key: cord-0695282-eyq4fpb5
authors: Díaz‐Resendiz, Karina Janice Guadalupe; Benitez‐Trinidad, Alma Betsaida; Covantes‐Rosales, Carlos Eduardo; Toledo‐Ibarra, Gladys Alejandra; Ortiz‐Lazareno, Pablo Cesar; Girón‐Pérez, Daniel Alberto; Bueno‐Durán, Adela Yolanda; Pérez‐Díaz, Daniela Alejandra; Barcelos‐García, Rocío Guadalupe; Girón‐Pérez, Manuel Iván
title: Loss of mitochondrial membrane potential (ΔΨ (m)) in leucocytes as post‐COVID‐19 sequelae
date: 2022-03-31
journal: J Leukoc Biol
DOI: 10.1002/jlb.3ma0322-279rrr
sha: ff04ad7e417294e18e6b83ca3e25cc067106a100
doc_id: 695282
cord_uid: eyq4fpb5

The mitochondrial membrane potential (ΔΨ (m)) is a parameter often used to determine mitochondrial function; therefore, it can be used to determine the integrity and functionality of cells. A decrement of ΔΨ (m) is implicated in several inflammatory‐related pathologies, such phenomena can be related to COVID‐19 infection. The present work aimed to compare the ΔΨ (m) in leucocytes (human PBMCs; HPBMC) isolated from healthy control (HC) subjects, patients with COVID‐19 (C‐19), recovered subjects at 40 ± 13 (R1) and 335 ± 20 (R2) days after infection (dai). Obtained data showed that ΔΨ (m) decreased in HPBMC of subjects with C‐19, R1, and R2 compared with HC. When analyzing the ΔΨ (m) data by sex, in females, a significant decrease was observed in R1 and R2 groups versus HC. Regarding men, a significant decrease of ΔΨ (m) was observed in R1, with respect to HC, contrary to R2 group, who reestablished this parameter. Obtained results suggest that the loss of ΔΨ (m) could be related to the long‐COVID.

gia, and fatigue) and generally have a good prognosis. [5] [6] [7] However, a significant number of patients develop severe fatal consequences related to inflammatory processes largely promoted by exacerbated cytokine production, which involves major systemic disturbances. 2, 8, 9 These include iron dysregulation that manifests as hyperferritinemia associated with disease severity. Iron dysregulation induces the production of reactive oxygen species and promotes oxidative stress. The elevated inflammatory/oxidative state can lead to mitochondrial dysfunction leading to cell death. 2,10-13 Patients recovering from acute As described above, mitochondrial integrity can determine differential vulnerability to SARS-CoV-2 infection, so some authors have proposed using mitochondrial function as a potential biomarker for developing severe COVID-19. 2, [17] [18] [19] An important indicator of mitochondrial integrity is mitochondrial membrane potential (ΔΨ m ); this parameter can provide critical data related to the physiologic state of the cell and mitochondrial function of COVID-19 patients. 20 Currently, there is information on the effects of SARS-CoV-2 on functional parameters of mitochondria in patients with active COVID-19 [12] [13] [14] [15] [16] [17] [18] ; however, there is no information on post-COVID effects on mitochondrial functionality. Therefore, the present research aimed to evaluate ΔΨ m in human PBMCs (HPBMCs) in patients with , subjects recently recovered from COVID-19 (R1) at 40 ± 13 days after infection (dai) and recovered subjects at 11 months postinfection (R2, at 335 ± 20 dai), as well as healthy control (HC) subjects. Patients who smoked or those with comorbidities (asthma, diabetes, hypertension, and neurodegenerative diseases) were excluded.

For oropharynx/nasopharynx swab sampling, 2 flexible swabs were 

RNA extraction was performed with the QIAamp RNA viral Mini Kit (Qiagen; Cat No/ID: 1020953 USA, Germantown). Samples were processed following the RT-qPCR methodology indicated in the Berlin protocol with modifications (InDRE protocol), using the StarQ One-

Step RT-qPCR kit (Qiagen; Cat No/ID: 210210, USA, Germantown).

The genes to be amplified in each sample were the E gene (E_Sarbeco_F: 

Venous blood from volunteers was placed in EDTA tubes. Blood samples were diluted with phosphate buffer saline (PBS, pH 7.2) in a 1:1 ratio, deposited on a Ficoll-Histopaque 1.077 g/ml column (1:2 ratio;

Histopaque:blood), and centrifuged at 10,000 g for 25 min at room temperature. The cell ring was recovered and washed with PBS (pH 7.2) at 12,000 g for 7 min. The cell button was then resuspended in RPMI-1640 supplemented with 10% FBS and 1% antibiotic (streptomycin/penicillin).

HPBMC were characterized by size (forward side scatter) and granularity (forward scatter). Once the study population was established in the BD Accuri C6 software template, cell counting proceeded. Then, cell viability was determined through propidium iodide staining; a 95% viability in HPBMC was considered for cell culture. In brief, 1 × 10 6 HPBMC/ml resuspended in RPMI medium supplemented with FBS (10%) and streptomycin/penicillin (1%) were placed in each well at 37 • C and 5% CO 2 in 24-well plates, for a period of 24 h.

The determination of ΔΨ m was performed using a cationic cyanine dye, 3,3′-dihexyloxacarbocyanine iodide (DiOC 6 (3) (Invitrogen™, Cat

No. D273). 23 

Mean fluorescence intensity (MFI) of DiOC6 (3) 

In this work, mitochondrial membrane potential ( Table 1) .

The patients with C-19 active indicated that they had not required hospitalization; however, 60% of the participants mentioned having had treatment prescribed by a physician with azithromycin (15%), ivermectin (10%), vitamins (8%), antipyretics (8%), anticoagulants (7%), corticosteroids (5%), anti-inflammatory drugs (5%), and others (2%). All COVID-19 participants were categorized as ambulatory patients (having a mild disease) since no one stated that they had been hospitalized, and no evidence of pneumonia or hypoxia (SpO 2 ≥ 94%) was ever observed. 24, 25 As for the 35 For R2 group, data indicate that these subjects restore ΔΨ m as compared with the R1 group (Figure 1(B) ); however, with respect to HC, the ΔΨ m is still significantly decreased (p < 0.05). These results suggest that the loss of ΔΨ m in subjects recovered 335 ± 20 days after SARS-CoV-2 infection can be associated with the so-called long-COVID, since of the 18 individuals who participated, 85% reported symptoms such as weariness, difficulty sleeping, fatigue, dyspnea, memory problems, anxiety, arthralgia, headache, dry cough, chest pain, myalgia, and anosmia ( Figure 2 ).

Furthermore, some authors have suggested that mitochondrial dysfunction is a predisposing factor for COVID-19 severity. diseases. [25] [26] [27] [30] [31] [32] [33] Regarding the ΔΨ m in HPBMC between males and females from the analyzed groups (HC, C-19, R1, and R2). In general, the results indicate that female HC have higher ΔΨ m than male HC ( Figure 3 ). In this sense, some authors report that between both sexes there are differences in mitochondrial function, in this way, compared with men, women present higher levels of enzymes (citrate synthase activity) ATP, and antioxidant compounds, 34,35 due to a higher mitochondrial activity, facts related to a longer life expectancy, 36 and a lower occurrence of diseases and aging. 37 On the other hand, the obtained data indicate that both sexes decrease significantly ΔΨ m in C-19 and R1 groups (Figure 3 ). fatigue, arthralgia, or headache) ( Figure 2 ). This agrees with Ortona et al., 38 who indicate that women seem to be twice as likely to develop long-COVID as men, due to hormonal factors are stronger in women than in men.

On the other hand, some authors have compared SARS-CoV-2 infection between men and women, indicating a high mortality rate in older men; suggesting that women conduct a more effective viral clearance, perhaps since females show more robust innate interferon antiviral response, in addition to greater adaptive immunity towards viral antigens. 4, 17, 38, 39 Besides, it has been reported that outbreaks of SARS and the Middle East respiratory syndrome have also shown a male predominance in disease susceptibility, a feature also observed in SARS-CoV-2. [40] [41] [42] In this regard, Channappanavar et al., 43 experimentally infected male and female mice with SARS-CoV, observing that males were more susceptible than females; however, ovariectomy or estrogen receptor antagonists increased the mortality of females, concluding that estrogens could play a protective effect against coronavirus infection. [42] [43] [44] Similarly, other studies have reported that ovariectomy decreases mitochondrial oxidative phosphorylation and increases oxidative stress. 33, 45 In this sense, the present study results suggest that the decrease of ΔΨ m in HPBMC from infected and recovered female patients to SARS-CoV-2 could be related to an estro-

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