Cell-free DNA levels associate with COPD exacerbations and mortality.

THE QUESTION ADDRESSED BY THE STUDY: Good biological indicators capable of predicting chronic obstructive pulmonary disease (COPD) phenotypes and clinical trajectories are lacking. Because nuclear and mitochondrial genomes are damaged and released by cigarette smoke exposure, plasma cell-free mitochondrial and nuclear DNA (cf-mtDNA and cf-nDNA) levels could potentially integrate disease physiology and clinical phenotypes in COPD. This study aimed to determine whether plasma cf-mtDNA and cf-nDNA levels are associated with COPD disease severity, exacerbations, and mortality risk. MATERIALS AND METHODS: We quantified mtDNA and nDNA copy numbers in plasma from participants enrolled in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE, n = 2,702) study and determined associations with relevant clinical parameters. RESULTS: Of the 2,128 participants with COPD, 65% were male and the median age was 64 (interquartile range, 59-69) years. During the baseline visit, cf-mtDNA levels positively correlated with future exacerbation rates in subjects with mild/moderate and severe disease (Global Initiative for Obstructive Lung Disease [GOLD] I/II and III, respectively) or with high eosinophil count (≥ 300). cf-nDNA positively associated with an increased mortality risk (hazard ratio, 1.33 [95% confidence interval, 1.01-1.74] per each natural log of cf-nDNA copy number). Additional analysis revealed that individuals with low cf-mtDNA and high cf-nDNA abundance further increased the mortality risk (hazard ratio, 1.62 [95% confidence interval, 1.16-2.25] per each natural log of cf-nDNA copy number). ANSWER TO THE QUESTION: Plasma cf-mtDNA and cf-nDNA, when integrated into quantitative clinical measurements, may aid in improving COPD severity and progression assessment.

Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death.

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.

Extracellular Release of Mitochondrial DNA: Triggered by Cigarette Smoke and Detected in COPD.

Cigarette smoke (CS) is the most common risk factor for chronic obstructive pulmonary disease (COPD). The present study aimed to elucidate whether mtDNA is released upon CS exposure and is detected in the plasma of former smokers affected by COPD as a possible consequence of airway damage. We measured cell-free mtDNA (cf-mtDNA) and nuclear DNA (cf-nDNA) in COPD patient plasma and mouse serum with CS-induced emphysema. The plasma of patients with COPD and serum of mice with CS-induced emphysema showed increased cf-mtDNA levels. In cell culture, exposure to a sublethal dose of CSE decreased mitochondrial membrane potential, increased oxidative stress, dysregulated mitochondrial dynamics, and triggered mtDNA release in extracellular vesicles (EVs). Mitochondrial DNA release into EVs occurred concomitantly with increased expression of markers that associate with DNA damage responses, including DNase III, DNA-sensing receptors (cGAS and NLRP3), proinflammatory cytokines (IL-1ß, IL-6, IL-8, IL-18, and CXCL2), and markers of senescence (p16 and p21); the majority of the responses are also triggered by cytosolic DNA delivery in vitro. Exposure to a lethal CSE dose preferentially induced mtDNA and nDNA release in the cell debris. Collectively, the results of this study associate markers of mitochondrial stress, inflammation, and senescence with mtDNA release induced by CSE exposure. Because high cf-mtDNA is detected in the plasma of COPD patients and serum of mice with emphysema, our findings support the future study of cf-mtDNA as a marker of mitochondrial stress in response to CS exposure and COPD pathology.

An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma.

Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of ∼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.

Commercial 4-dimensional echocardiography for murine heart volumetric evaluation after myocardial infarction.

BACKGROUND: Traditional preclinical echocardiography (ECHO) modalities, including 1-dimensional motion-mode (M-Mode) and 2-dimensional long axis (2D-US), rely on geometric and temporal assumptions about the heart for volumetric measurements. Surgical animal models, such as the mouse coronary artery ligation (CAL) model of myocardial infarction, result in morphologic changes that do not fit these geometric assumptions. New ECHO technology, including 4-dimensional ultrasound (4D-US), improves on these traditional models. This paper aims to compare commercially available 4D-US to M-mode and 2D-US in a mouse model of CAL. METHODS: 37 mice underwent CAL surgery, of which 32 survived to a 4 week post-operative time point. ECHO was completed at baseline, 1 week, and 4 weeks after CAL. M-mode, 2D-US, and 4D-US were taken at each time point and evaluated by two separate echocardiographers. At 4 weeks, a subset (n = 12) of mice underwent cardiac magnetic resonance (CMR) imaging to serve as a reference standard. End systolic volume (ESV), end diastolic volume (EDV), and ejection fraction (EF) were compared among imaging modalities. Hearts were also collected for histologic evaluation of scar size (n = 16) and compared to ECHO-derived wall motion severity index (WMSI) and global longitudinal strain as well as gadolinium-enhanced CMR to compare scar assessment modalities. RESULTS: 4D-US provides close agreement of ESV (Bias: -2.55%, LOA: - 61.55 to 66.66) and EF (US Bias: 11.23%, LOA - 43.10 to 102.8) 4 weeks after CAL when compared to CMR, outperforming 2D-US and M-mode estimations. 4D-US has lower inter-user variability as measured by intraclass correlation (ICC) in the evaluation of EDV (0.91) and ESV (0.93) when compared to other modalities. 4D-US also allows for rapid assessment of WMSI, which correlates strongly with infarct size by histology (r = 0.77). CONCLUSION: 4D-US outperforms M-Mode and 2D-US for volumetric analysis 4 weeks after CAL and has higher inter-user reliability. 4D-US allows for rapid calculation of WMSI, which correlates well with histologic scar size.

Mitochondrial Damage and Activation of the STING Pathway Lead to Renal Inflammation and Fibrosis.

Fibrosis is the final common pathway leading to end-stage renal failure. By analyzing the kidneys of patients and animal models with fibrosis, we observed a significant mitochondrial defect, including the loss of the mitochondrial transcription factor A (TFAM) in kidney tubule cells. Here, we generated mice with tubule-specific deletion of TFAM (Ksp-Cre/Tfamflox/flox). While these mice developed severe mitochondrial loss and energetic deficit by 6 weeks of age, kidney fibrosis, immune cell infiltration, and progressive azotemia causing death were only observed around 12 weeks of age. In renal cells of TFAM KO (knockout) mice, aberrant packaging of the mitochondrial DNA (mtDNA) resulted in its cytosolic translocation, activation of the cytosolic cGAS-stimulator of interferon genes (STING) DNA sensing pathway, and thus cytokine expression and immune cell recruitment. Ablation of STING ameliorated kidney fibrosis in mouse models of chronic kidney disease, demonstrating how TFAM sequesters mtDNA to limit the inflammation leading to fibrosis.

PINK1 attenuates mtDNA release in alveolar epithelial cells and TLR9 mediated profibrotic responses.

We have previously shown that endoplasmic reticulum stress (ER stress) represses the PTEN inducible kinase 1 (PINK1) in lung type II alveolar epithelial cells (AECII) reducing mitophagy and increasing the susceptibility to lung fibrosis. Although increased circulating mitochondrial DNA (mtDNA) has been reported in chronic lung diseases, the contribution of mitophagy in the modulation of mitochondrial DAMP release and activation of profibrotic responses is unknown. In this study, we show that ER stress and PINK1 deficiency in AECII led to mitochondrial stress with significant oxidation and damage of mtDNA and subsequent extracellular release. Extracellular mtDNA was recognized by TLR9 in AECII by an endocytic-dependent pathway. PINK1 deficiency-dependent mtDNA release promoted activation of TLR9 and triggered secretion of the profibrotic factor TGF-ß which was rescued by PINK1 overexpression. Enhanced mtDNA oxidation and damage were found in aging and IPF human lungs and, in concordance, levels of circulating mtDNA were significantly elevated in plasma and bronchoalveolar lavage (BAL) from patients with IPF. Free mtDNA was found elevated in other ILDs with low expression of PINK1 including hypersensitivity pneumonitis and autoimmune interstitial lung diseases. These results support a role for PINK1 mediated mitophagy in the attenuation of mitochondrial damage associated molecular patterns (DAMP) release and control of TGF-ß mediated profibrotic responses.

Nox1/Ref-1-mediated activation of CREB promotes Gremlin1-driven endothelial cell proliferation and migration.

Pulmonary arterial hypertension (PAH) is a complex degenerative disorder marked by aberrant vascular remodeling associated with hyperproliferation and migration of endothelial cells (ECs). Previous reports implicated bone morphogenetic protein antagonist Gremlin 1 in this process; however, little is known of the molecular mechanisms involved. The current study was designed to test whether redox signaling initiated by NADPH oxidase 1 (Nox1) could promote transcription factor CREB activation by redox factor 1 (Ref-1), transactivation of Gremlin1 transcription, EC migration, and proliferation. Human pulmonary arterial EC (HPAECs) exposed in vitro to hypoxia to recapitulate PAH signaling displayed induced Nox1 expression, reactive oxygen species (ROS) production, PKA activity, CREB phosphorylation, and CREB:CRE motif binding. These responses were abrogated by selective Nox1 inhibitor NoxA1ds and/or siRNA Nox1. Nox1-activated CREB migrated to the nucleus and bound to Ref-1 leading to CREB:CRE binding and Gremlin1 transcription. CHiP assay and CREB gene-silencing illustrated that CREB is pivotal for hypoxia-induced Gremlin1, which, in turn, stimulates EC proliferation and migration. In vivo, participation of Nox1, CREB, and Gremlin1, as well as CREB:CRE binding was corroborated in a rat PAH model. Activation of a previously unidentified Nox1-PKA-CREB/Ref-1 signaling pathway in pulmonary endothelial cells leads to Gremlin1 transactivation, proliferation and migration. These findings reveal a new signaling pathway by which Nox1 via induction of CREB and Gremlin1 signaling contributes to vascular remodeling and provide preclinical indication of its significance in PAH.

Mitochondrial DNA, nuclear context, and the risk for carcinogenesis.

The inheritance of mitochondrial DNA (mtDNA) from mother to child is complicated by differences in the stability of the mitochondrial genome. Although the germ line mtDNA is protected through the minimization of replication between generations, sequence variation can occur either through mutation or due to changes in the ratio between distinct genomes that are present in the mother (known as heteroplasmy). Thus, the unpredictability in transgenerational inheritance of mtDNA may cause the emergence of pathogenic mitochondrial and cellular phenotypes in offspring. Studies of the role of mitochondrial metabolism in cancer have a long and rich history, but recent evidence strongly suggests that changes in mitochondrial genotype and phenotype play a significant role in the initiation, progression and treatment of cancer. At the intersection of these two fields lies the potential for emerging mtDNA mutations to drive carcinogenesis in the offspring. In this review, we suggest that this facet of transgenerational carcinogenesis remains underexplored and is a potentially important contributor to cancer. Environ. Mol. Mutagen. 60:455-462, 2019. © 2018 Wiley Periodicals, Inc.

hnRNPA2 mediated acetylation reduces telomere length in response to mitochondrial dysfunction.

Telomeres protect against chromosomal damage. Accelerated telomere loss has been associated with premature aging syndromes such as Werner's syndrome and Dyskeratosis Congenita, while, progressive telomere loss activates a DNA damage response leading to chromosomal instability, typically observed in cancer cells and senescent cells. Therefore, identifying mechanisms of telomere length maintenance is critical for understanding human pathologies. In this paper we demonstrate that mitochondrial dysfunction plays a causal role in telomere shortening. Furthermore, hnRNPA2, a mitochondrial stress responsive lysine acetyltransferase (KAT) acetylates telomere histone H4at lysine 8 of (H4K8) and this acetylation is associated with telomere attrition. Cells containing dysfunctional mitochondria have higher telomere H4K8 acetylation and shorter telomeres independent of cell proliferation rates. Ectopic expression of KAT mutant hnRNPA2 rescued telomere length possibly due to impaired H4K8 acetylation coupled with inability to activate telomerase expression. The phenotypic outcome of telomere shortening in immortalized cells included chromosomal instability (end-fusions) and telomerase activation, typical of an oncogenic transformation; while in non-telomerase expressing fibroblasts, mitochondrial dysfunction induced-telomere attrition resulted in senescence. Our findings provide a mechanistic association between dysfunctional mitochondria and telomere loss and therefore describe a novel epigenetic signal for telomere length maintenance.

Aggressive triple negative breast cancers have unique molecular signature on the basis of mitochondrial genetic and functional defects.

Metastatic breast cancer is a leading cause of cancer-related deaths in women worldwide. Patients with triple negative breast cancer (TNBCs), a highly aggressive tumor subtype, have a particularly poor prognosis. Multiple reports demonstrate that altered content of the multicopy mitochondrial genome (mtDNA) in primary breast tumors correlates with poor prognosis. We earlier reported that mtDNA copy number reduction in breast cancer cell lines induces an epithelial-mesenchymal transition associated with metastasis. However, it is unknown whether the breast tumor subtypes (TNBC, Luminal and HER2+) differ in the nature and amount of mitochondrial defects and if mitochondrial defects can be used as a marker to identify tumors at risk for metastasis. By analyzing human primary tumors, cell lines and the TCGA dataset, we demonstrate a high degree of variability in mitochondrial defects among the tumor subtypes and TNBCs, in particular, exhibit higher frequency of mitochondrial defects, including reduced mtDNA content, mtDNA sequence imbalance (mtRNR1:ND4), impaired mitochondrial respiration and metabolic switch to glycolysis which is associated with tumorigenicity. We identified that genes involved in maintenance of mitochondrial structural and functional integrity are differentially expressed in TNBCs compared to non-TNBC tumors. Furthermore, we identified a subset of TNBC tumors that contain lower expression of epithelial splicing regulatory protein (ESRP)-1, typical of metastasizing cells. The overall impact of our findings reported here is that mitochondrial heterogeneity among TNBCs can be used to identify TNBC patients at risk of metastasis and the altered metabolism and metabolic genes can be targeted to improve chemotherapeutic response.

HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression.

Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies.

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.

Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU.

Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+ -permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+ -regulated transcription factor CREB (cyclic adenosine monophosphate response element-binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+ -dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.

Association of G-quadruplex forming sequences with human mtDNA deletion breakpoints.

BACKGROUND: Mitochondrial DNA (mtDNA) deletions cause disease and accumulate during aging, yet our understanding of the molecular mechanisms underlying their formation remains rudimentary. Guanine-quadruplex (GQ) DNA structures are associated with nuclear DNA instability in cancer; recent evidence indicates they can also form in mitochondrial nucleic acids, suggesting that these non-B DNA structures could be associated with mtDNA deletions. Currently, the multiple types of GQ sequences and their association with human mtDNA stability are unknown. RESULTS: Here, we show an association between human mtDNA deletion breakpoint locations (sites where DNA ends rejoin after deletion of a section) and sequences with G-quadruplex forming potential (QFP), and establish the ability of selected sequences to form GQ in vitro. QFP contain four runs of either two or three consecutive guanines (2G and 3G, respectively), and we identified four types of QFP for subsequent analysis: intrastrand 2G, intrastrand 3G, duplex derived interstrand (ddi) 2G, and ddi 3G QFP sequences. We analyzed the position of each motif set relative to either 5' or 3' unique mtDNA deletion breakpoints, and found that intrastrand QFP sequences, but not ddi QFP sequences, showed significant association with mtDNA deletion breakpoint locations. Moreover, a large proportion of these QFP sequences occur at smaller distances to breakpoints relative to distribution-matched controls. The positive association of 2G QFP sequences persisted when breakpoints were divided into clinical subgroups. We tested in vitro GQ formation of representative mtDNA sequences containing these 2G QFP sequences and detected robust GQ structures by UV-VIS and CD spectroscopy. Notably, the most frequent deletion breakpoints, including those of the "common deletion", are bounded by 2G QFP sequence motifs. CONCLUSIONS: The potential for GQ to influence mitochondrial genome stability supports a high-priority investigation of these structures and their regulation in normal and pathological mitochondrial biology. These findings emphasize the potential importance of helicases that subsequently resolve GQ to maintain the stability of the mitochondrial genome.