Crucial Role of Lysine-Specific Histone Demethylase 1 in RANKL-Mediated Osteoclast Differentiation.

Epigenetic regulators are involved in osteoclast differentiation. This study proposes that the inhibitors of epigenetic regulators could be effective in the treatment of osteoporosis. This study identified GSK2879552, a lysine-specific histone demethylase 1 (LSD1) inhibitor, as a candidate for the treatment of osteoporosis from epigenetic modulator inhibitors. We investigate the function of LSD1 during RANKL-induced osteoclast formation. LSD1 small-molecule inhibitors effectively inhibit the RANKL-induced osteoclast differentiation in a dose-dependent manner. LSD1 gene knockout in macrophage cell line Raw 264.7 also inhibits RANKL-mediated osteoclastogenesis. LSD1-inhibitor-treated primary macrophage cells and LSD1 gene knockout Raw 264.7 cells failed to show actin ring formation. LSD1 inhibitors prevent the expression of RANKL-induced osteoclast-specific genes. They also downregulated the protein expression of osteoclast-related markers in osteoclastogeneses, such as Cathepsin K, c-Src, and NFATc1. Although LSD1 inhibitors were shown to reduce the in vitro demethylation activity of LSD1, they did not modulate the methylation of Histone 3 K4 and K9 during osteoclastogenesis. The ovariectomy (OVX)-induced osteoporosis model revealed that GSK2879552 slightly restores OVX-induced cortical bone loss. LSD1 can be employed as a positive regulator to promote osteoclast formation. Hence, inhibition of LSD1 activities is a potential target for preventing bone diseases characterized by excessive osteoclast activities.

(S)-2-(Cyclobutylamino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)isonicotinamide Attenuates RANKL-Induced Osteoclast Differentiation by Inhibiting NF-κB Nuclear Translocation.

Osteoporosis is a common skeletal disease; however, effective pharmacological treatments still need to be discovered. This study aimed to identify new drug candidates for the treatment of osteoporosis. Here, we investigated the effect of EPZ compounds, protein arginine methyltransferase 5 (PRMT5) inhibitors, on RANKL-induced osteoclast differentiation via molecular mechanisms by in vitro experiments. EPZ015866 attenuated RANKL-induced osteoclast differentiation, and its inhibitory effect was more significant than EPZ015666. EPZ015866 suppressed the F-actin ring formation and bone resorption during osteoclastogenesis. In addition, EPZ015866 significantly decreased the protein expression of Cathepsin K, NFATc1, and PU.1 compared with the EPZ015666 group. Both EPZ compounds inhibited the nuclear translocation of NF-κB by inhibiting the dimethylation of the p65 subunit, which eventually prevented osteoclast differentiation and bone resorption. Hence, EPZ015866 may be a potential drug candidate for the treatment of osteoporosis.

Value-Added Products from Coffee Waste: A Review.

Coffee waste is often viewed as a problem, but it can be converted into value-added products if managed with clean technologies and long-term waste management strategies. Several compounds, including lipids, lignin, cellulose and hemicelluloses, tannins, antioxidants, caffeine, polyphenols, carotenoids, flavonoids, and biofuel can be extracted or produced through recycling, recovery, or energy valorization. In this review, we will discuss the potential uses of by-products generated from the waste derived from coffee production, including coffee leaves and flowers from cultivation; coffee pulps, husks, and silverskin from coffee processing; and spent coffee grounds (SCGs) from post-consumption. The full utilization of these coffee by-products can be achieved by establishing suitable infrastructure and building networks between scientists, business organizations, and policymakers, thus reducing the economic and environmental burdens of coffee processing in a sustainable manner.

Identification of Novel Genes for Cell Fusion during Osteoclast Formation.

Osteoclasts are derived from hematopoietic stem cells. Monocyte preosteoclasts obtain resorbing activity via cell-cell fusion to generate multinucleated cells. However, the mechanisms and molecules involved in the fusion process are poorly understood. In this study, we performed RNA sequencing with single nucleated cells (SNCs) and multinucleated cells (MNCs) to identify the fusion-specific genes. The SNCs and MNCs were isolated under the same conditions during osteoclastogenesis with the receptor activator of nuclear factor-κB ligand (RANKL) administration. Based on this analysis, the expression of seven genes was found to be significantly increased in MNCs but decreased in SNCs, compared to that in bone marrow-derived macrophages (BMMs). We then generated knockout macrophage cell lines using a CRISPR-Cas9 genome-editing tool to examine their function during osteoclastogenesis. Calcrl-, Marco-, or Ube3a-deficient cells could not develop multinucleated giant osteoclasts upon RANKL stimulation. However, Tmem26-deficient cells fused more efficiently than control cells. Our findings demonstrate that Calcrl, Marco, and Ube3a are novel determinants of osteoclastogenesis, especially with respect to cell fusion, and highlight potential targets for osteoporosis therapy.

N-[2-(4-benzoyl-1-piperazinyl)phenyl]-2-(4-chlorophenoxy) acetamide is a novel inhibitor of resorptive bone loss in mice.

The dynamic balance between bone formation and bone resorption is vital for the retention of bone mass. The abnormal activation of osteoclasts, unique cells that degrade the bone matrix, may result in many bone diseases such as osteoporosis. Osteoporosis, a bone metabolism disease, occurs when extreme osteoclast-mediated bone resorption outstrips osteoblast-related bone synthesis. Therefore, it is of great interest to identify agents that can regulate the activity of osteoclasts and prevent bone loss-induced bone diseases. In this study, we found that N-[2-(4-benzoyl-1-piperazinyl)phenyl]-2-(4-chlorophenoxy) acetamide (PPOAC-Bz) exerted a strong inhibitory effect on osteoclastogenesis. PPOAC-Bz altered the mRNA expressions of several osteoclast-specific marker genes and blocked the formation of mature osteoclasts, suppressing F-actin belt formation and bone resorption activity in vitro. In addition, PPOAC-Bz prevented OVX-induced bone loss in vivo. These findings highlighted the potential of PPOAC-Bz as a prospective drug for the treatment of osteolytic disorders.

Myocardial-specific ablation of Jumonji and AT-rich interaction domain-containing 2 (Jarid2) leads to dilated cardiomyopathy in mice.

Cardiomyopathy is a common myocardial disease that can lead to sudden death. However, molecular mechanisms underlying cardiomyopathy remain unclear. Jumonji and AT-rich interaction domain-containing 2 (Jarid2) is necessary for embryonic heart development, but functions of Jarid2 after birth remain to be elucidated. Here, we report that myocardial-specific deletion of Jarid2 using αMHC::Cre mice (Jarid2αMHC) causes dilated cardiomyopathy (DCM) and premature death 6-9 months after birth. To determine functions of Jarid2 in the adult heart and DCM, we analyzed gene expression in the heart at postnatal day (p)10 (neonatal) and 7 months (DCM). Pathway analyses revealed that dysregulated genes in Jarid2αMHC hearts at p10, prior to cardiomyopathy, represented heart development and muscle contraction pathways. At 7 months, down-regulated genes in Jarid2αMHC hearts were enriched in metabolic process and ion channel activity pathways and up-regulated genes in extracellular matrix components. In normal hearts, expression levels of contractile genes were increased from p10 to 7 months but were not sufficiently increased in Jarid2αMHC hearts. Moreover, Jarid2 was also necessary to repress fetal contractile genes such as TroponinI1, slow skeletal type (Tnni1) and Actin alpha 2, smooth muscle (Acta2) in neonatal stages through ErbB2-receptor tyrosine kinase 4 (ErbB4) signaling. Interestingly, Ankyrin repeat domain 1 (Ankrd1) and Neuregulin 1 (Nrg1), whose expression levels are known to be increased in the failing heart, were already elevated in Jarid2αMHC hearts within 1 month of birth. Thus, we demonstrate that ablation of Jarid2 in cardiomyocytes results in DCM and suggest that Jarid2 plays important roles in cardiomyocyte maturation during neonatal stages.

Max-depth-range technique for faster full-color hologram generation.

In this paper, a max-depth-range method is proposed to determine the optimum length of depth range for faster generation of full-color holograms. For each color channel, objects are divided by a fixed length to create a temporary depth range, and the wavefront recording plane (WRP) is placed in the middle of all layers within the temporary depth range. The proposed method is used to calculate full-color holograms significantly faster than a conventional multiple-WRP method but with almost the same reconstructed image quality. The feasibility of the proposed method was confirmed using numerical and optical experiments for various scenes containing multiple real objects.

Cardiac-specific developmental and epigenetic functions of Jarid2 during embryonic development.

Epigenetic regulation is critical in normal cardiac development. We have demonstrated that the deletion of Jarid2 (Jumonji (Jmj) A/T-rich interaction domain 2) in mice results in cardiac malformations recapitulating human congenital cardiac disease and dysregulation of gene expression. However, the precise developmental and epigenetic functions of Jarid2 within the developing heart remain to be elucidated. Here, we determined the cardiac-specific functions of Jarid2 and the genetic networks regulated by Jarid2. Jarid2 was deleted using different cardiac-specific Cre mice. The deletion of Jarid2 by Nkx2.5-Cre mice (Jarid2Nkx) caused cardiac malformations including ventricular septal defects, thin myocardium, hypertrabeculation, and neonatal lethality. Jarid2Nkx mice exhibited elevated expression of neural genes, cardiac jelly, and other key factors including Isl1 and Bmp10 in the developing heart. By employing combinatorial genome-wide approaches and molecular analyses, we showed that Jarid2 in the myocardium regulates a subset of Jarid2 target gene expression and H3K27me3 enrichment during heart development. Specifically, Jarid2 was required for PRC2 occupancy and H3K27me3 at the Isl1 promoter locus, leading to the proper repression of Isl1 expression. In contrast, Jarid2 deletion in differentiated cardiomyocytes by cTnt-Cre mice caused no gross morphological defects or neonatal lethality. Thus, the early deletion of Jarid2 in cardiac progenitors, prior to the differentiation of cardiac progenitors into cardiomyocytes, results in morphogenetic defects manifested later in development. Our studies reveal that there is a critical window during early cardiac progenitor differentiation when Jarid2 is crucial to establish the epigenetic landscape at later stages of development.

A Full-Visible-Spectrum Invisibility Cloak for Mesoscopic Metal Wires.

Structured metals can sustain a very large scattering cross-section that is induced by localized surface plasmons, which often has an adverse effect on their use as transparent electrodes in displays, touch screens, and smart windows due to an issue of low clarity. Here, we report a broadband optical cloaking strategy for the network of mesoscopic metal wires with submicrometer to micrometer diameters, which is exploited for manufacturing and application of high-clarity metal-wires-based transparent electrodes. We prepare electrospun Ag wires with 300-1800 nm in diameter and perform a facile surface oxidation process to form Ag/Ag2O core/shell heterogeneous structures. The absorptive Ag2O shell, together with the coating of a dielectric cover, leads to the cancellation of electric multipole moments in Ag wires, thereby drastically suppressing plasmon-mediated scattering over the full visible spectrum and rendering Ag wires to be invisible. Simultaneously with the effect of invisibility, the transmittance of Ag/Ag2O wires is significantly improved compared to bare Ag wires, despite the formation of an absorptive Ag2O shell. As an application example, we demonstrate that these invisible Ag wires serve as a high-clarity, high-transmittance, and high-speed defroster for automotive windshields.

Inhibitory Effects of N-[2-(4-acetyl-1-piperazinyl) phenyl]-2-(2-chlorophenoxy) acetamide on Osteoclast Differentiation In Vitro via the Downregulation of TRAF6.

Osteoclasts are poly-nuclear cells that resorb mineral components from old or damaged bone tissue. Primary mononuclear cells are activated by receptor activator of nuclear factor kappa-Β ligand (RANKL) and differentiate into large multinucleated cells. Dysregulation of osteoclast differentiation can lead to pathological bone loss and destruction. Many studies have focused on the development of new molecules to regulate RANKL-mediated signaling. In this study, N-[2-(4-acetyl-1-piperazinyl)phenyl]-2-(2-chlorophenoxy) acetamide (PPOA-N-Ac-2-Cl) led to a significant decrease in the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells in a dose-dependent manner, without inducing significant cytotoxicity. PPOA-N-Ac-2-Cl affected the expression of osteoclast-specific marker genes, such as TRAF6, c-fos, DC-STAMP, NFATc1, MMP9, CtsK, and TRAP (Acp5), during RANKL-mediated osteoclastogenesis. Moreover, PPOA-N-Ac-2-Cl significantly attenuated the protein levels of CtsK, a critical protease involved in bone resorption. Accordingly, bone resorption activity and F-actin ring formation decreased in the presence of PPOA-N-Ac-2-Cl. In conclusion, this study shows that PPOA-N-Ac-2-Cl acts as an inhibitor of osteoclast differentiation and may serve as a potential candidate agent for the treatment of osteoclast-related bone diseases by virtue of attenuating bone resorption.

PSTP-3,5-Me Inhibits Osteoclast Differentiation and Bone Resorption.

Osteogenesis is an orchestrated process regulated by osteoclastogenesis and osteoblastogenesis. Excessive osteoclastogenesis causes bone diseases, such as osteoporosis. Although a few drugs are effective in osteoporosis treatment, these drugs lead to side effects, including cellulitis, flatulence, and hypocalcemia. In this study, we reported a 2-(N-Phenylmethylsulfonamido)-N-(2-(phenylthio)phenyl)propanamide (PSTP) compound, PSTP-3,5-Me, as a potential therapeutic agent for osteoporosis. Mouse bone marrow-derived macrophages (BMMs) were differentiated into osteoclasts by receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in the presence of PSTP-3,5-Me. PSTP-3,5-Me inhibited osteoclast differentiation by reduced tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and suppressed the expression of osteoclast marker genes, such as cathepsin K (Ctsk) and TRAP (Acp5). We investigated signaling pathways mediated by RANKL and its receptor, RANK, and found that PSTP-3,5-Me inhibits nucleus translocation of nuclear factor of activated T cell cytoplasmic-1 (NFATc1). Moreover, PSTP-3,5-Me inhibited F-actin ring formation and mineral resorption. Overall, our data suggests that PSTP-3,5-Me attenuates osteoclast differentiation by blocking the activation of NFATc1.

Influence of physicochemical characteristics of flour on pancake quality attributes.

This study was conducted to identify the influence of flour characteristics of 13 Korean wheat cultivars on quality attributes of pancake. Pancake diameter showed negative correlation with SRC with distilled water and pancake height and positive correlation with springiness of cooked pancake. Springiness of baked pancake was under negative correlation with protein content, SDSS, dry gluten content, and positive correlation with final viscosity and setback in pasting properties of flour. Springiness and cohesiveness of baked pancake were under negative correlation with mixing time of Mixograph. Hardness of baked pancake was correlated with amylose content and breakdown of flour. Regression and principal component analysis indicated that pancake diameter, and springiness and cohesiveness of baked pancake can be explained by protein quantity and quality parameters, protein content, SDSS, mixing time of Mixograph, and SRCs related to protein content. Hardness of baked pancake can be predicted from amylose content and breakdown of flour. Batter viscosity as well as texture of cooked pancake could be influenced by protein quality and quantity according to grain hardness.

Geographic Differences in the Contribution of ubiA Mutations to High-Level Ethambutol Resistance in Mycobacterium tuberculosis.

Ethambutol (EMB) resistance can evolve through a multistep process, and mutations in the ubiA (Rv3806c) gene appear to be responsible for high-level EMB resistance in Mycobacterium tuberculosis We evaluated the prevalence of ubiA and embB (Rv3795) mutations in EMB-resistant strains originating from Africa and South Korea. No differences in embB mutation frequencies were observed between strains from both origins. However, ubiA mutations were present in 45.5% ± 6.5% of the African EMB-resistant isolates but in only 9.5% ± 1.5% of the South Korean EMB-resistant isolates. The ubiA mutations associated with EMB resistance were localized to regions encoding the transmembrane domains of the protein, whereas the embB mutations were localized to regions encoding the extramembrane domains. Larger studies are needed to investigate the causes of increased ubiA mutations as a pathway to high-level EMB resistance in African countries, such as extended EMB usage during tuberculosis treatment.

Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging.

Successful development of ultra-sensitive molecular imaging nanoprobes for the detection of targeted biological objects is a challenging task. Although magnetic nanoprobes have the potential to perform such a role, the results from probes that are currently available have been far from optimal. Here we used artificial engineering approaches to develop innovative magnetic nanoprobes, through a process that involved the systematic evaluation of the magnetic spin, size and type of spinel metal ferrites. These magnetism-engineered iron oxide (MEIO) nanoprobes, when conjugated with antibodies, showed enhanced magnetic resonance imaging (MRI) sensitivity for the detection of cancer markers compared with probes currently available. Also, we successfully visualized small tumors implanted in a mouse. Such high-performance, nanotechnology-based molecular probes could enhance the ability to visualize other biological events critical to diagnostics and therapeutics.

Characterisation of runs of homozygosity and inbreeding coefficients in the red-brown Korean native chickens.

OBJECTIVE: The analysis of runs of homozygosity (ROH) has been applied to assess the level of inbreeding and identify selection signatures in various livestock species. The objectives of this study were to characterize the ROH pattern, estimate the rate of inbreeding, and identify signatures of selection in the red-brown Korean native chickens. METHODS: The Illumina 60K single nucleotide polymorphism chip data of 651 chickens was used in the analysis. Runs of homozygosity were analysed using the PLINK v1.9 software. Inbreeding coefficients were estimated using the GCTA software and their correlations were examined. Genomic regions with high levels of ROH were explored to identify selection signatures. RESULTS: A total of 32,176 ROH segments were detected in this study. The majority of the ROH segments were shorter than 4 Mb. The average ROH inbreeding coefficients (FROH) varied with the length of ROH segments. The means of inbreeding coefficients calculated from different methods were also variable. The correlations between different inbreeding coefficients were positive and highly variable (r = 0.18-1). Five ROH islands harbouring important quantitative trait loci were identified. CONCLUSION: This study assessed the level of inbreeding and patterns of homozygosity in Red-brown native Korean chickens. The results of this study suggest that the level of recent inbreeding is low which indicates substantial progress in the conservation of red-brown Korean native chickens. Additionally, Candidate genomic regions associated with important production traits were detected in homozygous regions.

Association of HNMT gene polymorphisms with carnosine content in red-brown Korean native chickens.

Objective: Carnosine and anserine affect the meat flavor. The contents of carnosine and anserine in meat are affected by genetic and environmental factors. This study aimed to discover the single-nucleotide polymorphisms (SNPs) in the HNMT and HNMT-like genes and to associate them with the content of carnosine and anserine in Korean native chicken-red brown line (KNC-R ). Methods: This study used a total of 384 birds (males, n=192; females, n=192) aged 10 weeks old, for genotyping HNMT and HNMT-like genes. One synonymous SNP (rs29009298C/T) of the HNMT gene was genotyped by PCR-RFLP methods whereas four missense SNPs (rs734406537G/A; rs736514667A/G; rs15881680G/A and rs316765035T/C) of the HNMT gene, and one missense SNP rs737657949A/C of the HNMT-like gene were genotyped by PACE genotyping technology. Two-way ANOVA of the R program was used to associate HNMT genotypes with the contents of carnosine and anserine in KNC- R chickens. Results: There were significant associations (p<0.05) between the genotypes of the synonymous SNP:rs29009298C/T, missense SNP rs736514667A/G of the HNMT gene and the content of carnosine in KNC-Rs. This study also reported the sex effect on the carnosine content, where females had more content of carnosine compared to that of male KNC-R. Conclusion: Two SNPs (synonymous: rs735769522C/T) and missense: rs736514667A/G) in the HNMT gene might be used as genetic markers in the selection and breeding of chickens with better taste and high-flavored meat.

Genome-wide association studies of anserine and carnosine contents in the breast meat of Korean native chickens.

Histidine-containing dipeptides (HCDs), such as anserine and carnosine, are enormously beneficial to human health and contribute to the meat flavor in chickens. Meat quality traits, including flavor, are polygenic traits with medium to high heritability. Polygenic traits can be improved through a better understanding of their genetic mechanisms. Genome-wide association studies (GWAS) constitute an effective genomic tool to identify the significant single-nucleotide polymorphisms (SNPs) and potential candidate genes related to various traits of interest in chickens. This study identified potential candidate genes influencing the anserine and carnosine contents in chicken meat through GWAS. We performed GWAS of anserine and carnosine using the Illumina chicken 60K SNP chip (Illumina Inc., San Diego, CA) in 637 Korean native chicken-red-brown line (KNC-R) birds consisting of 228 males and 409 females. The contents of anserine and carnosine in breast meat of KNC-R chickens were investigated. The mean value of the anserine and carnosine are 29.12 mM/g and 10.69 mM/g respectively. The genomic heritabilities were moderate (0.24) for anserine and high (0.43) for carnosine contents. Four and nine SNPs were significantly (P < 0.05) associated with anserine and carnosine, respectively. Based on the GWAS result, the 30.6 to 31.9 Mb region on chicken chromosome 7 was commonly associated with both anserine and carnosine. Through the functional annotation analysis, we identified HNMT and HNMT-like genes as potential candidate genes associated with both anserine and carnosine. The results presented here will contribute to the ongoing improvement of meat quality to satisfy current consumer demands, which are based on healthier, better-flavored, and higher-quality chicken meat.

Major histocompatibility complex genes exhibit a potential immunological role in mixed Eimeria-infected broiler cecum analyzed using RNA sequencing.

OBJECTIVE: This study was conducted to investigate the differential expression of the major histocompatibility complex (MHC) gene region in Eimeria-infected broiler. METHODS: We profiled gene expression of Eimeria-infected and uninfected ceca of broilers sampled at 4, 7, and 21 days post-infection (dpi) using RNA sequencing. Differentially expressed genes (DEGs) between two sample groups were identified at each time point. DEGs located on chicken chromosome 16 were used for further analysis. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis was conducted for the functional annotation of DEGs. RESULTS: Fourteen significant (false discovery rate <0.1) DEGs were identified at 4 and 7 dpi and categorized into three groups: MHC-Y class I genes, MHC-B region genes, and non-MHC genes. In Eimeria-infected broilers, MHC-Y class I genes were upregulated at 4 dpi but downregulated at 7 dpi. This result implies that MHC-Y class I genes initially activated an immune response, which was then suppressed by Eimeria. Of the MHC-B region genes, the DMB1 gene was upregulated, and TAP-related genes significantly implemented antigen processing for MHC class I at 4 dpi, which was supported by KEGG pathway analysis. CONCLUSION: This study is the first to investigate MHC gene responses to coccidia infection in chickens using RNA sequencing. MHC-B and MHC-Y genes showed their immune responses in reaction to Eimeria infection. These findings are valuable for understanding chicken MHC gene function.

Identification of new major histocompatibility complex-B Haplotypes in Bangladesh native chickens.

OBJECTIVE: The major histocompatibility complex in chicken demonstrates a great range of variations within varities, breeds, populations and that can eventually influence their immuneresponses. The preset study was conducted to understand the major histocompatibility complex-B (MHC-B) variability in five major populations of Bangladesh native chicken: Aseel, Hilly, Junglefowl, Non-descript Deshi, and Naked Neck. METHODS: These five major populations of Bangladesh native chicken were analyzed with a subset of 89 single nucleotide polymorphisms (SNPs) in the high-density MHC-B SNP panel and Kompetitive Allele-Specific polymerase chain reaction genotyping was applied. To explore haplotype diversity within these populations, the results were analyzed both manually and computationally using PHASE 2.1 program. The phylogenetic investigations were also performed using MrBayes program. RESULTS: A total of 136 unique haplotypes were identified within these five Bangladesh chicken populations, and only one was shared (between Hilly and Naked Neck). Phylogenetic analysis showed no distinct haplotype clustering among the five populations, although they were shared in distinct clades; notably, the first clade lacked Naked Neck haplotypes. CONCLUSION: The present study discovered a set of unique MHC-B haplotypes in Bangladesh chickens that could possibly cause varied immune reponses. However, further investigations are required to evaluate their relationships with global chicken populations.

Lrrc10 is a novel cardiac-specific target gene of Nkx2-5 and GATA4.

Cardiac gene expression is precisely regulated and its perturbation causes developmental defects and heart disease. Leucine-rich repeat containing 10 (Lrrc10) is a cardiac-specific factor that is crucial for proper cardiac development and deletion of Lrrc10 in mice results in dilated cardiomyopathy. However, the mechanisms regulating Lrrc10 expression in cardiomyocytes remain unknown. Therefore, we set out to determine trans-acting factors and cis-elements critical for mediating Lrrc10 expression. We identify Lrrc10 as a transcriptional target of Nkx2-5 and GATA4. The Lrrc10 promoter region contains two highly conserved cardiac regulatory elements, which are functional in cardiomyocytes but not in fibroblasts. In vivo, Nkx2-5 and GATA4 endogenously occupy the proximal and distal cardiac regulatory elements of Lrrc10 in the heart. Moreover, embryonic hearts of Nkx2-5 knockout mice have dramatically reduced expression of Lrrc10. These data demonstrate the importance of Nkx2-5 and GATA4 in regulation of Lrrc10 expression in vivo. The proximal cardiac regulatory element located at around -200bp is synergistically activated by Nkx2-5 and GATA4 while the distal cardiac regulatory element present around -3kb requires SRF in addition to Nkx2-5 and GATA4 for synergistic activation. Mutational analyses identify a pair of adjacent Nkx2-5 and GATA binding sites within the proximal cardiac regulatory element that are necessary to induce expression of Lrrc10. In contrast, only the GATA site is functional in the distal regulatory element. Taken together, our data demonstrate that the transcription factors Nkx2-5 and GATA4 cooperatively regulate cardiac-specific expression of Lrrc10.