4. Discussion.
The main finding of our study was a significantly increased frequency of length heteroplasmy at the poly-C tract in the mtDNA region that contains the replication start site of the H strand. This sequence, between nucleotides 16184-16193, contains a poly-C tract interrupted by C>T changes, and the presence of C could results in replication instability with multiple poly-C sequences.
The poly-C heteroplasmy might be inherited from the mother with cellular heterogeneity between different tissues in the same individual. In addition, the degree of heteroplasmy might increase with age or under exposure to environmental toxics, drugs, bacterial and viral infections, among others. An increased length heteroplasmy at this poly-C region has been reported among cancer, diabetes and coronary artery disease patients [Zhao et al., 2010; Mueller et al., 2011;Shen et al., 2015]. This associations might be explained by a mechanism that links the poly-C tract with an impaired replication of the mtDNA that could result in a reduction of the number of mtDNA copies and the loss of mitochondrial functions. In a study with blood samples from 837 healthy adults Liou et al. found that the number of mtDNA copies was significantly reduced among individuals with uninterrupted poly-C [Liou et al, 2010]. Thus, variants that increased the amount of length heteroplasmy might cause alteration of mtDNA copy number in human blood cells compared to T-interrupted tracts [Amo et al., 2017]. Interestingly, length heteroplasmy at the control region was also associated with significantly lower copy number of mtDNA in leukocytes from breast cancer patients [Zhao et al., 2010 ].
The number of mitochondria and mtDNA copies vary between cells and tissues and might be critical for a proper physiological response to situations that require a high energy demand and other mitochondrial-mediated responses [Stefano et al., 2022;He et al., 2010; Stefano et al., 2017]. A high degree of heteroplasmy might impairs the maintenance of ATP levels in response to strong physiological demand, such as the required by the immunological cells after infections [Koshiba 2013;Moore and Ting , 2008; Schilf et al., 2021;Shenoy 2020]. The effect of heteroplasmy in mtDNA copy number and mitochondrial function has been validated in mouse strains with different mtDNA variants [Hu et al., 2019 ]. These engineered mice showed a non-random segregation of mtDNA copy numbers that suggested a pressure to reduce the degree of heteroplasmy when this was detrimental for covering the cellular demands. Situations that increase the degree of heteroplasmy, such as aging, exposure to environmental compounds, infections, etc, might result in an impaired capacity to respond through mitochondrial pathways, increasing the risk for disease.
Our study was based on whole blood cells and would thus be representative of the degree of heteroplasmy among leukocytes in a disease (COVID-19) characterised by an exacerbated inflammatory response with over-activation of immune-cells to respond to the infection. These cells are thus under strong metabolic demand that would require an increase in the number of mitochondria and mtDNA copies. It is tempting to speculate that a similar dynamic of mtDNA length heteroplasmy occurs in other cells and tissues such as lung, brain, heart. This possibility might be investigated in the context of persistent (long-COVID) symptoms.
The role of mitochondria in the pathophysiology of acute and chronic inflammation has been extensively studied. Immune-competent cells such as monocytes, macrophages, antigen-presenting cells, are under programmed changes in mitochondrial bioenergetics in response to innate and adaptive immunological processes [Angajala et al., 2018;Zuo et al., 2019]. The degree of mtDNA length heteroplasmy might contribute to define the extent of these immune-mediated responses, making some individuals more susceptible to an impaired capacity to fight infection or to regulate the extent of the proinflammatory stimuli [Lechuga-Vieco et al., 2020;Pollara et al., 2018; Zhu et al., 2018]. Several studies have shown the capacity of viruses, including SARS-CoV-2, to hijack oxidative phosphorylation, ATP production, and other mitochondrial functions [Stefano et al., 2022;Stefano et al., 2020]. Key mitochondrial mediated processes might be also affected by viral proteins that reduced the antiviral innate immune responses, thus promoting the extent of infection and disease severity [Chen et al, 2007; Yoshizumi et al., 2014; Choi et al., 2018]. Some authors have suggested that SARS-CoV-2 infection and replication was improved by the takeover of mitochondrial processes, and viral proteins would suppress mitochondrial functions reducing the innate and adaptive immune responses [Singh et al., 2020]. In this context, heteroplasmy at the poly-C tract might reduce the individual´s capacity to exhibit a proper mitochondria mediated immune response increasing the risk for severe COVID-19.
A functional link between 16189C and increased risk for disease has been suggested by some authors. Park et al. identified 16189C as a risk factor for diabetes and used chromatin immunoprecipitation in cybrid cells to identify the mitochondrial single-stranded DNA-binding protein (mtSSB) as a candidate protein bound to the 16189 region [Park et al., 2008]. MtSSB has a lower binding affinity for the 16189C variant and because this nucleotide lies in the control region of mtDNA replication and transcription the variant might affect mtDNA replication and recovery after mitochondrial damage [Takamatsu et al., 2002; Park et al., 2008]. In this context, several studies have reported increased circulating mtDNA as a marker of COVID-19 severity and mortality [Scozzi et al., 2021; Valdés-Aguayo et al., 2022; Streng et al., 2022]. Studies to determine whether mtDNA variants and leght heteroplasmy were associated with reduced number of mtDNA copies and increased circulating mtDNA would be of special interest.
A limitation of our study was the absence of over-time comparison of the degree of poly-C heteroplasmy within the same individual. Heteroplasmy is commonly inherited and could thus be present in all the individual´s cells, but it is well known that de degree of heteroplasmy might increase with age or in response to environmental exposures. It is possible that SARS-CoV-2 proteins that target the mitochondria enhance the miss-replication of mtDNA promoting impairment of innate and acquired immunity and increasing the risk for critical COVID-19. To verify this hypothesis it is necessary to determine the degree of heteroplasmy before and after infection.
Finally, the risk of developing severe COVID-19 has been associated with some mitochondrial haplogroups [Wu et al., 2021;Vázquez-Coto et al., 2022]. We previously reported a decreased frequency of the common European haplogroup H (7028 C) that could be protective for critical disease at younger age. In addition, 16223 T might be associated with an increased risk of severe disease. Interestingly, 16189 C is more common among individuals with haplogroups with 16223 T (such as the European X) than among haplogroup H (characterised by 16223 C) (supplementary table ) [Laricchia et al., 2022]. Thus, the association of haplogroups with several diseases might be explained by its linkage to 16189 C and other variants that could increase the risk for poly-C length heteroplasmy.
In conclusion, we report an increased frequency of poly-C length heteroplasmy at the mtDNA control region that contains the replication start site of the H-strand among patients with critical COVID-19. Length heteroplasmy at this region has been associated with a higher risk of cancer, diabetes, coronary artery disease, and viral diseases, among others. The poly-C heteroplasmy could increase mtDNA instability and reduction of the mtDNA copies per cell, that might contribute to manifest an exacerbated inflammatory response and impaired activation of the innate and acquired immunological response to viral infection. Further research to confirm the association are necessary, as well as functional studies to uncover the linkage between mtDNA length heteroplasmy and viral disease.
Acknowledgements. This work was supported by a grant from the Spanish Plan Nacional de I+D+I Ministerio de Economía y Competitividad and the European FEDER, grants ISCIII-PI21/00971 (E.C.), RICOR2040- RD21/0005/0011 (E.C.), and PI22/00705 (J.G.).
Competing interests. None of the authors have competing interests related to this work.
AUTHOR CONTRIBUTIONS . Lead researchers : EC, GMA, JG;study design : EC, GMA, JG; patient assessment and data acquisition : GMA, LAR, MGC, ECLL, JG; database: DVC, EC, GMA, JG; genotyping: DVC, CGL, MGF, LVC, EC; data filtering and analysis: EC, DVC; statistical analysis: EC, DVC;analysis of results: EC, DVC; writing the manuscript:EC; revision of manuscript: all the authors.
ETHICS AND CONSENT. This study was approved by the clinical research ethics committee of Hospital Universitario Central Asturias (HUCA) (project approval id ISCIII-PI21/00971). All the participants or they next of kin gave written or verbal consent. Data were handled in observance of Spanish legislation on data protection. The study complies with the principles of the Declaration of Helsinki (“Recommendations guiding doctors in biomedical research involving human subjects”).
DATA AVAILABILITY STATEMENT . The data that support the findings of this study are available from the corresponding author upon reasonable request. An Excel file with the raw data would be available for meta-analysis research.
Table 1. Main values in the critical COVID-19 patients aged ≤60 years and older.
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