Nucleotide composition bias in mitochondrial DNA (mtDNA) makes the heavy strand prone to form a DNA secondary structure called a guanine quadruplex (G4). This secondary structure has been shown to inhibit polymerase processivity in vitro. We previously identified pathogenic mtDNA variants that lead to increased G4-forming propensity, including a T to C mutation at m.10191 (m.10191 T > C) that causes Leigh syndrome. Cells treated with G4 binding agent (G4BA) berberine show a reduction in m.10191C pathogenic heteroplasmy levels. To help better understand the underlying mechanism behind berberine-induced heteroplasmy shift, we examined the relationship between mitochondrial fission and berberine-mediated shift. Here we show that knockdown of the fission factor DNM1L leads to an accelerated heteroplasmy shift towards the healthy mtDNA allele, lowering m.10191C by 10% in 3 weeks, compared to the 5 weeks required for berberine alone. The specific mechanism involves ATG7, as knockdown of ATG7 is able to partially delay this accelerated heteroplasmy shift. Taken together, we show that DNM1L knockdown is able to accelerate berberine-induced m.10191C heteroplasmy shifting through an autophagy-related mechanism.

Biological heterogeneity in host inflammatory responses to severe pneumonia predicts clinical outcomes and may influence the effectiveness of immunomodulatory therapy. The upstream drivers of this heterogeneity remain poorly defined. We hypothesized that microbial translocation from the lungs to the bloodstream, detectable via multi-compartment metagenomic analysis, contributes to divergent host responses in pneumonia. In this nested case–control study of mechanically ventilated patients with severe pneumonia, we collected paired plasma and endotracheal aspirate samples at baseline. Plasma samples underwent microbial cell-free DNA (mcfDNA) sequencing, and endotracheal aspirates were analyzed by Nanopore metagenomic sequencing. Host-response biomarkers were measured in both plasma and endotracheal aspirate samples. Microbial translocation of pulmonary origin was defined by the genus-level concordance of detectable taxa between matched endotracheal aspirate and plasma samples. Among 98 patients (76 pneumonia, 22 controls), plasma mcfDNA was markedly higher in microbiologically confirmed pneumonia compared with culture-negative pneumonia (median 4015 vs. 210 molecules/μL, p = 0.0006). Pulmonary microbial translocation was identified in 31 (41

Cardiometabolic heart failure with preserved ejection fraction (HFpEF) is a heterogenous metabolic disease, which in the heart presents as left ventricle diastolic dysfunction, ventricular stiffness, and myocardial structural remodeling. Deleterious changes in cardiac metabolism are central to HFpEF pathophysiology, and proposed treatments for the disease have focused on repairing these defects. In the present study, we used a preclinical mouse model that recapitulates cardiometabolic HFpEF to elucidate the molecular mechanisms driving cardiac dysfunction and tested whether recombinant adropin (Adr.) (a liver- and brain-derived endogenous peptide hormone) could reverse observed defects. We show that long-term treatment with Adr. reversed multiple markers of HFpEF-related cardiac dysfunction (including fibrosis, diastolic dysfunction, and cardiomyocyte hypertrophy). Using untargeted metabolomics, we found that Adr. treatment reduced hexosamine biosynthesis pathway activity, leading to a reduction in the O-GlcNAcylation of the cardiac fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD). Reducing LCAD O-GlcNAcylation increased LCAD activity in vitro and reduced the accumulation of long-chain acylcarnitines in HFpEF mouse hearts in vivo. Our results suggest that Adr. may restore cardiac metabolic function in HFpEF and that targeting this pathway may be a novel therapeutic avenue for this disease.

Inositol-requiring enzyme 1a (IRE1a) is a canonical signaling factor in the unfolded protein response (UPR). In addition to this essential role (which prevents the accumulation of misfolded proteins in the endoplasmic reticulum), the endoribonuclease activity of IRE1a targets multiple mRNAs for degradation through a process called Regulated IRE1a-Dependent Decay (RIDD). The products of over 50 genes have been identified as RIDD targets; however, the biological significance of this process remains underexplored. Using publicly available datasets, we examined the fate of 27 well-characterized RIDD targets in the septal wall of heart failure patients, and in mice subject to pressure overload-induced heart failure. We show that decreased mRNA abundance from these RIDD substrate genes - an outcome consistent with RIDD induction - is commonly observed in heart failure.

Single-cell transcriptomic profiling of chronic obstructive pulmonary disease (COPD) lungs identified QKI, an RNA-binding protein, as a candidate emphysema-associated gene, but its epithelial role in COPD pathobiology remains unclear. We show that QKI expression is reduced in human COPD lungs and that alveolar type 2 epithelial (AT2) cell QKI protein levels correlate strongly with spirometric indices and diffusing capacity (DL CO ). Lung epithelium-specific QKI knockout mice (QKI Δ/Δ ) developed spontaneous airspace enlargement with emphysema-like mechanics, and QKI-deficient AT2 cells showed impaired spheroid colony formation and increased apoptosis. Integrated transcriptomic and proteomic analyses of primary AT2 cells revealed a selective reduction in functional mitochondrial (respiratory-chain and metabolic) protein abundance despite relatively preserved transcript levels, consistent with mitochondrial transcriptome-proteome discordance. QKI loss increased mtDNA abundance and TOMM20 staining but decreased ATP5A, indicating accumulation of structurally increased but functionally dysfunctional mitochondria. In human epithelial cells, CRISPR-mediated QKI deficiency reduced oxidative respiration, increased glycolytic reliance, elevated mitochondrial ROS and membrane potential, and increased apoptosis; these phenotypes were partially rescued by QKI re-expression. These findings identify epithelial QKI as a regulator of mitochondrial integrity and stress tolerance in COPD.

Idiopathic pulmonary fibrosis (IPF) is characterized by failed alveolar epithelial repair and progressive fibrotic remodeling. Although aberrant reprogramming of alveolar type 2 (AT2) cells and accumulation of transitional AT2 states are increasing recognized as central features of IPF, the epithelial-intrinsic mechanisms that initiate these pathogenic states remain incompletely understood. Here, we identify mitochondrial transcription factor A (TFAM), a regulator of mitochondrial DNA maintenance, as a critical regulator of AT2 cell homeostasis. TFAM expression was reduced in AT2 cells from human IPF lungs. Inducible AT2 cell-specific Tfam deletion in mice caused spontaneous fibrotic remodeling and increased susceptibility to bleomycin-induced lung injury. TFAM-deficient AT2 cells acquired KRT8+ transitional and p21+ senescence-associated features before the onset of fibrotic transformation, accompanied by impaired oxidative phosphorylation, redox imbalance, mitochondrial superoxide accumulation, repression of mtDNA-encoded respiratory genes, and disrupted mitochondrial ultrastructure. TFAM-deficient AT2 cells developed a profibrotic secretory program that promoted extracellular matrix deposition and fibroblast activation. We further identified insulin-like growth factor-binding protein 2 (IGFBP2) as a secreted mediator induced in TFAM-deficient AT2 cells. IGFBP2 was elevated in AT2 cells in human IPF lung tissue and bronchoalveolar lavage fluid (BALF) from patients with IPF. IGFBP2 was detected in supernatants from fibrotic human precision-cut lung slices (hPCLS). IGFBP2 neutralization attenuated profibrotic remodeling in fibrotic hPCLS. Collectively, our findings identify TFAM-dependent mitochondrial homeostasis as an epithelial checkpoint linking AT2 cell-state stability to impaired epithelial-mesenchymal crosstalk driving pulmonary fibrosis.

Growth Differentiation Factor 15 (GDF15) is a protein that reflects mitochondrial energetic stress and is linked to physical and mental health symptoms, aging, and mortality. Here, we tested the hypothesis that GDF15 is a stress-responsive biomarker through a series of observational and experimental studies. We report four main findings. First, in the UK Biobank (n=53,026) and Framingham Heart Study (FHS) Offspring (n=3,460) cohorts, plasma GDF15 levels were elevated in individuals with symptoms of depression and anxiety. In the FHS cohort, GDF15 was also higher in participants exposed to chronic psychosocial stressors, including lower educational attainment, lower family income, and higher job strain. Second, plasma GDF15 levels in the FHS cohort correlated positively with epigenetic clocks measuring biological aging and effect sizes of GDF15 associations with psychosocial stressors were comparable to those observed for the clocks. Third, in a two-participant intensive-sampling study (n=112 days), saliva GDF15 showed a robust awakening response similar to established stress-related hormones. However, it exhibited a distinct negative pattern, peaking at waking and declining by 42-92% within 30-45 minutes. Finally, in two laboratory experiments (n=148), acute social-evaluative stress significant increased GDF15 levels in plasma and saliva within minutes. Together, these findings suggest that psychosocial stress may contribute to mitochondrial energetic stress indexed by GDF15, with implications for aging and health. This work opens new avenues for using GDF15 as a non-invasive biomarker to study the biological embedding of stress and its impact on aging trajectories.

ABSTRACTMitochondrial damage is a hallmark of metabolic diseases, including diabetes and metabolic dysfunction-associated steatotic liver disease, yet the consequences of impaired mitochondria in metabolic tissues are often unclear. Here, we report that dysfunctional mitochondrial quality control engages a retrograde (mitonuclear) signaling program that impairs cellular identity and maturity across multiple metabolic tissues. Surprisingly, we demonstrate that defects in the mitochondrial quality control machinery, which we observe in pancreatic β cells of humans with type 2 diabetes, cause reductions of β cell mass due to dedifferentiation, rather than apoptosis. Utilizing transcriptomic profiling, lineage tracing, and assessments of chromatin accessibility, we find that targeted deficiency anywhere in the mitochondrial quality control pathway (e.g., genome integrity, dynamics, or turnover) activate the mitochondrial integrated stress response and promote cellular immaturity in β cells, hepatocytes, and brown adipocytes. Intriguingly, pharmacologic blockade of mitochondrial retrograde signalingin vivorestores β cell mass and identity to ameliorate hyperglycemia following mitochondrial damage. Thus, we observe that a shared mitochondrial retrograde response controls cellular identity across metabolic tissues and may be a promising target to treat or prevent metabolic disorders.

The prevalence of cardiometabolic heart failure with preserved ejection fraction (HFpEF) continues to grow, representing over half of heart failure cases in the United States. As no specific medication for HFpEF exists, treatment guidelines focus on the management of comorbidities related to metabolic syndrome (e.g. obesity, diabetes, hypertension) that promote the disease1. These same comorbidities also drive pathology in non-cardiac tissues, and the links between cardiometabolic disease presentations in different organs are increasingly being recognized. Preclinical studies on the potential crosstalk between HFpEF and metabolic disease in the liver (e.g. metabolic dysfunction-associated liver disease; MASLD) have focused on how liver dysfunction may affect the heart, particularly through the release of secreted liver proteins. This may reflect the situation in the clinic, where incident MASLD is a risk factor for future HFpEF development. Here, in contrast to this developing paradigm of liver-initiated cardiac disease, we report for the first time a defect in cardiac metabolism related to the mitochondrial metabolic protein GCN5L1 that drives hepatic steatosis and MASLD in HFpEF.

Mitochondrial damage is a hallmark of metabolic diseases, including diabetes, yet the consequences of compromised mitochondria in metabolic tissues are often unclear. Here, we report that dysfunctional mitochondrial quality control engages a retrograde (mitonuclear) signaling program that impairs cellular identity and maturity in β-cells, hepatocytes, and brown adipocytes. Targeted deficiency throughout the mitochondrial quality control pathway, including genome integrity, dynamics, or turnover, impaired the oxidative phosphorylation machinery, activating the mitochondrial integrated stress response, eliciting chromatin remodeling, and promoting cellular immaturity rather than apoptosis to yield metabolic dysfunction. Indeed, pharmacologic blockade of the integrated stress response in vivo restored β-cell identity following loss of mitochondrial quality control. Targeting mitochondrial retrograde signaling may therefore be promising in the treatment or prevention of metabolic disorders.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is an age-related disease with remodeling of alveolar epithelial cells. Transitional alveolar epithelial type 2 (AT2) cells become senescent, contributing to tissue remodeling. Mitochondrial hemostasis is pivotal for epithelial cell fate, and its dysfunction drives fibrosis after lung injury. Transcription Factor A, Mitochondrial (TFAM), is essential for mitochondrial DNA integrity (mtDNA). We hypothesize that TFAM deficiency in AT2 leads to the accumulation of transitional AT2, promoting lung fibrosis. METHODS: Datasets from Lung Genomics Research Consortium (LGRC) microarray and IPF Atlas single-cell RNA sequencing (scRNA-seq) were analyzed to assess TFAM expression. TFAM in IPF and bleomycin-treated mouse lungs were quantified and localized via immunofluorescence. qPCR and Western blotting measured Tfam in primary mouse AT2 (pmAT2) cells isolated from bleomycin-treated mice. Tfam was deleted in pmAT2 via adenovirus-mediated Cre recombination. Scratch assays assessed fibroblast proliferation and migration in response to supernatants from Tfam-deficient cells, and cytokine arrays identified cytokine profile changes. Senescence was evaluated with immunofluorescence and SA-β-gal staining. Tfam conditional knockout (cKO) mice (Sftpc ERT2-Cre/WT; Tfam fl/fl) and controls (Sftpc ERT2-Cre/WT; Tfam WT/WT) were subjected to bleomycin injury. Lung function was measured by flexiVent, and tissues were collected for histology and senescence/fibrotic marker assessment. RESULTS: Compared to healthy donors (n=137) and COPD patients (n=220), IPF patients (n=255) showed significantly lower TFAM expression in lung tissue in the LGRC datasets. IPF Atlas scRNA-seq data demonstrated a significant TFAM reduction in AT2. The reduction of TFAM in AT2 of IPF lungs was further confirmed via immunofluorescence. Similarly, in the bleomycin mouse model, Tfam in AT2 was reduced at RNA and protein levels 14 days post-injury. Deleting Tfam from pmAT2 promoted mitochondrial dysfunction, mtDNA alterations, and emergence of transitional AT2 markers, including senescence markers like increased SA-β-gal staining, P21+ cells, and an expression of Krt8+, among others. These cells secreted distinct profibrotic and pro-senescence cytokines, including IGFBP2, GDF-15, VEGF and CXCL10. Conditioned medium from Tfam-deficient AT2 stimulated fibroblast activation and migration. In vivo, Tfam cKO mice exhibited high mortality (92.9%) post-bleomycin injury. Notably, Tfam cKO mice developed spontaneous distal lung fibrosis without injury as early as 18 weeks, assessed by lung function decline, lung histology, and fibrosis markers. Baseline assessments of Tfam cKO mice revealed increased transitional AT2 cells, marked by P21 and Krt8 expression. CONCLUSION: Our findings suggest that Tfam deficiency drives the emergence of transitional AT2 cells, which contribute to impaired epithelial-mesenchymal crosstalk, fibroblast activation, and lung fibrosis development.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and resulting coronavirus disease (COVID-19) cause placental dysfunction, which increases the risk of adverse pregnancy outcomes. While abnormal placental pathology resulting from COVID-19 is common, direct infection of the placenta is rare. This suggests that pathophysiology associated with maternal COVID-19, rather than direct placental infection, is responsible for placental dysfunction. We hypothesized that maternal circulating extracellular vesicles (EVs), altered by COVID-19 during pregnancy, contribute to placental dysfunction. To examine this hypothesis, we characterized circulating EVs from pregnancies complicated by COVID-19 and tested their effects on trophoblast cell physiology in vitro. Trophoblast exposure to EVs isolated from patients with an active infection (AI), but not controls, altered key trophoblast functions including hormone production and invasion. Thus, circulating EVs from participants with an AI, both symptomatic and asymptomatic cases, can disrupt vital trophoblast functions. EV cargo differed between participants with COVID-19, depending on the gestational timing of infection, and Controls, which may contribute to the disruption of the placental transcriptome and morphology. Our findings show that COVID-19 can have effects throughout pregnancy on circulating EVs, and circulating EVs are likely to participate in placental dysfunction induced by COVID-19.

ABSTRACTThe prevalence of cardiometabolic heart failure with preserved ejection fraction (HFpEF) continues to grow, representing over half of heart failure cases in the United States. As no specific medication for HFpEF exists, treatment guidelines focus on the management of comorbidities related to metabolic syndrome (e.g. obesity, diabetes, hypertension) that promote the disease. These same comorbidities also drive pathology in non-cardiac tissues, and the links between cardiometabolic disease presentations in different organs are increasingly being recognized. Preclinical studies on the potential crosstalk between HFpEF and metabolic disease in the liver (e.g. metabolic-associated fatty liver disease; MAFLD) have focused on how liver dysfunction may affect the heart, particularly through the release of secreted liver proteins. This may reflect the situation in the clinic, where incident MAFLD is a risk factor for future HFpEF development. Here, in contrast to this developing paradigm of liver-initiated cardiac disease, we report for the first time a defect in cardiac metabolism related to the mitochondrial metabolic protein GCN5L1 that drives hepatic steatosis and MAFLD in HFpEF.

There has been a recent expansion in our understanding of DNA-sensing mechanisms. Mitochondrial dysfunction, oxidative and proteostatic stresses, instability and impaired disposal of nucleoids cause the release of mitochondrial DNA (mtDNA) from the mitochondria in several human diseases, as well as in cell culture and animal models. Mitochondrial DNA mislocalized to the cytosol and/or the extracellular compartments can trigger innate immune and inflammation responses by binding DNA-sensing receptors (DSRs). Here, we define the features that make mtDNA highly immunogenic and the mechanisms of its release from the mitochondria into the cytosol and the extracellular compartments. We describe the major DSRs that bind mtDNA such as cyclic guanosine-monophosphate-adenosine-monophosphate synthase (cGAS), Z-DNA-binding protein 1 (ZBP1), NOD-, LRR-, and PYD- domain-containing protein 3 receptor (NLRP3), absent in melanoma 2 (AIM2) and toll-like receptor 9 (TLR9), and their downstream signaling cascades. We summarize the key findings, novelties, and gaps of mislocalized mtDNA as a driving signal of immune responses in vascular, metabolic, kidney, lung, and neurodegenerative diseases, as well as viral and bacterial infections. Finally, we define common strategies to induce or inhibit mtDNA release and propose challenges to advance the field.

In response to acute stress, prior studies have found an increase in circulating cell-free mitochondrial DNA (cf-mtDNA) and pro-inflammatory cytokines, highlighting two potential inter-related mechanisms by which stressors can get under the skin. However, prior studies lacked a resting control condition to isolate the effect of psychological stress from other aspects related to laboratory procedures. Here, we conducted a crossover experimental trial examining responses to a socio-evaluative stressor under laboratory conditions. 72 volunteers (age 20-50, 48% women) were tested on two occasions, counterbalanced, separated by at least a month. On one occasion, they were exposed to a 5-min socio-evaluative stressor (speech task), and on the other occasion, rested for the same period. Blood samples were obtained at 10 timepoints from pre- to 2 hours post-exposure to assess neuroendocrine (cortisol, catecholamines), pro-inflammatory cytokine (IL-6, IL-10, TNF-ɑ), and both plasma and serum cf-mtDNA responses. Compared to the control visit, the stressor significantly increased anxiety, heart rate, blood pressure, cortisol, and norepinephrine ( p 's<0.05-0.0001), confirming the psychobiological impact of the stressor. Unexpectedly, IL-6 and plasma cf-mtDNA increased (time effect p <0.0001) in both the stress and control conditions. While no significant effect of time was found for serum cf-mtDNA, plasma cf-mtDNA showed a bi-phasic response with an initial 22-24% increase at 5-10 min (g=0.07, stress-control visits), followed by a decrease and another 70-81% increase from 45 to 75 min (g=0.59 (stress visit), g=0.41 (control visit)). There were no significant associations between the pro-inflammatory and cf-mtDNA responses, pointing to their independent regulation. While mood, cardiovascular, and neuroendocrine reactivity were selectively induced by socio-evaluative stress, IL-6 and blood cf-mtDNA increased across both the stress and control conditions, suggesting that these biomarkers may reflect non-specific responses to the laboratory protocols (e.g., blood draw) rather than to socio-evaluative stress itself.

ABSTRACTThe right and left ventricle of the heart have distinctly different developmental origins and are affected differently by similar pathological stimuli. Though it is well established that the heart relies almost entirely on mitochondrial function to sustain energy production, it remains unclear whether bioenergetics differ in the two ventricles. Herein, we define a novel methodology to optimize the isolation of intact cardiomyocytes from the right versus the left ventricle. We demonstrate that this segmental Langendorff-free methodology yields viable cardiomyocytes with intact mitochondrial function. Further, we compare bioenergetics in right versus left ventricle cardiomyocytes and show that cardiomyocytes from the right ventricle have a greater maximal capacity for respiration and enhanced glycolytic rate. This increase in respiration was concomitant with increased fatty acid oxidation and levels of fatty acid oxidation proteins, but no change in mitochondrial electron transport complex expression. These data validate a potentially powerful tool to evaluate differences in right and left ventricular function and advance the understanding of cardiac bioenergetic differences. These data will be discussed in the context of differential responses by the right versus ventricle in pathology.

Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage. In space radiation-exposed cells, targeting specific microRNAs with antagomirs can reduce cardiovascular damage and improve cellular function. Here the authors describe a reduction in inflammation and DNA double-strand break activity within these cells upon antagomir treatment.

The prevalence of cardiometabolic heart failure with preserved ejection fraction (HFpEF) continues to grow worldwide, and now represents over half of current heart failure cases in the United States ([1][1]). Due to a lack of specific approved therapies, current treatment guidelines focus on the management of comorbidities related to metabolic syndrome (e.g. obesity, diabetes, hypertension) that promote HFpEF progression ([1][1]). The same comorbidities also drive cardiometabolic disease in non-cardiac tissues, and links between disease presentations in different organs are increasingly being recognized in the clinic. However, mechanistic studies examining the underlying pathophysiological connections have not kept pace, particularly in the cardio-hepatic disease axis ([2][2]). To address this, we used a recently developed and validated preclinical model of HFpEF ([3][3]) to examine how this disease impacts the liver. The development of HFpEF in mice leads to the simultaneous development of widespread hepatic steatosis that is consistent with human non-alcoholic fatty liver disease (NAFLD). Mechanistically, we show that the liver steatosis observed is driven by excess glucogenic amino acid entry into the TCA cycle, which promotes hepatic glucose production and de novo lipogenesis. Our findings suggest that HFpEF development is a multi-organ event, with implications for both preclinical and translational research. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3

Mitochondrial DNA (mtDNA) is inherited almost exclusively from the maternal lineage. Paternal destruction of either mtDNA or whole mitochondria has been the dominant model for mtDNA transmission. Recently, Lee et al. provided evidence for mitochondrial transcription factor A (TFAM) import sequence regulation as a potential cause for mtDNA depletion in human sperm before fertilization.