Potential exhaled breath biomarkers identified in chlorine-exposed mice.

Exhaled breath (EB) contains various volatile organic compounds (VOCs) that can indicate specific biological or pathological processes in the body. Analytical techniques like gas chromatography-mass spectrometry (GC-MS) can be used to detect and measure these exhaled biomarkers. In this study, the objective was to develop a non-invasive method of EB sampling in animals that were awake, as well as to analyze EB for volatile biomarkers specific for chlorine exposure and/or diagnostic biomarkers for chlorine-induced acute lung injury (ALI). To achieve this, a custom-made sampling device was used to collect EB samples from 19 female Balb/c mice. EB was sampled both pre-exposure (serving as internal control) and 30 min after exposure to chlorine. EB was collected on thermal desorption tubes and subsequently analyzed for VOCs by GC-MS. The following day, the extent of airway injury was assessed in the animals by examining neutrophils in the bronchoalveolar lavage fluid. VOC analysis revealed alterations in the EB biomarker pattern post-chlorine exposure, with eight biomarkers displaying increased levels and six exhibiting decreased levels following exposure. Four chlorinated compounds: trichloromethane, chloroacetone, 1,1-dichloroacetone and dichloroacetonitrile, were increased in chlorine-exposed mice, suggesting their specificity as chlorine EB biomarkers. Furthermore, chlorine-exposed mice displayed a neutrophilic inflammatory response and body weight loss 24 h following exposure. In conclusion, all animals developed an airway inflammation characterized by neutrophil infiltration and a specific EB pattern that could be extracted after chlorine exposure. Monitoring EB samples can readily and non-invasively provide valuable information on biomarkers for diagnosis of chlorine-induced ALI, confirming chlorine exposures.

Protocol to separate small and large extracellular vesicles from mouse and human cardiac tissues.

Extracellular vesicles (EVs) are secreted by cells under various conditions and can contribute to the disease progression in tissues. Here, we present a protocol to separate small and large EVs from mouse hearts and cardiac tissues collected from patients. We describe steps for utilizing enzymatic digestion for release of EVs from interstitial space followed by differential centrifugation and immunoaffinity purification. The isolated EVs can be used for various experiments to gain insight into their in vivo functions. For complete details on the use and execution of this protocol, please refer to Liang et al. (2023).1.

Cardiovascular aging: the mitochondrial influence.

Age-associated cardiovascular disease is becoming progressively prevalent due to the increased lifespan of the population. However, the fundamental mechanisms underlying the aging process and the corresponding decline in tissue functions are still poorly understood. The heart has a very high energy demand and the cellular energy needed to sustain contraction is primarily generated by mitochondrial oxidative phosphorylation. Mitochondria are also involved in supporting various metabolic processes, as well as activation of the innate immune response and cell death pathways. Given the central role of mitochondria in energy metabolism and cell survival, the heart is highly susceptible to the effects of mitochondrial dysfunction. These key organelles have been implicated as underlying drivers of cardiac aging. Here, we review the evidence demonstrating the mitochondrial contribution to the cardiac aging process and disease susceptibility. We also discuss the potential mechanisms responsible for the age-related decline in mitochondrial function.

Mitochondria are secreted in extracellular vesicles when lysosomal function is impaired.

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.

Cancer-cell-secreted extracellular vesicles target p53 to impair mitochondrial function in muscle.

Skeletal muscle loss and weakness are associated with bad prognosis and poorer quality of life in cancer patients. Tumor-derived factors have been implicated in muscle dysregulation by inducing cachexia and apoptosis. Here, we show that extracellular vesicles secreted by breast cancer cells impair mitochondrial homeostasis and function in skeletal muscle, leading to decreased mitochondrial content and energy production and increased oxidative stress. Mechanistically, miR-122-5p in cancer-cell-secreted EVs is transferred to myocytes, where it targets the tumor suppressor TP53 to decrease the expression of TP53 target genes involved in mitochondrial regulation, including Tfam, Pgc-1α, Sco2, and 16S rRNA. Restoration of Tp53 in muscle abolishes mitochondrial myopathology in mice carrying breast tumors and partially rescues their impaired running capacity without significantly affecting muscle mass. We conclude that extracellular vesicles from breast cancer cells mediate skeletal muscle mitochondrial dysfunction in cancer and may contribute to muscle weakness in some cancer patients.

Deciphering functional roles and interplay between Beclin1 and Beclin2 in autophagosome formation and mitophagy.

The degradation of macromolecules and organelles by the process of autophagy is critical for cellular homeostasis and is often compromised during aging and disease. Beclin1 and Beclin2 are implicated in autophagy induction, and these homologs share a high degree of amino acid sequence similarity but have divergent N-terminal regions. Here, we investigated the functions of the Beclin homologs in regulating autophagy and mitophagy, a specialized form of autophagy that targets mitochondria. Both Beclin homologs contributed to autophagosome formation, but a mechanism of autophagosome formation independent of either Beclin homolog occurred in response to starvation or mitochondrial damage. Mitophagy was compromised only in Beclin1-deficient HeLa cells and mouse embryonic fibroblasts because of defective autophagosomal engulfment of mitochondria, and the function of Beclin1 in mitophagy required the phosphorylation of the conserved Ser15 residue by the kinase Ulk1. Mitochondria-ER-associated membranes (MAMs) are important sites of autophagosome formation during mitophagy, and Beclin1, but not Beclin2 or a Beclin1 mutant that could not be phosphorylated at Ser15, localized to MAMs during mitophagy. Our findings establish a regulatory role for Beclin1 in selective mitophagy by initiating autophagosome formation adjacent to mitochondria, a function facilitated by Ulk1-mediated phosphorylation of Ser15 in its distinct N-terminal region.

From "crisis to recovery": A complete insight into the mechanisms of chlorine injury in the lung.

Chlorine (Cl2) gas is a toxic industrial chemical (TIC) that poses a hazard to human health following accidental and/or intentional (e.g. terrorist) release. By using a murine model of sub-lethal Cl2 exposure we have examined the airway hyper responsiveness, cellular infiltrates, transcriptomic and proteomic responses of the lung. In the "crisis" phase at 2 h and 6 h there is a significant decreases in leukocytes within bronchoalveolar lavage fluid accompanied by an upregulation within the proteome of immune pathways ultimately resulting in neutrophil influx at 24 h. A flip towards "repair" in the transcriptome and proteome occurs at 24 h, neutrophil influx and an associated drop in the lung function persisting until 14 d post-exposure and subsequent "recovery" after 28 days. Collectively, this research provides new insights into the mechanisms of damage, early global responses and processes of repair induced in the lung following the inhalation of Cl2.

Bioavailability of inhaled or ingested PFOA adsorbed to house dust.

Indoor environments may impact human health due to chemical pollutants in the indoor air and house dust. This study aimed at comparing the bioavailability and distribution of PFOA following both an inhalation and an oral exposure to PFOA coated house dust in rats. In addition, extractable organofluorine (EOF) was measured in different tissue samples to assess any potential influence of other organofluorine compounds in the experimental house dust. Blood samples were collected at sequential time points after exposure and at the time of termination; the lungs, liver, and kidney were collected for quantification of PFOA and EOF. The concentration of PFOA in plasma increased rapidly in both exposure groups attaining a Cmax at 3 h post exposure. The Cmax following inhalation was four times higher compared to oral exposures. At 48 h post exposure, the levels of PFOA in the plasma, liver, and kidney were twice as high from inhalation exposures. This shows that PFOA is readily bioavailable and has a rapid systemic distribution following an inhalation or oral exposure to house dust coated with PFOA. The proportion of PFOA to EOF corresponded to 65-71% and 74-87% in plasma and tissues, respectively. The mass balance between EOF and target PFOA indicates that there might be other unknown PFAS precursor and/or fluorinated compounds that co-existed in the house dust sample that can have accumulated in rats.

Estimated daily intake of per- and polyfluoroalkyl substances related to different particle size fractions of house dust.

Indoor environmental pollutants are a threat to human health. In the current study, we analysed 25 per- and polyfluoroalkyl substances (PFASs) in seven different size fraction of house dust including the two relevant for exposure via ingestion and inhalation. The highest PFAS concentration is found in the inhalable particulate fraction which is explained by the increased surface area as the particulate's sizes decrease. The estimated daily intake (EDI) of the individual PFAS and exposure pathways were calculated for children and adults. In addition, the total EDI for PFOA and its precursors was estimated. The polyfluoroalkyl phosphoric acid diesters (diPAP), followed by PFOA and PFHxA fluortelomer, showed the highest concentrations of PFAS analysed. The cumulative EDI of PFAS for children was 3.0 ng/kg bw per day, a worst-case scenario, which is 17 times higher than the calculated EDI for adults. For children, ingestion of dust was found to result in 800 times higher PFOA exposure than via inhalation. The contribution from PFOA precursors corresponded to only 1% of the EDI from dust indicating PFOA as the main source of exposure. The EDI's of PFOA and PFOS from dust were lower than the calculated EDI's from food ingestion reported by the Swedish Food Agency. Our data indicate that the EDI for the sum of four PFASs: PFOA, PFNA, PFHxS and PFOS from dust intake alone is close to the established tolerable weakly intake of 4.4 ng/kg bw in children, set by European Food Safety Authority (EFSA) in 2020. The combined EDI levels PFOA and PFOS from both dust and food exceeded the EFSA TWI for both children and adults. This study demonstrates that dust is a relevant exposure pathway for PFAS intake and that analysis of relevant particle size fractions is important for evaluation of dust as an exposure pathway.

The role of mitochondrial fission in cardiovascular health and disease.

Mitochondria are organelles involved in the regulation of various important cellular processes, ranging from ATP generation to immune activation. A healthy mitochondrial network is essential for cardiovascular function and adaptation to pathological stressors. Mitochondria undergo fission or fusion in response to various environmental cues, and these dynamic changes are vital for mitochondrial function and health. In particular, mitochondrial fission is closely coordinated with the cell cycle and is linked to changes in mitochondrial respiration and membrane permeability. Another key function of fission is the segregation of damaged mitochondrial components for degradation by mitochondrial autophagy (mitophagy). Mitochondrial fission is induced by the large GTPase dynamin-related protein 1 (DRP1) and is subject to sophisticated regulation. Activation requires various post-translational modifications of DRP1, actin polymerization and the involvement of other organelles such as the endoplasmic reticulum, Golgi apparatus and lysosomes. A decrease in mitochondrial fusion can also shift the balance towards mitochondrial fission. Although mitochondrial fission is necessary for cellular homeostasis, this process is often aberrantly activated in cardiovascular disease. Indeed, strong evidence exists that abnormal mitochondrial fission directly contributes to disease development. In this Review, we compare the physiological and pathophysiological roles of mitochondrial fission and discuss the therapeutic potential of preventing excessive mitochondrial fission in the heart and vasculature.

Mcl-1 Differentially Regulates Autophagy in Response to Changes in Energy Status and Mitochondrial Damage.

Myeloid cell leukemia-1 (Mcl-1) is a unique antiapoptotic Bcl-2 member that is critical for mitochondrial homeostasis. Recent studies have demonstrated that Mcl-1's functions extend beyond its traditional role in preventing apoptotic cell death. Specifically, data suggest that Mcl-1 plays a regulatory role in autophagy, an essential degradation pathway involved in recycling and eliminating dysfunctional organelles. Here, we investigated whether Mcl-1 regulates autophagy in the heart. We found that cardiac-specific overexpression of Mcl-1 had little effect on baseline autophagic activity but strongly suppressed starvation-induced autophagy. In contrast, Mcl-1 did not inhibit activation of autophagy during myocardial infarction or mitochondrial depolarization. Instead, overexpression of Mcl-1 increased the clearance of depolarized mitochondria by mitophagy independent of Parkin. The increase in mitophagy was partially mediated via Mcl-1's LC3-interacting regions and mutation of these sites significantly reduced Mcl-1-mediated mitochondrial clearance. We also found that Mcl-1 interacted with the mitophagy receptor Bnip3 and that the interaction was increased in response to mitochondrial stress. Overall, these findings suggest that Mcl-1 suppresses nonselective autophagy during nutrient limiting conditions, whereas it enhances selective autophagy of dysfunctional mitochondria by functioning as a mitophagy receptor.

Chlorine exposure induces Caspase-3 independent cell death in human lung epithelial cells.

Chlorine (Cl2) is a common toxic industrial gas and human inhalation exposure causes tissue damage with symptoms ranging from wheezing to more severe symptoms such as lung injury or even death. Because the mechanism behind Cl2-induced cell death is not clearly understood, the present study aimed to study the cellular effects in vitro after Cl2 exposure of human A549 lung epithelial cells. In addition, the possible treatment effects of the anti-inflammatory antioxidant N-acetyl cysteine (NAC) were evaluated. Exposure of A549 cells to Cl2 (100-1000 ppm) in the cell medium induced cell damage and toxicity within 1 h in a dose-dependent manner. The results showed that 250 ppm Cl2 increased cell death and formation of apoptotic-like bodies, while 500 ppm Cl2 exposure resulted in predominantly necrotic death. Pre-treatment with NAC was efficient to prevent cell damage at lower Cl2 concentrations in part by averting the formation of apoptotic-like bodies and increasing the expression of the anti-apoptotic proteins clusterin and phosphorylated tumour protein p53(S46). Analysis showed that Cl2 induced cell death by a possibly caspase-independent mechanism, since no cleavage of caspase-3 could be detected after exposure to 250 ppm. Currently, these results justifies further research into new treatment strategies for Cl2-induced lung injury.

Mitochondrial quality surveillance: mitophagy in cardiovascular health and disease.

Selective autophagy of mitochondria, known as mitophagy, is a major quality control pathway in the heart that is involved in removing unwanted or dysfunctional mitochondria from the cell. Baseline mitophagy is critical for maintaining fitness of the mitochondrial network by continuous turnover of aged and less-functional mitochondria. Mitophagy is also critical in adapting to stress associated with mitochondrial damage or dysfunction. The removal of damaged mitochondria prevents reactive oxygen species-mediated damage to proteins and DNA and suppresses activation of inflammation and cell death. Impairments in mitophagy are associated with the pathogenesis of many diseases, including cancers, inflammatory diseases, neurodegeneration, and cardiovascular disease. Mitophagy is a highly regulated and complex process that requires the coordination of labeling dysfunctional mitochondria for degradation while simultaneously promoting de novo autophagosome biogenesis adjacent to the cargo. In this review, we provide an update on our current understanding of these steps in mitophagy induction and discuss the physiological and pathophysiological consequences of altered mitophagy in the heart.

Cardiolipin Remodeling Defects Impair Mitochondrial Architecture and Function in a Murine Model of Barth Syndrome Cardiomyopathy.

BACKGROUND: Cardiomyopathy is a major clinical feature in Barth syndrome (BTHS), an X-linked mitochondrial lipid disorder caused by mutations in Tafazzin (TAZ), encoding a mitochondrial acyltransferase required for cardiolipin remodeling. Despite recent description of a mouse model of BTHS cardiomyopathy, an in-depth analysis of specific lipid abnormalities and mitochondrial form and function in an in vivo BTHS cardiomyopathy model is lacking. METHODS: We performed in-depth assessment of cardiac function, cardiolipin species profiles, and mitochondrial structure and function in our newly generated Taz cardiomyocyte-specific knockout mice and Cre-negative control mice (n≥3 per group). RESULTS: Taz cardiomyocyte-specific knockout mice recapitulate typical features of BTHS and mitochondrial cardiomyopathy. Fewer than 5% of cardiomyocyte-specific knockout mice exhibited lethality before 2 months of age, with significantly enlarged hearts. More than 80% of cardiomyocyte-specific knockout displayed ventricular dilation at 16 weeks of age and survived until 50 weeks of age. Full parameter analysis of cardiac cardiolipin profiles demonstrated lower total cardiolipin concentration, abnormal cardiolipin fatty acyl composition, and elevated monolysocardiolipin to cardiolipin ratios in Taz cardiomyocyte-specific knockout, relative to controls. Mitochondrial contact site and cristae organizing system and F1F0-ATP synthase complexes, required for cristae morphogenesis, were abnormal, resulting in onion-shaped mitochondria. Organization of high molecular weight respiratory chain supercomplexes was also impaired. In keeping with observed mitochondrial abnormalities, seahorse experiments demonstrated impaired mitochondrial respiration capacity. CONCLUSIONS: Our mouse model mirrors multiple physiological and biochemical aspects of BTHS cardiomyopathy. Our results give important insights into the underlying cause of BTHS cardiomyopathy and provide a framework for testing therapeutic approaches to BTHS cardiomyopathy, or other mitochondrial-related cardiomyopathies.

Loss of Parkin Results in Altered Muscle Stem Cell Differentiation during Regeneration.

The high capacity of the skeletal muscle to regenerate is due to the presence of muscle stem cells (MuSCs, or satellite cells). The E3 ubiquitin ligase Parkin is a key regulator of mitophagy and is recruited to mitochondria during differentiation of mouse myoblast cell line. However, the function of mitophagy during regeneration has not been investigated in vivo. Here, we have utilized Parkin deficient (Parkin-/-) mice to investigate the role of Parkin in skeletal muscle regeneration. We found a persistent deficiency in skeletal muscle regeneration in Parkin-/- mice after cardiotoxin (CTX) injury with increased area of fibrosis and decreased cross-sectional area (CSA) of myofibres post-injury. There was also a significant modulation of MuSCs differentiation and mitophagic markers, with altered mitochondrial proteins during skeletal muscle regeneration in Parkin-/- mice. Our data suggest that Parkin-mediated mitophagy plays a key role in skeletal muscle regeneration and is necessary for MuSCs differentiation.