Clues toward the Cause of Long COVID

I previously shared some of the amazing data from the paper "Muscle abnormalities worsen after post-exertional malaise in long COVID" by Appelman et al. 

The paper will undoubtable become a classic in the field of Long COVID, providing a fascinating series of clues that the researchers followed past several dead-ends to their interesting implications. 

First, and most importantly, the researchers confirmed beyond a shadow of doubt that Post-Exertional Malaise (PEM) is a real disease, with myriad muscle and metabolic abnormalities in the Long COVID patients following intense exercise.  Metabolomics provided additional key findings, including the first clue: a possible blockage of glycolysis in Long COVID (see diagrams in previous post).  

A Clue: Glycolysis Blockage

In the glycolysis pathway, the phosphoenolpyruvate (PEP) levels of Long COVID patients were increased, while pyruvate levels were decreased, indicating a disruption or imbalance in enzyme activities within the pathway.   This could be due to a decreased activity of the enzyme pyruvate kinase (PK), which converts PEP to pyruvate in the final step of glycolysis.  Reduced PK activity would result in a buildup of PEP.  With less PEP being converted to pyruvate, the downstream levels of pyruvate would be lower.  

There are several interconnected regulatory pathways that can reduce the activity of pyruvate kinase (PK), the two most relevant being Oxidative Stress and Hypoxia.  Increased levels of reactive oxygen species (ROS) or oxidative stress can lead to the oxidation and inactivation of PK.  Under hypoxic conditions (low oxygen levels), the transcription factor HIF-1 (Hypoxia-Inducible Factor 1) can be activated, which can lead to the downregulation of PK expression and activity.  This is part of the cellular adaptation to hypoxia, where glycolysis is regulated to favor the production of metabolic intermediates for other pathways.

Hypoxia?

In the context of Long Covid and post-exertional malaise (PEM), hypoxia from microclots has been suggested to increase lactic acid production. However, in this study the metabolomics showed decreased* lactic acid, because glycolysis was shut down at PEP by loss of PK activity, not at pyruvate by loss of pyruvate dehydrogenase (PDH).  

The researchers looked for but did not find decreased muscle oxygen perfusion or any differences in microvasculature.  The researchers noted decreased oxygen utilization, but this could be due to anything that disrupts metabolism and does not indicate hypoxia as a specific issue.  

They noted amyloid plaques in the extracellular matrix; the plaques were not blocking the microcapillaries and it is unclear what role they play in the pathophysiology of Long COVID: are they a cause of PEM, or a consequence?  

Their observation that Long COVID patients' muscle force was not dependent on The Citric Acid (TCA) cycle enzyme succinate dehydrogenase (SDH) can also be explained by impaired metabolism upstream of TCA Cycle, i.e. in glycolysis.

Oxidative Stress

Therefore, it seems likely that the regulatory pathway most likely to contribute to reduced pyruvate kinase (PK) expression and activity is the oxidative stress pathway.  

Long Covid patients have been reported to exhibit higher levels of oxidative stress markers, such as lipid peroxidation products and decreased antioxidant levels, compared to healthy individuals or those who have recovered from acute COVID-19 infection.  Physical exertion and exercise can lead to an acute increase in reactive oxygen species (ROS) production, potentially exacerbating oxidative stress in individuals with Long Covid and triggering PEM symptoms.

Some studies have suggested that Long Covid patients may experience mitochondrial dysfunction, which can further contribute to increased ROS generation and oxidative stress.

Next Steps

The paper concluded with these results, but the logical next step would be to use metabolomics to assess free radical concentrations.  One theory is that COVID spike proteins form "pores", or holes in the mitochondrial membranes, disrupting the mitochondria and releasing free radicals into the cell.  

The researchers did look at COVID nucleocapsid protein, but found it in both the control and Long COVID groups in equal concentrations, suggesting that remnant viral protein doesn't explain the pathophysiology of Long COVID.  But maybe remnant virus affects the Long COVID patients differently?  

The researchers noted immune cell infiltration into muscle tissue, which could be in response to a signal from excess free radicals, or could be due to some other reason like persistent COVID infection/expression.  

Lactate?

This study seems to indicate the lactate is not an important variable for the pathophysiology of Long COVID.  However, the details about how lactate was measured limit these conclusions. The researchers measured lactate from three different sources:  metabolomic blood (venous) and muscle lactate measured before and after PEM, and capillary (i.e. finger prick) lactate measured during exercise.  Venous lactate measured one week after PEM induction showed a slightly increased level in Long COVID patients, but none of the other metabolomic lactate measurements showed any difference.  The capillary lactate also showed a slightly elevated blood lactate level before exercise in the Long COVID patients, but the difference was not significantly different.  

However, the baseline measurements were not taken in a fasted condition, and because eating normally raises resting lactate it cannot be determined from this study if fasted lactate might show other differences that are important to Long COVID.