Abstract Background: Targeting rare cell types such as circulating tumor cells (CTCs), considered to be metastatic precursors in cancer progression, may provide long-term clinical benefits by enhancing patient outcomes. The study of CTCs comes with technical challenges due to their heterogeneity and low cell numbers, leading to difficulties in genomic characterization. This study aims to overcome these technical challenges by developing a comprehensive gene expression profiling workflow using qPCR and RNA sequencing (RNA-Seq) on rare cells enriched with Genesis System with Celselect SlidesTM. Methods: To mimic CTCs in blood, we created a rare cell model sample by spiking A549 non-small cell lung cancer cells at concentrations of 50, 100, 500 and 1000 cells/ml in whole blood from healthy donors. The Genesis System, which can capture rare cells of 8µm-30µm, isolated 83.73% of spiked A549 cells in whole blood. Using the same blood samples without A549 spiked in, we enriched cells using the Genesis and it was used as a baseline for gene expression controls. High quality RNA was extracted from each of the enriched samples and used for both qPCR and RNA-Seq to detect the gene expression signature of the non-small cell lung cancer cells. Results: Our analyses revealed significant differential expression of cancer-related genes (p < 0.05) in all A549-spiked samples when compared to the blood-only controls. Notably, in the 50 cells/ml dilution, RNA-Seq analysis identified over 3,000 differentially expressed genes, encompassing protein-coding genes, long non-coding RNAs, and small non-coding RNAs. Among these, 759 genes were consistently present across all enriched samples, suggesting a common expression pattern. To investigate the potential utility of these genes as non-small cell lung cancer biomarkers, we constructed a custom qPCR array comprising 38 of these genes and pre-amplified their targets using 50pg of RNA from the enriched cell samples. Notably, six genes from this panel (MET, TOP2A, NF1, SPP1, GAPDH, and ANXA5) exhibited significant differential expression (+/- 4-fold, p<0.05) in the enriched samples. MET and TOP2A, in particular, are well-known biomarkers for non-small cell lung cancer. Conclusion: Our liquid biopsy workflow demonstrates the recovery of high-quality RNA from enriched rare cells, including CTCs. It enables comprehensive profiling of the tumor transcriptome, encompassing RNAs of various sizes, including miRNAs. Our approach has the potential to facilitate the identification of patient-specific biomarkers, enabling swift monitoring using preamplification and qPCR in advancement of targeted oncology therapy and research. Citation Format: Srikanth Perike, Angelica Olcott, Cissy Jiang, Nish Kumar, Nathan Knapp, Jennifer Placek, Candice Cox, Yoon-Tae Kang, Linda Lingelbach, Elizabeth Dreskin. Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7501.

Background: Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods: Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results: In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls with > 40% reduction in MLC2a phosphorylation. 12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls, with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusions: AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key regulator of atrial contractility in AF.

BACKGROUND: Atrial fibrillation (AF)—the most common sustained cardiac arrhythmia—increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)—the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)—causes hypophosphorylation of MLC2a and results in atrial hypocontractility. METHODS: Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies. RESULTS: In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls ( P =2.0×10 −2 ; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation ( P =1.4×10 −6 ; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF ( P =2.9×10 −2 and 6.7×10 −3 , respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls ( P =5.0×10 −6 ; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls ( P =1.8×10 −2 and 4.1×10 −2 , respectively; n=6, 6, and 5). CONCLUSIONS: Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Obesity-induced atrial fibrillation (AF) is modulated increased fatty infiltration in the atrial myocardium leading to increased exposure to free fatty acids (FFA). Mass spectrometry studies of obese mice have shown increased saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA) compared to lean controls with the highest increase in the PUFA, linoleic acid. While some reports suggest that some SFAs and MUFAs induce ventricular arrhythmias in mice, the differential electrophysiological effects of SFAs, MUFAs, and PUFAs on the atria remain unclear.

Background: Increased free fatty acids (FFA) and FA infiltration, both major hallmarks of obesity can individually modulate changes leading to AF. For example, mass spectrometry on obese mouse heart tissues have shown that saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA) are all significantly increased compared to controls. The highest increase was shown to be in linoleic acid (LA), a PUFA followed by palmitic acid (PA, SFA) and oleic acid (OA, MUFA). Although reports suggest that some FAs induce ventricular arrhythmias in mice, the differential electrophysiological effects of SFAs, MUFAs, and PUFAs on the atria remain unclear. Objective: To determine the individual roles of SFAs, MUFAs, and PUFAs in mediating atrial remodeling in obesity-induced AF using mature human induced pluripotent stem-derived atrial cardiomyocytes (iPSC-aCMs). Methods: We compared iPSC-aCMs and HL-1 cells treated with bovine serum albumin (BSA) with palmitic acid (PA), oleic acid (OA) and LA cells (600 uM each) for 24 hours and 1 week. Ion channel function were assessed using optical voltage mapping and whole-cell patch clamping, and we assessed atrial remodeling using qPCR, and western blotting. Results: iPSC-aCMs treated with PA, OA, and LA displayed higher expression of FABP3 and CPT1A suggesting increased FA metabolism (Figure A-C). In HL1 cells, protein expression of Cpt1a was increased in all groups after FA treatment (Figure D). PA differentially modulated expression of Kv7.1 and NCX1 in HL1 cells (Figure E-F) and ion channel mRNA expression in iPSC-aCMs (Figure G-L). After 1 week PA and LA treatment b-adrenergic signaling was significantly increased in iPSC-aCMs while OA decreased it compared to controls (Figure M-O). Lastly, 24-hour PA, OA, LA treatment differentially modulated the atrial action potential (AP). While PA and LA significantly shorten the action potential, there was no significant changes to the AP after OA treatment. Conclusions: Using mature iPSC-aCMs, we showed for the first time that SFA, MUFA, and PUFA not only increased FA metabolism but differentially affected b-adrenergic signaling and ion channel remodeling leading to differential effects on the atrial AP. Our findings may have important implications for the management of obesity-induced AF in patients.

Introduction: Diet induced obese (DIO) mice display increased inducible atrial fibrillation (AF) and an overall increase in reactive oxygen species (ROS) production. NADPH oxidase 2 (NOX2), a major source of cytosolic ROS production in human atria, has been implicated in AF independent of obesity and is significantly increased in the atria of DIO mice. Although treatment with MitoTEMPO, a mitochondrial specific antioxidant reduced AF burden in DIO mice, the actual role of NOX2 in increasing ROS production and atrial remodeling in the context of obesity-induced AF remains unclear. Hypothesis: To test the hypothesis that increased NOX2 modulates atrial remodeling in obesity-induced AF, we used control mice, DIO and Nox2 -KO mice fed with a 60% HFD for 10 weeks (DIO-KO) and DIO mice treated with a NOX2 blocker, apocynin (DIO-Apocynin). Methods: Trans-esophageal rapid (TE) pacing was used to look at the AF phenotype. Cellular electrophysiology (EP), Western blotting, whole-cell patch clamping were performed to study ion channel remodeling and ROS production. Results: All three DIO mouse groups displayed significantly greater body weight compared to their respective controls (Figure A) After TE pacing, DIO-Apocynin mice displayed 28.26 ± 25.40 s and DIO-KO mice displayed 17.43 ± 31.80 s compared to 167.3 ± 168.9 s in DIO mice (Figure B). NOX2 inhibition reversed obesity-induced ion channel remodeling of potassium channels such as KCNQ1 and KCNE1 encoding for the IKs current and KCNA5 encoding for the IKur current and also reduced mediators of oxidative stress. (Figure C-M) Lastly, voltage clamp in DIO-KO mice showed that NOX2 inhibition reverses obesity-induced IK current increase. (Figure N-O) Conclusions: Thus, these results prove that antioxidant therapy targeting Nox2 abrogated ion channel remodeling and reversed the obesity-induced AF burden. Our findings show the importance of targeting specific antioxidant pathways to manage the AF in patients with obesity.

Introduction: 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. PPP1R12CPP1c 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 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.

MNPs were prepared with modification of an MRI agent (FerroTrace, Ferronova) currently undergoing a phase 1 clinical trial. In vitro binding was evaluated in PSMA and FAP positive cell lines. To form orthotopic tumours, LNCaP cells (PSMAþ human prostate cancer) were injected into the prostate. After 4-6 weeks of tumour growth, mice underwent 16.4T T2-weighted 3D MRI 24 hours after intravenous injection of contrast agents. Enhancement of MRI contrast in tumours was quantified by determining the proportion of pixels with low signal intensity throughout regions of interest. Accumulation of MNPs in prostate tumours at 24 hours post injection was confirmed ex vivo by Prussian blue staining. RESULTS: PSMA and FAP-MNPs demonstrated specific binding in vitro. MRI contrast of tumours was increased in FAP (p<0.001) and PSMA-MNPs (p<0.05) but not control MNPs (p>0.05), relative to orthotopic tumours with no injected MNPs. FAP-MNPs provided increased contrast relative to PSMA-MNPs (w10% enhancement, p<0.05). Increased accumulation of FAP-MNPs in prostate tumours was observed on tissue sections compared to PSMA-MNPs. Control-MNPs showed minimal tumour accumulation. CONCLUSIONS: These results suggest that FAP-targeting MNPs could enhance the MRI of prostate tumours and assist in the delivery of precise focal treatment.

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.

Background: Inducible atrial fibrillation (AF) in diet-induced obese (DIO) mice is mediated in part by a combined effect of ion channel remodeling and atrial fibrosis. NADPH oxidase 2 (NOX2), a major source of reactive oxygen species (ROS) production in human atria, is increased in DIO mice. Although a mitochondrial antioxidant (MitoTEMPO) reduced AF burden, and reversed ion channel and structural remodeling, the role of NOX2 in increased ROS production and atrial remodeling in obesity-induced AF remains unclear. Objective: To test the hypothesis that increased NOX2 modulates atrial remodeling in obesity-induced AF, we treated DIO mice with apocynin, NOX2 inhibitor, and fed a 60% high fat diet (HFD) to Nox2 -KO mice. Methods: Weight, BP, plasma glucose, trans-esophageal rapid (TE) pacing and F 2 -isoprostanes were measured in DIO mice and compared to controls. Echocardiography, electrophysiology (EP), immunohistochemistry, Western blotting, cellular patch clamping and optical mapping studies were performed. Results: The average weight of DIO mice treated with apocynin (DIO-A) and vehicle control mice was 38.4 ± 3.8 g versus 44.0 ± 7.5 g versus 31.7 ± 1.19 g respectively (P≤0.0001). Both groups of DIO mice displayed progressive increase in weight over 10 weeks of HFD + drug treatment compared to controls (P≤0.0001). After TE pacing, DIO mice treated with apocynin showed significantly reduced pacing-induced AF burden when compared to DIO mice treated with vehicle. DIO mice treated with apocynin displayed 20.2 ± 26.1 sec versus 162.3 ± 133.7 sec and 18.3 ± 18.1 sec in DIO mice treated with vehicle and control mice respectively (P≤0.0001). Western blotting experiments showed that potassium channels, Kv7.1 and Kv1.5 protein expression is restored in Nox2 -KO HFD mice compared to DIO mice thus indicating restoration of ion channel remodeling upon Nox2 inhibition. Conclusions: We showed that genetic and pharmacological inhibition of NOX2 abrogates ion channel remodeling and reverses obesity-induced AF burden. Our findings may have important implications for the management of obesity-mediated AF in patients.

Background: Obesity-induced atrial fibrillation (AF) is modulated in part by increased serum free fatty acids (FFA) and FA infiltration of the heart. Mass spectrometry data on obese mice have shown increased saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA) compared to lean controls with the largest increase in the PUFA, linoleic acid (LA). Although reports suggest that some FAs induce ventricular arrhythmias in mice, the differential electrophysiological (EP) effects of SFAs, MUFAs, and PUFAs on the atria remain unclear. Objective: To determine if SFAs, MUFAs, and PUFAs differentially modulate atrial remodeling using mature human induced pluripotent stem-derived atrial cardiomyocytes (iPSC-aCMs) and HL1 cells. . Methods: We compared iPSC-aCMs and HL-1 cells treated with bovine serum albumin (BSA) with palmitic acid (PA), oleic acid (OA) and LA (600 uM each) for 1 week. Ion channel function was assessed using whole-cell patch clamping and we assessed β-oxidation and atrial remodeling with using ELISA, qPCR, and Western blotting. Results: iPSC-aCMs treated with PA, OA, and LA displayed higher expression of FABP3 , ANGPTL4 , and CPT1A suggesting increased FA metabolism and β-oxidation (Figure A-C). In HL1 cells, protein expression of Cpt1a was increased in all groups after FA treatment (Figure D). PA differentially modulated expression of Kv7.1 and NCX1 in HL1 cells (Figure E-F) and ion channel mRNA expression in iPSC-aCMs (Figure G-L). After 1 week PA and LA treatment β-adrenergic signaling was significantly increased in iPSC-aCMs while OA decreased it compared to controls (Figure M-O). Conclusions: Using mature iPSC-aCMs, we showed for the first time that SFA, MUFA, and PUFA not only increased FA metabolism but also differentially modulated ion channel and structural remodeling and β-adrenergic signaling in the atria. Our findings may have important implications for the management of obesity-induced AF in patients.

INTRODUCTION AND OBJECTIVE:Prostate cancer (PCa) is the most frequently diagnosed malignancy and second leading cause of cancer-specific deaths in men in Western countries. Early stages of PCa can ...

Background: Prostate cancer (PCa) is one of the most frequently diagnosed malignancies in men, and its incidence and mortality continue to be a significant clinical problem. Recent evidence from our lab identified MEIS1, an important HOX protein cofactor, as a potential tumor suppressor. Patients bearing MEIS1-positive prostate tumors were less likely to have biochemical recurrence and metastasis compared to men bearing MEIS-negative tumors. Androgen receptor (AR), the major oncogene in PCa, has been shown to interact with HOXB13 to promote prostate cancer progression, but the function of MEIS proteins to antagonize AR/HOXB13 interactions are unknown. We hypothesize that MEIS1 proteins interact with HOXB13 to suppress cancer initiation and progression, and loss of MEIS1 expression in a portion of prostate tumors enables oncogenic AR/HOXB13 interactions. Methods: We determined the impact of MEIS1 expression and dependency of HOXB13 on AR signaling using cell lines ectopically expressing MEIS1 and/or CRISPR-mediated HOXB13 deletion in both androgen-sensitive LAPC4 and castration-resistant CWR22Rv1 cells. Western blots, qPCR, Proximity Ligation Assay and co-IPs were performed to evaluate the relationship among MEIS1, HOXB13 and AR. Hormonally-intact and castrated male nude mice were used to test the in vivo capability of MEIS1-mediated tumor formation and rate of tumor growth in the presence and absence of AR ligand. Results: We found that AR expression was significantly increased when MEIS1 was ectopically expressed compared to controls and HOXB13 knock-out lines. Re-expression of MEIS1 enhanced the binding between HOXB13-MEIS and AR-MEIS, and reduced the AR-HOXB13 interaction. In castrated mice, re-expression of MEIS1

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.

BACKGROUND:Epidemiological studies have established obesity as an independent risk factor for atrial fibrillation (AF), but the underlying pathophysiological mechanisms remain unclear. Reduced cardiac sodium channel expression is a known causal mechanism in AF. We hypothesized that obesity decreases Nav1.5 expression via enhanced oxidative stress, thus reducing INa, and enhancing susceptibility to AF.METHODS:To elucidate the underlying electrophysiological mechanisms a diet-induced obese mouse model was used. Weight, blood pressure, glucose, F2-isoprostanes, NOX2 (NADPH oxidase 2), and PKC (protein kinase C) were measured in obese mice and compared with lean controls. Invasive electrophysiological, immunohistochemistry, Western blotting, and patch clamping of membrane potentials was performed to evaluate the molecular and electrophysiological phenotype of atrial myocytes.RESULTS:Pacing-induced AF in 100% of diet-induced obese mice versus 25% in controls (P<0.01) with increased AF burden. Cardiac sodium channel expression, INa and atrial action potential duration were reduced and potassium channel expression (Kv1.5) and current (IKur) and F2-isoprostanes, NOX2, and PKC-α/δ expression and atrial fibrosis were significantly increased in diet-induced obese mice as compared with controls. A mitochondrial antioxidant reduced AF burden, restored INa, ICa,L, IKur, action potential duration, and reversed atrial fibrosis in diet-induced obese mice as compared with controls.CONCLUSIONS:Inducible AF in obese mice is mediated, in part, by a combined effect of sodium, potassium, and calcium channel remodeling and atrial fibrosis. Mitochondrial antioxidant therapy abrogated the ion channel and structural remodeling and reversed the obesity-induced AF burden. Our findings have important implications for the management of obesity-mediated AF in patients. Graphic Abstract: A graphic abstract is available for this article.

The molecular roles of HOX transcriptional activity in human prostate epithelial cells remain unclear, impeding the implementation of new treatment strategies for cancer prevention and therapy. MEIS proteins are transcription factors that bind and direct HOX protein activity. MEIS proteins are putative tumor suppressors that are frequently silenced in aggressive forms of prostate cancer. Here we show that MEIS1 expression is sufficient to decrease proliferation and metastasis of prostate cancer cells in vitro and in vivo murine xenograft models. HOXB13 deletion demonstrates that the tumor-suppressive activity of MEIS1 is dependent on HOXB13. Integration of ChIP-seq and RNA-seq data revealed direct and HOXB13-dependent regulation of proteoglycans including decorin (DCN) as a mechanism of MEIS1-driven tumor suppression. These results define and underscore the importance of MEIS1-HOXB13 transcriptional regulation in suppressing prostate cancer progression and provide a mechanistic framework for the investigation of HOXB13 mutants and oncogenic cofactors when MEIS1/2 are silenced.

Background: Atrial fibrillation (AF) increases stroke risk five-fold. Atrial hypocontractility from atrial myosin light chain (MLC2a) dephosphorylation contributes to stroke risk in AF. Recent proteomic data has shown increased protein phosphatase 1 subunit 12C (PPP1R12C) targeting to MLC2a in AF. However, it is unclear whether PPP1R12C causes MLC2a dephosphorylation in AF. Objective: Determine whether increased PPP1R12C expression causes MLC2a dephosphorylation and increases AF risk. Methods: Western blots and co-IPs were performed to evaluate the relationship among PPP1R12C, PP1c and MLC2a in human atrial tissues (AF vs SR). Mice with either a knockout (KO) or lentiviral (LV) cardiac overexpression of PPP1R12C were evaluated with invasive EP studies for AF inducibility vs WT controls. Results: In human AF, PPP1R12C was increased 4-fold ( P <0.005, n=6) with an 88% reduction in S-19-MLC2a phosphorylation ( P <0.05, n=4). PPP1R12C-PP1c and PPP1R12C-MLC2a binding was increased 2-fold in AF ( P <0.05, n=6). AF burden in LV-12C mice increased nearly tenfold vs. KO and WT mice ( P <0.05, n=6). Conclusion: In human AF, increased PPP1R12C expression is associated with reduced P-MLC2a through enhanced binding with the PP1c catalytic subunit. This dephosphorylation is a likely contributor to atrial hypocontractility and stroke risk in AF. Additionally, increased PPP1R12C expression in mice increases AF risk. Future studies will examine the effects of increased PPP1R12C expression upon atrial contractile function in mice.