats)was applied to knockout CALM1 in ESCC cell lines KYSE150, Eca109 and TE-1. MTT, flow cytometry, Transwell migration, scratch wound-healing and colony formation assays were performed to assay the combined effect of knock-out CALM1 and EGFR inhibitor on ESCC cell proliferation and migration. In addition, nude mice xenograft model was used to observe the synergistic inhibition of knock-out CALM1 and Afatinib. RESULTS: Both CALM1 and EGFR were found to be significantly over-expressed in ESCC compared with paired normal control. Over-expressed CALM1 and EGFR were significantly associated with clinical stage, T classification and poor overall prognosis, respectively. In vitro, the combined effect of knock-out CALM1 mediated by the lentivirus and EGFR inhibitor was shown to be capable of inhibiting the proliferation, inducing cell cycle arrest at G1/S stage and increasing apoptosis of KYSE-150 and Eca109 cells; invasion and migration were also suppressed. In vivo, the results of tumor weight and total fluorescence were markedly reduced compared with the sgCtrl-infected group and sgCAML1 group. CONCLUSION: Our data demonstrated that knock-out of CALM1 could sensitize ESCC cells to EGFR inhibitor, and it may exert oncogenic role via promotion of EMT. Taken together, CALM1 may be a tempting target to overcome Afatinib resistance.

14q32.11 microdeletion including CALM1, TTC7B, PSMC1, and RPS6KA5: A new potential cause of developmental and language delay in three unrelated patients.

Three unrelated patients with similar microdeletions of chromosome 14q32.11 with shared phenotypes including language and developmental delay, and four overlapping genes -CALM1, TTC7B, PSMC1, and RPS6KA5 have been presented. All four genes are expressed in the brain and have haploinsufficiency scores, which reflect low tolerance to loss of function variation. An insight on the genes in the overlapping region, which may influence the resulting phenotype has been provided. Given the three patients' similar phenotypes and lack of normal variation in this region, it was suggested that this microdeletion may be associated with developmental and language delay.

Tribbles Pseudokinase 2 (TRIB2) Regulates Expression of Binding Partners in Bovine Granulosa Cells.

Members of the Tribbles (TRIB) family of pseudokinases are critical components of intracellular signal transduction pathways in physiological and pathological processes. TRIBs, including TRIB2, have been previously shown as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling network associated with TRIB2 and its binding partners in granulosa cells during ovarian follicular development is not fully defined. We previously reported that TRIB2 is differentially expressed in growing dominant follicles while downregulated in ovulatory follicles following the luteinizing hormone (LH) surge or human chorionic gonadotropin (hCG) injection. In the present study, we used the yeast two-hybrid screening system and in vitro coimmunoprecipitation assays to identify and confirm TRIB2 interactions in granulosa cells (GCs) of dominant ovarian follicles (DFs), which yielded individual candidate binding partners including calmodulin 1 (CALM1), inhibin subunit beta A (INHBA), inositol polyphosphate phosphatase-like 1 (INPPL1), 5'-nucleotidase ecto (NT5E), stearoyl-CoA desaturase (SCD), succinate dehydrogenase complex iron sulfur subunit B (SDHB) and Ras-associated protein 14 (RAB14). Further analyses showed that all TRIB2 binding partners are expressed in GCs of dominant follicles but are differentially regulated throughout the different stages of follicular development. CRISPR/Cas9-driven inhibition along with pQE-driven overexpression of TRIB2 showed that TRIB2 differently regulates expression of binding partners, which reveals the importance of TRIB2 in the control of gene expression linked to various biological processes such as proliferation, differentiation, cell migration, apoptosis, calcium signaling and metabolism. These data provide a larger view of potential TRIB2-regulated signal transduction pathways in GCs and provide strong evidence that TRIB2 may act as a regulator of target genes during ovarian follicular development.

Calm1 signaling pathway is essential for the migration of mouse precerebellar neurons.

The calcium ion regulates many aspects of neuronal migration, which is an indispensable process in the development of the nervous system. Calmodulin (CaM) is a multifunctional calcium ion sensor that transduces much of the signal. To better understand the role of Ca(2+)-CaM in neuronal migration, we investigated mouse precerebellar neurons (PCNs), which undergo stereotyped, long-distance migration to reach their final position in the developing hindbrain. In mammals, CaM is encoded by three non-allelic CaM (Calm) genes (Calm1, Calm2 and Calm3), which produce an identical protein with no amino acid substitutions. We found that these CaM genes are expressed in migrating PCNs. When the expression of CaM from this multigene family was inhibited by RNAi-mediated acute knockdown, inhibition of Calm1 but not the other two genes caused defective PCN migration. Many PCNs treated with Calm1 shRNA failed to complete their circumferential tangential migration and thus failed to reach their prospective target position. Those that did reach the target position failed to invade the depth of the hindbrain through the required radial migration. Overall, our results suggest the participation of CaM in both the tangential and radial migration of PCNs.

MiR-202-3p Targets Calm1 and Suppresses Inflammation in a Mouse Model of Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is regarded as a type of respiratory failure. Emerging evidence has demonstrated the significant roles of microRNAs in various disorders. Nevertheless, the role of miR-202-3p in ARDS is unclear. Forty male C57BL/6 mice treated with phosphate buffer saline/lipopolysaccharide (PBS/LPS) and administrated with NC/miR-202-3p agomir were divided into four groups. A reverse transcription-quantitative polymerase chain reaction was used to evaluate the level of miR-202-3p, its target genes, and proinflammatory factors. Hematoxylin­eosin was utilized for histological observation of the lung tissues. The Wet/Dry ratio, myeloperoxidase activity, and total protein concentration in bronchoalveolar lavage fluid were assessed to determine pulmonary edema. Western blotting was used for quantifying protein levels of proinflammatory factors, nuclear factor kappa B (NF-κB), and NLR family pyrin domain containing 3 (NLRP3) signaling-associated proteins. Calmodulin 1 (Calm1) protein expression in murine lung tissues was evaluated by immunohistochemistry. The binding relation between miR-202-3p and Calm1 was assessed by luciferase reporter assay. The results showed that miR-202-3p was lowly expressed in the lung tissues of ARDS mice. Overexpressed miR-202-3p relieved LPS-induced edema, reduced proinflammatory factors, and inactivated NF-κB/NLRP3 signaling in murine lung tissues. Calm1 was targeted by miR-202-3p and displayed a high level of LPS-induced ARDS. In conclusion, miR-202-3p targets Calm1 and suppresses inflammation in LPS-induced ARDS, thereby inhibiting the pathogenesis of ARDS in a mouse model.

Sequence variations and accessory proteins adapt TMC functions to distinct sensory modalities.

Transmembrane channel-like (TMC) proteins are expressed throughout the animal kingdom and are thought to encode components of ion channels. Mammals express eight TMCs (mTMC1-8), two of which (mTMC1 and mTMC2) are subunits of mechanotransduction channels. C. elegans expresses two TMCs (TMC-1 and TMC-2), which mediate mechanosensation, egg laying, and alkaline sensing. The mechanisms by which nematode TMCs contribute to such diverse physiological processes and their functional relationship to mammalian mTMCs is unclear. Here, we show that association with accessory proteins tunes nematode TMC-1 to divergent sensory functions. In addition, distinct TMC-1 domains enable touch and alkaline sensing. Strikingly, these domains are segregated in mammals between mTMC1 and mTMC3. Consistent with these findings, mammalian mTMC1 can mediate mechanosensation in nematodes, while mTMC3 can mediate alkaline sensation. We conclude that sequence diversification and association with accessory proteins has led to the emergence of TMC protein complexes with diverse properties and physiological functions.

Changes in urinary exosomal protein CALM1 may serve as an early noninvasive biomarker for diagnosing diabetic kidney disease.

BACKGROUND: The risk of the development and progression of diabetic kidney disease (DKD) was increased by abnormal calcium release. However, it is still unknown whether calcium signal pathway-related proteins are changed in urinary exosomes. This study aims to explore the changes in urinary exosomal proteins, which may provide novel biomarkers for diagnosing DKD. METHODS: Urinary exosomes were isolated from 132 participants by size exclusion chromatography method and 72 participants were tested by LC-MS/MS (Discovery phase). Correlation and multivariate logistics analysis were applied to evaluate selected urinary proteins. Western blot and ELISA were used to validate the selected protein (Validation phase: n = 60). The diagnostic performance of the selected biomarker was evaluated by receiver operating characteristic curve analyses between the discovery and validation phases. RESULTS: Sixteen calcium signal pathway-related proteins were identified, however, only Calmodulin-1(CALM1) was continuously increased. Different expression of CALM1 was found in patients with different level of estimated glomerular filtration rate (eGFR) in two cohorts. The level of CALM1 was correlated with eGFR and serum creatinine levels in two cohorts. Multivariate analysis revealed that serum albumin (ALB) levels and CALM1 were independent risk factors for DKD. A diagnostic model based on CALM1 and serum ALB levels that could significantly distinguish DKD was established and validated. CONCLUSIONS: Significant changes in calcium signal pathway-related urinary exosomal proteins were observed. The CALM1 may serve as an early noninvasive biomarker for diagnosing DKD.

CALM1 rs3179089 polymorphism might contribute to coronary artery disease susceptibility in Chinese male: a case-control study.

BACKGROUND: Calmodulin 1 (CALM1) mutations are involved in the development of coronary artery disease (CAD). However, the relationship of CALM1 rs3179089 polymorphism with CAD is unknown. OBJECTIVE: This study aimed to identify the relationship of CALM1 rs3179089 polymorphism with CAD susceptibility, CALM1 expression, blood pressure, blood glucose, blood coagulation and serum lipid levels of CAD patients. METHODS: 550 CAD patients and 550 control subjects were genotyped for CALM1 using Sequenom MassARRAY technology. CALM1 expression level was measured by quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: CALM1 mRNA expression was higher in CAD patients than that in control subjects (P < 0.001). CAD patients with CC genotype had higher CALM1 mRNA expression level than control subjects with CC genotype (P = 0.006). Genotypic frequency of rs3179089 was different between male patients of CAD and control subjects (P = 0.045). Rs3179089 polymorphism was related to CAD risk of males in recessive model (P = 0.039). Moreover, rs3179089 polymorphism was associated with systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting plasma glucose (FPG), and D-Dimer (D-D) level of patients with CAD in recessive model (P = 0.013 for SBP; P = 0.034 for DBP; P = 0.004 for FPG; P = 0.046 for D-D). In addition, rs3179089 polymorphism was correlated with low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) serum levels of patients with CAD in both addictive (P = 0.025 for LDL-C; P = 0.001 for TC) and recessive models (P = 0.001 for LDL-C; P = 0.001 for TC). CONCLUSION: CALM1 expression is associated with development of CAD. CALM1 rs3179089 polymorphism affects CAD susceptibility in males, and blood pressure, blood glucose, blood coagulation and serum lipid of CAD patients.

Cardio-omentopexy requires a cardioprotective innate immune response to promote myocardial angiogenesis in mice.

Objective: The pedicled greater omentum, when applied onto stressed hearts using omentopexy, has been shown to be protective in humans and animals. The mechanisms underlying cardioprotection using omentopexy remain elusive. This study examined whether macrophage-mediated angiogenesis accounts for the cardioprotective effect of omentopexy in mice. Methods: C57BL/6 mice were subjected to minimally invasive transverse aortic constriction for 6 weeks and subsequent cardio-omentopexy for 8 weeks. Control mice underwent the same surgical procedures without aortic constriction or cardio-omentopexy. Results: Transverse aortic constriction led to left ventricular concentric hypertrophy, reduced mitral E/A ratio, increased cardiomyocyte size, and myocardial fibrosis in the mice that underwent sham cardio-omentopexy surgery. The negative effects of transverse aortic constriction were prevented by cardio-omentopexy. Myocardial microvessel density was elevated in the mice that underwent aortic constriction and sham cardio-omentopexy surgery, and cardio-omentopexy further enhanced angiogenesis. Nanostring gene array analysis uncovered the activation of angiogenesis gene networks by cardio-omentopexy. Flow cytometric analysis revealed that cardio-omentopexy triggered the accumulation of cardiac MHCIIloLyve1+TimD4+ (Major histocompatibility complex class IIlow lymphatic vessel endothelial hyaluronan receptor 1+ T cell immunoglobulin and mucin domain conataining 4+) resident macrophages at the omental-cardiac interface. Intriguingly, the depletion of macrophages with clodronate-liposome resulted in the failure of cardio-omentopexy to protect the heart and promote angiogenesis. Conclusions: Cardio-omentopexy protects the heart from pressure overload-elicited left ventricular hypertrophy and dysfunction by promoting myocardial angiogenesis. Cardiac MHCIIloLyve1+TimD4+ resident macrophages play a critical role in the cardioprotective effect and angiogenesis of cardio-omentopexy.

SRC fine-tunes ADAM10 shedding activity to promote pituitary adenoma cell progression.

A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a metalloproteinase known to modulate the progression of several types of tumor. However, the role played by ADAM10 in pituitary adenomas is currently unknown, and what factors orchestrate the activation of ADAM10 in this kind of tumor is also unclear. Here, we found that SRC kinase is an ADAM10-interacting partner and that SRC kinase activity is required for this interaction. As a new positive regulator promoting the shedding activity of ADAM10, SRC could compete with calmodulin 1 (CALM1) for ADAM10 binding in a mutually exclusive manner. Strikingly, the interaction between ADAM10 and CALM1 is regulated by SRC activity. Furthermore, we proved that the cytoplasmic region of ADAM10 is required for the shedding activity of ADAM10 upon SRC activation. As a proof-of-concept, we discovered that the combination of ADAM10 and SRC inhibitors can inhibit cell proliferation and migration to a great extent. Thus, our findings shed light on a novel therapeutic strategy to block the tumorigenesis and migration of pituitary adenoma.

Association between the polymorphisms of CALM1 gene and osteoarthritis risk: a meta-analysis based on observational studies.

The existing studies on the association between polymorphisms of Calmodulin 1 (CALM1) gene and the risk of osteoarthritis (OA, a complex multifactorial disease and a major degenerative form of arthritis) in different populations have yielded conflicting findings. Therefore, we conducted a meta-analysis by systematically searching PubMed, Embase, Medline, Cochrane Library and Google Scholar, and assessing this association by calculating pooled odds ratios with 95% confidence intervals. Subgroup analyses stratified by ethnicity, OA type, and genotype were also conducted. Six studies (2752 cases and 3259 controls) involving six single nucleotide polymorphisms were included. Our data suggested that the T allele and genotype TT of the rs12885713 polymorphism, and the C allele of the rs2300496 polymorphism in the CALM1 gene all increased the risk of OA. The pooled results revealed no significant association between the CALM1 rs3213718 polymorphism and the risk of OA. Stratification analyses by ethnicity and OA type showed that the rs12885713 polymorphism increased the risk of OA among Asians and in knee OA, respectively. In conclusion, the rs12885713 and rs2300496 polymorphisms of the CALM1 gene may both increase the risk of OA. Owing to the limitations of the present study, this finding should be further confirmed in future well-designed studies.

Association of CALM1 rs3179089 Polymorphism with Ischemic Stroke in Chinese Han Population.

A quantitative transcriptomics analysis has reported that Calmodulin 1 (CALM1) is highly expressed in human brain tissues. This study aims to evaluate the relationship between CALM1 rs3179089 polymorphism and ischemic stroke (IS) in Chinese Han population. A total of 550 patients with IS and 550 control subjects were recruited and genotyped using Sequenom MassArray technology. The mRNA expression of CALM1 was measured using quantitative real-time polymerase chain reaction. CALM1 mRNA expression was significantly higher in patients with IS than that in control subjects (P = 0.006). The genomic frequency distribution was significantly different between female patients with IS and female controls (χ2 = 6.043, P = 0.047). In recessive model, CALM1 rs3179089 polymorphism was associated with the risk of IS in female patients. GG genotype significantly increased the risk of IS compared with the CC+GC genotype in females (OR 8.68, P = 0.042; adjusted OR 8.72, Padj = 0.042). Rs3179089 polymorphism was associated positively with plasmas D-Dimer of patients with IS in recessive model (ßa = 3.24, P = 0.018; ßb = 3.20, Padj = 0.019). Moreover, rs3179089 polymorphism was related positively to thrombin time of patients with IS in addictive (ßa = 2.32, P = 0.005, ßb = 2.26, Padj=0.006) and recessive model (ßa = 11.19, P = 0.001, ßb = 11.13, Padj = 0.001). CALM1 expression was involved in the development of IS. CALM1 rs3179089 polymorphism was associated with IS risk in Chinese females, and related to blood coagulation of IS patients.

Human Calmodulin Mutations.

Fluxes of calcium (Ca2+) across cell membranes enable fast cellular responses. Calmodulin (CaM) senses local changes in Ca2+ concentration and relays the information to numerous interaction partners. The critical role of accurate Ca2+ signaling on cellular function is underscored by the fact that there are three independent CaM genes (CALM1-3) in the human genome. All three genes are functional and encode the exact same CaM protein. Moreover, CaM has a completely conserved amino acid sequence across all vertebrates. Given this degree of conservation, it was long thought that mutations in CaM were incompatible with life. It was therefore a big surprise when the first CaM mutations in humans were identified six years ago. Today, more than a dozen human CaM missense mutations have been described, all found in patients with severe cardiac arrhythmias. Biochemical studies have demonstrated differential effects on Ca2+ binding affinities for these CaM variants. Moreover, CaM regulation of central cardiac ion channels is impaired, including the voltage-gated Ca2+ channel, CaV1.2, and the sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor isoform 2, RyR2. Currently, no non-cardiac phenotypes have been described for CaM variant carriers. However, sequencing of large human cohorts reveals a cumulative frequency of additional rare CaM mutations that raise the possibility of CaM variants not exclusively causing severe cardiac arrhythmias. Here, we provide an overview of the identified CaM variants and their known consequences for target regulation and cardiac disease phenotype. We discuss experimental data, patient genotypes and phenotypes as well as which questions remain open to understand this complexity.

The control of precerebellar neuron migration by RNA-binding protein Csde1.

Neuronal migration during brain development sets the position of neurons for the subsequent wiring of neural circuits. To understand the molecular mechanism regulating the migrating process, we considered the migration of mouse precerebellar neurons. Precerebellar neurons originate in the rhombic lip of the hindbrain and show stereotypic, long-distance tangential migration along the circumference of the hindbrain to form precerebellar nuclei at discrete locations. To identify the molecular components underlying this navigation, we screened for genes expressed in the migrating precerebellar neurons. As a result, we identified the following three genes through the screening; Calm1, Septin 11, and Csde1. We report here functional analysis of one of these genes, Csde1, an RNA-binding protein implicated in the post-transcriptional regulation of a subset of cellular mRNA, by examining its participation in precerebellar neuronal migration. We found that shRNA-mediated inhibition of Csde1 expression resulted in a failure of precerebellar neurons to complete their migration into their prospective target regions, with many neurons remaining in migratory paths. Furthermore, those that did reach their destination failed to invade the depth of the hindbrain via radial migration. These results have uncovered a crucial role of Csde1 in the proper control of both radial and tangential migration of precerebellar neurons.