Constanza Morén

Mitochondrial Injury in Human Acute Carbon Monoxide Poisoning: The Effect of Oxygen Treatment

The best oxygen therapy for acute carbon monoxide poisoning (ACOP) remains unestablished. Reported mitochondrial complex IV (mtCIV) inhibition, together with carboxyhaemoglobin (COHb)-induced hypoxia, may influence acute clinical symptoms and outcome. To “mitochondrially” evaluate treatment efficacy, we correlated intoxication severity and symptoms with mitochondrial function (mtCIV activity) and oxidative stress (lipid peroxidation) in 60 poisoned patients and determined ACOP recovery depending on either normobaric or hyperbaric oxygen therapy along a 3-month follow-up. In the present article we positively evaluate mtCIV as a good marker of ACOP recovery, treatment effectiveness, and late neurological syndrome development, which advocates for hyperbaric oxygen therapy as the treatment of choice. However, we discourage its usefulness as a severity marker because of its excessive sensitivity. We additionally evaluate oxidative stress role and prognostic factors for neurological sequelae development.

Molecular basis of reduced birth weight in smoking pregnant women: mitochondrial dysfunction and apoptosis

In utero exposure of fetuses to tobacco is associated with reduced birth weight. We hypothesized that this may be due to the toxic effect of carbon monoxide (CO) from tobacco, which has previously been described to damage mitochondria in non‐pregnant adult smokers. Maternal peripheral blood mononuclear cells (PBMCs), newborn cord blood mononuclear cells (CBMCs) and placenta were collected from 30 smoking pregnant women and their newborns and classified as moderate and severe smoking groups, and compared to a cohort of 21 non‐smoking controls. A biomarker for tobacco consumption (cotinine) was assessed by ELISA (enzyme‐linked immunosorbent assay). The following parameters were measured in all tissues: mitochondrial chain complex IV [cytochrome c oxidase (COX)] activity by spectrophotometry, mitochondrial DNA levels by reverse transcription polymerase chain reaction, oxidative stress by spectrophotometric lipid peroxide quantification, mitochondrial mass through citrate synthase spectrophotometric activity and apoptosis by Western blot parallelly confirmed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) assay in placenta. Newborns from smoking pregnant women presented reduced birth weight by 10.75 percent. Materno‐fetal mitochondrial and apoptotic PBMC and CBMC parameters showed altered and correlated values regarding COX activity, mitochondrial DNA, oxidative stress and apoptosis. Placenta partially compensated this dysfunction by increasing mitochondrial number; even so ratios of oxidative stress and apoptosis were increased. A CO‐induced mitotoxic and apoptotic fingerprint is present in smoking pregnant women and their newborn, with a lack of filtering effect from the placenta. Tobacco consumption correlated with a reduction in birth weight and mitochondrial and apoptotic impairment, suggesting that both could be the cause of the reduced birth weight in smoking pregnant women.

Study of oxidative, enzymatic mitochondrial respiratory chain function and apoptosis in perinatally HIV-infected pediatric patients.

Abstract Mitochondrial toxicity in perinatally human immunodeficiency virus (HIV)-infected pediatric patients has been scarcely investigated. Limited data are available about HIV or antiretroviral (ARV)-mediated mitochondrial damage in this population group, specifically, regarding oxygen consumption and apoptosis approach. We aimed to elucidate whether a given mitochondrial DNA depletion is reflected at downstream levels, to gain insight on the pathology of HIV and highly active antiretroviral therapy (HAART) in perinatally HIV-infected pediatric patients. We studied 10 healthy control participants and 20 perinatally HIV-infected pediatric patients (10 under ARV treatment and 10 off treatment). We determined mitochondrial mass, subunits II and IV of complex IV, global and specific mitochondrial enzymatic and oxidative activities, and apoptosis from peripheral blood mononuclear cells. Global oxygen consumption was significantly compromised in HIV-infected untreated patients, compared to the control group (0.76 ± 0.01 versus 1.59 ± 0.15; P = 0.014). Apoptosis showed a trend to increase in untreated patients as well. The overall complex (C) CI-III-IV activity of the mitochondrial respiratory chain (MRC) was significantly decreased in HIV-infected treated patients with respect to the control group (1.52 ± 0.38 versus 6.38 ± 1.53; P = 0.02). No statistically significant differences were found between untreated and HAART-treated patients. These findings suggest the pathogenic role of both HIV and HAART in mitochondrial dysfunction in vertical infection. The abnormalities in mitochondrial genome may be downstream reflected through a global alteration of the MRC. Mitochondrial impairment associated with HIV and HAART was generalized, rather than localized, in this series of perinatally HIV-infected patients.

Mitochondrial toxicity in human pregnancy: an update on clinical and experimental approaches in the last 10 years.

Mitochondrial toxicity can be one of the most dreadful consequences of exposure to a wide range of external agents including pathogens, therapeutic agents, abuse drugs, toxic gases and other harmful chemical substances. However, little is known about the effects of mitochondrial toxicity on pregnant women exposed to these agents that may exert transplacental activity and condition fetal remodeling. It has been hypothesized that mitochondrial toxicity may be involved in some adverse obstetric outcomes. In the present study, we investigated the association between exposure to mitochondrial toxic agents and pathologic conditions ranging from fertility defects, detrimental fetal development and impaired newborn health due to intra-uterine exposure. We have reviewed data from studies in human subjects to propose mechanisms of mitochondrial toxicity that could be associated with the symptoms present in both exposed pregnant and fetal patients. Since some therapeutic interventions or accidental exposure cannot be avoided, further research is needed to gain insight into the molecular pathways leading to mitochondrial toxicity during pregnancy. The ultimate objective of these studies should be to reduce the mitochondrial toxicity of these agents and establish biomarkers for gestational monitoring of harmful effects.

Mitochondrial DNA disturbances and deregulated expression of oxidative phosphorylation and mitochondrial fusion proteins in sporadic inclusion body myositis.

Sporadic inclusion body myositis (sIBM) is one of the most common myopathies in elderly people. Mitochondrial abnormalities at the histological level are present in these patients. We hypothesize that mitochondrial dysfunction may play a role in disease aetiology. We took the following measurements of muscle and peripheral blood mononuclear cells (PBMCs) from 30 sIBM patients and 38 age- and gender-paired controls: mitochondrial DNA (mtDNA) deletions, amount of mtDNA and mtRNA, mitochondrial protein synthesis, mitochondrial respiratory chain (MRC) complex I and IV enzymatic activity, mitochondrial mass, oxidative stress and mitochondrial dynamics (mitofusin 2 and optic atrophy 1 levels). Depletion of mtDNA was present in muscle from sIBM patients and PBMCs showed deregulated expression of mitochondrial proteins in oxidative phosphorylation. MRC complex IV/citrate synthase activity was significantly decreased in both tissues and mitochondrial dynamics were affected in muscle. Depletion of mtDNA was significantly more severe in patients with mtDNA deletions, which also presented deregulation of mitochondrial fusion proteins. Imbalance in mitochondrial dynamics in muscle was associated with increased mitochondrial genetic disturbances (both depletion and deletions), demonstrating that proper mitochondrial turnover is essential for mitochondrial homoeostasis and muscle function in these patients.

Mitochondrial DNA (mtDNA) variants in the European haplogroups HV, JT, and U do not have a major role in schizophrenia

It has been reported that certain genetic factors involved in schizophrenia could be located in the mitochondrial DNA (mtDNA). Therefore, we hypothesized that mtDNA mutations and/or variants would be present in schizophrenia patients and may be related to schizophrenia characteristics and mitochondrial function. This study was performed in three steps: (1) identification of pathogenic mutations and variants in 14 schizophrenia patients with an apparent maternal inheritance of the disease by sequencing the entire mtDNA; (2) case‐control association study of 23 variants identified in step 1 (16 missense, 3 rRNA, and 4 tRNA variants) in 495 patients and 615 controls, and (3) analyses of the associated variants according to the clinical, psychopathological, and neuropsychological characteristics and according to the oxidative and enzymatic activities of the mitochondrial respiratory chain. We did not identify pathogenic mtDNA mutations in the 14 sequenced patients. Two known variants were nominally associated with schizophrenia and were further studied. The MT‐RNR2 1811A > G variant likely does not play a major role in schizophrenia, as it was not associated with clinical, psychopathological, or neuropsychological variables, and the MT‐ATP6 9110T > C p.Ile195Thr variant did not result in differences in the oxidative and enzymatic functions of the mitochondrial respiratory chain. The patients with apparent maternal inheritance of schizophrenia did not exhibit any mutations in their mtDNA. The variants nominally associated with schizophrenia in the present study were not related either to phenotypic characteristics or to mitochondrial function. We did not find evidence pointing to a role for mtDNA sequence variation in schizophrenia.

Hyperbaric oxygen therapy for carbon monoxide poisoning.

Dear Editor, Annane et al.’s article on carbon monoxide (CO) poisoning remarks on the lack of clinical benefits of hyperbaric oxygen therapy based on the results of two randomized trials comparing treatment with normobaric (NBO) versus hyperbaric (HBO) oxygen or one versus two sessions of HBO therapy [1]. We recently published a similar study on COpoisoned patients trying to establish the therapy of choice for CO intoxication based, in addition to clinical symptoms, on mitochondrial functionality [2]. It is well known that CO inhibits mitochondrial cytochrome c oxidase (COX) function, and such inhibition could participate in acute symptoms and clinical outcome of CO poisoning, classically attributed to carboxyhemoglobin (COHb)-mediated hypoxia [3–5]. We studied COX activity in 35 severely CO-intoxicated patients (mean COHb 23 ± 1.4%) randomly treated with one (n = 21) or two (n = 14) HBO sessions [2]. After intoxication, COX activity was decreased by 42.5% compared with 30 healthy individuals matched by age and sex (54.80 versus 95.29 nmol/min/mg protein) and progressively recovered after treatment, independently of the number of sessions administered (one versus two HBO sessions; Fig. 1). Our mitochondrial results in severely poisoned patients were in accordance with the lack of clinical benefits of one versus two HBO sessions reported by Annane and collaborators [1, 2]. We also studied COX function in 25 moderately CO-intoxicated patients (mean COHb 12.4 ± 1.0) randomly treated with NBO (n = 11) or HBO (n = 14) [2]. After intoxication, COX activity was decreased by 34.3% compared with ageand sex-matched controls (62.64 versus 95.29 nmol/ min/mg protein) and exclusively recovered its function in HBO-treated subjects (Fig. 1). Our mitochondrial results in moderately poisoned patients recommend usage of HBO with respect to NBO, in comparison with Annane’s clinical results reporting lack of beneficial effects of HBO therapy [1, 2]. Molecular data in CO poisoning suggest a role for COX inhibition. Its slow recovery in the persistence of symptoms and delayed sequelae recommends HBO therapy, probably one single session, independently of intoxication severity [2]. Neither our findings nor those reported by Annane et al. support the usefulness of multiple-HBO-session therapy. However, both findings highlight divergences on the usefulness of HBO which may be due to distinct molecular or clinical effects of CO poisoning, different types of patients included in each study, different conditions of HBO administration, or differences in elapsed time before COHb assessment or before treatment randomization [1, 2]. Thus, the ideal treatment for CO poisoning remains controversial.

Mitochondrial and apoptotic in vitro modelling of differential HIV-1 progression and antiretroviral toxicity

OBJECTIVES Ex vivo analysis of mitochondrial function may reveal HIV progression and the impact of ART. We propose a mitochondrial and apoptotic in vitro model using Jurkat T cells incubated with plasma. The objectives of this study were to evaluate mitochondrial and apoptotic lesions in this model in relation to HIV progression, and to assess the effect of >1 year of standard non-thymidine-containing therapy. METHODS This was a cross-sectional comparison among three age- and gender-matched groups (n = 19 × 3): healthy non-HIV-infected participants, HIV-infected long-term non-progressors (LTNPs) and standard antiretroviral-naive chronically infected patients [standard progressors (Sps)], longitudinally evaluated before (Sp1) and after (Sp2) >1 year of efavirenz + tenofovir + emtricitabine therapy. We analysed mitochondrial DNA content by RT-PCR, mitochondrial function by spectrophotometry, mitochondrial protein synthesis by western blot analysis, mitochondrial dynamics by western blot analysis (MFN2), apoptotic transition pore formation by western blot analysis (VDAC-1) and mitochondrial membrane potential and annexin V/propidium iodide fluorescence by flow cytometry. RESULTS There was a decreasing non-significant trend towards lower mitochondrial parameters for HIV-infected values with respect to uninfected control reference values. HIV progression (LTNP versus Sp1) was associated with decreased mitochondrial genetic, functional and translational parameters, which partially recovered after treatment intervention (Sp2). Mitochondrial fusion showed a trend to decrease non-significantly in Sp patients compared with LTNP patients, especially after therapy. All apoptotic parameters showed a trend to increase in Sp1 with respect to LTNP, followed by recovery in Sp2. CONCLUSIONS We proposed an in vitro model for mitochondrial and apoptotic assessment to test the effects of HIV infection and its therapy, resembling in vivo conditions. This model could be useful for clinical research purposes.

Exhaustion of mitochondrial and autophagic reserve may contribute to the development of LRRK2 G2019S -Parkinson’s disease

BackgroundMutations in leucine rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD). Mitochondrial and autophagic dysfunction has been described as etiologic factors in different experimental models of PD. We aimed to study the role of mitochondria and autophagy in LRRK2G2019S-mutation, and its relationship with the presence of PD-symptoms.MethodsFibroblasts from six non-manifesting LRRK2G2019S-carriers (NM-LRRK2G2019S) and seven patients with LRRK2G2019S-associated PD (PD-LRRK2G2019S) were compared to eight healthy controls (C). An exhaustive assessment of mitochondrial performance and autophagy was performed after 24-h exposure to standard (glucose) or mitochondrial-challenging environment (galactose), where mitochondrial and autophagy impairment may be heightened.ResultsA similar mitochondrial phenotype of NM-LRRK2G2019S and controls, except for an early mitochondrial depolarization (54.14% increased, p = 0.04), was shown in glucose. In response to galactose, mitochondrial dynamics of NM-LRRK2G2019S improved (− 17.54% circularity, p = 0.002 and + 42.53% form factor, p = 0.051), probably to maintain ATP levels over controls. A compromised bioenergetic function was suggested in PD-LRRK2G2019S when compared to controls in glucose media. An inefficient response to galactose and worsened mitochondrial dynamics (− 37.7% mitochondrial elongation, p = 0.053) was shown, leading to increased oxidative stress. Autophagy initiation (SQTSM/P62) was upregulated in NM-LRRK2G2019S when compared to controls (glucose + 118.4%, p = 0.014; galactose + 114.44%, p = 0.009,) and autophagosome formation increased in glucose media. Despite of elevated SQSTM1/P62 levels of PD-NMG2019S when compared to controls (glucose + 226.14%, p = 0.04; galactose + 78.5%, p = 0.02), autophagosome formation was deficient in PD-LRRK2G2019S when compared to NM-LRRK2G2019S (− 71.26%, p = 0.022).ConclusionsEnhanced mitochondrial performance of NM-LRRK2G2019S in mitochondrial-challenging conditions and upregulation of autophagy suggests that an exhaustion of mitochondrial bioenergetic and autophagic reserve, may contribute to the development of PD in LRRK2G2019S mutation carriers.

OC03.05: Mitochondrial toxicity and fetal cardiac remodelling in HIV‐infected pregnancies

Objectives: To establish z-score model for fetal tricuspid annular plane systolic excursion (FAM-TAPSE)and mitral annular plane systolic excursion (FAM-MAPSE) based on gestational age (GA), then to evaluate the ventricle systolic function of fetus with heart failure. Methods: 1012 normal fetuses and 24 fetuses with heart failure were involved. FAM-TAPSE and FAM-MAPSE were measured by free angle M-mode echocardiography,normal fetuses FAM-TAPSE and FAM-MAPSE z-score models were constructed by using first standard regression analysis using GA as independent variable. The fetuses with heart failure were divided into left heart failure (LHF)group and right heart failure (RHF) group by Tei index. Subsequently, the two parameters between normal and fetuses with heart failure were compared. Results: The models used to calculate Z-score for FAM-TAPSE and FAM-MAPSE were constructed, and GA had close correlation with them. Compared with normal fetuses, the mean Z-scores of FAM-TAPSE and FAM-MAPSE were statistically different in fetuses with heart failure(p<0.001). The FAM-MAPSE z-score of LHF and the FAM-MAPSE z-score of RHF were all less than -2zscores. Conclusions: The FAM-TAPSE and FAM-MAPSE Z-score declined in fetuses with heart failure and they can provide quantitative evidence in evaluation of heart systolic function, FAM-TAPSE and FAM-MAPSE Z-score would be markers for assessing heart systolic function in fetuses with heart failure.