SARS-CoV-2/COVID's major underlying cause: Mitochondrial dysfunction, loss of membrane potential (ΔΨ m) and impaired OXPHOS under hypoxic conditions

Mitochondria are located inside the cell. Their function is to maintain cellular homeostasis and "aerobic respiration": by generating Adenosine Triphosphate (ATP) from ADP, mitochondria supply cells with energy. Through generation of low levels of Reactive Oxygen Species (ROS), mitochondria are involved in cell signaling. Imbalance in one of the processes involved in energy supply and ROS generation contributes to pathology, as is the case with infectious diseases such as COVID. Mitochondrial dysfunction is also involved in systemic disease. 

In this part of the "Deterioration in SARS-CoV-2 series", I will discuss the key role of mitochondria in the energy production process and ROS generation. As is known since SARS-CoV-1 (2003), the proteins of the coronavirus are able to "hack" mitochondria and get access to host cells via the "hijacking of mitochondria". I will offer insight into the properties of the virus that impair mitochondrial health which by doing so, contribute to viral replication and suppression of the immune response. I will also discuss possible treatment options to effectuate "mitochondrial redox".

1.   Mitochondrial function
1.1 Substrate feeding through the TCA and OXPHOS
1.2 What impaired mitochondrial function amounts to
1.3 The Warburg effect

2.  COVID-mitochondriopathy and hypoxia
2.1 Hypoxic conditions in COVID
2.2 The Warburg Effect in COVID via PI3K/AKT/mTOR and MAPK/ERK pathways induce microthrombosis
2.3 Inhibition of Bcl-2 family members leads to necrotic cell death
2.4 NSP4 and ORF9b of SARS-CoV-2 damage mitochondrial membrane potential (ΔΨ _m) and induce release of mtDNA into the cytosol

2.5 Fibrosis and mitochondrial dysfunction
2.5.1 Alleviating pulmonary fibrosis through restoration of mitochondrial integrity and REDOX (Metformin)
2.5.2 Mitophagy impairment in spite of enhanced PINK1/Parkin
2.6 Mitochondrial damage leading to mismatched ventilation/perfusion (V/Q) in COVID
3. Mitchondrial DNA and TLR-9 contribute to SARS-endothelial damage

1. Mitochondrial function

Mitochondria are located on and in most cells, except for mature erythrocytes (COVID-19 sepsis: revisiting mitochondrial dysfunction in pathogenesis, aging, inflammation and mortality, Inflammation Research, 7 August 2020). A 2015 study revealed that mitochondria located on the edge of muscle cells are optimized to generate membrane voltage (power supply), while interconnected mitochondria inside muscle cells are optimized to use voltage in order to produce ATP (High-resolution 3D images reveal the muscle mitochondrial power grid, NIH News, 30 July 2015).

Mitochondria contribute to cellular homeostasis through generation of ATP and low levels of ROS, required for cell signaling. Endothelial cells are supplied with ATP by glycolysis, whilst ROS generation towards endothelial cells depends on mitochondria.

1.1 Substrate feeding through the TCA and OXPHOS
Glucose (metabolized via glycolysis and pyruvate oxidation), fatty acids (metabolized via fatty acid-beta-oxidation) and amino acids (via oxidative deamination) feed into the TCA cycle (or Krebs cycle) before entering the Electron Transport Chain (ETC) within the mitochondrial matrix, in order to undergo Oxidative Phosphorylation (OXPHOS).

Under normoxic conditions, cells metabolize glucose into pyruvate in order to feed the TCA. The TCA cycle produces Nicotinamide adenine dinucleotide (NADH) to substrate OXPHOS in order to generate ATP. Beta-oxidation of fatty acids or pyruvate form substances to produce Acetyl-CoA within the mitochondrial matrix. Citrate synthase converts acetyl-CoA to citrate. Succinyl-CoA is hydrolized (Mitochondrial ETC: OXPHOS, oxidant production and methods of measurement, Redox Biology 37 (2020)).

Five protein complexes are located inside the inner mitochondrial membrane, close to the TCA cycle. Complexes I-IV make up the Electron Transport Chain; Complex V is part of the ATP Synthase. The TCA cycle provides NADH and FADH2 to the ETC. NADH and FADH2 donate a pair of electrons to Complex I and II of the Electron Transport Chain. Through a coupling synthase, Complex V is tied to the generation of ATP from ADP (Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production and methods of measurement, Redox Biology 37 (2020); Feeding Mitochondria: Potential Role of nutritional components to improve critical illness convalescence, Clinical Nutrition 38 (2019)).

1.2 What impaired mitochondrial function amounts to
Mitochondrial function is essential for neurological functions. Age-related neurodegeneration is a consequence of age-affected mitochondrial degeneration. Pathogens, such as viruses, impair mitochondrial health as well. During the ATP synthase through oxidative phosphorylation (OXPHOS), oxidative stress is generated. Excess Reactive Oxygen Species (ROS) is harmful to mitochondria. ROS damages lipids, proteins and nucleic acids, leading to impaired metabolism, cell death, impaired glucose metabolism, impaired calcium homeostasis and DNA damage. Damage Associated Patterns (DAMP) generate ROS in response to the detection of viral DNA. Oxidized mitochondrial DNA, cardiolipin and cytochrome from damaged mitochondria are released into the cytoplasm,leading to enduring systemic inflammation (COVID: a Mitochondrial Perspective, DNA and Cell Biology Vol. 40 Number 6, 2021).

Hyperferritinemia, accumulation of iron occurring due to COVID, induces ROS formation. Iron homeostasis dysregulation incites platelet destruction. The cytokine cascade contributes to ROS formation through TNF-α, IFN-, IL-6 and IL-10, stimulating the pro-inflammatory profile. IL-6 and TNF-α impair the processes of ATP and OXPHOS. This amounts to the loss of mitochondrial membrane potential, increasing mitochondrial permeability ("leaky mitochondria").

Upon losing integrity, mitochondrial DNA is released into the intracellular fluid, inducing an inflammatory profile of IL1-β and IL-6 through stimulation of the inflammasome NLRP3. Damage to mitochondria in epithelial alveolar cells contributes to the release of inflammatory CXCL-8, CCL3, CCL4, CCL20, IL-6 and IL-12. Hyperferritinemia drives mitochondrial respiration from aerobic to an anaerobic state.

In SARS-CoV-2 infection, TCA cycle/Krebs cycle metabolites citrate, malate, fumarate and aconitate are shown to be decreased through depression of TCA cycle genes. TCA cycle and OXPHOS are depressed in COVID, indicating altered cellular metabolism. Furthermore, choline uptake is increased by polarized macrophages during SARS-CoV-2 infection, subsequently leading to choline downregulation. On the other hand, homocysteine upregulation in COVID contributes to endothelial damage (Metabolic reprogramming and epigenetic changes of vital organs in SARS-CoV-2-induced systemic toxicity, JCI Insight 2021; 6(2)).

1.3 The Warburg effect
The Warburg Effect marks a shift from the TCA cycle and oxidative phosphorylation to aerobic glycolysis by glucose uptake and increased fermentation of glucose to lactose, even in the presence of abundant oxygen (Neoplasia, Robbins & Cotran Pathologic Basis of Disease, 2021). Aerobic glycolysis is exploited by rapidly replicating cells and necessary for cell proliferation, viral replication and drug resistance. Initially, aerobic glycolysis is necessary for proliferation of neutrophils and M1 macrophage activation. As a first line of defense, neutrophils and M1 macrophages depend on glycolysis and Fatty Acid Synthesis (FAS).

2. COVID-mitochondriopathy and hypoxia

2.1 Hypoxic conditions in COVID
Under hypoxic conditions, pyruvate is converted to lactate. Hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1a and HIF-1-beta, shifts metabolism from mitochondrial respiration towards aerobic glycolysis. COVID/SARS-CoV-2-infection is marked by increased levels of pyruvate, pyruvate kinase and lactate dehydrogenase (LDH), indicating glycolysis with lactate fermentation.

Hypoxia occurs when leukocytes are activated in response to Interferon activation, as well as in response to Pathogen-Associated Patterns (PAMP) and Damage-Associated Patterns (DAMP). DAMP activate the inflammatory NF-kB-pathway and STING (Interferon Genes) pathway. Moreover, fragmented damaged mitochondria are secreted as DAMP. Vascular Endothelial Growth Factor (VEGF) is upregulated under hypoxic conditions, notably by HIF-1-alpha. HIF-1-alpha can accumulate in inflammatory cells through the Prolyl Hydroxylase-pathway or TLR4-mTOR.

Increased glycolysis in monocytes sustains inflammatory cytokine production (IL-1β, TNF-α and IL-6), T cell impairment and pulmonary epithelial cell death (Metabolic reprogramming in COVID, International Journal of Molecular Sciences 2021, 22, 11475).

The hyper-inflammatory stage of hypoxia is marked by activation of NF-kB, NLRP3, mTOR and MAPK (mitogen-activated protein kinase), which induce the cytokine cascade. The NAD+ (Nicotinamide Adenine Dinucleotide) mitochondrial homeostatic regulators Sirtuins 3, 4 and 5 are downregulated. Downregulated Sirtuins enhance ROS formation, which in turn downregulates PHD, a regulator of HIF-1-alpha. HIF-1-alpha thus stabilizes. Enzymes Glut1, LDH, PDHK, HK and COX-2 indicate the shift to anabolic glycolysis (COVID sepsis: revisiting mitochondrial dysfunction in pathogenesis, aging and inflammation, Inflammation Research, 3 August 2020). Glut2 uptake is stimulated by the EGFR.

In addition, activation of complement C5a is involved in activating pro-inflammatory neutrophils and macrophages by activation of PI3K/Akt and MAPK pathways, stimulating endothelial damage and thrombosis. HIF-1a in alveolar epithelial cells activates the NF-kB pathway, mediates cell inflammation through CD4+ and CD8+ and enhances inflammatory cytokines IL-2 and TNF-a, driving complement-activated endothelial damage (COVID-driven endothelial damage: complement, HIF-1 and ABL2 are potential pathways of damage and targets for cure, Annals of Hematology 9 June 2020).

Cells under oxygen deprivation induce adaptive responses through AMP-kinase. When presented with high O2 tension, Prolyl Hydroxylases (PHDs) oxidize HIF-1a under normoxic conditions. Under low O2 tension, impaired succinate dehydrogenase leads to accumulation of succinate with subsequent inhibition of PHDs. Disproportional lactate production through glycolysis lowers cellular pH. HIF-1 activates transcription of PDK1, encoding a kinase that inactivates pyruvate dehydrogenase. Since pyruvate dehydrogenase is essential for metabolizing acetyl-CoA in the mitochondrial matrix, mitochondrial respiration is decreased (Hypoxia and mitochondrial oxidative metabolism, Biochimica et Biophysica Acta 1797 (2010)).

2.2 The Warburg Effect in COVID via PI3K/AKT/mTOR and MAPK/ERK pathways induce microthrombosis
Pyruvate Dehydrogenase (PDH) inhibition by Pyruvate kinase dehydrogenase 1 (PDK1) is stimulated by HIF-1a, PI3K/AKT/mTOR and the MAPK/ERK pathway, activated by loss of P53. This means that the PI3K/AKT pathway, by activating PDK1, blocks off pyruvate from feeding into mitochondria.

PDK1 and ERK in platelets stimulate aerobic glycolysis, which leads to thromboxane activation and microthrombosis, while Platelet-derived Growth Factor (PDGF) activates glycolysis via PI3K and HIF-1a. The PI3K/AKT pathway activates ATP Citrate Lyase (ACLY), thereby enhancing Acetyl-CoA to sustain Fatty Acid Synthesis (FAS). Acetyl-CoA Carboxylase (ACC) sustains arachidonic acid synthesis, necessary for generation of thromboxane. 

AMP-activated protein kinase (AMPK) counteracts the Warburg effect and the PI3K/AKT/mTOR pathway. In addition, AMPK acts on production of the vasodilator Ang 1-7 in endothelial cells and stabilizes ACE2, decreasing vasoconstriction and platelet-derived microthrombosis (The Key role of the Warburg Effect in SARS-CoV-2 replication and associated inflammatory response, Biochimie 180 (2021)).

2.3 Inhibition of Bcl-2 family members leads to necrotic cell death
Bcl-2 family members tightly regulate Bax/Bak activation. Through Bax/Bak activation, Bcl-2 family members regulate apoptosis by regulating mitochondrial outer membrane permeability (BAX/BAK macropores regulate the mtDNA extrusion). Upon permeabilization of the mitochondrial outer membrane, cytochrome-C is released in order to activate Caspase, leading to necrotic cell death. Right after SARS-CoV-2 infection, pro-apoptotic genes are upregulated, among which the BCL2L11, leading to apoptosis by inhibition of anti-apoptotic Bcl-2 and activation of Bax-Bak (SARS-CoV-2 Mitochondriopathy in COVID-19 Pneumonia Exacerbates Hypoxemia, Redox Biology 58 (2022)).

2.4. NSP4 and ORF9b of SARS-CoV-2 damage mitochondrial membrane potential (ΔΨ _m) and induce release of mtDNA into the cytosol
Mitochondrial DNA (mtDNA) released into the cytosol enhances inflammation. mtDNA is the result of mitochondrial damage following loss of mitochondrial membrane potential. mtDNA are DAMPs, therefore promoting an inflammatory cascade through the cGAS/STING1-mediated interferon signaling.

It was found that transfection with SARS-CoV-2 proteins NSP2, NSP4, NSP6, NSP8, ORF3a, ORF6 and ORF9b upregulate mitochondrial Reactive Oxygen Species and downregulate mitochondrial membrane potential. Notably, NSP4, ORF6 and ORF9b induce mtDNA release from epithelial cells.

MCL-1 regulates inner membrane vesicle formation and packaging of mtDNA. Vesicles enclosing mtDNA are derived from the inner membrane and extruded through BAX/BAK macropores. NSP4 acts on BAX/BAK to induce macropore formation. ORF9b acts on MCL-1 to impair the inhibitory (protective) effect on BAX/BAK macropore formation and to impair the regulatory effect of MCL-1 on inner membrane stability and vesicle formation (NSP4 and ORF9b of SARS-CoV-2 induce pro-inflammatory mitochondrial DNA release in inner membrane-derived vesicles, Cells 2022, 11).

Extracellular mitochondrial DNA induces the release of pro-inflammatory cytokines (PICs). The loss of mitochondrial (membrane) integrity and the release of mtDNA induce a highly inflammatory IL1-β release. Moreover, loss of ΔΨ m and mtDNA circulating in the cytosol contributes to chronic inflammation, one of the appearances of Post-Acute SARS Syndrome/Long COVID.

2.5 Fibrosis and mitochondrial dysfunction
Excess ROS generation under hypoxia, leads to damage of mitochondrial DNA, proteins and lipids. The transition pore in the mitochondrial inner membrane loses integrity, inducing mitochondrial depolarization and swelling and loss of Electric Chain Transport (energy generation).
Increased mitochondrial permeability enhance the release of Cytochrome C into the cytosol.

Tissue Growth Factor-beta (TGF-β), induced by anti-inflammatory macrophages, stimulates fibroblasts through Smad signaling. Fibroblasts activate fibro-collagen, Extracellular Matrix Molecules (ECM) and inhibit the degradation of ECM molecules. The deletion of mitochondrial DNA, induced by Angiotensin II, is reported in cardiac fibrosis. Mitochondrial damage in alveolar epithelial cells contributes to pulmonary fibrosis. Impaired mitophagy, the removal of dysfunctional and defective mitochondria, contributes to fibrotic disease through activation of the platelet PDGFR/PI3K/AKT pathway (Mitochondrial function in fibrotic diseases, Cell Death Discovery (2020)6:80).

2.5.1 Alleviating pulmonary fibrosis through restoration of mitochondrial integrity and REDOX
Targeting mitochondrial Sirtuin-3 (SIRT3) could alleviate pulmonary fibrosis (Mitochondrial Sirtuin 3: New emerging biological function and therapeutic target, Theranostics 2020;10(18)). Furthermore, as hydrogen peroxide generation dependent on NOX4 contributes to fibroblast formation, LYCAT and NOX4-inhibition through activation of the AMPK-pathway could attenuate lung fibrosis.

Metformin attenuates lung fibrosis through activation of the AMPK-pathway, which inhibits TGF-β induced NOX4 expression and ROS. Furthermore, AMPK activation attenuates mTOR-activation (Metformin attenuates lung fibrosis development via NOX4 suppression, Respiratory Research 17, Art. 107(2016)). Mitoquinone is an antioxidant that inhibits TGF-β and NOX4 expression; mitoquinone also prevents Nrf2 downregulation (Mitoquinone ameliorates pressure overload-induced cardiac
fibrosis and left ventricular dysfunction in mice, Redox Biology 21, 101100 (2019)).

2.5.2 Mitophagy impairment in spite of enhanced PINK1/Parkin
Infection with SARS-CoV-2 causes mitochondrial lesion. In spite of PINK1/Parkin activation and mitochondrial P62 accumulation, the normal process of mitophagy of damaged mitochondria was inhibited. SARS-CoV-2 was found to inhibit P62 and LC3 binding. The mitochondrial outer membrane protein Tom20 was found to be used by SARS-CoV-2 to provide SARS-CoV-2 dsRNA entry. Interestingly, Chaperonin HSP60 remained high following SARS-CoV-2 infection, indicative of impaired clearance.

Knockdown of Tom20 and application of Cyclosporin D inhibitor Cyclosporin A, each decrease viral replication, indicating that loss of loss of mitochondrial membrane integrity and MPTP opening are vital in progression towards COVID (SARS-CoV-2 causes Mitochondrial Dysfunction and Mitophagy Impairment, Frontiers in Microbiology Vol. 12 (ahead of print)).

2.6 Mitochondrial damage leading to mismatched ventilation/perfusion (V/Q) in COVID
SARS-CoV-2 damages airway epithelial mitochondria (AEC) and pulmonary artery smooth muscle cells (PASMC), thereby triggering apoptosis and impairing hypoxic pulmonary vasoconstriction (HPV).

The M protein of SARS-CoV-2 was found to depolarize the mitochondrial membrane potential and increase the opening of the mitochondrial permeability transition pore (mPTP). Nsp7, Nsp9 and the M protein induced apoptosis of airway epithelial cells. While Ca2+ levels rise in response to hypoxia, M and Nsp9 were found to inhibit the rise of Ca2+, reducing an accurate HPV under hypoxic circumstances.

Increased Drp1-mediated fission and Nsp7-induced inhibition of ETC electron transport chain Complex I oxidative metabolism by SARS-CoV-2, contribute to mitochondrial dysfunction in COVID. Through expression of Caspase7 and Annexin V, apoptosis is induced. These factors contribute to impairment of oxygen-sensing. As a result, impaired HPV leads to mismatched ventilation/perfusion (V/Q) and leakage into the capillary (SARS-CoV-2 mitochondriopathy in COVID pneumonia exacerbates hypoxemia, Redox Biology 58 (2022) 102508).

3. Mitchondrial DNA and TLR-9 contribute to SARS-endothelial damage
SARS-CoV-2 was found to infect HUVECs, endothelial cells, through their expression of ACE2 and TMPRSS2. The sera of COVID patients evidenced release of mitochondrial cytochrome B and NAD dehydrogenase. Mitochondrial Complex I protein levels were significantly decreased in COVID. Complex I is necessary to maintain healthy ROS levels. A reduction of Complex I levels induces mitochondrial damage. Abnormal levels of intracellular Ca2+ concentrations indicate endothelial and mitochondrial dysfunction. Reduction of Ca2+ disrupts vasodilation and induces endothelial damage.

From SARS-CoV-1 (2003), it was known that the mitochondrial antiviral signaling protein (MAVS) was disabled in order to manipulate mitochondrial function.
The E-ORF and ORF10 of SARS-CoV-2 activates TLR-9 through multiple cells.

TLR-9 activates a hyperinflammatory state. Through an increase of mtDNA release in HUVECs, TLR-9 becomes activated, which increases the inflammatory pathways My88 and NF-kB, decreases eNOS and induces the release of high levels of cytokine IL-6, thus creating a feedback loop into endothelial damage and thrombosis (Mitochondrial DNA and TLR-9 activation contribute to SARS-CoV-2 induced endothelial cell damage, Vascular Pharmacology 142(2022)).