bims-kimdis Biomed News
on Ketones, inflammation and mitochondria in disease
Issue of 2024–11–17
nineteen papers selected by
Matías Javier Monsalves Álvarez, Universidad Andrés Bello



  1. Front Pharmacol. 2024 ;15 1463381
      Heart failure (HF) is a life-threatening cardiac syndrome characterized by high morbidity and mortality, but current anti-heart failure therapies have limited efficacy, necessitating the urgent development of new treatment drugs. Exogenous ketone supplementation helps prevent heart failure development in HF models, but therapeutic ketosis in failing hearts has not been systematically elucidated, limiting the use of ketones to treat HF. Here, we summarize current evidence supporting ketotherapy in HF, emphasizing ketone metabolism in the failing heart, metabolic and non-metabolic therapeutic effects, and mechanisms of ketotherapy in HF, involving the dynamics within the mitochondria. We also discuss clinical strategies for therapeutic ketosis, aiming to deepen the understanding of the characteristics of ketone metabolism, including mitochondrial involvement, and its clinical therapeutic potential in HF.
    Keywords:  cardiovascular protection; energy metabolism; heart failure; ketogenic treatment; ketone bodies; mechanisms of ketotherapy
    DOI:  https://doi.org/10.3389/fphar.2024.1463381
  2. Cell. 2024 Nov 07. pii: S0092-8674(24)01214-5. [Epub ahead of print]
      β-Hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve the interconversion of BHB and primary energy intermediates. Here, we identify a previously undescribed BHB secondary metabolic pathway via CNDP2-dependent enzymatic conjugation of BHB and free amino acids. This BHB shunt pathway generates a family of anti-obesity ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. The most abundant BHB-amino acid, BHB-Phe, is a ketosis-inducible congener of Lac-Phe that activates hypothalamic and brainstem neurons and suppresses feeding. Conversely, CNDP2-KO mice exhibit increased food intake and body weight following exogenous ketone ester supplementation or a ketogenic diet. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, enzymatic amino acid BHB-ylation defines a ketone shunt pathway and bioactive ketone metabolites linked to energy balance.
    Keywords:  BHB; enzyme; ketone; metabolite; metabolomics; obesity
    DOI:  https://doi.org/10.1016/j.cell.2024.10.032
  3. Cells. 2024 Oct 22. pii: 1746. [Epub ahead of print]13(21):
      Spinal cord injury (SCI) pathology and pathophysiology can be attributed to both primary physical injury and secondary injury cascades. Secondary injury cascades involve dysregulated metabolism and energetic deficits directly linked to compromised mitochondrial bioenergetics. Rescuing mitochondrial function and reducing oxidative stress are associated with neuroprotection. In this regard, ketosis after traumatic brain injury (TBI), or after SCI, improves secondary neuropathology by decreasing oxidative stress, increasing antioxidants, reducing inflammation, and improving mitochondrial bioenergetics. Here, we follow up on our previous study and have used an exogenous ketone monoester, (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KE), as an alternative to a ketogenic diet, focusing on mitochondrial function between 1 and 14 days after injury. Starting 3 h following a cervical level 5 (C5) hemi-contusion injury, animals were fed either a standard control diet (SD) or a ketone ester diet (KED) combined with KE administered orally (OKE). We found that mitochondrial function was reduced after SCI at all times post-SCI, accompanied by reduced expression of most of the components of the electron transport chain (ETC). The KE rescued some of the bioenergetic parameters 1 day after SCI when D-β-Hydroxybutyrate (BHB) concentrations were ~2 mM. Still, most of the beneficial effects were observed 14 days after injury, with BHB concentrations reaching values of 4-6 mM. To our knowledge, this is the first report to show the beneficial effects of KE in rescuing mitochondrial function after SCI and demonstrates the suitability of KE in ameliorating the metabolic dysregulation that occurs after traumatic SCI without requiring a restrictive dietary regime.
    Keywords:  D-β-hydroxybutyrate; dietary treatment; metabolism; neurotrauma; respirometry
    DOI:  https://doi.org/10.3390/cells13211746
  4. Front Med (Lausanne). 2024 ;11 1432717
      Ketogenic diet (KD) is a high-fat, low-carbohydrate (CHO) diet, designed to induce a metabolic state of ketosis in which the body metabolizes primarily lipids for energy production. Various forms of KD are being promoted as promising treatments for numerous health conditions from chronic headaches to weight-loss and even different forms of cancer and are becoming increasingly more popular. KD appears to be an efficacious approach for weight-loss, and maintenance, improved glycemia, cognitive function and cancer prognosis. However, there is a controversy regarding the safety of KD, and the potential health risks that might be associated with long-term exposure to KD. There is a gap between the acceptance and utilization of KD in individuals with health conditions and the criticism and negative attitudes toward KD by some clinicians. Many individuals choose to follow KD and are encouraged by the positive results they experience. Although the medical establishment does not endorse KD as a first line of treatment, clinicians need to be informed about KD, and offer support and medical supervision for patients who self-select to follow KD. This can ensure that within the boundaries of KD, patients will make good and healthy dietary choices and prevent clinical disengagement in extreme cases. To that end, there is an urgent need for good quality research to address the issues of long-term safety of KD in different clinical populations and for standardization of KD both in research and in the clinic.
    Keywords:  cancer; diabetes; intractable pediatric epilepsy; neurodegenerative disease; obesity; very-low-carbohydrate-diet; weight-loss
    DOI:  https://doi.org/10.3389/fmed.2024.1432717
  5. Eur J Neurosci. 2024 Nov 11.
      Parkinson's disease (PD) is a progressive, neurodegenerative disease characterized by motor dysfunction and dopamine deficits. The MitoPark (MP) mouse model of PD recapitulates several facets of Parkinson's disease, including gradual development of motor deficits, which enables the study of potential therapeutic interventions. One therapeutic strategy involves decreasing the mitochondrial metabolic load by inducing ketosis and providing an alternative energy source for neurons, leading to decreased neuronal oxidative stress. Thus, we hypothesized that administration of a ketone ester-enriched diet (KEED) would improve motor and dopamine release deficits in MP mice. Motor function (rotarod and open field tests), dopamine release (fast-scan cyclic voltammetry), tissue dopamine levels (gas chromatography-mass spectrometry) and dopamine neurons and axons (immunofluorescence) were assessed in MP, and control mice fed either the standard or a KEED. When started on the ketone diet before motor dysfunction onset, MP mice had improved motor function relative to standard diet (SD) MP mice. While the KEED did not preserve dopamine neurons or striatal dopamine axons, dopamine release in ketone diet MP mice was greater than SD MP mice but less than control mice. In a follow-up experiment, we began the ketone diet after motor dysfunction onset and observed a modest preservation of motor function in ketone diet MP mice relative to SD MP mice. The improvement in motor dysfunction indicates that a KEED or ketone supplement may have a beneficial effect on delaying motor deficit progression in Parkinson's disease.
    Keywords:  Parkinson's disease; dopamine neurons; ketosis; neuroprotection; striatum
    DOI:  https://doi.org/10.1111/ejn.16601
  6. FASEB J. 2024 Nov 15. 38(21): e70157
      This study investigated the effects of 14 days low energy availability (LEA) versus optimal energy availability (OEA) in endurance-trained females on substrate utilization, insulin sensitivity, and skeletal muscle mitochondrial oxidative capacity; and the impact of metabolic changes on exercise performance. Twelve endurance-trained females (V̇O2max 55.2 ± 5.1 mL × min-1 × kg-1) completed two 14-day randomized, blinded, cross-over, controlled dietary interventions: (1) OEA (51.9 ± 2.0 kcal × kg fat-free mass (FFM)-1 × day-1) and (2) LEA (22.3 ± 1.5 kcal × kg FFM-1 × day-1), followed by 3 days OEA. Participants maintained their exercise training volume during both interventions (approx. 8 h × week-1 at 79% heart rate max). Skeletal muscle mitochondrial respiratory capacity, glycogen, and maximal activity of CS, HAD, and PFK were unaltered with LEA. 20-min time trial endurance performance was impaired by 7.8% (Δ -16.8 W, 95% CI: -23.3 to -10.4, p < .001) which persisted following 3 days refueling post-LEA (p < .001). Fat utilization was increased post-LEA as evidenced by: (1) 99.4% (p < .001) increase in resting plasma free fatty acids (FFA); (2) 270% (p = .007) larger reduction in FFA in response to acute exercise; and (3) 28.2% (p = .015) increase in resting fat oxidation which persisted during submaximal exercise (p < .001). These responses were reversed with 3 days refueling. Daily glucose control (via CGM), HOMA-IR, HOMA-β, were unaffected by LEA. Skeletal muscle O2 utilization and carbohydrate availability were not limiting factors for aerobic exercise capacity and performance; therefore, whether LEA per se affects aspects of training quality/recovery requires investigation.
    Keywords:  caloric restriction; fat oxidation; insulin sensitivity; metabolism; mitochondrial oxidative capacity
    DOI:  https://doi.org/10.1096/fj.202401780R
  7. J Physiol. 2024 Nov 13.
      
    Keywords:  muscle memory; muscle plasticity; myonuclear permanence; myonuclei; performance‐enhancing drugs; resistance training; skeletal muscle fibre
    DOI:  https://doi.org/10.1113/JP287606
  8. BMC Med. 2024 Nov 13. 22(1): 529
       BACKGROUND: Intermittent fasting (IF) holds promise for enhancing metabolic health. However, the optimum IF forms and their superiority over continuous energy restriction (CER) remain unclear due to disconnected findings.
    METHODS: We systematically searched PubMed, Embase, and the Cochrane databases for meta-analyses of randomized controlled trials (RCTs) investigating the association between IF and metabolic health outcomes. Subsequently, we performed an umbrella review and network meta-analysis (NMA) to evaluate the efficacy of different forms of IF (time-restricted eating (TRE), alternate-day fasting (ADF), and 5:2 diet (regular eating for 5 days and energy restriction for 2 days per week)) compared to CER and usual diets on metabolic health outcomes. To assess the certainty of both direct and indirect estimates, we employed the Confidence in Network Meta-Analysis (CINeMA) approach. Additionally, we calculated the surface under the cumulative ranking curve (SUCRA) for each dietary strategy to determine their ranking in terms of metabolic health benefits.
    RESULTS: Ten of the best and non-redundant meta-analysis studies, involving 153 original studies and 9846 participants, were included. When considering direct evidence only, all IF forms significantly reduced body weight compared to usual diets. In NMA incorporating indirect evidence, all IF regimens also significantly reduced body weight compared to usual diets. In the SUCRA of NMA, IF ranked higher than usual diets or CER in 85.4% and 56.1% of the outcomes, respectively. ADF had the highest overall ranking for improving metabolic health (ranked first: 64.3%, ranked second: 14.3%).
    CONCLUSIONS: Overall, all IF forms demonstrate potentials to improve metabolic health, with ADF appearing to produce better outcomes across investigated outcomes. Further high-quality trials are warranted to confirm the (relative) efficacy of IF on metabolic health.
    TRIAL REGISTRATION: PROSPERO (record no: CRD42022302690).
    Keywords:  Alternate-day fasting; Continuous energy restriction; Intermittent fasting; Metabolic health; Time-restricted eating
    DOI:  https://doi.org/10.1186/s12916-024-03716-1
  9. Trends Cell Biol. 2024 Nov 08. pii: S0962-8924(24)00211-3. [Epub ahead of print]
      Pyroptosis is a lytic, proinflammatory type of programmed cell death crucial for the immune response to pathogen infections and internal danger signals. Gasdermin D (GSDMD) acts as the pore-forming protein in pyroptosis following inflammasome activation. While recent research has improved our understanding of pyroptosis activation and execution, many aspects regarding the molecular mechanisms controlling inflammasome and GSDMD activation remain to be elucidated. A growing body of literature has shown that S-palmitoylation, a reversible post-translational modification (PTM) that attaches palmitate to cysteine residues, contributes to multi-layered regulation of pyroptosis. This review summarizes the emerging roles of S-palmitoylation in pyroptosis research with a focus on mechanisms that regulate NLRP3 inflammasome and GSDMD activation.
    Keywords:  GSDMD; NLRP3; gasdermin; inflammasome; palmitoylation; pyroptosis
    DOI:  https://doi.org/10.1016/j.tcb.2024.10.005
  10. Sci Rep. 2024 Nov 14. 14(1): 28079
      Desmin intermediate filaments play a crucial role in stress transmission and mechano-protection. The loss of its integrity triggers myofibril breakdown and muscle atrophy for which desmin phosphorylation (pDes) is a priming factor. We investigated whether eccentric accentuated resistance exercise (RE) influences the regulation of pDes, effecting its susceptibility to cleavage. Ten healthy persons performed 14 RE-sessions (2 per week). Muscle biopsies were collected in both untrained and trained conditions at rest (pre 1, pre 14) and one hour after RE (post 1, post 14). Western blotting and immunohistochemistry were utilized to assess desmin content, phosphorylation at several sites and susceptibility to cleavage. In untrained condition (pre 1, post 1), RE induced dephosphorylation of serin 31 and 60. Trained muscle exhibited more pronounced dephosphorylation at Serin 31 post-RE. Dephosphorylation was accompanied by reduced susceptibility of desmin to cleavage. Additionally, training increased total desmin content, upregulated baseline serine 31 phosphorylation and attenuated pDes at serine 60 and threonine 17. Our findings suggest that acute and repeated RE changes the phosphorylation pattern of desmin and its susceptibility to cleavage, highlighting pDes as an adaptive mechanism in skeletal muscle, contributing to the proteostatic regulation in response to recurring stress.
    Keywords:  Desmin; Exercise; Human skeletal muscle; Intermediate filaments
    DOI:  https://doi.org/10.1038/s41598-024-79385-0
  11. J Gen Physiol. 2024 Dec 02. pii: e202313485. [Epub ahead of print]156(12):
      Mitochondrial fission is a key trigger of cardiac ischemia-reperfusion injuries (IR). Exercise training is an efficient cardioprotective strategy, but its impact on mitochondrial fragmentation during IR remains unknown. Using isolated rat hearts, we found that exercise training limited the activation of dynamin-like protein 1 and limited mitochondrial fragmentation during IR. These results support the hypothesis that exercise training contributes to cardioprotection through its capacity to modulate the mitochondrial fragmentation during IR.
    DOI:  https://doi.org/10.1085/jgp.202313485
  12. Front Nutr. 2024 ;11 1485632
      Intermittent fasting (IF) has demonstrated extensive health benefits through the regulation of fatty acid metabolism and modulation of the neuroimmune microenvironment, primarily via the activation of key signaling pathways such as AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1). IF not only facilitates fatty acid oxidation and improves metabolic health, but also enhances mitochondrial function, mitigates oxidative stress, promotes autophagy, and inhibits apoptosis and ferroptosis. These mechanisms contribute to its substantial preventive and therapeutic potential in various conditions, including neurodegenerative disorders such as Alzheimer's and Parkinson's diseases, autoimmune diseases, and neurotraumatic conditions. While supportive evidence has been obtained from animal models and preliminary clinical studies, further large-scale, long-term randomized controlled trials are imperative to establish its safety and evaluate its clinical efficacy comprehensively.
    Keywords:  AMPK; SIRT1; autophagy; fatty acid metabolism; intermittent fasting; ketone bodies; neuroimmuno microenvironment
    DOI:  https://doi.org/10.3389/fnut.2024.1485632
  13. Pflugers Arch. 2024 Nov 14.
      The Na+-K+-ATPase is a critical regulator of ion homeostasis during contraction, buffering interstitial K+ accumulation, which is linked to muscle fatigue during intense exercise. Within this context, we adopted a recently reported methodology to examine exercise-induced alterations in maximal Na+-K+-ATPase activity. Eighteen trained healthy young males completed a repeated high-intensity cycling protocol consisting of three periods (EX1-EX3) of intermittent exercise. Each period comprised 10 × 45-s cycling at ~ 105% Wmax and a repeated sprint test. Muscle biopsies were sampled at baseline and after EX3 for determination of maximal in vitro Na+-K+-ATPase activity. Blood was drawn after each period and in association with a 2-min cycling test at a standardized high intensity (~ 90% Wmax) performed before and after the session to assess plasma K+ accumulation. Further, a 5-h recovery period with the ingestion of carbohydrate or placebo supplementation was implemented to explore potential effects of carbohydrate availability before sampling a final biopsy and repeating all tests. A ~ 12% reduction in maximal Na+-K+-ATPase activity was demonstrated following EX3 compared to baseline (25.2 ± 3.9 vs. 22.4 ± 4.8 μmol·min-1·g-1 protein, P = 0.039), which was sustained at the recovery time point (~ 15% decrease compared to baseline to 21.6 ± 5.9 μmol·min-1·g-1 protein, P = 0.008). No significant effect of carbohydrate supplementation was observed on maximal Na+-K+-ATPase activity after recovery (P = 0.078). In conclusion, we demonstrate an exercise-induced depression of maximal Na+-K+-ATPase activity following high-intensity intermittent exercise, which was sustained during a 5-h recovery period and unrelated to carbohydrate availability under the present experimental conditions. This was shown using a novel NADH coupled assay and confirms previous findings using other methodological approaches.
    Keywords:  Carbohydrate; Excitability; Excitation–contraction coupling; Fatigue; Glycogen; Potassium
    DOI:  https://doi.org/10.1007/s00424-024-03036-6
  14. Life Sci. 2024 Nov 09. pii: S0024-3205(24)00815-4. [Epub ahead of print]359 123225
      Chronic diseases linked with sedentary lifestyles and poor dietary habits are increasingly common in modern society. Exercise is widely acknowledged to have a plethora of health benefits, including its role in primary prevention of various chronic conditions like type 2 diabetes mellitus, obesity, cardiovascular disease, and several musculoskeletal as well as degenerative disorders. Regular physical activity induces numerous physiological adaptations that contribute to these positive effects, primarily observed in skeletal muscle but also impacting other tissues. There is a growing interest among researchers in developing pharmaceutical interventions that mimic the beneficial effects of exercise for therapeutic applications. Exercise mimetic medications have the potential to be helpful aids in enhancing functional outcomes for patients with metabolic dysfunction, neuromuscular and musculoskeletal disorders. Some of the potential targets for exercise mimetics include pathways involved in metabolism, mitochondrial function, inflammation, and tissue regeneration. The present review aims to provide an exhaustive overview of the current understanding of exercise physiology, the role of exerkines and biomolecular pathways, and the potential applications of exercise mimetic drugs for the treatment of several diseases.
    Keywords:  Exercise; Exercise mimetic drugs; Exerkines; Metabolic dysfunction; Musculoskeletal disorders; Tissue regeneration
    DOI:  https://doi.org/10.1016/j.lfs.2024.123225
  15. Front Physiol. 2024 ;15 1501362
      Wooden breast (WB) is a myopathy that occurs in pectoralis major (PM) muscles, predominately affecting large, fast-growing broilers. Severe myodegeneration, increased hypoxia, reduced blood flow, and increased collagen deposition are hallmark characteristics of WB that culminate in unsatisfactory fresh meat quality attributes, such as poor water-holding capacity, tenderness, and processing characteristics. Therefore, WB meat is often downgraded resulting in economic losses for the United States poultry industry. Although WB has been well characterized, its etiology remains undefined. As the scientific community continues to resolve mechanisms responsible for WB onset, understanding biochemical changes associated with WB may facilitate solutions to negate its poor meat quality attributes. Given changes in metabolism of living muscle can alter biochemical processes during the conversion of muscle to meat, this review aims to summarize and discuss the current knowledge of WB muscle and meat biochemistry. For example, it appears metabolic pathways that support combating stress are upregulated in WB muscle at the expense of glycolytic flux, which presumably contributes to the high ultimate pH of WB meat. Further, perturbed function of WB mitochondria, such as altered calcium handling, impacts aspects of postmortem metabolism and proteolysis. Collectively, metabolic dysfunction of WB muscle alters the biochemical processes that occur during the conversion of muscle to meat, and thus contributes to the poor WB meat quality.
    Keywords:  fresh meat quality; myopathy; poultry; skeletal muscle metabolism; wooden breast
    DOI:  https://doi.org/10.3389/fphys.2024.1501362
  16. FEBS J. 2024 Nov 14.
      Recent experimental studies indicate that mitochondria in mammalian cells are maintained at temperatures of at least 50 °C. While acknowledging the limitations of current experimental methods and their interpretation, we here consider the ramifications of this finding for cellular functions and for evolution. We consider whether mitochondria as heat-producing organelles had a role in the origin of eukaryotes and in the emergence of homeotherms. The homeostatic responses of mitochondrial temperature to externally applied heat imply the existence of a molecular heat-sensing system in mitochondria. While current findings indicate high temperatures for the innermost compartments of mitochondria, those of the mitochondrial surface and of the immediately surrounding cytosol remain to be determined. We ask whether some aspects of mitochondrial dynamics and motility could reflect changes in the supply and demand for mitochondrial heat, and whether mitochondrial heat production could be a factor in diseases and immunity.
    Keywords:  cold‐shock; eukaryote origins; heat‐shock; homeothermy; immunity; mitochondria; mitochondrial disease; mitochondrial dynamics; temperature gradients; thermogenesis
    DOI:  https://doi.org/10.1111/febs.17316
  17. J Appl Physiol (1985). 2024 Nov 15.
      Eccentric contractions (ECC) are accompanied by accumulation of intracellular calcium ions ([Ca2+]i) and induce skeletal muscle damage. Suppressed muscle damage in repeated bouts of ECC is well characterized, however, whether it is mediated by altered Ca2+ profiles remains unknown. PURPOSE: We tested the hypothesis that repeated ECC suppresses Ca2+ accumulation via adaptions in Ca2+ regulation. METHODS: Male Wistar rats were divided into two groups: ECC single bout (ECC-SB) and repeated bout (ECC-RB). Tibialis anterior (TA) muscles were subjected to ECC (40 times, 5 sets) once (ECC-SB), or twice 14 days apart (ECC-RB). Under anesthesia, the TA muscle was loaded with Ca2+ indicator Fura-2 AM and the 340/380 nm ratio was evaluated as [Ca2+]i. Ca2+ handling proteins were measured by western blots. RESULTS: ECC induced [Ca2+]i increase in both groups, but ECC-RB evinced a markedly suppressed [Ca2+]i (Time: P < 0.01, Group: P = 0.0357). 5 hours post-ECC, in contrast to the localized [Ca2+]i accumulation in ECC-SB, ECC-RB exhibited lower and more uniform [Ca2+]i (P < 0.01). In ECC-RB mitochondria Ca2+ uniporter complex components, MCU and MICU2, were significantly increased pre-second ECC bout (P < 0.01) and both SERCA1 and MICU1 were better preserved after contractions (P < 0.01). CONCLUSION: 14 days after novel ECC skeletal muscle mitochondrial Ca2+ regulating proteins were elevated. Following subsequent ECC [Ca2+]i accumulation and muscle damage were suppressed and SERCA1 and MICU1 preserved. These findings suggest that tolerance to a subsequent ECC bout is driven, at least in part, by enhanced mitochondrial and SR Ca2+ regulation.
    Keywords:  Calcium ion; mitochondria; muscle damage; repeated bout effect
    DOI:  https://doi.org/10.1152/japplphysiol.00164.2024
  18. ACS Nano. 2024 Nov 15.
      Pulmonary fibrosis (PF) is an interstitial lung disease tightly associated with the disruption of mitochondrial pool homeostasis, a delicate balance influenced by functional and dysfunctional mitochondria within lung cells. Mitochondrial transfer is an emerging technology to increase functional mitochondria via exogenous mitochondrial delivery; however, the therapeutic effect on mitochondrial transfer is hampered during the PF process by the persistence of dysfunctional mitochondria, which is attributed to impaired mitophagy. Herein, we reported engineering mitochondria mediated by mitophagy-enhanced nanoparticle (Mito-MEN), which promoted synchronal regulation of functional and dysfunctional mitochondria for treating PF. Mitophagy-enhanced nanoparticles (MENs) were fabricated through the encapsulation of Parkin mRNA, and the electrostatic interaction favored MENs to anchor isolated healthy mitochondria for the construction of Mito-MEN. Mito-MEN increased the load of functional exogenous mitochondria by enhancing mitochondrial delivery efficiency and promoted mitophagy of dysfunctional endogenous mitochondria. In a bleomycin (BLM)-induced PF mouse model, Mito-MEN repaired mitochondrial function and efficiently relieved PF-related phenotypes. This study provides a powerful tool for synchronal adjustment of mitochondrial pool homeostasis and offers a translational approach for pan-mitochondrial disease therapies.
    Keywords:  alveolar epithelial cells; mitophagy; nanoengineered mitochondria; nanoparticle; pulmonary fibrosis
    DOI:  https://doi.org/10.1021/acsnano.4c10328
  19. Methods Mol Biol. 2025 ;2878 99-116
      Reactive oxygen species (ROS) play important roles in physiological and pathological processes. Mitochondria, particularly in skeletal and cardiac myocytes, are recognized as the primary site of ROS generation. Detecting oxidative modifications of intracellular or circulating molecules, such as lipids, proteins, and nucleic acids, is commonly employed to indicate ROS formation. However, this approach is indirect and provides limited insights into the spatiotemporal aspects of ROS generation. Understanding these aspects is crucial for comprehending the role of ROS in various pathophysiological conditions. To address this, fluorescent probes can be employed to measure ROS formation, offering a means to investigate ROS generation in both isolated mitochondria and intact cells. This chapter outlines three prominent examples for the use of fluorescent sensors to evaluate mitochondrial ROS formation in either isolated organelles or intact cells. The methods are explained in detail, and an analysis of the limitations of each technique is provided, underscoring potential sources of errors during the assay and the subsequent interpretation of results.
    Keywords:  Amplex Red; Fluorescence; HyPer7; MitoTracker Red CM-H2XRos; Mitochondria; Reactive oxygen species
    DOI:  https://doi.org/10.1007/978-1-0716-4264-1_6