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.
.