Macrophages-derived exosomal lncRNA LIFR-AS1 promotes osteosarcoma cell progression via miR-29a/NFIA axis

Abstract Background Osteosarcoma (OS) is the most common primary malignant bone tumor in young people. Tumor-associated macrophages (TAMs) have been reported to play an important role in the development of osteosarcoma. However, the detailed molecular mechanisms remain largely unknown and need to be elucidated. Recently, exosomes have been reported as the crucial mediator between tumor cells and the tumor microenvironment. And a lot of lncRNAs have been reported to act as either oncogenes or tumor suppressors in osteosarcoma. In this research, we aim to explore the role of macrophages-derived exosomal lncRNA in osteosarcoma development and further elucidated the potential molecular mechanisms involved. Methods TAMs were differentiated from human mononuclear cells THP-1, and a high-throughput microarray assay was used to analyze the dysregulated lncRNAs and miRNAs in osteosarcoma cells co-cultured with macrophages-derived exosomes. Western blot, qRT-PCR assays, and Dual-luciferase reporter assay were used to verify the interaction among LIFR-AS1, miR-29a, and NFIA. Cck-8, EdU, colony formation assay, wound-healing, and transwell assay were performed to explore the characterize the proliferation and metastasis ability of OS cells. And qPCR, Western blots, immunohistochemistry, and cell immunofluorescence were used to detect the expression of relative genes or proteins. Results In this study, we found that THP-1-induced macrophage-derived exosomes could facilitate osteosarcoma cell progression both in vitro and in vivo. Then, the results of the high-throughput microarray assay showed that LIFR-AS1 was highly expressed and miR-29a was lowly expressed. Furthermore, LIFR-AS1 was identified as a miR-29a sponge, and NFIA was validated as a direct target of miR-29a. Functional assays demonstrated that knockdown of exosomal LIFR-AS1 could attenuate the promotion effects of macrophages-derived exosomes on osteosarcoma cell progression and miR-29a inhibition could reserve the effect of LIFR-AS1-knockdown exosomes. Correspondingly, NFIA-knockdown could partially reverse the tumor inhibition effect of miR-29a on osteosarcoma cells. Conclusions Taken together, macrophages-derived exosomal lncRNA LIFR-AS1 can promote osteosarcoma cell proliferation, invasion, and restrain cell apoptosis via miR-29a/NFIA axis, which can act as a potential novel therapeutic target for osteosarcoma therapy.

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LncRNA SNHG15 contributes to doxorubicin resistance of osteosarcoma cells through targeting the miR-381-3p/GFRA1 axis

Open Life Sciences ◽

10.1515/biol-2020-0086 ◽

2020 ◽

Vol 15 (1) ◽

pp. 871-883

Author(s):

Jinshan Zhang ◽

Dan Rao ◽

Haibo Ma ◽

Defeng Kong ◽

Xiaoming Xu ◽

...

Keyword(s):

Cell Lines ◽

Noncoding Rna ◽

Xenograft Model ◽

Inhibitory Effect ◽

Cell Counting ◽

Luciferase Reporter ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells

AbstractBackgroundOsteosarcoma is a common primary malignant bone cancer. Long noncoding RNA small nucleolar RNA host gene 15 (SNHG15) has been reported to play an oncogenic role in many cancers. Nevertheless, the role of SNHG15 in the doxorubicin (DXR) resistance of osteosarcoma cells has not been fully addressed.MethodsCell Counting Kit-8 assay was conducted to measure the half-maximal inhibitory concentration value of DXR in osteosarcoma cells. Western blotting was carried out to examine the levels of autophagy-related proteins and GDNF family receptor alpha-1 (GFRA1). Quantitative reverse transcription-polymerase chain reaction was performed to determine the levels of SNHG15, miR-381-3p, and GFRA1. The proliferation of osteosarcoma cells was measured by MTT assay. The binding sites between miR-381-3p and SNHG15 or GFRA1 were predicted by Starbase bioinformatics software, and the interaction was confirmed by dual-luciferase reporter assay. Murine xenograft model was established to validate the function of SNHG15 in vivo.ResultsAutophagy inhibitor 3-methyladenine sensitized DXR-resistant osteosarcoma cell lines to DXR. SNHG15 was upregulated in DXR-resistant osteosarcoma tissues and cell lines. SNHG15 knockdown inhibited the proliferation, DXR resistance, and autophagy of osteosarcoma cells. MiR-381-3p was a direct target of SNHG15, and GFRA1 bound to miR-381-3p in osteosarcoma cells. SNHG15 contributed to DXR resistance through the miR-381-3p/GFRA1 axis in vitro. SNHG15 depletion contributed to the inhibitory effect of DXR on osteosarcoma tumor growth through the miR-381-3p/GFRA1 axis in vivo.ConclusionsSNHG15 enhanced the DXR resistance of osteosarcoma cells through elevating the autophagy via targeting the miR-381-3p/GFRA1 axis. Restoration of miR-381-3p expression might be an underlying therapeutic strategy to overcome the DXR resistance of osteosarcoma.

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Exosomal miR-199a-5p Inhibits Tumorigenesis and Angiogenesis by Targeting VEGFA in Osteosarcoma

10.21203/rs.3.rs-912512/v1 ◽

2021 ◽

Author(s):

Lu Zhang ◽

Hongxin Cao ◽

Guanghui Gu ◽

Dehui Hou ◽

Yunhao You ◽

...

Keyword(s):

Molecular Mechanisms ◽

Target Genes ◽

Enrichment Analysis ◽

Luciferase Reporter ◽

Pathway Enrichment Analysis ◽

Osteosarcoma Cell ◽

Plasma Samples ◽

Osteosarcoma Cells ◽

Hub Genes

Abstract Background: Osteosarcoma (OS) is the most common primary bone malignancy in children and adolescents. microRNAs have been found to play a vital role in tumor angiogenesis. Here, we investigated the effects of miR-199a-5p on tumor growth and angiogenesis in osteosarcoma. Furthermore, the underlying molecular mechanisms and signaling pathways were explored.Methods: The datasets were extracted from the Gene Expression Omnibus and the differentially expressed miRNAs (DEmiRNAs) were screened out by the GEO2R online platform. The potential target genes were predicted using the miRTarBase database. The predicted target genes were further analyzed by Gene Ontology and pathway enrichment analysis and a regulatory network of DEmiRNAs and their target genes was constructed. In addition, the effects of osteosarcoma cell derived exosomal miR-199a-5p on the proliferation, migration and neovascularization of HUVECs were evaluated by conducting EdU assays, Transwell experiments and tube formation assays. A dual-luciferase reporter assay was performed to detect whether VEGFA was the direct target of miR-199a-5p. Furthermore, in vivo xenograft models were established to further investigate the intrinsic role of miR-199a-5p in osteosarcoma tumorigenesis and angiogenesis. Results: A total of 149 DE-miRNAs were screened out, including 136 upregulated miRNAs and 13 downregulated miRNAs in human osteosarcoma plasma samples compared with normal plasma samples. A total of 1313 target genes of the top three upregulated and downregulated miRNAs were predicted. In the PPI network, the top 10 hub nodes with higher degrees were identified as hub genes, such as TP53 and VEGFA. By constructing the miRNA-hub gene network, we found that most of hub genes could be potentially modulated by miR-663a, miR-199a-5p and miR-223-3p. In addition, we found that the expression level of miR-199a-5p in exosomes derived from osteosarcoma cells was remarkably higher than the osteosarcoma cells, and the exosomes derived from osteosarcoma cells were transported to HUVECs. Overexpression of miR-199a-5p could significantly inhibited HUVEC proliferation, migration and neovascularization, whereas downregulation of miR-199a-5p expression exerted the opposite effect. Moreover, the in vivo results verified that overexpression of miR-199a-5p in osteosarcoma cells could suppress the growth and angiogenesis of tumors. Conclusion: Our results demonstrated that miR-199a-5p could be transported from osteosarcoma cells to HUVECs through exosomes, subsequently targeting VEGFA and inhibiting the growth and angiogenesis of osteosarcoma. Therefore, miR-199a-5p may act as a biomarker in the diagnosis and treatment of osteosarcoma.

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The proliferation and invasion of osteosarcoma are inhibited by miR-101 via targetting ZEB2

Bioscience Reports ◽

10.1042/bsr20181283 ◽

2019 ◽

Vol 39 (2) ◽

Cited By ~ 5

Author(s):

Haopeng Lin ◽

Xiaodong Zheng ◽

Ting Lu ◽

Yang Gu ◽

Canhao Zheng ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Molecular Mechanisms ◽

Bone Cell ◽

Luciferase Reporter ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Human Osteosarcoma ◽

Direct Target ◽

Proliferation And Invasion

AbstractHaving a better grasp of the molecular mechanisms underlying carcinogenesis and progression in osteosarcoma would be helpful to find novel therapeutic targets. Different types of cancers have presented abnormal expression of miRNA-101 (miR-101). Nevertheless, we still could not figure out what expression of miR-101 in human osteosarcoma is and its biological function. Thus, we conducted the present study to identify its expression, function, and molecular mechanism in osteosarcoma. We detected the expression of miR-101 in osteosarcoma samples and cell lines. The effects of miR-101 on osteosarcoma cells’ proliferation and invasion were evaluated. Luciferase reporter assay was applied to identify the direct target of miR-101. Compared with adjacent normal specimens and normal bone cell line by using qPCR, the expression levels of miR-101 in osteosarcoma specimens and human osteosarcoma cell lines distinctly decreased. According to function assays, we found that overexpression of miR-101 significantly inhibited the cell proliferation and invasion in osteosarcoma cells. Moreover, we confirmed that zinc finger E-box binding homeobox 2 (ZEB2) was a direct target of miR-101. In addition, overexpression of ZEB2 could rescue the inhibition effect of proliferation and invasion induced by miR-101 in osteosarcoma cells. MiR-101 has been proved to be down-regulated in osteosarcoma and has the ability to suppress osteosarcoma cell proliferation and invasion by directly targetting ZEB2.

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Silencing Herg1 by shRNA inhibits the proliferation and invasion of osteosarcoma in vivo and in vitro

10.21203/rs.3.rs-331716/v1 ◽

2021 ◽

Author(s):

Zhida Chen ◽

Chao Song ◽

Yunping Chen ◽

Lili Xiao ◽

Yuanjie Jiang ◽

...

Keyword(s):

Transcriptional Activation ◽

Nuclear Translocation ◽

Affinity Purification ◽

Luciferase Reporter ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Nude Mouse Model ◽

Proliferation And Invasion

Abstract Background: Osteosarcoma is the most common malignant bone tumor of childhood, and improvements in the survival rates have reached a plateau phase. This study was aimed to explore the the importance of the human ether-a-go-go-related potassium channel (Herg1) in the proliferation and invasion of osteosarcoma.Methods: The effects of Herg1 silenced on osteosarcoma cell proliferation and invasion were detected by were detected by CCK-8 assay, wound healing assay and transwell assay, respectively. Tandem affinity purification, mass spectrometry and Dual luciferase reporter assay were used to find out possible molecules responsible for the action of Herg1 on osteosarcoma cells. Nude mouse model was used to investigate the in vivo functions of Herg1. Results: Herg1 silencing by shRNA significantly suppressed the proliferation and invasion of osteosarcoma cells in vitro as well as tumorigenicity and metastasis in nude mice. Moreover, Herg1 promoted osteosarcoma progression via turning off Hippo signaling pathway, leading to the activation of Yes-associated protein (YAP). Mechanistically, Herg1 co-localized and interacted with NF2 (also called Merlin), and this interaction probably caused the de-phosphorylation of MST1/2 and LATS1/2, which drove YAP nuclear translocation and transcriptional activation. Conclusion: Herg1 acts as an oncogene in osteosarcoma and may therefore serve as a useful therapeutic target for osteosarcoma patients.

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Long non-coding RNA PGM5-AS1 modulates epithelial-mesenchymal transition and invasion and metastasis of osteosarcoma cells by impairing miR-140-5p-mediated FBN1 inhibition

10.21203/rs.2.19800/v1 ◽

2019 ◽

Author(s):

Wei Liu ◽

Pengcheng Liu ◽

Hang Gao ◽

Xu Wang ◽

Ming Yan

Keyword(s):

Epithelial Mesenchymal Transition ◽

Differentially Expressed ◽

Luciferase Reporter ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Luciferase Reporter Gene ◽

Mesenchymal Transition ◽

Therapeutic Modalities ◽

Gene Assay

Abstract Background Osteosarcoma represents a uncommon tumor occurring in bone possessing elevated rate of incidence and reduced rate of healing. Hence, developing effective therapeutic modalities to treat osteosarcoma is essential. Various long non-coding RNAs (lncRNAs) are engaged in prognostic and diagnostic processes of diseases. Thus, we identified the molecular mechanism of lncRNA PGM5-AS1 in progression of osteosarcoma.Methods Microarray-based analysis was employed to screen the osteosarcoma-related differentially expressed lncRNA. PGM5-AS1 level in osteosarcoma tissues and cells as well as the levels of microRNA-140-5p (miR-140-5p) and fibrillin-1 (FBN1) in osteosarcoma tissues were determined. Dual-luciferase reporter gene assay, RNA pull-down assay, and RNA immunoprecipitation assay were done to find out the relation among PGM5-AS1, miR-140-5p, and FBN1. shRNA, mimic, or inhibitor was applied to alter the levels of PGM5-AS1, miR-140-5p, and FBN1 in osteosarcoma cells to investigate how they regulated migration, invasion, and epithelial-mesenchymal transition (EMT) of osteosarcoma cells in vitro. The nude mice were injected with osteosarcoma cell line with depleted PGM5-AS1 to detect the role of PGM5-AS1 in tumorigenesis in vivo.Results PGM5-AS1 possessing elevated level in osteosarcoma was identified as the differentially expressed lncRNA in osteosarcoma. Osteosarcoma tissues exhibited low miR-140-5p expression and high FBN1 expression. Results confirmed that PGM5-AS competitively bound to miR-140-5p to upregulate FBN1. Also, hindering PGM5-AS1 and FBN1 or enforcing miR-140-5p dampened migration, invasion, and EMT of osteosarcoma cells in vitro. Furthermore, silencing PGM5-AS1 markedly inhibited tumorigenesis in vivo.Conclusion All in all, PGM5-AS1 depletion causes FBN1 reduction to retard osteosarcoma processes by negatively modulating miR-140-5p, highlighting PGM5-AS1 silencing as a biomarker for osteosarcoma management.

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Platelet-derived lysophosphatidic acid mediated LPAR1 activation as a therapeutic target for osteosarcoma metastasis

Oncogene ◽

10.1038/s41388-021-01956-6 ◽

2021 ◽

Author(s):

Satoshi Takagi ◽

Yuki Sasaki ◽

Sumie Koike ◽

Ai Takemoto ◽

Yosuke Seto ◽

...

Keyword(s):

Platelet Activation ◽

Pulmonary Metastasis ◽

Lysophosphatidic Acid ◽

Therapeutic Intervention ◽

Molecular Mechanisms ◽

Morphological Changes ◽

Osteosarcoma Cells ◽

Invasion And Metastasis ◽

Platelet Releasate

AbstractOsteosarcoma is the most common primary malignant bone cancer, with high rates of pulmonary metastasis. Osteosarcoma patients with pulmonary metastasis have worse prognosis than those with localized disease, leading to dramatically reduced survival rates. Therefore, understanding the biological characteristics of metastatic osteosarcoma and the molecular mechanisms of invasion and metastasis of osteosarcoma cells will lead to the development of innovative therapeutic intervention for advanced osteosarcoma. Here, we identified that osteosarcoma cells commonly exhibit high platelet activation-inducing characteristics, and molecules released from activated platelets promote the invasiveness of osteosarcoma cells. Given that heat-denatured platelet releasate maintained the ability to promote osteosarcoma invasion, we focused on heat-tolerant molecules, such as lipid mediators in the platelet releasate. Osteosarcoma-induced platelet activation leads to abundant lysophosphatidic acid (LPA) release. Exposure to LPA or platelet releasate induced morphological changes and increased invasiveness of osteosarcoma cells. By analyzing publicly available transcriptome datasets and our in-house osteosarcoma patient-derived xenograft tumors, we found that LPA receptor 1 (LPAR1) is notably upregulated in osteosarcoma. LPAR1 gene KO in osteosarcoma cells abolished the platelet-mediated osteosarcoma invasion in vitro and the formation of early pulmonary metastatic foci in experimental pulmonary metastasis models. Of note, the pharmacological inhibition of LPAR1 by the orally available LPAR1 antagonist, ONO-7300243, prevented pulmonary metastasis of osteosarcoma in the mouse models. These results indicate that the LPA–LPAR1 axis is essential for the osteosarcoma invasion and metastasis, and targeting LPAR1 would be a promising therapeutic intervention for advanced osteosarcoma.

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Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18073332 ◽

2021 ◽

Vol 18 (7) ◽

pp. 3332

Author(s):

Olga V. Naidenko ◽

David Q. Andrews ◽

Alexis M. Temkin ◽

Tasha Stoiber ◽

Uloma Igara Uche ◽

...

Keyword(s):

Immune System ◽

High Throughput ◽

Environmental Protection Agency ◽

High Throughput Screening ◽

Molecular Mechanisms ◽

Specific Activity ◽

Laboratory Animals ◽

In Vitro Assays ◽

Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

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miR-182-5p and miR-378a-3p regulate ferroptosis in I/R-induced renal injury

Cell Death and Disease ◽

10.1038/s41419-020-03135-z ◽

2020 ◽

Vol 11 (10) ◽

Author(s):

Chenguang Ding ◽

Xiaoming Ding ◽

Jin Zheng ◽

Bo Wang ◽

Yang Li ◽

...

Keyword(s):

Cell Death ◽

Renal Injury ◽

Molecular Mechanisms ◽

Ischemia Reperfusion ◽

Kidney Injury ◽

Luciferase Reporter ◽

Renal Tubular Cell ◽

In Cells

Abstract Renal tubular cell death is the key factor of the pathogenesis of ischemia/reperfusion (I/R) kidney injury. Ferroptosis is a type of regulated cell death (RCD) found in various diseases. However, the underlying molecular mechanisms related to ferroptosis in renal I/R injury remain unclear. In the present study, we investigated the regulatory role of microRNAs on ferroptosis in I/R-induced renal injury. We established the I/R-induced renal injury model in rats, and H/R induced HK-2 cells injury in vitro. CCK-8 was used to measure cell viability. Fe2+ and ROS levels were assayed to evaluate the activation of ferroptosis. We performed RNA sequencing to profile the miRNAs expression in H/R-induced injury and ferroptosis. Western blot analysis was used to detect the protein expression. qRT-PCR was used to detect the mRNA and miRNA levels in cells and tissues. We further used luciferase reporter assay to verify the direct targeting effect of miRNA. We found that ischemia/reperfusion-induced ferroptosis in rat’s kidney. We identified that miR-182-5p and miR-378a-3p were upregulated in the ferroptosis and H/R-induced injury, and correlates reversely with glutathione peroxidases 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression in renal I/R injury tissues, respectively. In vitro studies showed that miR-182-5p and miR-378a-3p induced ferroptosis in cells. We further found that miR-182-5p and miR-378a-3p regulated the expression of GPX4 and SLC7A11 negatively by directly binding to the 3′UTR of GPX4 and SLC7A11 mRNA. In vivo study showed that silencing miR-182-5p and miR-378a-3p alleviated the I/R-induced renal injury in rats. In conclusion, we demonstrated that I/R induced upregulation of miR-182-5p and miR-378a-3p, leading to activation of ferroptosis in renal injury through downregulation of GPX4 and SLC7A11.

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Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽

10.1161/str.52.suppl_1.p780 ◽

2021 ◽

Vol 52 (Suppl_1) ◽

Author(s):

Francisco J Gonzalez-Gonzalez ◽

Perike Srikanth ◽

Andrielle E Capote ◽

Alsina Katherina M ◽

Benjamin Levin ◽

...

Keyword(s):

Atrial Fibrillation ◽

Light Chain ◽

Myosin Light Chain ◽

Molecular Mechanisms ◽

Contractile Function ◽

Right Atrial ◽

Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

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Abstract P458: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atrial Hypocontractility In Atrial Fibrillation

Circulation Research ◽

10.1161/res.129.suppl_1.p458 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Francisco J Gonzalez-Gonzalez ◽

Srikanth Perike ◽

Frederick Damen ◽

Andrielle Capote ◽

Katherina M Alsina ◽

...

Keyword(s):

Atrial Fibrillation ◽

Light Chain ◽

Myosin Light Chain ◽

Molecular Mechanisms ◽

Contractile Function ◽

Right Atrial ◽

Western Blots

Introduction: Atrial fibrillation (AF), is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 2.7 million to 6.1 million and is predictive to increase to 12.1 million in 2030. AF increases the chances of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. Objective: The overexpression of PPP1R12C, causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), decreasing atrial contractility. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluated the role of PP1c-PPP1R12C interaction in MLC2a de-phosphorylation we used Western blots, coimmunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The Overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, that cause a reduction in atrial contractility and increases AF inducibility. All these discoveries advocate that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

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