Possible Pathogenesis and Prevention of Long COVID: SARS-CoV-2-Induced Mitochondrial Disorder

The primary symptom of coronavirus disease 2019 (COVID-19) is a respiratory illness [ 1 ]. However, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can also cause gastrointestinal symptoms and inflammation, which can affect the lungs and immune response through cytokines in the bloodstream [ 2 ]. People with post-COVID-19 illness may experience a variety of symptoms that can persist for weeks, months, or even years after infection [ 3 ]. Sometimes, symptoms even disappear and reappear. These symptoms can be difficult to explain and manage. Long COVID has been defined as “signs, symptoms, and conditions that continue or develop after initial SARS-CoV-2 infection” [ 4 ]. Clinical evaluations and the assessment of results of routine blood work, chest X-rays, and electrocardiograms may be normal [ 5 ]. The most commonly experienced symptoms of post-COVID-19 conditions include an exaggerated hyperventilation response during exercise, tiredness or weakness that interferes with daily life, malaise after exertion, fever, and more [ 6 , 7 ]. The peak oxygen uptake (VO 2peak ) in patients with long COVID-19 has been found to be significantly lower than normal [ 8 ]. In the first few months after infection, due to chronotropic insufficiency and reduced cardiac output, the cardiovascular system can lead to low VO 2peak through lower- than-normal cardiac output [ 8 ]. Alternatively, peripheral factors such as muscle mass, strength and perfusion, mitochondrial function, or arteriovenous oxygen difference may also contribute to low VO 2peak [ 8 ]. These symptoms are similar to those reported by patients with myalgic encephalomyelitis/chronic fatigue syndrome and people with genetic mitochondrial diseases [ 7 , 9 ]. Co-localization of the SARS-CoV-2 genome and mitochondria in SARS-CoV-2-infected tissues has been reported [ 10 ]. Mitochondria can be extremely vulnerable to physiological and pathological stimuli such as viral infections. The integrity and normal function of mitochondria can be altered during SARS-CoV-2 infection, thereby modulating cellular responses. The replication of SARS-CoV-2 begins with virus-induced double-membrane vesicles (DMV) that originate from the endoplasmic reticulum and eventually integrate to form a complex membrane network [ 11 ]. SARS-CoV-2 proteins are known to selectively target organelle compartments such as the endoplasmic reticulum and mitochondria, and they can reside in the host mitochondrial matrix [ 12 ]. This results in mitochondrial dysfunction and increased oxidative stress, ultimately leading to the loss of mitochondrial integrity and cell death [ 13 ]. These proteins also combine with non-specific and selective channel mitochondrial permeability transition pore (mPTP) complexes on mitochondria, which could alter the mitochondrial morphology and function, disrupt the Ca 2+ cycle, inhibit voltage-gated calcium channel (VDCC) activity, and lead to ion homeostasis disorder and mitochondrial dysfunction, thereby disrupting the cellular Ca 2+ cycle and cell viability [ 14 ]. Furthermore, hyperactivation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome results in hyper-responsiveness in classically activated macrophages when mitochondrial function is compromised, increasing mitochondrial reactive oxygen species (mtROS), and free circulating mitochondrial DNA (mtDNA). Oxidized mtDNA, cardiolipin, and cytochrome c from the damaged mitochondria, when released into the cytoplasm, activate damage-associated molecular patterns (DAMPs), leading to sustained local and systemic inflammatory responses [ 15 , 16 , 17 ]. This article reviews hypotheses regarding the changes in mitochondrial status under SARS-CoV-2 infection, including mitochondrial redox homeostasis, regulation of inflammation, and antiviral signaling, among others, and discusses why mitochondrial damage may contribute to long COVID.

When the immune system senses tissue damage, certain molecules are released to trigger an immune response via DAMPs [ 53 ]. DAMPs and related pathogen-associated molecular patterns (PAMPs) trigger inflammasome assembly, activate caspase-1 [ 54 ], and induce mitochondrial damage [ 55 ] ( Figure 1 ). Toll-like receptors (TLRs) are expressed on both innate and non-immune cells. The activation of TLRs plays a dual role [ 56 ], including the recognition and elimination of PAMPs in bacteria, viruses, and other pathogens, and the involvement of pathogens in the recognition and elimination of autogenous DAMPs released from dying or lysed cells [ 57 ]. Typical PAMPs are nucleic acids, including viral RNA and DNA, but also include surface-exposed glycoproteins, lipoproteins, and various membrane components [ 57 ]. The engagement of PAMPs and/or DAMPs can activate pattern recognition receptors (PRRs), such as TLRs or nucleotide-binding oligomerization domain-containing protein 2 (NOD2), resulting in NFκB activation and the activation of gene transcription. It was earlier reported that double-stranded RNA (dsRNA) viral PAMPs are known to activate TLR3 [ 58 ], whereas single-stranded RNA (ssRNA) viral PAMPs mainly activate TLR7/8 [ 59 ]. Changes in the distribution of charged amino acids in the spike protein of Omicron variants interfere with the recognition of TLRs by PRRs, thereby reducing activation of the NF-κB pathway and related signaling pathways, consequently inhibiting viral replication and systemic immune hyperactivation [ 60 ].

Both SARS-CoV-2-induced and inactivity-induced changes may increase mitochondrial dysfunction and myofibril breakdown, and decrease mitochondrial biogenesis and muscle synthesis [ 82 ]. Cells infected with SARS-CoV-2 exhibit abnormally swollen mitochondrial cristae [ 83 ]. Mitochondria in SARS-CoV-2-infected cells are markedly thinned, and the mitochondria are translocated and clustered around viral dsRNA containing DMV [ 84 ]. NSP4 and ORF9b cause structural changes in mitochondria, as well as the formation of outer membrane macropores and release of mtDNA-laden inner membrane vesicles [ 85 ]. In addition, genes related to mitochondria are predominantly downregulated in SARS-CoV-2-infected cells [ 86 ]; for example, the expression of mtDNA, antioxidants (e.g., catalase, glutathione synthetase), and mitochondrial respiratory chain proteins (NDUFA9, NADH: ubiquinone oxidoreductase subunit A9; SDHA, succinate dehydrogenase complex flavoprotein subunit A; COX4I1, cytochrome c oxidase subunit 4I1) were significantly reduced in SARS-CoV-2-infected placentas [ 87 ]. SARS-CoV ORF7a, ORF8a, and ORF9b are known to be located in the mitochondria, which can inhibit retinoic acid-inducible gene I-mitochondrial antiviral signaling protein (RIG1-MAVS)-dependent interferon signaling, enhance viral replication, and destroy mitochondrial function [ 88 ]. Mitochondrial dysfunction is one of the earliest and most prominent neurodegenerative features of SARS-CoV-2-induced neuropathology [ 89 ]. In addition, metabolic syndromes such as diabetes, obesity, and cardiovascular and liver diseases —which are related to mitochondria—also lead to susceptibility and adverse outcomes of SARS-CoV-2 infection [ 90 , 91 , 92 , 93 ], significantly increasing the mortality rate of SARS-CoV-2 infection [ 94 , 95 ]. Many environmental chemicals, malnutrition, and exacerbated socio-economic stress can also cause mitochondrial damage, which can negatively affect the prognosis of COVID-19 [ 96 ]. Furthermore, cardiac mitochondrial disruption after infection, ROS production, and energetic stress lead to mitochondrial alterations and cardiovascular dysfunction in COVID-19 patients, and the incidence of adverse cardiovascular events increases in recovered COVID-19 patients [ 97 ]. 3.1. Alteration of Mitochondrial Ca 2+ Signaling The host Ca 2+ channel affects every process of infection by the virus, which invades the host Ca 2+ channel to disrupt homeostasis and benefit its lifecycle at the expense of host survival. When inhibiting the activity of store-operated Ca 2+ channels, viral shedding is reduced, thereby reducing the infectivity of many enveloped RNA viruses [ 98 , 99 ]. During viral infection, infectious agents are also known to trigger dysregulation of host cell signaling cascades that affect cellular calcium dynamics [ 100 , 101 ]. This can lead to an imbalance in calcium concentrations, which can have an impact on mitochondrial function. Furthermore, six specific acidic residues (E819, D820, D830, D839, D843, and D848) on the SARS-CoV-2 spike proteins may bind calcium, thereby regulating the ability of the virus to insert into lipid membranes, which, in turn, affects oxidative phosphorylation (OXPHOS) and mitochondrial calcium sequestration [ 14 , 102 , 103 ]. The endoplasmic reticulum, mitochondria, and plasma membrane are all involved in the coordination of calcium dynamics, and there is evidence that viruses themselves may cause alterations in mitochondrial calcium signaling. ORF3a is known to assemble into dimeric folds and form non-selective Ca 2+ permeable cation channels [ 104 ]. When ORF3 is expressed, the mitochondria-associated membrane (MAM) formation is increased and cytoplasmic calcium is transported to mitochondria through the endoplasmic reticulum, as ORF3a is a calcium ion transporter [ 104 ]. NSP2 and NSP4 interact with the endoplasmic reticulum lipid raft-associated protein 1/2 (ERLIN1/2) complex to regulate Ca 2+ signaling from the endoplasmic reticulum to the mitochondria [ 105 ]. Furthermore, the calcium influx due to ORF3a activates the switch for calcium-dependent caspases and apoptosis which, in turn, trigger programmed cell death [ 106 ]. SARS-CoV-2 proteins significantly disrupt the baseline oscillations of cellular calcium, block L-type calcium channel activity, and cause cellular damage [ 14 ]. SARS-CoV-2 proteins alter mPTP, causing it to open and trigger cell death [ 14 ]. The mPTP complex components SPG7 matrix AAA peptidase subunit (SPG7) and cyclophilin D are upregulated in cells infected with SARS-CoV-2 [ 107 ]. SPG7, peptidylprolyl isomerase F (PPIF), and mitochondrial matrix import factor 23 (MIX23) are known to interact. The knockdown of MIX23 in mitochondria significantly enhanced the ability of mitochondria to sequester calcium. In contrast, an mPTP blocker cyclosporin A is known to restore mitochondrial Ca 2+ retention and cell viability after viral infection [ 14 ]. ORF3a, ORF9b and 9c, ORF10, and

The fusion and fission dynamics of mitochondria remodel the ultrastructure of the membrane [ 122 ], allowing for the removal of damaged mitochondria [ 123 ]. The mitochondrial morphology is altered or destabilized in the normal physiological division–fusion dynamics of cells, which plays a role in many pathological conditions [ 124 ]. For example, inhibiting the mitochondrial fusion-essential protein mitofusin-2 (Mfn2) increased ROS production and increased c-Jun N-terminal kinase (JNK) and nuclear factor kappa-light chain enhancer (NFκB) in activated B cell signaling, resulting in decreased insulin signaling and glucose uptake [ 125 ]. Additionally, the aberrant expression of the dynein-related protein 1 (DRP1, mitochondrial fission protein) increased H 2 O 2 production and damaged mitochondria [ 126 ]. The ORF9b protein mediates the degradation of the DRP1, leading to the abnormal lengthening of mitochondria [ 46 ]. Moreover, infected cells present a down-regulation of genes associated with mitochondrial ribosome synthesis, mitochondrial complex I synthesis, translocase, mitochondrial fission process 1 (MTFP1, mitochondrial fission-promoting protein), and suppressor of cytokine signaling 6 (SOCS6) [ 86 ]. The down-regulation of the mechanistic target of rapamycin complex 1 (mTORC1) resulted in decreased expression of MTFP1 and complex I [ 127 , 128 ]. Reduced expression of MTFP1 activates nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and SOCS6, leading to mitochondrial hyper fusion [ 129 ].

The activation of MOMP leads to cell death through cytochrome c release, apoptosis protease activating factor-1 (APAF-1)-dependent, and deoxyadenosine triphosphate (dATP)-dependent assembly, followed by caspase activation and the involvement of proinflammatory signaling functions. ORF3a is involved in necrotic cell death and lysosomal damage [ 28 ], and influences viral replication and virulence [ 26 ]. ORF6 and ORF7a have been shown to display the highest potency for cytotoxicity [ 33 ]. SARS-CoV-2 infects mitochondria and forms double-membrane vesicles which, in turn, leads to the release of mtDNA into circulation and the activation of the immune response, resulting in a severe pro-inflammatory state [ 139 ]. The mtDNA variation in COVID-19 patients can predict the degree of disease and the risk of developing severe disease [ 140 , 141 ]. After apoptosis, mtDNA is released, further aggravating local and systemic inflammatory responses [ 142 ]. Positive correlations between mtDNA levels and pro-inflammatory markers have been determined in the plasma of COVID-19 patients [ 143 , 144 ]. This is now used an indicator of COVID-19 severity or a predictor of mortality. Moreover, when mtDNA is released into the cytosol, it acts as a DAMP, which, in turn, causes a “cytokine storm” and inhibits cellular regulation of excessive inflammatory responses [ 143 ].

During SARS-CoV-2 infection, TLRs first bind their ligands and then recruit adapter proteins such as myeloid differentiation primary response 88 (MyD88) and adapter-inducible interferon-β (TRIF)-containing the TIR domain, which activate downstream immune molecular pathways [ 163 ], thus protecting the body from invading antigens. However, excessive activation of TLRs can lead to severe inflammatory responses, resulting in injury and even death [ 56 , 164 ]. Furthermore, when mtROS are increased, it leads to mitochondrial dysfunction and mtDNA leakage, which induces TLR9 and NFκB activation and the release of inflammatory cytokines [ 165 ]. TLR7 can be activated by ssRNAs rich in guanosine (G) and uridine (U) [ 166 ]. Two GU-rich ssRNA sequences in SARS-CoV-2 can activate TLR7 and its downstream MyD88-related pathway in human dendritic cells [ 167 ]. TLR7 recognizes SARS-CoV-2 in immune cells, triggering the production of type I interferon and pro-inflammatory cytokines [ 168 , 169 ]. TLR9 can recognize cytosine-guanine (CpG)-rich sequences in viral and mtDNA [ 170 ], and CpG in the SARS-CoV-2 envelope protein and ORF10 coding region can be recognized by TLR9 as a ligand [ 171 ]. TLR9 promotes inflammation through NFκB signaling and IL-6 production, and it impairs endothelial cell function by reducing endothelial nitric oxide synthase (eNOS) expression [ 165 ].

In people with long COVID and chronic fatigue syndrome, reducing the viral load and improving mitochondrial health may be beneficial ( Figure 2 ). First, direct-acting antiviral drugs—especially analogs such as remdesivir—can effectively inhibit viral replication by blocking the activity of viral polymerase [ 181 ]. In addition to reducing viral load in cells, they may also reduce sources of chronic inflammation that lead to severe sepsis, multi-organ failure, and mitochondrial damage. However, the recent results of remdesivir treatment showed no benefit for long COVID-19 patients [ 182 , 183 ]. After one year of follow-up, one in six survivors considered recovery from COVID-19 incomplete, and one in four was troubled by fatigue [ 182 ]. Another study has reported that one-third of patients experienced fatigue 6 months after COVID-19 [ 183 ]. Generalized antioxidants, such as N-acetylcysteine [ 184 ], glutathione [ 185 ], and catalase [ 186 ], can effectively reduce mitochondrial changes, as they restore and protect mitochondrial function [ 187 ], thereby reducing the spread of the virus SARS-CoV-2. Mitochondrial oxidative stress can also be reduced using mitochondrion-targeted catalytic antioxidants such as MnTBAP [ 188 ], EUK-8, and EUK-134 [ 189 ], as well as Mito-TEMPO and mitoquinol [ 112 , 162 , 190 ]. In addition, mitochondrial quinone/mitochondrial phenol mesylate (Mito-MES) has been demonstrated to possess significant antiviral activity against SARS-CoV-2 and can reduce excessive inflammation in the host [ 191 ]. Effective treatment can also be obtained with mPTP inhibitors such as NIM811 [ 192 , 193 ]. IL-6R and IL-1 receptor blockers are useful in the treatment of cytokine release syndrome [ 194 , 195 ]. For example, the IL-6R-blocking antibody Tocilizumab can be used to treat severe cytokine release syndrome [ 196 , 197 ] and SARS-CoV-2 infection [ 198 ]. Likewise, the IL-1 receptor antagonist anakinra significantly improved survival in SARS-CoV-infected mice with over-active NLRP3 inflammasome [ 195 , 199 , 200 ]. Increased survival and clinical improvement were observed in patients with COVID-19 adult respiratory distress syndrome receiving high-dose intravenous anakinra [ 201 ]. In addition, SARS-CoV-2-infected cells mainly rely on glycolysis to meet their energy demands, due to mitochondrial respiratory dysfunction. Inhibition of glycolysis in infected cells has been found to inhibit viral proliferation [ 107 ]. Ruthenium red—a mitochondrial calcium uniporter (MCU) inhibitor—also normalizes mitochondrial morphology and function in HIV-infected cells [ 202 ]. Other known MCU inhibitors include ruthenium 265, mitoxantrone, and the antibiotic doxycycline [ 203 ]. All of these strategies may help to improve mitochondrial health in people with long COVID or chronic fatigue syndrome.