Biology subjects

Arora, R.

Publications and source records attributed to Arora, R..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Merkel cell polyomavirus regulates miR183 cluster and piR62011 in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer, a major subset of which is caused by the clonal integration of Merkel Cell Polyomavirus (MCV). Recent studies by Cheng et al. (2017) reported that virus-derived small T antigen protein-bound EP400 complex drives expression of genes essential for cellular transformation. On close analysis of their ChIP-Seq data, we uncovered that the complex binds to the promoter region of the microRNA-183 cluster. The miRNA183 cluster is a cluster of 3 miRNAs (miR183, 182 & 96) expressed and regulated together. These miRNAs are conserved across species, highly expressed in human embryonic stem cells and necessary for sensory/ mechanosensory organ development. We hypothesized that the MCV oncoproteins regulate host miRNA expression directly; an interaction novel in polyomaviruses. We tested miRNA expression via qPCR in both virus positive and negative MCC cell lines and found the former showed a much higher level. Further, fibroblasts expressing T antigens displayed an increase in miR182 expression in comparison to control. Knock-down of T antigens in MCC cells correspondingly decreased miR182 levels. To investigate its regulation we performed luciferase assays for the miRNA predicted promoter that showed increased activity in the presence of T antigens. Intriguingly, the seed sequence of miR182 completely matches to a piRNA called piR62011. Upon reanalysis of a MCC small RNA library, piR62011 emerged as the highest expressed. We found it expressed in MKL-1, a MCV positive cell line as well. Finally, to translate our findings into therapy for MCC, we screened small molecule (CMBL) library by performing surface plasmon resonance (SPR) assay and identified small molecules that binds to pre-miRNA182 and are testing them for their activity to kill MCC.

molecular biology↗

bioRxiv2021

Pre-implantation mouse embryo movement under hormonally altered conditions

Precise regulation of embryo movement is crucial to successful implantation, but the role of ovarian hormones in this process is not understood. We ascertain the effects of altered hormonal environment on embryo movement using two delayed implantation models: Natural lactational Diapause (ND), a naturally occurring alternate model of pregnancy, and Artificially induced Diapause (AD), a laboratory version of ND. Our previous work suggests that embryos in a natural pregnancy (NP) first display unidirectional clustered embryo movement, followed by bidirectional scattering and spacing movement. In contrast, in the ND model, embryos are present as clusters near the oviductal-uterine junction for ~24-hours longer than NP, followed by locations consistent with a unidirectional scattering and spacing movement. Intriguingly, the AD model closely resembles embryo location in NP and not ND. Further, unlike the popular paradigm of reduced estrogen (E2) levels in diapause E2 levels are comparable across NP, ND, and AD, while progesterone (P4) levels are reduced in ND and highly increased in AD when compared to NP. Exogenous administration of E2 or P4 modifies the unidirectional clustered embryo movement, while E2 treatment causes a reduction in P4 and affects the bidirectional phase of embryo movement. Taken together, our data suggest embryo movement can be modulated by both P4 and E2. Understanding natural hormonal adaptation in diapause provides an opportunity to determine key players regulating embryo movement and implantation success. This knowledge can be leveraged to understand pregnancy survival and implantation success in hormonally altered conditions in the clinic.

developmental biology↗

bioRxiv2021

Sequestration of LINE-1 in novel cytosolic bodies by MOV10 restricts retrotransposition

LINE-1 (L1) are autonomous retroelements that have retained their ability to mobilize. Mechanisms regulating L1 mobility include DNA methylation in somatic cells and the Piwi-interacting RNA pathway in the germline. During pre-implantation stages of mouse embryonic development, however, both pathways are inactivated leading to a critical window necessitating alternate means of L1 regulation. We previously reported an increase in L1 levels in Dicer_KO mouse embryonic stem cells (mESCs). Intriguingly this was accompanied by only a marginal increase in retrotransposition, suggestive of additional mechanisms suppressing L1 mobility. Here, we demonstrate that L1 Ribonucleoprotein complexes (L1 RNP) accumulate as aggregates in Dicer_KO cytoplasm along with the RNA helicase MOV10. The combined overexpression of L1 RNAs and MOV10 is sufficient to create L1 RNP aggregates in stem cells. In Dicer_KO mESCs, MOV10 is upregulated due to the loss of its direct regulation by miRNAs. The newly discovered post-transcriptional regulation of Mov10 expression, and its role in preventing L1 retrotransposition by driving novel cytosolic aggregation affords alternate routes to explore for therapy and disease progression.

cell biology↗

bioRxiv2021

AGO1 regulates major satellite transcripts and H3K9me3 distribution at pericentromeric regions in mESCs

Argonaute proteins (AGOs), that play an essential role in cytosolic post-transcriptional gene silencing, have been also reported to function in nuclear processes like transcriptional activation or repression, alternative splicing and, chromatin organization. As most of these studies have been conducted in human cancer cell lines, the relevance of AGOs nuclear functions in the context of mouse early embryonic development remains uninvestigated. Here, we examined a possible role of the AGO1 protein on the distribution of constitutive heterochromatin in mouse Embryonic Stem Cells (mESCs). We observed a specific redistribution of the repressive histone mark H3K9me3 and the heterochromatin protein HP1, away from pericentromeric regions upon Ago1 depletion. Furthermore, we demonstrated that major satellite transcripts are strongly upregulated in Ago1_KO mESCs and that their levels are partially restored upon AGO1 rescue. We also observed a similar redistribution of H3K9me3 and HP1 in Drosha_KO mESCs, suggesting a role for microRNAs (miRNAs) in the regulation of heterochromatin distribution in mESCs. Finally, we showed that specific miRNAs with complementarity to major satellites can partially regulate the expression of these transcripts. Summary blurbDepletion of AGO1 in mESCs leads to a redistribution of H3K9me3 and HP1 away from pericentromeric regions and is accompanied by an upregulation of major satellites transcripts.

cell biology↗

bioRxiv2021

An Immune Cell Atlas Reveals Dynamic COVID-19 Specific Neutrophil Programming Amendable to Dexamethasone Therapy

SARS-CoV-2 is a novel coronavirus that causes acute respiratory distress syndrome (ARDS), death and long-term sequelae. Innate immune cells are critical for host defense but are also the primary drivers of ARDS. The relationships between innate cellular responses in ARDS resulting from COVID-19 compared to other causes of ARDS, such as bacterial sepsis is unclear. Moreover, the beneficial effects of dexamethasone therapy during severe COVID-19 remain speculative, but understanding the mechanistic effects could improve evidence-based therapeutic interventions. To interrogate these relationships, we developed an scRNA-Seq and plasma proteomics atlas (biernaskielab.ca/COVID_neutrophil). We discovered that compared to bacterial ARDS, COVID-19 was associated with distinct neutrophil polarization characterized by either interferon (IFN) or prostaglandin (PG) active states. Neutrophils from bacterial ARDS had higher expression of antibacterial molecules such as PLAC8 and CD83. Dexamethasone therapy in COVID patients rapidly altered the IFNactive state, downregulated interferon responsive genes, and activated IL1R2+ve neutrophils. Dexamethasone also induced the emergence of immature neutrophils expressing immunosuppressive molecules ARG1 and ANXA1, which were not present in healthy controls. Moreover, dexamethasone remodeled global cellular interactions by changing neutrophils from information receivers into information providers. Importantly, male patients had higher proportions of IFNactive neutrophils, a greater degree of steroid-induced immature neutrophil expansion, and increased mortality benefit compared to females in the dexamethasone era. Indeed, the highest proportion of IFNactive neutrophils was associated with mortality. These results define neutrophil states unique to COVID-19 when contextualized to other life-threatening infections, thereby enhancing the relevance of our findings at the bedside. Furthermore, the molecular benefits of dexamethasone therapy are also defined, and the identified pathways and plasma proteins can now be targeted to develop improved therapeutics.

immunology↗

bioRxiv2021

TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells.

Alternative Lengthening of Telomeres (ALT) is a Break-Induced Replication (BIR)-based mechanism elongating telomeres in a subset of human cancer cells. While the notion that spontaneous DNA damage at telomeres is required for ALT to occur, the molecular triggers of this physiological telomere instability are largely unknown. We previously proposed that the telomeric long noncoding RNA TERRA may represent one such trigger; however, given the lack of tools to suppress TERRA transcription in cells, our hypothesis remained speculative. We have now developed Transcription Activator-Like Effectors able to rapidly inhibit TERRA transcription from multiple chromosome ends in an ALT cell line. TERRA transcription inhibition decreases marks of DNA replication stress and DNA damage at telomeres and impairs ALT activity and telomere length maintenance. We conclude that TERRA transcription actively destabilizes telomere integrity in ALT cells, thereby initiating BIR and supporting telomere elongation. Our data point to TERRA transcription manipulation as a potentially useful target for therapy.

cell biology↗

bioRxiv2021

CRISPR/Cas9 targeting of MCPyV T antigen in Merkel tumors

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCPyV) T antigen gene expression, post integration ([~]80% cases), or by UV mediated DNA damage. Viral-positive Merkel tumors are not only caused by but also oncogenically addicted to tumor antigen expression. In this study we used CRISPR-Cas9 based gene-editing to develop a potential therapeutic tool for MCPyV positive MCC. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a genome editing technology whereby a guide RNA (gRNA) molecule, targets a Cas endonuclease to a specific genomic site, using sequence homology, and induces a double strand break. To target MCPyV T antigens, we designed a strategy using 2 gRNAs targeting the T antigen genomic region that would cut off a substantial portion of the gene thereby rendering it dysfunctional. We validated the MCPYV T antigen targeting efficiency of our gRNAs, both individually and together by in vitro cleavage assays. Finally, to translate this finding, we delivered this CRISPR system in patient-derived MCC cell lines and show reduction in T antigen gene expression. Our proof-of-concept study shows that 2 MCPyV targeting CRISPR/Cas gRNAs in combination can knock out MCPyV T antigen, thus, being of therapeutic importance. We hope that this CRISPR system can be potentially delivered in vivo for advancing MCPyV positive MCC treatment in the future.

molecular biology↗

bioRxiv2020

Regions of Highly Recurrent Electrogram Morphology at Sites of Low Cycle Length Accurately Reflect Arrhythmogenic Substrate for Atrial Fibrillation - Implications For a New, Mechanism Guided Therapeutic Approach for Atrial Fibrillation

BackgroundAlthough atrial electrograms (EGMs) are thought to reflect pathophysiological substrate for atrial fibrillation (AF), it is not known which electrograms are suitable targets during AF ablation. We hypothesized that electrogram morphology recurrence (EMR) better reflects arrhythmogenic AF substrate than traditional frequency and complexity measures of AF. In a canine rapid atrial pacing (RAP) model of AF, we assessed the relationship between EMR and traditional AF electrogram measures, rotational activity in the atria, fibrosis, myofiber orientation and parasympathetic innervation. MethodsPersistent AF was induced in 13 dogs by RAP for 6-8 weeks. High-density epicardial mapping (117 electrodes) was performed in six atrial sub-regions. EMR measures Recurrence percentage (Rec%) and cycle length of the most frequent electrogram morphology (CLR), Fractionated Interval (FI), Organization Index (OI), Dominant Frequency (DF) and Shannons Entropy (ShEn) were analyzed before and after atropine administration. Myocyte fiber orientation, amount of fibrosis and spatial distribution of parasympathetic nerve fibers were quantified. ResultsRec% was greatest in the appendages, and CLR was lowest in the posterior left atrium. Rec%/CLR correlated with FI, OI and the complexity measure ShEn, but not with DF. All electrogram measures were poorly correlated with fibrosis and myofiber anisotropy. Rec% correlated closely with stability of rotational activity. Unlike other measures, Rec% correlated closely with spatial heterogeneity of parasympathetic nerve fibers; this was reflected in CLR response to atropine. ConclusionEMR correlates closely with stability of rotational activity and with the pattern of atrial parasympathetic innervation. CLR may therefore be a viable therapeutic target in persistent AF.

physiology↗

bioRxiv2020

Endometrial epithelial ARID1A is critical for uterine gland function in early pregnancy establishment

Though endometriosis and infertility are clearly associated, the pathophysiological mechanism remains unclear. Previous work has linked endometrial ARID1A loss to endometriosis-related endometrial non-receptivity. Here, we show in mice that ARID1A binds and regulates transcription of the Foxa2 gene required for endometrial gland function. Uterine specific deletion of Arid1a compromises gland development and diminishes Foxa2 and Lif expression. Deletion of Arid1a with Ltf-iCre in the adult mouse endometrial epithelium preserves gland development while still compromising gland function. Mice lacking endometrial epithelial Arid1a are severely sub-fertile due to defects in implantation, decidualization, and endometrial receptivity from disruption of the LIF-STAT3-EGR1 pathway. FOXA2 is also reduced in the endometrium of women with endometriosis in correlation with diminished ARID1A, and both ARID1A and FOXA2 are reduced in non-human primates induced with endometriosis. Our findings describe a role for ARID1A in the endometrial epithelium supporting early pregnancy establishment through the maintenance of gland function.

physiology↗

bioRxiv2020

INTEGRINS MEDIATE PLACENTAL EXTRACELLULAR VESICLE TRAFFICKING TO LUNG AND LIVER IN VIVO

Membrane-bound extracellular vesicles (EVs) mediate intercellular communication in all organisms, and those produced by placental mammals have become increasingly recognized as significant mediators of fetal-maternal communication. Here, we aimed to identify maternal cells targeted by placental EVs and elucidate the mechanisms by which they traffic to these cells. Exogenously administered pregnancy-associated EVs traffic specifically to the lung; further, placental EVs associate with lung interstitial macrophages and liver Kupffer cells in an integrin-dependent manner. Localization of EV to maternal lungs was confirmed in unmanipulated pregnancy using a transgenic reporter mouse model, which also provided in situ and in vitro evidence that fetally-derived EVs, rarely, may cause genetic alteration of maternal cells. These results provide for the first time direct in vivo evidence for targeting of placental EVs to maternal immune cells, and further, evidence that EVs can alter cellular phenotype.

cell biology↗

bioRxiv2020

3D synthetic microscaffolds promote homogenous expression of NANOG in mouse embryonic stem cells

The development of in vitro models, which accurately recapitulate early embryonic development, is one of the fundamental challenges in stem cell research. Most of the currently employed approaches involve the culture of embryonic stem cells (ESCs) on two-dimensional (2D) surfaces. However, the monolayer nature of these cultures does not permit cells to grow and proliferate in realistic three-dimensional (3D) microenvironments, as in an early embryo. In this paper, novel 3D synthetic scaffold arrays, fabricated by two-photon polymerization photolithography, are utilized to mimic tissue-specific architecture, enabling cell-to-matrix interaction and cell-to-cell communication in vitro. Mouse ESCs (mESCs) are able to grow and proliferate on these structures and maintain their pluripotent state. Furthermore, the 3D microscaffold arrays are integrated into a microscopy slide allowing the evaluation of the expression of key pluripotency factors at the single-cell level. Comparing 2D and 3D surfaces, mESCs grown in serum+LIF on 3D microscaffolds exhibit a stronger and more homogenous expression of NANOG and OCT4 pluripotency factors, than cells cultivated in 2i media, demonstrating that 3D microscaffolds capture naive pluripotency in vitro. Thus, the slide affords a novel and unique tool to model and study mammalian early development with greater physiological relevance than conventional 2D cultures.

bioengineering↗

bioRxiv2020

Merkel cell polyomavirus in Merkel cell carcinoma: Integration sites and involvement of the KMT2D tumor suppressor gene

Merkel cell carcinoma (MCC) is an uncommon, lethal cancer of the skin caused by either Merkel cell polyomavirus (MCV) or UV-linked mutations. MCV is found integrated into MCC tumor genomes, accompanied by truncation mutations that render the MCV large T antigen replication incompetent. We used the open access HPV Detector/ Cancervirus Detector tool to determine the MCV integration sites in whole exome sequencing data from 5 MCC cases, thereby adding to the limited published MCV integration site junction data. We also systematically reviewed published data on integration for MCV in the human genome, presenting a collation of 123 MCC cases and their linked chromosomal sites. We confirm that there are no highly recurrent specific sites of integration. We found that, chromosome 5 is the chromosome most frequently involved by MCV integration and that integration sites are significantly enriched for genes with binding sites for oncogenic transcription factors such as LEF1 and ZEB1, suggesting the possibility of increased open chromatin in these gene sets. Additionally, in one case we found integration involving the tumor suppressor gene KMT2D for the first time, adding to previous reports of rare MCV integration into host tumor suppressor genes in MCC.

cancer biology↗

bioRxiv2020

Private Antibody Repertoires Are Public

When faced with a given pathogen, the antibody response generally functions similarly across different people,1-4 but the source of this similarity has been unclear. One hypothesis was that people share a high proportion of the same VDJ-recombined antibody genes, but this has been disproven.5,6 An alternative is that people share a high proportion of functionally similar antibodies,7,8 but testing this hypothesis requires a method for measuring functional similarity that scales to the millions of antibodies per repertoire and across multiple repertoires, which is impossible experimentally. We recently described a framework for doing so computationally,9 which revealed that repertoires consist of loose overlapping functional classes of antibodies with similar antigen-binding capacities;10-12 this framework allowed us to estimate a repertoires antigen-binding capacity,{tau} , for the ideal target of any given antibody. Here, we show that this framework supports the second hypothesis, and provide the first comprehensive demonstration of overwhelming functional overlap between repertoires from 20 different individuals directly from sequence, without need of binding studies. Overlap is highest among the young and falls with age, due to the selective loss of antibodies that represent a core set of shared or "public" antigen-binding capacities. We reveal considerable heterogeneity in antigen-binding capacities for antibodies against influenza, HIV, and SARS-CoV-2, and show that while some of these classes shrink with age, others persist across individuals. These discoveries change our understanding of repertoire diversity and have implications for vaccine and therapeutic-antibody development, especially for the aged.

immunology↗

bioRxiv2020

SARS-CoV2 Testing: The Limit of Detection Matters

Resolving the COVID-19 pandemic requires diagnostic testing to determine which individuals are infected and which are not. The current gold standard is to perform RT-PCR on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of ~100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss more infected patients, resulting in more false negatives. However, the false-negative rate for a given LoD remains unknown. Here we address this question using over 27,500 test results for patients from across our healthcare network tested using the Abbott RealTime SARS-CoV-2 EUA. These results suggest that each 10-fold increase in LoD is expected to increase the false negative rate by 13%, missing an additional one in eight infected patients. The highest LoDs on the market will miss a majority of infected patients, with false negative rates as high as 70%. These results suggest that choice of assay has meaningful clinical and epidemiological consequences. The limit of detection matters.

microbiology↗

bioRxiv2020

Mechanical and signaling mechanisms that guide pre-implantation embryo movement.

How a mammalian embryo determines and arrives at its site of attachment is a mystery that has puzzled researchers for decades. Additionally, in multiparous species, embryos face a unique challenge of achieving adequate spacing to avoid competition for maternal resources. Using our enhanced confocal imaging and 3D image reconstruction technology, we evaluate murine embryo location in the uterus along the longitudinal oviductal-cervical axis. Our analysis reveals three distinct pre-implantation stages: a) Embryo entry; b) Unidirectional movement of embryo clusters; and c) Bidirectional scattering and spacing of embryos. We show that unidirectional movement of embryo clusters is facilitated by a mechanical stimulus of the embryo as a physical object and is regulated by adrenergic uterine smooth muscle contractions. Embryo scattering, on the other hand, relies on embryo-uterine communication reliant on the LPAR3 signaling pathway and is independent of adrenergic muscle contractions. We propose that the presence of embryo clusters in the uterine horn provides an opportunity for the uterus to sense and count the embryos, followed by scattering and spacing these embryos along the given length of the horn. Thus, uterine implantation sites in mice are neither random nor predetermined but are guided by the number of embryos entering the uterine lumen. These studies have implications for understanding how embryo-uterine communication is key to determining an optimal implantation site, which is necessary for the success of a pregnancy. Significance StatementIn mammals that carry multiple offspring in one gestation, embryos seemingly acquire even embryo spacing. Such even distribution would imply a guided interaction between the mother and the fetus very early on in pregnancy to allow favorable pregnancy outcomes. Thus, it is essential to understand quantitatively if and when such a uniform distribution of embryos is established. Further, uncovering the physical and biological mechanisms that allow for such equal distribution of embryos, will improve our understanding of early pregnancy events and provide for novel targets for improving pregnancy success in case of infertility and artificial reproductive technologies as well as to develop non-hormonal therapies for contraception.

developmental biology↗

bioRxiv2020

A metabolomics study of ascorbic acid-induced in situ freezing tolerance in spinach (Spinacia oleracea L.)

Freeze-thaw stress is one of the major environmental constraints that limit plant growth and reduces productivity and quality. Plants exhibit a variety of cellular dysfunction following freeze-thaw stress, including accumulation of reactive oxygen species (ROS). This means that enhancement of antioxidant capacity by exogenous application of antioxidants could potentially be one of major strategies for improving freezing tolerance (FT) of plants. Exogenous application of ascorbic acid (AsA), as an antioxidant, has been shown to improve plant tolerance against abiotic stresses but its effect on FT has not been investigated. We evaluated the effect of AsA-feeding on FT of spinach (Spinacia oleracea L.) at whole-plant and excised leaf level, and conducted metabolite profiling of leaves before and after AsA-treatment to explore metabolic explanation for change in FT. AsA-application did not impede leaf-growth; instead slightly promoted it. Temperature-controlled freeze-thaw tests revealed AsA-fed plants were more freezing tolerant as indicated by: 1) less visual damage/mortality; 2) lower ion-leakage; and 3) less oxidative-injury, lower abundance of free radicals (O2 *- and H2O2). Comparative leaf metabolic profiling revealed clear separation of metabolic phenotypes for control vs. AsA-fed leaves. Specifically, AsA-fed leaves had greater abundance of antioxidants (AsA, glutathione, alpha-& gamma-tocopherol) and compatible solutes (proline, galactinol, myo-inositol). AsA-fed leaves also had higher activity of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, catalase). These changes, together, may improve FT via alleviating freeze-induced oxidative stress as well as protecting membranes from freeze-desiccation. Additionally, improved FT by AsA-feeding may potentially include enhanced cell wall/lignin augmentation and bolstered secondary metabolism as indicated by diminished level of phenylalanine and increased abundance of branched amino acids, respectively.

plant biology↗

bioRxiv2019

DETECTing Merkel cell Polyomavirus in Merkel Tumours

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCV) T antigen expression, post integration ([~]80% cases), or by UV mediated DNA damage. Interestingly, overall survival of patients suffering from MCV positive Merkel cell carcinoma is better, making this differential information of significant diagnostic and prognostic value. Also, MCV as a causative agent also provides a direct target for therapy in virus positive MCC patients. Currently, the methods used for diagnosis of MCV in tumours are often tedious, discordant and unreliable. In this study we used a guided molecular scissors based - DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) technique to develop an in vitro molecular diagnostic tool for MCV positive MCC. DETECTR couples recombinase polymerase based amplification of target MCV DNA with Cas12a mediated detection. CRISPR diagnostics couple specific detection followed by cutting of the pathogenic DNA by the Cas enzyme - gRNA complex, with non-specific cutting of ssDNA that provides a measurable visual cue. To detect MCV DNA in MCC tumours, we designed Cas12a gRNAs targeting the MCV DNA and tested their targeting efficiency, and sensitivity using a fluorophore quencher labeled reporter assay. We show that this sophisticated MCV DETECTR system can detect MCV integrated in Merkel tumour rapidly, specifically and with femto-molar sensitivity. This new MCV DNA detecting system is promising and we hope it can be coupled with histopathological and immunohistochemical studies to diagnose the viral status of MCC in clinics in the near future.

molecular biology↗

bioRxiv2019

Disruption of NOX2-dependent Oxidative Injury with a Targeted Gene-Therapy Approach Prevents Atrial Fibrillation in a Canine Model

Atrial fibrillation is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal as they are not targeted to the molecular mechanisms underlying AF. In this study, we demonstrated using a novel gene-based strategy in a clinically relevant large animal of AF that oxidative injury is a key mechanism underlying the onset and perpetuation of AF. First, we demonstrated that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through induction of NADPH oxidase 2 (NOX2) and generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electrical remodeling in AF by upregulating a constitutively active form of acetylcholine-dependent K+ current (IKACh) - called IKH - by a mechanism involving frequency-dependent activation of protein kinase C epsilon (PKC{varepsilon}). To understand the mechanism by which oxidative injury promotes the genesis and/or maintenance of AF, we performed targeted injection of NOX2 shRNA in atria of normal dogs followed by rapid atrial pacing. The time to onset of non-sustained AF increased by more than 5-fold in NOX2 shRNA treated dogs. Furthermore, animals treated with NOX2 shRNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, with attenuated activation of PKC{varepsilon}, a likely molecular mechanism underlying this beneficial electrophysiological remodeling. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.\n\nOne Sentence SummaryTargeted disruption of NOX2-dependent oxidative injury with a novel gene therapy approach prevents onset as well as perpetuation of atrial fibrillation.

physiology↗