Tissue-specific differences in Ca2+ sensitivity of the mitochondrial permeability transition pore (PTP). Experiments in male rat liver and heart.

Permeability transition pore (PTP) opening dissipates ion and electron gradients across the internal mitochondrial membrane (IMM), including excess Ca2+ in the mitochondrial matrix. After opening, immediate PTP closure must follow to prevent outer membrane disruption, loss of cytochrome c, and eventual apoptosis. Flickering, defined as the rapid alternative opening/closing of PTP, has been reported in heart, which undergoes frequent, large variations in Ca2+. In contrast, in tissues that undergo depolarization events less often, such as the liver, PTP would not need to be as dynamic and thus these tissues would not be as resistant to stress. To evaluate this idea, it was decided to follow the reversibility of the permeability transition (PT) in isolated murine mitochondria from two different tissues: the very dynamic heart, and the liver, which suffers depolarizations less frequently. It was observed that in heart mitochondria PT remained reversible for longer periods and at higher Ca2+ loads than in liver mitochondria. In all cases, Ca2+ uptake was inhibited by ruthenium red and PT was delayed by Cyclosporine A. Characterization of this phenomenon included measuring the rate of oxygen consumption, organelle swelling and Ca2+ uptake and retention. Results strongly suggest that there are tissue-specific differences in PTP physiology, as it resists many more Ca2+ additions before opening in a highly active organ such as the heart than in an organ that seldom suffers Ca2+ loading, such as the liver.

Mfn2 regulates mitochondria and mitochondria-associated endoplasmic reticulum membrane function in neurodegeneration induced by repeated sevoflurane exposure.

Repeated sevoflurane exposure in neonatal mice can leads to neuronal apoptosis and mitochondrial dysfunction. The mitochondria are responsible for energy production to maintain homeostasis in the central nervous system. The mitochondria-associated endoplasmic reticulum membrane (MAM) is located between the mitochondria and endoplasmic reticulum (ER), and it is critical for mitochondrial function and cell survival. MAM malfunction contributes to neurodegeneration, however, whether it is involved in sevoflurane-induced neurotoxicity remains unknown. Our study demonstrated that repeated sevoflurane exposure induced mitochondrial dysfunction and dampened the MAM structure. The upregulated ER-mitochondria tethering enhanced Ca2+ transition from the cytosol to the mitochondria. Overload of mitochondrial Ca2+ contributed to opening of the mitochondrial permeability transition pore (mPTP), which caused neuronal apoptosis. Mitofusin 2(Mfn2), a key regulator of ER-mitochondria contacts, was found to be suppressed after repeated sevoflurane exposure, while restoration of Mfn2 expression alleviated cognitive dysfunction due to repeated sevoflurane exposure in the adult mice. These evidences suggest that sevoflurane-induced MAM malfunction is vulnerable to Mfn2 suppression, and the enhanced ER-mitochondria contacts promotes mitochondrial Ca2+ overload, contributing to mPTP opening and neuronal apoptosis. This paper sheds light on a novel mechanism of sevoflurane-induced neurotoxicity. Furthermore, targeting Mfn2-mediated regulation of the MAM structure and mitochondrial function may provide a therapeutic advantage in sevoflurane-induced neurodegeneration.

The mitochondrial carrier homolog 2 is involved in down-regulation of influenza A virus replication.

BACKGROUND: The mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein regulating mitochondrial metabolism and functions in lipid homeostasis and apoptosis. Experimental data on the interaction of MTCH2 with viral proteins in virus-infected cells are very limited. Here, the interaction of MTCH2 with PA subunit of influenza A virus RdRp and its effects on viral replication was investigated. METHODS: The human MTCH2 protein was identified as the influenza A virus PA-related cellular factor with the Y2H assay. The interaction between GST.MTCH2 and PA protein co-expressed in transfected HEK293 cells was evaluated by GST-pull down. The effect of MTCH2 on virus replication was determined by quantification of viral transcript and/or viral proteins in the cells transfected with MTCH2-encoding plasmid or MTCH2-siRNA. An interaction model of MTCH2 and PA was predicted with protein modeling/docking algorithms. RESULTS: It was observed that PA and GST.MTCH2 proteins expressed in HEK293 cells were co-precipitated by glutathione-agarose beads. The influenza A virus replication was stimulated in HeLa cells whose MTCH2 expression was suppressed with specific siRNA, whereas the increase of MTCH2 in transiently transfected HEK293 cells inhibited viral RdRp activity. The results of a Y2H assay and protein-protein docking analysis suggested that the amino terminal part of the viral PA (nPA) can bind to the cytoplasmic domain comprising amino acid residues 253 to 282 of the MTCH2. CONCLUSION: It is suggested that the host mitochondrial MTCH2 protein is probably involved in the interaction with the viral polymerase protein PA to cause negative regulatory effect on influenza A virus replication in infected cells.

Peptides Targeting the IF1-ATP Synthase Complex Modulate the Permeability Transition Pore in Cancer HeLa Cells.

The mitochondrial protein IF1 is upregulated in many tumors and acts as a pro-oncogenic protein through its interaction with the ATP synthase and the inhibition of apoptosis. We have recently characterized the molecular nature of the IF1-Oligomycin Sensitivity Conferring Protein (OSCP) subunit interaction; however, it remains to be determined whether this interaction could be targeted for novel anti-cancer therapeutic intervention. We generated mitochondria-targeting peptides to displace IF1 from the OSCP interaction. The use of one selective peptide led to displacement of the inhibitor IF1 from ATP synthase, as shown by immunoprecipitation. NMR spectroscopy analysis, aimed at clarifying whether these peptides were able to directly bind to the OSCP protein, identified a second peptide which showed affinity for the N-terminal region of this subunit overlapping the IF1 binding region. In situ treatment with the membrane-permeable derivatives of these peptides in HeLa cells, that are silenced for the IF1 inhibitor protein, showed significant inhibition in mitochondrial permeability transition and no effects on mitochondrial respiration. These peptides mimic the effects of the IF1 inhibitor protein in cancer HeLa cells and confirm that the IF1-OSCP interaction inhibits apoptosis. A third peptide was identified which counteracts the anti-apoptotic role of IF1, showing that OSCP is a promising target for anti-cancer therapies.

EFHD1 promotes osteosarcoma proliferation and drug resistance by inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP) by binding to ANT3.

Chemoresistance is the main obstacle in the clinical treatment of osteosarcoma (OS). In this study, we investigated the role of EF-hand domain-containing protein 1 (EFHD1) in OS chemotherapy resistance. We found that the expression of EFHD1 was highly correlated with the clinical outcome after chemotherapy. We overexpressed EFHD1 in 143B cells and found that it increased their resistance to cell death after drug treatment. Conversely, knockdown of EFHD1 in 143BR cells (a cisplatin-less-sensitive OS cell line derived from 143B cells) increased their sensitivity to treatment. Mechanistically, EFHD1 bound to adenine nucleotide translocase-3 (ANT3) and inhibited its conformational change, thereby inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP). This effect could maintain mitochondrial function, thereby favoring OS cell survival. The ANT3 conformational inhibitor carboxyatractyloside (CATR), which can promote mPTP opening, enhanced the chemosensitivity of EFHD1-overexpressing cells when combined with cisplatin. The ANT3 conformational inhibitor bongkrekic acid (BKA), which can inhibit mPTP opening, restored the resistance of EFHD1 knockdown cells. In conclusion, our results suggest that EFHD1-ANT3-mPTP might be a promising target for OS therapy in the future.

The ER-SURF pathway uses ER-mitochondria contact sites for protein targeting to mitochondria.

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER mitochondria encounter structure (ERMES) and Tom70, together with Djp1 and Lam6, are part of two parallel and partially redundant ER-to-mitochondria delivery routes. When ER-to-mitochondria transfer is prevented by loss of these two contact sites, many precursors of mitochondrial inner membrane proteins are left stranded on the ER membrane, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.

[Newborn screening, clinical features and genetic analysis for Citrin deficiency in Henan province].

OBJECTIVE: To explore the prevalence, clinical features, genetic characteristics and prognosis of Citrin deficiency in Henan province of China. METHODS: A total of 986 565 neonates screened by tandem mass spectrometry at the Third Affiliated Hospital of Zhengzhou University from January 2013 to December 2021 were retrospectively analyzed. Analysis of SLC25A13 gene variants and parental verification were carried out for neonates suspected for Citrin deficiency by next-generation sequencing. The clinical, biochemical and genetic characteristics of Citrin deficiency patients were integrated to guide the diet treatment and follow up the growth and development. Paired-t test was used to compare the amino acid levels in the peripheral blood samples before and after the treatment. RESULTS: Nine cases of Citrin deficiency were diagnosed among the 986 565 neonates. Specific elevation of citrulline was observed in all of the 9 cases. Six variants were detected by genetic sequencing, among which c.852_855delTATG, c.615+5G>A, c.550C>T and IVS16ins3kb were known pathogenic variants, whilst c.1111_1112delAT and c.837T>A were unreported previously. The detection rate for c. 852_855delTATG was the highest (61.6%, 11/18), followed by IVS16ins3kb (16.7%, 3/18). The clinical symptoms of all patients were relieved after the treatment, and the blood amino acid profile and biochemical parameters were significantly improved by gradually falling within the normal range. By June 2022, all patients had shown a good prognosis. CONCLUSION: The prevalence of Citrin deficiency among neonates from Henan Province by tandem mass spectrometry is 1/109 618, and the carrier rate for the pathogenic variants of the SLC25A13 gene was 1/166. The c.852_855delTATG may be a hot spot variant among the patients. Discovery of the novel variants has enriched the mutational spectrum of the SLC25A13 gene. Above results have provided a basis for the early diagnosis, treatment, prognosis and genetic counseling for the affected families.

Inhibition of MFN1 restores tamoxifen-induced apoptosis in resistant cells by disrupting aberrant mitochondrial fusion dynamics.

Tamoxifen (TAM) resistance presents a major clinical obstacle in the management of estrogen-sensitive breast cancer, highlighting the need to understand the underlying mechanisms and potential therapeutic approaches. We showed that dysregulated mitochondrial dynamics were involved in TAM resistance by protecting against mitochondrial apoptosis. The dysregulated mitochondrial dynamics were associated with increased mitochondrial fusion and decreased fission, thus preventing the release of mitochondrial cytochrome c to the cytoplasm following TAM treatment. Dynamin-related GTPase protein mitofusin 1 (MFN1), which promotes fusion, was upregulated in TAM-resistant cells, and high MFN1 expression indicated a poor prognosis in TAM-treated patients. Mitochondrial translocation of MFN1 and interaction between MFN1 and mitofusin 2 (MFN2) were enhanced to promote mitochondrial outer membrane fusion. The interaction of MFN1 and cristae-shaping protein optic atrophy 1 (OPA1) and OPA1 oligomerization were reduced due to augmented OPA1 proteolytic cleavage, and their apoptosis-promoting function was reduced due to cristae remodeling. Furthermore, the interaction of MFN1 and BAK were increased, which restrained BAK activation following TAM treatment. Knockdown or pharmacological inhibition of MFN1 blocked mitochondrial fusion, restored BAK oligomerization and cytochrome c release, and amplified activation of caspase-3/9, thus sensitizing resistant cells to apoptosis and facilitating the therapeutic effects of TAM both in vivo and in vitro. Conversely, overexpression of MFN1 alleviated TAM-induced mitochondrial apoptosis and promoted TAM resistance in sensitive cells. These results revealed that dysregulated mitochondrial dynamics contributes to the development of TAM resistance, suggesting that targeting MFN1-mediated mitochondrial fusion is a promising strategy to circumvent TAM resistance.

Psammaplin A and Its Analogs Attenuate Oxidative Stress in Neuronal Cells through Peroxisome Proliferator-Activated Receptor γ Activation.

Psammaplins are sulfur containing bromotyrosine alkaloids that have shown antitumor activity through the inhibition of class I histone deacetylases (HDACs). The cytotoxic properties of psammaplin A (1), the parent compound, are related to peroxisome proliferator-activated receptor γ (PPARγ) activation, but the mechanism of action of its analogs psammaplin K (2) and bisaprasin (3) has not been elucidated. In this study, the protective effects against oxidative stress of compounds 1-3, isolated from the sponge Aplysinella rhax, were evaluated in SH-SY5Y cells. The compounds improved cell survival, recovered glutathione (GSH) content, and reduced reactive oxygen species (ROS) release at nanomolar concentrations. Psammaplins restored mitochondrial membrane potential by blocking mitochondrial permeability transition pore opening and reducing cyclophilin D expression. This effect was mediated by the capacity of 1-3 to activate PPARγ, enhancing gene expression of the antioxidant enzymes catalase, nuclear factor E2-related factor 2 (Nrf2), and glutathione peroxidase. Finally, HDAC3 activity was reduced by 1-3 under oxidative stress conditions. This work is the first description of the neuroprotective activity of 1 at low concentrations and the mechanism of action of 2 and 3. Moreover, it links for the first time the previously described effects of 1 in HDAC3 and PPARγ signaling, opening a new research field for the therapeutic potential of this compound family.

Dual rare genetic diseases in five pediatric patients: insights from next-generation diagnostic methods.

BACKGROUND: Clinicians traditionally aim to identify a singular explanation for the clinical presentation of a patient; however, in some cases, the diagnosis may remain elusive or fail to comprehensively explain the clinical findings. In recent years, advancements in next-generation sequencing, including whole-exome sequencing, have led to the incidental identification of dual diagnoses in patients. Herein we present the cases of five pediatric patients diagnosed with dual rare genetic diseases. Their natural history and diagnostic process were explored, and lessons learned from utilizing next-generation diagnostic technologies have been reported. RESULTS: Five pediatric cases (3 boys, 2 girls) with dual diagnoses were reported. The age at diagnosis was from 3 months to 10 years. The main clinical presentations were psychomotor retardation and increased muscular tension, some accompanied with liver dysfunction, abnormal appearance, precocious puberty, dorsiflexion restriction and varus of both feet, etc. After whole-exome sequencing, nine diseases were confirmed in these patients: Angelman syndrome and Krabbe disease in case 1, Citrin deficiency and Kabuki syndrome in case 2, Homocysteinemia type 2 and Copy number variant in case 3, Isolated methylmalonic acidemia and Niemann-Pick disease type B in case 4, Isolated methylmalonic acidemia and 21-hydroxylase deficiency in case 5. Fifteen gene mutations and 2 CNVs were identified. Four novel mutations were observed, including c.15292de1A in KMT2D, c.159_164inv and c.1427G > A in SLC25A13, and c.591 C > G in MTHFR. CONCLUSIONS: Our findings underscore the importance of clinicians being vigilant about the significance of historical and physical examination. Comprehensive clinical experience is crucial for identifying atypical clinical features, particularly in cases involving dual rare genetic diseases.

Inhibition of the mPTP and Lipid Peroxidation Is Additively Protective Against I/R Injury.

BACKGROUND: During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca2+ and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined. METHODS: Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca2+, ROS, and Fe2+ to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective. RESULTS: In the absence of Ca2+, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca2+ in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca2+ synergize to induce mPTP opening. Furthermore, this synergistic form of Ca2+- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone. CONCLUSIONS: In the present study, we have investigated the relationship between Ca2+ and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca2+ mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca2+ and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.

Imeglimin attenuates NLRP3 inflammasome activation by restoring mitochondrial functions in macrophages.

Imeglimin is a novel oral antidiabetic drug for treating type 2 diabetes. However, the effect of imeglimin on NLRP3 inflammasome activation has not been investigated yet. Here, we aimed to investigate whether imeglimin reduces LPS-induced NLRP3 inflammasome activation in THP-1 macrophages and examine the associated underlying mechanisms. We analyzed the mRNA and protein expression levels of NLRP3 inflammasome components and IL-1ß secretion. Additionally, reactive oxygen species (ROS) generation, mitochondrial membrane potential, and mitochondrial permeability transition pore (mPTP) opening were measured by flow cytometry. Imeglimin inhibited NLRP3 inflammasome-mediated IL-1ß production in LPS-stimulated THP-1-derived macrophages. In addition, imeglimin reduced LPS-induced mitochondrial ROS production and mitogen-activated protein kinase phosphorylation. Furthermore, imeglimin restored the mitochondrial function by modulating mitochondrial membrane depolarization and mPTP opening. We demonstrated for the first time that imeglimin reduces LPS-induced NLRP3 inflammasome activation by inhibiting mPTP opening in THP-1 macrophages. These results suggest that imeglimin could be a promising new anti-inflammatory agent for treating diabetic complications.

New insights into the structure and dynamics of the TOM complex in mitochondria.

To date, there is no general physical model of the mechanism by which unfolded polypeptide chains with different properties are imported into the mitochondria. At the molecular level, it is still unclear how transit polypeptides approach, are captured by the protein translocation machinery in the outer mitochondrial membrane, and how they subsequently cross the entropic barrier of a protein translocation pore to enter the intermembrane space. This deficiency has been due to the lack of detailed structural and dynamic information about the membrane pores. In this review, we focus on the recently determined sub-nanometer cryo-EM structures and our current knowledge of the dynamics of the mitochondrial two-pore outer membrane protein translocation machinery (TOM core complex), which provide a starting point for addressing the above questions. Of particular interest are recent discoveries showing that the TOM core complex can act as a mechanosensor, where the pores close as a result of interaction with membrane-proximal structures. We highlight unusual and new correlations between the structural elements of the TOM complexes and their dynamic behavior in the membrane environment.

Association of poly(rC)-binding protein-2 with sideroflexin-3 through TOM20 as an iron entry pathway to mitochondria.

Iron is essential for all the lives and mitochondria integrate iron into heme and Fe-S clusters for diverse use as cofactors. Here, we screened mitochondrial proteins in KU812 human chronic myelogenous leukemia cells by glutathione S-transferase pulldown assay with PCBP2 to identify mitochondrial receptors for PCBP2, a major cytosolic Fe(II) chaperone. LC-MS analyses identified TOM20, sideroflexin-3 (SFXN3), SFXN1 and TOM70 in the affinity-score sequence. Stimulated emission depletion microscopy and proteinase-K digestion of mitochondria in HeLa cells revealed that TOM20 is located in the outer membrane of mitochondria whereas SFXN3 is located in the inner membrane. Although direct association was not observed between PCBP2 and SFXN3 with co-immunoprecipitation, proximity ligation assay demonstrated proximal localization of PCBP2 with TOM20 and there was a direct binding between TOM20 and SFXN3. Single knockdown either of PCBP2 and SFXN3 in K562 leukemia cells significantly decreased mitochondrial catalytic Fe(II) and mitochondrial maximal respiration. SFXN3 but not MFRN1 knockout (KO) in mouse embryonic fibroblasts decreased FBXL5 and heme oxygenase-1 (HO-1) but increased transferrin uptake and induced ferritin, indicating that mitochondrial iron entry through SFXN3 is distinct. MFRN1 KO revealed more intense mitochondrial Fe(II) deficiency than SFXN3 KO. Insufficient mitochondrial heme synthesis was evident under iron overload both with SFXN3 and MFRN KO, which was partially reversed by HO-1 inhibitor. Conversely, SFXN3 overexpression caused cytosolic iron deficiency with mitochondrial excess Fe(II), which further sensitized HeLa cells to RSL3-induced ferroptosis. In conclusion, we discovered a novel pathway of iron entry into mitochondria from cytosol through PCBP2-TOM20-SFXN3 axis.

Effects of aerobic exercise on the regulation of mitochondrial carrier homolog-2 and its influence on the catabolic and anabolic activity of lipids in the mesenteric adipose tissue of obese mice.

The aim was to understand the direct impact of aerobic short-term exercise on lipid metabolism, specifically in regulating the mitochondrial carrier homolog 2 (MTCH2) and how it interferes with lipid metabolism in mesenteric adipose tissue. Swiss mice were divided into three groups: control, sedentary obese, and exercised obese. The obese groups were induced into obesity for fourteen weeks of a high-fat diet, and the trained submitted to seven aerobic exercise sessions. The exercise proved the significant increase of the pPerilipin-1, a hormone-sensitive lipase gene, and modulates lipid metabolism by increasing the expression of Mtch2 and acetyl Co-A carboxylase, perhaps occurring as feedback to regulate lipid metabolism in adipose tissue. In conclusion, we demonstrate, for the first time, how aerobic physical exercise increases Mtch2 transcription in mesenteric adipose tissue. This increase was due to changes in energy demand caused by exercise, confirmed by observing the significant reduction in mesenteric adipose tissue mass in the exercised group. Also, we showed that physical exercise increased the phosphorylative capacity of PLIN1, a protein responsible for the degradation of fatty acids in the lipid droplet, providing acyl and glycerol for cellular metabolism. Although our findings demonstrate evidence of MTCH2 as a protein that regulates lipid homeostasis, scant knowledge exists concerning the signaling of the MTCH2 pathway in regulatingfatty acid metabolism. Therefore, unveiling the means of molecular signaling of MTCH2 demonstrates excellent potential for treating obesity.

Clinical, biochemical, and genotypical characteristics in urea cycle mitochondrial transporter disorders.

BACKGROUND: This study aimed to evaluate clinical, biochemical, and genotypic findings of patients diagnosed with urea cycle mitochondrial transporter disorders. CASE SERIES: In this study, patients followed up with the diagnosis of urea cycle mitochondrial transporter disorders in the pediatric metabolism outpatient clinic of Diyarbakir Children's Hospital were retrospectively examined. Height, weight, head circumference, gender, age at diagnosis, follow-up period, consanguinity history between parents, and treatments of the patients included in the study were evaluated. Eight patients suffering from urea cycle mitochondrial transporter disorders were enrolled in the study. Five patients were found to have biallelic variants of the SLC25A15 gene. Two patients were found to have biallelic variants of the SLC25A13 gene. Two of our patients presented with gait disturbances and were diagnosed with HHH syndrome. One patient presented with liver failure and was diagnosed with HHH syndrome. The other three patients were identified by family screening. Citrin deficiency was detected in two patients with cholestasis and hepatomegaly in the infantile period. Ornithine levels increased in three of our patients with HHH syndrome during the first month of treatment despite a protein-restricted diet and adequate caloric intake. CONCLUSIONS: Increasing patients' caloric intake with HHH syndrome improves their ornithine levels. Our patients with citrin deficiency recovered clinically and biochemically before seven months.

Sudden death with cardiac involvement in a neonate with carnitine-acylcarnitine translocase deficiency.

A female neonate born with normal Apgar scores at 38+2 weeks of gestational age unexpectedly passed away within less than 30 hours after birth. The situation mirrored her brother's earlier demise within 24 hours post-delivery, suggesting a possible genetic disorder. Gross examination revealed widespread cyanosis and distinct yellowish changes on the cardiac ventricles. Histopathological examination disclosed lipid accumulation in the liver, heart, and kidneys. Tandem mass spectrometry detected elevated levels of 10 amino acids and 14 carnitines in cardiac blood. Trio-whole genome sequencing (Trio-WGS) identified the SLC25A20 c.199-10T>G mutation associated with carnitine-acylcarnitine translocase disease (CACTD), a type of fatty acid oxidation disorders (FAODs) with a potential for sudden death. Further validation of gene expression confirmed the functional deficiency of SLC25A20, ultimately diagnosing CACTD as the underlying cause of the neonate's demise. This case highlights the importance of prenatal metabolic and genetic screening for prospective parents and emphasizes the need for forensic doctors to integrate metabolomic and genomic investigations into autopsies for suspected inherited metabolic diseases.

Food-grade titanium dioxide (E171) and zinc oxide nanoparticles induce mitochondrial permeability and cardiac damage after oral exposure in rats.

Food-grade titanium dioxide (E171) and zinc oxide nanoparticles (ZnO NPs) are found in diverse products for human use. E171 is used as whitening agent in food and cosmetics, and ZnO NPs in food packaging. Their potential multi-organ toxicity has raised concerns on their safety. Since mitochondrial dysfunction is a key aspect of cardio-pathologies, here, we evaluate the effect of chronic exposure to E171 and ZnO NPs in rats on cardiac mitochondria. Changes in cardiac electrophysiology and body weight were measured. E171 reduced body weight more than 10% after 5 weeks. Both E171 and ZnO NPs increased systolic blood pressure (SBP) from 110-120 to 120-140 mmHg after 45 days of treatment. Both NPs altered the mitochondrial permeability transition pore (mPTP), reducing calcium requirement for permeability by 60% and 93% in E171- and ZnO NPs-exposed rats, respectively. Treatments also affected conformational state of adenine nucleotide translocase (ANT). E171 reduced the binding of EMA to Cys 159 in 30% and ZnO NPs in 57%. Mitochondrial aconitase activity was reduced by roughly 50% with both NPs, indicating oxidative stress. Transmission electron microscopy (TEM) revealed changes in mitochondrial morphology including sarcomere discontinuity, edema, and hypertrophy in rats exposed to both NPs. In conclusion, chronic oral exposure to NPs induces functional and morphological damage in cardiac mitochondria, with ZnO NPs being more toxic than E171, possibly due to their dissociation in free Zn2+ ion form. Therefore, chronic intake of these food additives could increase risk of cardiovascular disease.

Emodin Blocks mPTP Opening and Improves LPS-Induced HMEC-1 Cell Injury by Upregulation of ATP5A1.

BACKGROUND: Emodin has been shown to exert anti-inflammatory and cytoprotective effects. Our study aimed to identify a novel anti-inflammatory mechanism of emodin. METHODS: An LPS-induced model of microvascular endothelial cell (HMEC-1) injury was constructed. Cell proliferation was examined using a CCK-8 assay. The effects of emodin on reactive oxygen species (ROS), cell migration, the mitochondrial membrane potential (MMP), and the opening of the mitochondrial permeability transition pore (mPTP) were evaluated. Actin-Tracker Green was used to examine the relationship between cell microfilament reconstruction and ATP5A1 expression. The effects of emodin on the expression of ATP5A1, NALP3, and TNF-α were determined. After treatment with emodin, ATP5A1 and inflammatory factors (TNF-α, IL-1, IL-6, IL-13 and IL-18) were examined by Western blotting. RESULTS: Emodin significantly increased HMEC-1 cell proliferation and migration, inhibited the production of ROS, increased the mitochondrial membrane potential, and blocked the opening of the mPTP. Moreover, emodin could increase ATP5A1 expression, ameliorate cell microfilament remodeling, and decrease the expression of inflammatory factors. In addition, when ATP5A1 was overexpressed, the regulatory effect of emodin on inflammatory factors was not significant. CONCLUSION: Our findings suggest that emodin can protect HMEC-1 cells against inflammatory injury. This process is modulated by the expression of ATP5A1.

MCU-independent Ca2+ uptake mediates mitochondrial Ca2+ overload and necrotic cell death in a mouse model of Duchenne muscular dystrophy.

Mitochondrial Ca2+ overload can mediate mitochondria-dependent cell death, a major contributor to several human diseases. Indeed, Duchenne muscular dystrophy (MD) is driven by dysfunctional Ca2+ influx across the sarcolemma that causes mitochondrial Ca2+ overload, organelle rupture, and muscle necrosis. The mitochondrial Ca2+ uniporter (MCU) complex is the primary characterized mechanism for acute mitochondrial Ca2+ uptake. One strategy for preventing mitochondrial Ca2+ overload is deletion of the Mcu gene, the pore forming subunit of the MCU-complex. Conversely, enhanced MCU-complex Ca2+ uptake is achieved by deleting the inhibitory Mcub gene. Here we show that myofiber-specific Mcu deletion was not protective in a mouse model of Duchenne MD. Specifically, Mcu gene deletion did not reduce muscle histopathology, did not improve muscle function, and did not prevent mitochondrial Ca2+ overload. Moreover, myofiber specific Mcub gene deletion did not augment Duchenne MD muscle pathology. Interestingly, we observed MCU-independent Ca2+ uptake in dystrophic mitochondria that was sufficient to drive mitochondrial permeability transition pore (MPTP) activation and skeletal muscle necrosis, and this same type of activity was observed in heart, liver, and brain mitochondria. These results demonstrate that mitochondria possess an uncharacterized MCU-independent Ca2+ uptake mechanism that is sufficient to drive MPTP-dependent necrosis in MD in vivo.