Biology subjects

O'Rourke, B.

Publications and source records attributed to O'Rourke, B..

16 recordsLinked to original sources

bioRxiv2026

SureQuant IS-PRM Enables Cross-Species Targeted Quantification of Retinoid Metabolism and Signaling Proteins in the Heart

Retinoic acid signaling is critical for cardiac development and homeostasis. Dysregulation of all-trans retinoic acid metabolism contributes to vascular atherogenesis, restenosis, calcification, and heart failure. Therefore, assessment of proteins involved in retinoid metabolism and signaling has gained interest for identifying potential biomarkers and therapeutic targets in cardiovascular disease. However, quantifying these proteins remains challenging due to limitations of antibody-based methods. We developed a targeted proteomics approach using SureQuant internal standard-triggered parallel reaction monitoring mass spectrometry to profile these proteins. We designed a panel of 80 stable isotope-labeled (heavy) peptides representing proteins involved in retinoid signaling and metabolism, with sequences applicable to human samples and conserved across multiple species. Survey experiments using directed data-dependent acquisition on the Orbitrap Fusion Lumos mass spectrometer determined precursor and product ion masses for each heavy peptide, which were programmed into the SureQuant method for continuous monitoring. Upon detection of these heavy internal standards, the instrument transitions to a targeted PRM acquisition mode in which repeated high-resolution MS/MS spectra of both endogenous (light) and heavy peptides are acquired. Using this method, retinoid pathway-associated proteins were quantified to as low as 10 attomoles for selected targets across multiple tissues and developmental stages. Distinct tissue-specific retinoid metabolic networks were identified across lung, liver, retinal cell lines and cardiac tissues. Developmental profiling of mouse and rat hearts revealed remodeling of retinoid pathway proteins from embryonic to postnatal and adult stages, suggesting a functional transition from retinoid-driven cardiac development toward maintenance of retinoid homeostasis in the mature heart.

biochemistry↗

bioRxiv2026

Formate reduces ischemic injury in the male heart by increasing protein S-nitrosation

Ischemic heart disease is a leading cause of death for both men and women in the United States. We and others have demonstrated that nitric oxide (NO) signaling and associated protein S-nitrosation (SNO) play a key role in reducing ischemic injury in the heart. We also find that while females typically exhibit endogenous protection from ischemic injury, this protection is abrogated with the loss of the formate-generating enzyme alcohol dehydrogenase 5 (ADH5), but formate supplementation provided a rescue. Here, we investigate the cardioprotective efficacy of formate in male hearts. Hearts were Langendorff-perfused and subjected to ischemia/reperfusion (I/R) injury with and without formate. Formate-mediated protection was also examined using an in vitro model of coverslip-induced ischemic injury to identify molecular underpinnings. We found that formate yields protection from I/R injury in ex vivo and in vitro models by increasing post-ischemic protein SNO levels, while NO synthase inhibition blocked this formate-mediated increase in protein SNO in vitro, and attenuated protection from I/R injury ex vivo. Moreover, post-ischemic levels of tetrahydrobiopterin (BH4), a cofactor necessary for NOS function, were preserved in formate-treated hearts. Furthermore, inhibition of dihydrofolate reductase (DHFR), a one-carbon enzyme critical for BH4 recycling, blunted formate-mediated protection ex vivo. Collectively, our findings suggest that formate is a potent cardioprotective agent that confers protection by preserving post-ischemic BH4 levels, and enhancing protein SNO levels through a NOS-dependent mechanism. These findings have significant implications for the clinical prevention and treatment of ischemic heart disease in males.

physiology↗

bioRxiv2025

Coumarins disrupt cell-cell communication for control of pathogenesis and virulence in ESKAPEEs and fungal opportunists

Intricate communication networks and sensing systems underpin the complexity of microbe-host interactions, enabling spatiotemporal control of optimised microbe-host consortia in a diverse range of ecosystems. A central component of these complex interactomes has been the signalling events that enable recognition of host and microbe, whether that niche be clinical or environmental, human or plant. Coumarins have emerged as significant plant derived signalling molecules shaping microbiome dynamics and pathogen behaviours from a broad spectrum of ecosystems. Here we explored the role of natural and synthetic coumarin compounds in signal interference and control of pathogenesis in bacterial and fungal pathogens, uncovering an important hydroxylation-motif in the specific inhibition of two Pseudomonas aeruginosa interspecies and interkingdom communication molecules. Characterisation of the anti-biofilm activity of coumarins revealed changes in exopolysaccharide production independent of the initial attachment phenotype. Molecular modelling provides an insight into the receptor binding dynamics of three closely related natural coumarins, suggesting an intricate and highly specific host-microbe interaction at the species level. As the very real threat of antimicrobial resistance continues to shadow our horizons, phytochemicals such as coumarins have potential to deliver an ecological solution to dysbiosis in the host-microbe interaction. ImportanceNatural ecosystems rely on homeostatic interactions between the kingdoms of life to ensure sustainable and balanced communities can persist. Plant-derived coumarins have recently emerged as playing an important role in shaping microbial communities, presenting a remarkable chemical diversity that can influence the behaviour of bacteria and fungi. At the same time, one of the major challenges to human health continues to be the spread of antimicrobial resistance and the parallel absence of a concerted effort to source and produce new antibiotics at industrial scale. Therefore, new approaches to the control of infection are required, and an ecosystem-level lens may offer one such innovative intervention. Coumarins have the potential to neutralise the very mechanisms used by bacteria and fungi to cause infection leading to morbidity and mortality in hosts ranging from plant to animals. Here we present a mechanistic insight into how effective these molecules can be in targeting keystone pathogens termed the ESKAPEEs and their fungal counterparts.

microbiology↗

bioRxiv2025

GSK3B inhibition partially reverses brain ethanol-induced transcriptomic changes in C57BL/6J mice: Expression network co-analysis with human genome-wide association studies

Alcohol use disorder (AUD) is a chronic behavioral disease with greater than 50% of its risk due to complex genetic contributions. Existing pharmacological and behavioral treatments for AUD are minimally effective and underutilized. Animal model behavioral genetics and human genome-wide association studies have begun to identify individual genes contributing to the progressive compulsive consumption of ethanol that occurs with AUD, promising possible new therapeutic targets. Our laboratory has previously identified Gsk3b as a central member in a network of ethanol-responsive genes in mouse prefrontal cortex, which altered ethanol consumption with genetic manipulation and was also significantly associated with risk for alcohol dependence in human genome-wide association studies. Here we perform detailed brain RNA sequencing transcriptomic studies to characterize a highly specific and clinically available GSK3B pharmacological inhibitor, tideglusib, as a possible therapeutic for clinical trials on treatment of AUD. A model of chronic intermittent ethanol consumption was used to study gene expression changes in prefrontal cortex and nucleus accumbens in the presence or absence of tideglusib treatment. Multivariate analysis of differentially expressed genes showed that tideglusib largely reversed ethanol- induced expression changes for two prominent clusters of genes in both prefrontal cortex and nucleus accumbens. Bioinformatic analysis showed these genes to have prominent roles in neuronal functioning and synaptic activity. Additionally, mouse brain differential gene expression data was analyzed together with human protein-protein interaction and genome-wide association studies on AUD to derive networks responding to tideglusib and relevant to human genetic risk for alcohol dependence. These studies identified discrete networks significantly enriched with genes provisionally associated with AUD, and provide key information on central hubs of such networks. Together these studies document tideglusib as a major modulator of chronic ethanol consumption-evoked brain gene expression signatures, and identify possible new targets for therapeutic modulation of AUD.

genomics↗

bioRxiv2024

Inhibition of Cardiac p38 Highlights the Role of the Phosphoproteome in Heart Failure Progression

Heart failure (HF) is a complex condition characterized by the inability of the heart to pump sufficient oxygen to the organs to meet their metabolic needs. Among the altered signal transduction pathways associated with HF pathogenesis, the p38 mitogen-activated protein kinase (p38 MAPK) pathway--activated in response to stress-- has attracted considerable attention for its potential role in HF progression and cardiac hypertrophy. However, the exact mechanisms by which p38 MAPK influences HF remain unclear. Addressing knowledge gaps may provide insight on why p38 inhibition has yielded inconsistent outcomes in clinical trials. Here we investigate the effects of p38 MAPK inhibition via SB203580 on cardiac remodeling in a guinea pig model of HF and sudden cardiac death. Using a well-established HF model with ascending aortic constriction and daily isoproterenol (ACi) administration, we assessed proteomic changes across three groups: sham-operated controls, untreated ACi, and ACi treated with SB203580 (ACiSB). Cardiac function was evaluated by M-mode echocardiography, while proteome and phosphoproteome profiles were analyzed using multiplexed tandem mass tag labeling and LC-MS/MS. Our findings demonstrate that chronic SB203580 treatment offers protection against progressive decline in cardiac function in HF. The proteomic data indicate that SB203580-treatment exerts broad protection of the cardiac phosphoproteome, beyond inhibiting maladaptive p38-dependent phosphorylation, extending to PKA and AMPK networks among others, ultimately protecting the phosphorylation status of critical myofibrillar and Ca2+-handling proteins. Though SB203580 had a more restricted impact on widespread protein changes in HF, its biosignature was consistent with preserved mitochondrial energetics as well as reduced oxidative and inflammatory stress.

molecular biology↗

bioRxiv2024

A selective GSK3β inhibitor, tideglusib, decreases intermittent access and binge ethanol self-administration in C57BL/6J mice

Over 10% of the US population over 12 years old meets criteria for Alcohol Use Disorder (AUD), yet few effective, long-term treatments are currently available. Glycogen synthase kinase 3 beta (GSK3{beta}) has been implicated in ethanol behaviors and poses as a potential therapeutic target in the treatment of AUD. Here we investigate the role of tideglusib, a selective GSK3{beta} inhibitor, in ethanol consumption and other behaviors. We have shown tideglusib decreases ethanol consumption in both a model of daily, progressive ethanol intake (two-bottle choice, intermittent ethanol access) and binge-like drinking behavior (drinking-in-the-dark) without effecting water intake. Further, we have shown tideglusib to have no effect on ethanol pharmacokinetics, taste preference, or anxiety-like behavior, though there was a transient increase in total locomotion following treatment. Additionally, we assessed liver health following treatment via serum levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase and showed no effect on aminotransferase levels though there was a decrease in alkaline phosphatase. RNA sequencing studies revealed a role of GSK3{beta} inhibition via tideglusib on the canonical Wnt signaling pathway, suggesting tideglusib may carry out its effects on ethanol consumption through effects on {beta}-catenin binding to the transcription factors TCF3 and LEF1. The data presented here further implicate GSK3{beta} in alcohol consumption and support the use of tideglusib as a potential therapeutic in the treatment of AUD.

pharmacology and toxicology↗

bioRxiv2024

Innate Immune Activation and Mitochondrial ROS Invoke Persistent Cardiac Conduction System Dysfunction after COVID-19

BackgroundCardiac risk rises during acute SARS-CoV-2 infection and in long COVID syndrome in humans, but the mechanisms behind COVID-19-linked arrhythmias are unknown. This study explores the acute and long term effects of SARS-CoV-2 on the cardiac conduction system (CCS) in a hamster model of COVID-19. MethodsRadiotelemetry in conscious animals was used to non-invasively record electrocardiograms and subpleural pressures after intranasal SARS-CoV-2 infection. Cardiac cytokines, interferon-stimulated gene expression, and macrophage infiltration of the CCS, were assessed at 4 days and 4 weeks post-infection. A double-stranded RNA mimetic, polyinosinic:polycytidylic acid (PIC), was used in vivo and in vitro to activate viral pattern recognition receptors in the absence of SARS-CoV-2 infection. ResultsCOVID-19 induced pronounced tachypnea and severe cardiac conduction system (CCS) dysfunction, spanning from bradycardia to persistent atrioventricular block, although no viral protein expression was detected in the heart. Arrhythmias developed rapidly, partially reversed, and then redeveloped after the pulmonary infection was resolved, indicating persistent CCS injury. Increased cardiac cytokines, interferon-stimulated gene expression, and macrophage remodeling in the CCS accompanied the electrophysiological abnormalities. Interestingly, the arrhythmia phenotype was reproduced by cardiac injection of PIC in the absence of virus, indicating that innate immune activation was sufficient to drive the response. PIC also strongly induced cytokine secretion and robust interferon signaling in hearts, human iPSC-derived cardiomyocytes (hiPSC-CMs), and engineered heart tissues, accompanied by alterations in electrical and Ca2+ handling properties. Importantly, the pulmonary and cardiac effects of COVID-19 were blunted by in vivo inhibition of JAK/STAT signaling or by a mitochondrially-targeted antioxidant. ConclusionsThe findings indicate that long term dysfunction and immune cell remodeling of the CCS is induced by COVID-19, arising indirectly from oxidative stress and excessive activation of cardiac innate immune responses during infection, with implications for long COVID Syndrome.

pathology↗

bioRxiv2023

Molecular and cellular mechanisms by which cardiac sympathetic denervation prevents heart failure and sudden cardiac arrest

RATIONALESudden cardiac arrest (SCA) and heart failure (HF) are leading causes of death. The underlying mechanisms are incompletely understood, limiting the design of new therapies. Whereas most autonomic modulation therapies have not shown clear benefit in HF patients, growing evidence indicates cardiac sympathetic denervation (CSD) exerts cardioprotective effects. The underlying molecular and cellular mechanisms remain unexplored. OBJECTIVEBased on the hypothesis that mitochondrial reactive oxygen species (mROS) drive the pathogenesis of HF and SCA, we investigated whether CSD prevents SCA and HF by improving mitochondrial antioxidant capacity and redox balance, to correct impaired Ca2+ handling and repolarization reserve. METHODS AND RESULTSWe interrogated CSD-specific responses in pressure-overload HF models with spontaneous SCA using in vivo echocardiographic and electrocardiographic studies and in vitro biochemical and functional studies including ratiometric measures of mROS, Ca2+ and sarcomere dynamics in left ventricular myocytes. Pressure-overloaded HF reduced mitochondrial antioxidant capacity and increased mROS, which impaired {beta}-adrenergic signaling and caused SR Ca2+ leak, reducing SR Ca2+ and increasing diastolic Ca2+, impaired myofilament contraction and further increased the sympathetic stress response. CSD improved contractile function and mitigated mROS-mediated diastolic Ca2+ overload, dispersion of repolarization, triggered activity and SCA by upregulating mitochondrial antioxidant and NADPH-producing enzymes. CONCLUSIONSOur findings support a fundamental role of sympathetic stress-induced downregulation of mROS scavenging enzymes and RyR-leak mediated diastolic Ca2+ overload in HF and SCA pathogenesis that are mitigated by CSD. This first report on the molecular and cellular mechanisms of CSD supports its evaluation in additional high-risk patient groups. BRIEF SUMMARYCardiac sympathetic denervation (CSD) confers cardioprotective effects via unclear mechanisms. In a guinea pig model that uniquely mimics human pressure-overload heart failure (HF) with spontaneous sudden cardiac arrest (SCA), we interrogated CSD-specific responses using echocardiographic, electrocardiographic and biochemical measures, and ratiometric measures of mitochondrial reactive oxygen species (mROS), Ca2+ and sarcomere dynamics. Consistent with our hypothesis, CSD rescued cardioprotection by upregulating mitochondrial antioxidant and NADPH-producing enzymes, which mitigate mROS-mediated Ca2+ derangements, repolarization lability, triggered activity, HF and SCA. Our findings provide the first molecular and cellular mechanistic basis for evaluating CSD therapy in a broader group of high-risk patients.

physiology↗

bioRxiv2021

PDE9 Inhibition Activates PPARa to Stimulate Mitochondrial Fat Metabolism and Reduce Cardiometabolic Syndrome

Central obesity with cardiometabolic syndrome (CMS) is a major global contributor to human disease, and effective therapies are needed. Here, we show inhibiting cyclic-GMP selective phosphodiesterase-9A (PDE9-I) suppresses established diet-induced obesity and CMS in ovariectomized female and male mice. PDE9-I reduces abdominal, hepatic, and myocardial fat accumulation, stimulates mitochondrial activity in brown and white fat, and improves CMS, without altering activity or food intake. PDE9 localizes to mitochondria, and its inhibition stimulates lipolysis and mitochondrial respiration coupled to PPAR-dependent gene regulation. PPAR upregulation is required for PDE9-I metabolic efficacy and is absent in non-ovariectomized females that also display no metabolic benefits from PDE9-I. The latter is compatible with estrogen receptor- altering PPAR chromatin binding identified by ChIPSeq. In humans with heart failure and preserved ejection fraction, myocardial expression of PPARA and its regulated genes is reduced versus control. These findings support testing PDE9-I to treat obesity/CMS in men and postmenopausal women. SummaryOral inhibition of phosphodiesterase type 9 stimulates mitochondrial fat metabolism and lipolysis, reducing central obesity without changing appetite

physiology↗

bioRxiv2021

Myocardial BDNF regulates cardiac bioenergetics through the transcription factor Yin Yang 1

Circulating Brain-derived Neurotrophic Factor (BDNF) is markedly decreased in heart failure patients. Both BDNF and its receptor, Tropomyosin Related Kinase Receptor (TrkB), are expressed in cardiomyocytes, however the role of myocardial BDNF signaling in cardiac pathophysiology is poorly understood. We found that cardiac-specific TrkB knockout (cTrkB KO) mice displayed a blunted adaptive cardiac response to exercise, with attenuated upregulation of transcription factor networks controlling mitochondrial biogenesis/metabolism, including Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1). The cTrkB KO mice developed an exacerbated heart failure progression with transaortic constriction. The downregulation of PGC-1 in cTrkB KO mice exposed to exercise or TAC resulted in decreased cardiac energetics. We further unraveled that BDNF induces PGC-1 upregulation and bioenergetics through a novel signaling pathway, the pleiotropic transcription factor Yin Yang 1 (YY1). Taken together, our findings suggest that myocardial BDNF plays a critical role in regulating cellular energetics in the cardiac stress response.

molecular biology↗

bioRxiv2020

Preclinical Mesenchymal Stromal Cell Delivery Via An Ex Vivo Bioreactor Attenuates Clot Formation For Intravascular Applications

While mesenchymal stromal cells (MSCs) are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID-19, trauma, or cancers. Here, we tested a novel ex vivo approach aimed at improving the safety of MSC systemic administration by use of a bioreactor. In this device, MSCs are seeded on the outside of a hollow-fiber filter, sequestering them behind a hemocompatible membrane, while still maintaining cross talk with blood cells and circulating signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against "free" MSCs, as a model of systemic administration, which were directly injected into the circuit. Our results showed that physical isolation of the MSCs via a bioreactor extends the time necessary for clot formation to occur when compared to "free" MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Further, application of this technology in two in vivo models did not require additional heparin to maintain target ACT levels relative to the acellular device. Taken together, the use of hollow fiber filters to house MSCs, if adopted clinically, could offer a novel method to control systemic MSC exposure and prolong clot formation time.

bioengineering↗

bioRxiv2020

Blood-derived mitochondrial DNA copy number is associated with gene expression across multiple tissues and is predictive for incident neurodegenerative disease

BackgroundMitochondrial DNA copy number (mtDNA-CN) can be used as a proxy for mitochondrial function and is associated with a number of aging-related diseases. However, it is unclear how mtDNA-CN measured in blood can reflect risk for diseases that primarily manifest in other tissues. Using the Genotype-Tissue Expression Project, we interrogated the relationships between mtDNA-CN measured in whole blood and gene expression from whole blood as well as 47 additional tissues. ResultsWe evaluated associations between blood-derived mtDNA-CN and gene expression in whole blood for 418 individuals, correcting for known confounders and surrogate variables derived from RNA-sequencing. Using a permutation-derived cutoff (p<2.70e-6), mtDNA-CN was significantly associated with expression for 721 genes in whole blood, including nuclear genes that are required for mitochondrial DNA replication. Significantly enriched pathways included splicing (p=1.03e-8) and ubiquitin-mediated proteolysis (p=2.4e-10). Genes with target sequences for the mitochondrial transcription factor NRF1 were also enriched (p=1.76e-35). In non-blood tissues, there were more significantly associated genes than expected in 30 out of 47 tested tissues, suggesting that global gene expression in those tissues is correlated with mtDNA-CN. Pathways that were associated in multiple tissues included RNA-binding, catalysis, and neurodegenerative disease. We evaluated the association between mtDNA-CN and incident neurodegenerative disease in an independent dataset, the UK Biobank, using a Cox proportional-hazards model. Higher mtDNA-CN was significantly associated with lower risk for incident neurodegenerative disease (HR=0.73, 95% CI= 0.66;0.90). ConclusionsThe observation that mtDNA-CN measured in whole blood is associated with gene expression in other tissues suggests that blood-derived mtDNA-CN can reflect metabolic health across multiple tissues. Key pathways in maintaining cellular homeostasis, including splicing, RNA binding, and catalytic genes were significantly associated with mtDNA-CN, reinforcing the importance of mitochondria in aging-related disease. As a specific example, genes involved in neurodegenerative disease were significantly enriched in multiple tissues. This finding, validated in a large independent cohort study showing an inverse association between mtDNA-CN and neurodegenerative disease, solidifies the link between blood-derived mtDNA-CN, altered gene expression in both blood and non-blood tissues, and aging-related disease.

genetics↗

bioRxiv2020

MitoWave: Spatiotemporal analysis of mitochondrial membrane potential fluctuations during ischemia-reperfusion

Mitochondria exhibit non-stationary unstable membrane potential ({Delta}{Psi}m) when subjected to stress, such as during Ischemia/Reperfusion (I/R). Understanding the mechanism of {Delta}{Psi}m instability involves characterizing and quantifying this phenomenon in response to I/R stress in an unbiased and reproducible manner. We designed a simple ImageJ-MATLAB-based workflow called MitoWave to unravel dynamic mitochondrial {Delta}{Psi}m changes that occur during ischemia and reperfusion. MitoWave employs MATLABs wavelet transform toolbox. In-vitro Ischemia was effected by placing a glass coverslip for 60 minutes on a monolayer of neonatal mouse ventricular myocytes (NMVMs). Removal of the coverslip allowed for reperfusion. {Delta}{Psi}m response to I/R was recorded on a confocal microscope using TMRM as the indicator. As proof-of-principle, we used MitoWave analysis on ten invitro I/R experiments. Visual observations corroborated quantitative MitoWave analysis results in classifying the ten I/R experiments into five outcomes that were observed based on the oscillatory state of {Delta}{Psi}m throughout the reperfusion time period. Statistical analysis of the distribution of oscillating mitochondrial clusters during reperfusion shows significant differences between five different outcomes (p< 0.001). Features such as time-points of {Delta}{Psi}m depolarization during I/R, area of mitochondrial clusters and time-resolved frequency components during reperfusion were determined per cell and per mitochondrial cluster. We found that mitochondria from NMVMs subjected to I/R oscillate in the frequency range of 8.6-45mHz, with a mean of 8.73{+/-}4.35mHz. Oscillating clusters had smaller areas ranging from 49.78{+/-}40.64 m2 while non-oscillating clusters had larger areas 65.97{+/-}42.07m2. A negative correlation between frequency and mitochondrial cluster area was seen. We also observed that late {Delta}{Psi}m loss during ischemia correlated with early {Delta}{Psi}m stabilization after oscillation on reperfusion. Thus, MitoWave analysis provides a way to quantify complex time-resolved mitochondrial behavior. It provides an easy to follow workflow to automate microscopy analysis and allows for unbiased, reproducible quantitation of complex nonstationary cellular phenomena. Statement of SignificanceUnderstanding mitochondrial instability in Ischemia Reperfusion injury is key to determining efficacy of interventions. The MitoWave analysis is a powerful yet simple tool that enables even beginner MATALAB-Image J users to automate analysis of time-series from microscopy data. While we used it to detect {Delta}{Psi}m changes during I/R, it can be adapted to detect any such spatio-temporal changes. It standardizes the quantitative analysis of complex biological signals, opens the door to in-depth screening of the genes, proteins and mechanisms underlying metabolic recovery after ischemia-reperfusion.

cell biology↗

bioRxiv2019

Mitochondrial DNA Copy Number and Incident Atrial Fibrillation

BackgroundMechanistic studies suggests that mitochondria DNA (mtDNA) dysfunction may be associated with increased risk of atrial fibrillation (AF). The association between mtDNA copy number (mtDNA-CN) and incident AF in the general population, however, remains unknown. MethodsWe conducted prospective analyses of 19,709 participants from the Atherosclerosis Risk in Communities Study (ARIC), the Multi-Ethnic Study of Atherosclerosis (MESA) and the Cardiovascular Health Study (CHS). mtDNA-CN from peripheral blood was calculated from probe intensities on the Affymetrix Genome-Wide Human single nucleotide polymorphisms (SNP) Array 6.0 in ARIC and MESA, and from multiplexed real time quantitative polymerase chain reaction (qPCR) in CHS. Incident AF cases were identified through electrocardiograms, review of hospital discharge codes, Medicare claims, and death certificates. ResultsThe median follow-up time was 21.4 years in ARIC, 12.9 years in MESA and 11.0 years in CHS, during which 4,021 participants developed incident atrial fibrillation (1,761 in ARIC, 790 in MESA, and 1,470 in CHS). The fully-adjusted pooled hazard ratio for incident atrial fibrillation comparing the 1st to the 5th quintile of mitochondria DNA copy number was 1.13 (1.01, 1.27). The fully-adjusted pooled hazard ratio comparing the 10th vs the 90th percentile of mitochondria DNA copy number was 1.13 (1.04, 1.24). Dose-response spline analysis also showed an inverse association between mitochondria DNA copy number and hazard for atrial fibrillation for all three cohorts. These associations were consistent across subgroups. ConclusionsMitochondria DNA copy number was inversely associated with the risk of AF independent of traditional cardiovascular risk factors. These findings implicate mitochondria DNA copy number as a novel risk factor for atrial fibrillation. Further research is warranted to understand the underlying mechanisms and to evaluate the role of mitochondria DNA copy number in the management of atrial fibrillation risk.

genetics↗

bioRxiv2019

Mitochondrial DNA Copy Number (mtDNA-CN) Can Influence Mortality and Cardiovascular Disease via Methylation of Nuclear DNA CpGs

BackgroundMitochondrial DNA copy number (mtDNA-CN) has been associated with a variety of aging-related diseases, including all-cause mortality. However, the mechanism by which mtDNA-CN influences disease is not currently understood. One such mechanism may be through regulation of nuclear gene expression via the modification of nuclear DNA (nDNA) methylation. MethodsTo investigate this hypothesis, we assessed the relationship between mtDNA-CN and nDNA methylation in 2,507 African American (AA) and European American (EA) participants from the Atherosclerosis Risk in Communities (ARIC) study. To validate our findings we assayed an additional 2,528 participants from the Cardiovascular Health Study (CHS) (N=533) and Framingham Heart Study (FHS) (N=1,995). We further assessed the effect of experimental modification of mtDNA-CN through knockout of TFAM, a regulator of mtDNA replication, via CRISPR-Cas9. ResultsThirty-four independent CpGs were associated with mtDNA-CN at genome-wide significance (P<5x10-8). Meta-analysis across all cohorts identified six mtDNA-CN associated CpGs at genome-wide significance (P<5x10-8). Additionally, over half of these CpGs were associated with phenotypes known to be associated with mtDNA-CN, including coronary heart disease, cardiovascular disease, and mortality. Experimental modification of mtDNA-CN demonstrated that modulation of mtDNA-CN directly drives changes in nDNA methylation and gene expression of specific CpGs and nearby transcripts. Strikingly, the neuroactive ligand receptor interaction KEGG pathway was found to be highly overrepresented in the ARIC cohort (P= 5.24x10-12), as well as the TFAM knockout methylation (P=4.41x10-4) and expression (P=4.30x10-4) studies. ConclusionsThese results demonstrate that changes in mtDNA-CN influence nDNA methylation at specific loci and result in differential expression of specific genes that may impact human health and disease via altered cell signaling.

genomics↗

bioRxiv2018

Precisely control mitochondrial membrane potential with light to manipulate cell fate decisions

Mitochondrial dysfunction has been implicated in many pathological conditions and diseases. The normal functioning of mitochondria relies on maintaining the inner mitochondrial membrane (IMM) potential (a.k.a. {Delta}{Psi}m) that is essential for ATP synthesis, Ca2+ homeostasis, redox balance and regulation of other key signaling pathways such as mitophagy and apoptosis. However, the detailed mechanisms by which {Delta}{Psi}m regulates cellular function remain incompletely understood, partially due to difficulty of manipulating {Delta}{Psi}m with spatiotemporal resolution, reversibility, or cell type specificity. To address this need, we have developed a next-generation optogenetic-based technique for controllable mitochondrial depolarization with light. We demonstrate successful targeting of the heterologous Channelrhodopsin-2 (ChR2) fusion protein to the IMM and formation of functional cationic channels capable of light-induced selective {Delta}{Psi}m depolarization and mitochondrial autophagy. Importantly, we for the first time show that optogenetic-mediated mitochondrial depolarization can be well-controlled to differentially influence the fate of cells expressing mitochondrial ChR2: while sustained moderate light illumination induces substantial apoptotic cell death, transient mild light illumination elicits cytoprotection via mitochondrial preconditioning. Finally, we show that Parkin overexpression exacerbates, instead of ameliorating, mitochondrial depolarization-mediated cell death in HeLa cells. In summary, we provide evidence that the described mitochondrial-targeted optogenetics may have a broad application for studying the role of mitochondria in regulating cell function and fate decision.

cell biology↗