Persistent symptoms with covid & spike – what about the calpains?

Disclaimer: this article explores possible mechanisms behind symptoms that many are now unfortunately suffering with following the pandemic. It represents a hypothesis that has not been borne out with robust scientific study and should not be relied upon to guide any healthcare decisions. If you or a loved one is suffering with symptoms mentioned in this article, be sure to talk with your doctor or other healthcare professional before making any changes. This article has been put up for education purposes only.

Could problems with calcium homeostasis and calpain hyperactivity be causing the persistent symptoms seen in some people after both covid infection and vaccination? In this article I’m going to explore this possibility. I won’t be analysing the various merits or otherwise of our attempts to bring covid under control but instead I’ll be looking at an interesting corner of our biology that could tie together much of what we have been seeing in the post pandemic period.

It turns out that the calcium-calpain system is at the core of many different physiological and pathological processes, and there are many lifestyle and environmental factors that can disrupt it such as poor metabolic health, exposure to pesticides and heavy metals as well as excessive EMFs. It also seems likely that viruses have evolved to gain an advantage by disrupting this system because calpains are also involved in viral entry, immune function and inflammation. Adding to this, some of the interventions we have seen such as melatonin and taurine may be working in part by inhibiting excessive calpain activity.

Even though the spike protein does represent many new challenges, the situation is not entirely new: post-viral symptoms such as fatigue and brain fog have been recognised for decades, and it is suspected that conditions such as ME/CFS are largely driven by viral infection which is never entirely cleared from the body. There is also a growing awareness of microbial factors in triggering many other conditions too, including heart disease, diabetes, cancer and Alzheimer’s among others. With the arrival of covid, there has been an important new drive to understand conditions like ME/CFS as well as ways in which long term immune activation against various microbes, their antigens & metabolites can push people further towards disease states. In order to understand this further, it is helpful to look at underlying mechanisms, and one such mechanism could be calpain disruption.

What are Calpains?

Calpains are a family of more than a dozen intracellular proteolytic enzymes, but the best known and characterised isoforms are calpain 1 and 2 (or μ-calpain and m-calpain). They are an important part of our physiology regulating cell growth, maturation, immunity, inflammation and tissue remodelling, but their dysregulation has been linked to many chronic conditions.

More specifically, calpains are calcium-dependent cysteine proteases and are activated by an increase in intracellular calcium ions, either entering the cell via calcium channels or being released inside the cell from the endoplasmic reticulum, mitochondria and calciosomes. They can also be activated by ERK phosphorylation [R]. Upon activation, they cleave or ‘cut’ other proteins so that an initial protein is transformed into something else. This means that they can activate or deactivate other molecules, and they have a large number of different substrates on which they act. For example, they are responsible for the creation of the inflammatory cytokine il-1α by activating its precursor, while also degrading and deactivating IκBα, a protein that is involved in the dampening down of inflammation by inhibiting NF-κB. In both of these cases, calpain activation leads to inflammation, but in two different ways. And in other cases, inflammation may be reduced by calpain as with some immune cells: there is evidence that constant calpain cleavage of proteins in macrophages and neutrophils may help to keep the cell in a deactivated and less inflammatory state until they are called on to react. This means that as with everything, it’s nearly always more complex than simply needing more or less of something – and we should always take the context into account as much as possible. Sometimes inflammation is essential and other times it’s damaging – the key is our ability to regulate it and maintain homeostasis. 

Back to calpain. The first calpain to be discovered and purified was calpain 2 in 1976 and got its name from two similar enzymes, calmodulin and papain from papaya. The family consists of two main isoforms: μ-calpain (mu) and m-calpain (or calpain I and II) as well as calpastatin, which inhibits their function.

Since then, more types were discovered: Calpains 1, 2, 4, 5 and 7 are expressed ubiquitously throughout the body whereas others are found in specific tissues: calpain 3 (skeletal muscle), calpain 6 (placenta), calpain 8 (smooth muscle), calpain 9 (stomach), calpain 11 (testes), calpain 12 (skin after birth) and calpain 13 (testes and lung)[R]. We also have calpastatin which inhibits the calpain enzymes.

Although I’ll focus more on functional changes from environmental stressors, there are also several types of muscular dystrophy associated with genetic mutations in calpain genes which are collectively called calpainopathy [R].

Physiological functions of calpain

Calpain activation is a normal part of our physiology, and even though research is at an early stage it is suggested to be essential for many processes. It is only when it becomes dysregulated that it becomes problematic. Calpains are inactive at normal physiological concentrations of Ca²⁺ but when this increases, these enzymes start working. As the names suggest, μ-Calpain is activated at micromolar concentrations and m-calpain is activated at millimolar concentrations, but the necessary Ca²⁺ activation concentrations decrease after autolysis, representing a possible first step in activation which may also be aided by phospholipids [R] including phosphatidylcholine (a hydrogenated derivative of which is also found in LNPs) 

As part of our physiological responses, there is evidence to suggest the calpains and calpastatin are important for: 

• Normal cell growth, proliferation, maturation [R]
• Apoptosis and caspase activation [R]
• Vascular homeostasis and platelet activation [R]
• Remodelling of cytoskeletons, vasculature, muscles and other tissues including collagen deposition. [R][R][R]
• Inflammation activation [R]
• Neuronal growth and pruning [R][R]
• Memory & fear consolidation [R]
• Macrophage & neutrophil activation [R]
• Natural killer activation [R]
• Th17 & STAT3 promotion via SOCS3 deactivation [R][R]

Bodily processes such as these exist in balance where too much or too little can cause issues – so they are closely controlled. For example, apoptosis – or programmed cell death –  is essential to get rid of old and damaged cells to help prevent cancer and keep our body working optimally, but too much has also been linked to things like neurodegeneration and heart issues. 

Usually our body does a great job of maintaining balance, but the problem is often that our modern way of life often puts unnatural pressure on these systems which can push them beyond limits that they never would have seen in pre-industrial times.

Now let’s take a look at the main stimulator of calpains: calcium.

The importance of calcium homeostasis

Calcium is the most abundant mineral in the human body, and although more than 99% of it is stored in the bones, the small amount of calcium that exists in blood and inside cells is one of the most important second messenger molecules. A second messenger is a signalling molecule that responds to a first messenger, such as a hormone or neurotransmitter binding to a cell receptor. 

The fundamental nature of calcium has been known for a long time: in the 1880s, Sydney Ringer was experimenting with artificial stimulation of toad hearts, and all solutions were failing until he tried the London tap water, which worked likely due to its high calcium content. Sydney Ringer is perhaps best known for Ringer solution, which is an isotonic mixture of water and salts used in medicine to restore fluids after dehydration.

His experiments led to the finding that calcium signalling is essential for the heartbeat and muscle contractions in general, but it is also essential for many other processes that sustain us throughout life, including, metabolism, mitosis, neuronal activity, neurotransmitter release and synaptic plasticity and much more. In fact, life starts with a pulsating calcium wave that activates the egg just 1-3 minutes after the sperm binds, and these dynamic bursts continue and accompany us in one way or another every second of our lives. It is amazing to think that every human endeavour, thought, emotion and reaction has all been made possible by calcium moving in and out of cells.

There are two broad actions of calcium. Firstly, calcium helps to maintain the resting membrane potential of our cells together with other essential ions such as potassium, sodium and chloride. A healthy cell has an electrical charge of around -70mV at rest, and calcium helps to stabilise this by regulating ion channels [R]. Secondly, sharp intracellular increases in calcium concentrations act like an on-off switch for a myriad of different cellular functions to do with cell growth, maturation, survival and apoptosis. It either comes from outside the cell, or it is released from within the cell from intracellular stores. These changes usually only last a few milliseconds before Ca²⁺ levels fall back to baseline, but in that time calcium binds to a host of enzymes, kinases and other proteins including calpains creating the physiological changes we associate with it.

The processes that control both extracellular and intracellular calcium are tightly controlled. Serum calcium levels are sensed by the parathyroid and thyroid glands. Low levels stimulate the release of PTH which increases blood levels by increasing calcium liberation from bones, increased absorption from the intestine and increased retention by the kidneys, acting only for a few minutes until levels are restored. Calcitonin released by the thyroid does the opposite – it lowers blood levels by stimulating renal release and increasing uptake by the bones, which is the main reason why thyroid disease is linked with osteoporosis. 

Intracellular levels are also tightly regulated by different processes including:

• An increase from outside the cell through calcium channels such as the voltage gated calcium channels.
• An increase from activation of receptors such as NMDA, AMPA & histamine receptors which also pulls calcium into the cell.
• An increase from release from intracellular stores including mitochondria and the endoplasmic reticulum via G protein-coupled receptor binding, phospholipase C activation and cleavage of PIP2 into DAG and IP3. IP3 goes on to bind to the endoplasmic reticulum and releases calcium into the cytosol.
• A decrease via calcium leaving the cells through the calcium channels as well as absorption by the endoplasmic reticulum and the mitochondria to get things back to baseline.

Increases in IP3 aren’t always stimulated by extracellular signals, but they can also come from within the cell, for example in cases of ER stress. ER stress is when the endoplasmic reticulum runs into problems with misfolded proteins, which increases IP3 and calcium dumping into the cytoplasm. Usually, the UFP response is able to resolve the issue of misfolded proteins, but if the situation is severe enough, calcium increases so much that the cell commits suicide in what is called apoptosis, before it can start to damage other cells. ER stress is important because it is stimulated in large part by oxidative stress and metabolic dysfunction, and the resulting misfolded proteins have been implicated in many diseases. 

The signalling capacity of these quick bursts are what makes calcium special. For example, in neurons, calcium regulates excitation and is important for the action potential, which is the way nerve signals propagate from one neuron to another. In muscles, calcium binds to calmodulin and this complex stimulates muscle contraction by stimulating myosin light-chain kinase which goes on to phosphorylate MLC[R].

But like all processes, calcium signalling can become dysregulated especially in our modern world. The system has evolved to work within certain limits, but when those limits are exceeded, it has difficulty maintaining homeostasis and symptoms emerge. For example, the main way in which MSG causes excitotoxicity and death of neurons is by binding the NMDA receptor and stimulating calcium influx [R]. MSG causes a completely unnatural spike in glutamate that no food from nature would be able to reproduce, and the modern world is unfortunately full of stimuli like this. And when intracellular calcium gets dysregulated, so do the downstream processes it activates including the calpain enzymes.

Calcium-calpain dysregulation

Although modern life has brought with it many benefits, the downside is that many of our bodily systems struggle to maintain balance under these conditions because they evolved in a very different world. Here are some things that likely throw calcium and calpains off balance: 

• Physiological factors in excess: ER stress & metabolic dysfunction [R][R], glutamate [R][R][R], histamine (H1R + H2R [R]), thrombin [R], adrenaline [R], NADPH oxidase [R], uric acid [R], angiotensin II [R][R], hyperglycemia [R], oxidised & glycated LDL [R], homocysteine activation of MMP-9 [R], ceramides [R], alcohol [R][R][R], excessive muscle contraction leading to muscle injury, magnesium deficiency [R], selenium deficiency [R].
  
• Pathological factors & elements of modern life: hypoxia [R][R], some viruses (including the spike protein) [R][R], some bacteria such as S.aureus [R] and S. pneumoniae (pneumolysin) [R], emfs effect on VGCCs [R][R], pesticides such as dichlorvos, malathion, glyphosate and rotenone [R][R][R], heavy metals such as mercury [R], cadmium [R] and aluminium [R], air pollution [R] and a range of other synthetics such as diatrizoic Acid (a contrast used in medical imaging) [R], flame retardants [R], bisphenols and phthalates [R][R][R], and drugs such as amphetamine [R] and methamphetamine [R], MSG, acrylamide [R].

One of the difficulties in studying the real world impact of these factors on calcium is their ubiquity. For example, the pesticide glyphosate is now found in water supplies everywhere including in rainwater, and even contaminating organic crops, some medications and even the clothes we wear. This means that total avoidance and therefore studying exposed versus non-exposed populations is near impossible. But that doesn’t mean that their effect can be ignored, and if indeed they cause significant calcium dysregulation, then it would not be a leap to imagine that they also increase covid risk as well as inhibited viral clearance and therefore long covid. For example, animal studies have clearly suggested that calcium disruption could be one of the main reasons behind glyphosate-induced neurotoxicity [R], a point of view shared by glyphsate expert and senior research scientist Dr Stephanie Seneff.

Another ubiquitous aspect in the modern world is metabolic dysfunction. ER stress, hyperglycemia, high blood pressure, oxidised lipids, excess uric acid, ceramides and thrombin are all hallmarks of metabolic dysfunction and to some degree present in nearly everyone living in modern societies thanks largely to our modern, industrialised lifestyle. Some studies suggest that upwards of 90% of Americans have some sign of metabolic dysfunction [R], with more than 1 in 10 having diabetes [R] with Europe not far behind.

Viral interactions: calcium, calpain & spike

It has been known for some time that dysregulated calcium is seen in some viral infections [R] and this seems to be happening to a significant extent in Sars-cov-2 infection [R]. This could also be the reason why the sense of smell is affected as calcium overloads olfactory neurons. 

The same study [R] showed that only the S1 protein was necessary for this effect by interfering with calcium channels, and another study [R] demonstrated that only the spike RBD is necessary for “acute-to-prolonged induction of the intracellular free calcium concentration”, which suggests that vaccination can do the same. Interestingly this same study shows that the effect decreases with Omicron BA.5.2 & XBB suggesting that decreasing virulence correlates with decreasing calcium disruption.

Several studies have also shown that lower blood calcium levels are a good indicator of covid severity [R], which suggests that the higher the viral load, the more calcium is being shunted from the blood into cells.

From the above, we can guess that this would be activating calpains, and indeed this has also been confirmed by Juibari et al [R], and it is possible that coronaviruses (and others) have evolved to interfere with calcium channels in order to benefit from calpain activity and gain a survival advantage. Not only does it help with viral entry but excessive calpain activation might make the host a nicer place to hang out by dampening down the immune response.

In terms of viral entry, the study and references suggest that calpains facilitate this via Talin cleavage so that TMPRSS2 is no longer needed, and they demonstrated that in vitro cell entry was blocked by 75%-92% by using a calpain inhibitor.

In terms of immune modulation, calpain activity appears to keep macrophages and neutrophils in a deactivated, rest state whereas the calpain inhibitor calpastatin has been shown to activate these cells in other studies [R]. The same mechanism is possible for natural killer cell cytotoxicity [R], and calpain cleavage of SOCS3 increases STAT3 hyperexpression [R] which can lead to a dysfunctional interferon response and increased il-6 as well as other negative outcomes for the immune system, especially if part of a positive feedback loop with PAI-1. Early on, an excellent paper by Matsuyama et al [R] suggested that STAT3 and PAI-1 positive feedback could be responsible for much of the covid pathology. PAI-1 is also higher in the elderly, obese and metabolically unhealthy patients, and also peaks during winter months. 

A fundamental part of our immune response to viruses are Interferons. These key immune molecules interfere with all stages of viral infection including reducing replication and increasing clearance, and higher levels have been observed with most covid infections even though sars-cov-2 is able to reduce interferon levels, potentially because it contains a protein called Mac1 [R] as part of nsp3. In a non serious infection, interferons and other immune chemicals rise and quickly overcome the virus before returning to normal, whereas in severe cases the interferon response becomes dysregulated, with prolonged lower interferon gamma [R], and higher levels of other interferons [R] that end up damaging the body. In long covid, some studies point to a switch to chronically raised interferon gamma from CD8+ T cells [R] which suggests that the immune system is not able to clear the persistent viral debris. Even though it lacks the Map1 peptide, Seneff et al draw on experimental evidence from other studies suggesting type I interferons are significantly reduced following vaccination [R]. They suggest that this could be because of spike protein and microRNAs (miR-148a and miR-590) leaving transfected cells in exosomes which reduce IRF9 levels and inhibit the interferon type I response in particular. Exosomes have also been associated with a greater pro-inflammatory response in covid infections as well as increased TLR expression [R], even though their contents may be different.

So what does this have to do with calcium and calpain? It turns out that stressed cells such as cancer cells shed significantly more exosomes than healthy cells [R][R][R], and shedding may be a way to rid the cell of toxic matter and oxidative stress [R]. This shedding is mediated by rises in intracellular calcium ions [R]. Calpain is also thought to be a key player as its capacity to remodel cytoskeleton proteins may be an essential part of increasing exosome budding [R]. Could it be that certain predisposing factors set people up for an exaggerated exosome response, reducing IRF9 and interferons and leading to these chronic, persistent infections that then become a vicious cycle?

Long term, persistent symptoms

While the interferon response has been shown to be dysregulated, there are other factors to consider including persistent spike protein, microclotting and autoimmunity which could lead to a whole range of issues such as immune dysregulation, chronic inflammation, mitochondrial dysfunction and dysautonomia. The persistent antigen theory suggests that fragments of viral proteins hang around long after acute symptoms have dissipated. I was first aware of this in Bruce Patterson’s research which found higher monocyte levels and spike fragments in a significant number of them up to 15 months post infection [R], together with differences in cytokine levels. His team’s latest research has found a similar pattern among vaccinated individuals experiencing longer term symptoms [R] and this joins a growing number of papers that have found mRNA and spike protein lasting much longer in the body than first thought. The mRNA has been found up to 60 days following vaccination [R] while another found recombinant spike sequences up to 187 days after vaccination [R], although this needs to be confirmed in larger studies.

Exciting research on microclots could also explain systemic symptoms. Resia Pretorius Ph.D. and her team from Stellenbosch University in South Africa have found evidence of microclotting present in long covid patients as well as ME/CFS patients to a lesser extent. Initial studies demonstrated that the S1 protein was capable of stimulating microclotting through platelet activation and fibrinogen interactions [R] and further studies have found evidence of this in long covid patients [R]. Dr Caroline Dalton of Sheffield Hallam University replicated and added to this research and the hope is that a standardised microclot test will be accepted into the NHS. They not only found higher microclots correlating with more severe symptoms but also higher levels of Von Willebrand’s factor and α2AP, which are key players in excessive coagulation.

Other animal studies have found that the spike protein competitively binds to heparin sulphate, increasing thrombin activity by reducing the binding of both antithrombin and heparin cofactor II [R]. 

The microclots found could explain two puzzling issues: firstly, symptoms may persist in time because the microclots seem to be extremely resistant to being broken down by fibrinolysis, and secondly they could explain the spatial aspect of symptoms travelling to areas of the body that weren’t affected directly by infection or transfection. If microclots form in the pulmonary or systemic circulation, they could travel around the body and accumulate in the capillaries, leading to vascular inflammation and hypoxia. Exercise intolerance, fatigue and brain fog have been flagged as significant symptoms, and this would be expected if there is any disruption to getting oxygen to tissues, especially the big muscles of the legs.

Together with Bruce Patterson’s research, these studies represent a leap forward because they have identified physical differences between long covid patients and controls, and made important links to ME/CFS. One of the issues is that many people still maintain that these conditions are purely psychological, but this assumption falls apart when physical changes can be demonstrated with lab testing.

Other clues come from similar conditions with a lot of overlap, including MCAS and dysautonomia. MCAS is characterised by hyperactive mast cells releasing excess histamine and other pro-inflammatory mediators, and dysautonomia is when the nervous system has difficulty in maintaining balance between sympathetic and parasympathetic branches. The spike protein has been implicated in MCAS as it seems to be able to directly stimulate mast cells either on its own or together with LPS. Chris Masterjohn also points out potential problems with iron and zinc [R]. Many of these aspects likely significantly activate calpains.

Conditions that may arise from calpain dysregulation

Below is a summary of some of the systems that could be affected by calpain hyperactivation, and although there is a striking overlap between existing calpain research and covid symptoms, it is important to point out that most of these studies are in vitro and animal studies, and many use calpain inhibitors to confirm benefits as an indirect measure. This means that although these studies have big implications, direct evidence from human studies is lacking.

Another consideration is that although this evidence points the finger at calpains, they are really just mediators responding to their environment. If calpains are raised due to diabetic hyperglycemia for example, or persistant spike protein, then it is those triggers that need to be addressed. Nevertheless, if calpains represent a fundamental checkpoint as it seems they do, their manipulation could be an important tool to improve symptoms while root causes are dealt with as well as reducing positive feedback loops such as thrombosis generating hypoxia > hypoxia activating calpain > calpain generating thrombosis as is probably the case with microcirculation issues.

With that said, let’s take a look:

• Mitochondrial dysfunction: mitochondria are responsible for the majority of our cellular energy in most cells as well as many other functions such as immune signalling and steroidogenesis. Many symptoms associated with long covid and spike pathology could arise from mitochondrial dysfunction, particularly heart issues, and transgenic studies examining mitochondrial calpain overexpression have shown dilated heart failure [R]. When Ca²⁺ overloads the cell, mitochondrial calpain increases ROS production and cleaves the Na⁺/Ca²⁺ exchanger which may be the most significant cause of increased calcium uptake into the mitochondria [R]. This can stimulate apoptosis as it does in the cell, and calpain also cleaves mitochondrial AIF which also induces apoptosis. Calpain 10 also cleaves complex 1, so this calpain has also been found to directly impair mitochondrial energy generation, mitophagy, fission and survival and calpains in general have been linked to worsened cardiac injury and dysfunction after ischemia/reperfusion injury [R][R]. ER stress also increases mitochondrial Ca²⁺ accumulation [R].
  
  Long covid exercise intolerance studies have found that as well as cardiopulmonary  problems with oxygen delivery, oxygen extraction at the cellular level is reduced [R]. If less oxygen is being used by the cell, less will diffuse into the cell due to increased pO2. This suggests mitochondrial dysfunction and a potential switch to anaerobic lactate metabolism. This also leads to lower CO2 levels, which can further decrease oxygen extraction from HbO2 via the Bohr effect.
  
• Chronic inflammation & neuroinflammation: Calpains activate the NLRP3 inflammasome linked to myocarditis [R], neuroinflammation [R] and endothelial dysfunction potentially contributing towards atherosclerosis [R]. Calpains also activate IL-1 alpha [R] (but not il-1 beta), and animal models suggest that calpains also indirectly activate NF-kB by inactivating IkBα [R][R]. Calpains also cause an increase in TNF-a [R] and vice versa [R] although this may be more related to oxidative stress and mast cells in heart tissue [R]. Significant NLRP3 activation has been noted from the start in covid.
  
• Coagulation issues: calpain has been implicated in thrombotic thrombocytopenic purpura (TTP), an extremely rare condition that has been seen both after covid and vaccination. One study suggested this was due to calpain’s proteolysis of von Willebrand factor [R] while another linked it to platelet microparticles [R]. Generally, calpain is known to play a role in platelet activation [R]. This is an important part of the healing response after surgery & injury, but in excess leads to thrombosis. Calpain mediated platelet activation and thrombosis has also been confirmed in hypoxic conditions [R]. In sepsis, calpain is implicated in negative coagulation outcomes via platelet apoptosis and release of procoagulant microparticles [R]. HMGB1 is also implicated in thrombosis and correlated with calpain levels [R]. Neutrophils have also been implicated in thrombosis when generating excessive NETs. The process of NETosis depends on PAD4 and calpain synergy [R].
  
• Endothelial dysfunction: endothelial dysfunction is a key feature of post spike symptoms, both for major blood vessels and the microcirculation, and this could be especially relevant for pre-existing metabolic issues such as hyperglycemia. Nitric oxide is essential for healthy endothelial function but eNOS is degraded by calpain. Animal models have found that this is one reason why hyperglycemia induces endothelial dysfunction, because it activates the Na⁺/H⁺ exchanger which leads to Ca²⁺ overload [R]. However, eNOS is usually protected from this degradation by hsp90, but cellular studies have shown that prolonged calpain activation might overcome this, showing significantly lower eNOS and NO levels [R]. This negative effect of hyperglycemia on the endothelium is also increased by homocysteine via calpain activation [R]. Another way calpain contributes to endothelial dysfunction is by stimulating the release of the microparticles from hyperreactive platelets. Here, animal studies show that calpain-1 cleaves PAR-1 on endothelial cells, which activates TACE. TACE generates TNF-a which has long been implicated in endothelial inflammation, as well as protein C receptor degradation which is an anticoagulant serine protease [R]. Platelet activation and subsequent microparticles are a well known complication of diabetes and insulin resistance, but are also central to thrombosis and inflammation induced by infections, including sars-cov-2 [R]. This seems to persist to give longer term symptoms [R], and although PAR-1 wasn’t implicated, increased TNF-a along with other inflammatory cytokines have been found to be a hallmark of long covid [R] also present in vascular inflammation and atherosclerosis. Calpain has also been shown to cleave CCL5 into a more active form. CCL5 deposits onto the endothelium where it attracts monocytes and has been implicated into the progression of vascular disease [R].
  
• Pulmonary arterial hypertension (PAH): pulmonary remodelling is a key alteration leading to PAH, and calpains represent an important step in this tissue remodelling by controlling collagen synthesis. The following in vitro study suggested that calpain inhibition reduced pulmonary remodelling and pulmonary hypertension [R], whereas inhibiting calpastatin increased it. Increases in PDGF, serotonin, H2O2, endothelin-1 and interleukin-6 all increased calpain activity via serine50 phosphorylation without changing the levels, which lead to increased cell proliferation and increased collagen-1 deposition.
  
• Exercise intolerance: post-exertion fatigue is one of the main symptoms associated with long covid and describes extreme fatigue following physical, mental or emotional exertion. Many people notice that with physical exercise, they now suffer with severe fatigue the day after exercising which can last for days. Some reasons put forward for its prevalence range from mitochondrial dysfunction to a disrupted acid-base balance to microclots impairing microcirculation leading to tissue hypoxia. Exercise has been found to activate calpains, which under normal conditions help the body heal and regenerate by stimulating protein breakdown and remodelling, but in excess may be behind muscle soreness and injury [R][R]. As we have already seen, calpain dysregulation has been linked with mitochondrial dysfunction as well as a prothrombotic state that may go on to damage microcirculation. This microcirculation damage is likely a key factor, because it goes on to cause hypoxia in tissues which will increase Ca²⁺ influx and further calpain activation. Increased inflammation has also been linked to mitochondrial dysfunction, and just like microclots, it can start in one area of the body and cause issues in another – which may explain why muscles, particularly leg muscles which are usually far away from any direct infection, can still be involved. While people are often used to ‘pushing through’, this may be a time where it would be wiser to hold back, as overexertion will likely make things worse.
  
• Atrial fibrillation: historically atrial fibrillation has been linked to Ca²⁺ overload which causes atrial electrical remodelling. Recent data suggest that the activation of μ-calpain may lead to the destruction of contractile filaments in fibrillating atria, with total calpain enzymatic capacity found to be more than doubled [R]. NF-kB activation may also play a part, leading to increased il-8, TNF-a and adhesion molecules. Calpain inhibitors have shown reductions in NF-kB, and in vitro studies have also shown that inhibiting calpain can reduce the breakdown of muscle fibres that can perpetuate atrial fibrillation as well as reducing apoptosis [R]. Having said this, some studies suggest that calpain inhibition may make things worse by increasing the possibility of necrotic cell death [R] which again suggests that simply blocking calpains may be shortsighted without trying to address what is at the root.
  
• Myocarditis: research has linked several viral infections as well as covid vaccination to myocarditis. Some studies link myocarditis to an inflammatory response from T cell infiltration against spike protein, whereas others have suggested causes such as autoantibodies and autoimmunity. Cardiomyocyte death is a key feature, and Calpains have also been implicated in this in animal studies, with their inhibition showing benefit [R]. One study found that T cell and antibody responses were essentially the same in myocarditis cases and asymptomatic controls, but interestingly, the difference between the two groups was the level of free, unbound spike in circulation [R] which could be activating the calcium channels.
  
• Neurodegeneration: neuroinflammation, hypoxia, poor circulation and mitochondrial dysfunction have all been linked to neurodegeneration and Alzheimer’s with amyloid plaque formation and tau hyperphosphorylation, and animal models have demonstrated that calpain inhibition can reduce the toxicity of amyloid plaques [R]. Calpain activation may also increase Aβ amyloid by increasing BACE1 activity, and BACE1 has been found to be higher in Alzheimer’s patients together with lower levels of the calpain inhibitor, calpastatin [R]. The same review also suggested that Aβ amyloid can activate calpain, yet calpain inhibition could also increase amyloid build up [R]. This speaks to the idea of dealing with root causes rather than simply inhibiting calpain enzymes, which in Alzheimer’s likely includes glucose metabolism issues and chronic infection. Other studies have highlighted calpain’s role in the aggregation of α-synuclein, a keyfeature of Parkinson’s [R], and previous studies mentioned have shown NLRP3 activation, a potent cause of neuroinflammation in the CNS.
  
  Another link between neurodegeneration and calpains is excessive GSK-3β activity, which has been suggested as being a key driver in Alzheimer’s disease [R]. Calpains have been found to increase GSK-3β activity by cleaving it into a more active form, which is another reason calpain inhibitors are being researched for Alzheimer’s prevention [R] and why there is renewed interest in lithium – not only for its effect on GSK-3β [R] but also as a potential calpain inhibitor [R].
  
• Retinal issues: neurodegeneration also extends to the eyes, and μ-calpain activation has been implicated in neuron damage in the retina [R]. Calpain overactivity has been linked to several eye diseases via inflammation and neovascularization [R].
  

Calpain, or lack of it’s inhibition, has also been implicated in ischemia/reperfusion injury in kidneys [R], in multiple sclerosis [R], in IgE mediated mast cell activation [R] and many other conditions.

Covid outcomes worsened by comorbidities

It has been evident throughout that the biggest factors in covid severity have been certain comorbidities and age. While it’s true that these comorbidities like diabetes and hypertension have been repeatedly linked with numerous immune system issues like immunosenescence & lower vitamin D, another reason behind increased susceptibility may be due to some level of calpain dysregulation that is already present thanks to these pre-existing chronic diseases. For example, higher angiotensin II in hypertensive patients has been shown to stimulate calpain activation as well as uric acid, which is found at higher levels in people with metabolic dysfunction like diabetes [R]. Hyperglycemia doesn’t just cause endothelial dysfunction via calpain activity, it may raise calpain expression too [R], together with high levels of homocysteine. Hypoxia is another important stimulator of calpain activation, and elevated calpain 1 has also been found in covid patients with ARDS [R].

While it is clear that calpains are not the cause in these cases, we have seen how they represent an important checkpoint on the road to a pro-inflammatory, prothrombotic state that not only results in increased susceptibility to infections, but many other conditions too.

Returning to a state of calpain homeostasis

I started off this article as an exploration of calpains with no intention of adding any remediation strategies. The majority of studies cited are animal or in vitro studies, so it is impossible to say for certain that this situation translates to humans, and there is no way to test the level of calpain activation or calcium dysregulation.

This means that any drug approaches are probably many years away, if indeed they’d be helpful at all.

But in researching this article, I came across a lot of lifestyle factors that are worth a mention because they not only appear to ameliorate calpain dysregulation, but they are also generally healthy and often something we should all be aiming for anyway. There are also several supplements mentioned which have proven safety track records and well-studied effects on calcium channels even if their direct effect on calpain remains unknown.

So with that said, let’s look at some generally healthy things that might also help with calpain issues. (Be sure to ask your doctor before making any changes to your diet or lifestyle)

• Improve metabolic health and reduce ER stress: metabolic health can be greatly improved by removing all processed foods from the diet, exercising regularly, improving circadian rhythms and reducing psychological stress where possible. This is of course advice that we hear often, but one of the main benefits could be in regulating ER stress, calcium and calpains. Insulin resistance is an important driver of many of the negative markers we saw earlier including hypertension and hyperglycemia. Intermittent fasting (started gradually) or the fasting mimicking diet is a great way to improve many of these markers. Walking after meals, avoiding overeating, eating carbohydrates last, starting a meal with a salad and some apple cider vinegar dressing and including more berries such as blueberries or aronia in the diet are some good, general steps to improving blood sugar.
  
• Increase magnesium levels: magnesium is perhaps the most common nutrient deficiency around today, and it also helps regulate calcium channels, reducing calcium influx and NMDA and AMPA receptor activity. It may also help to keep intracellular calcium lower by decreasing TRPM7 [R], a protein that acts both as an enzyme and an ion channel. It also helps reduce blood sugar, blood pressure and normalise thousands of other bodily reactions [R][R], and stress and excess dietary phosphorus and calcium increase our need for magnesium.
  
• Reduce exposure to air pollution, pesticides, heavy metals, bisphenols and phthalates: we have seen how air pollution, certain pesticides and heavy metals could dysregulate calcium channels, and there is extensive literature about other negative effects, so there has never been a better time to reduce exposure where possible and support the detox systems of the body. One of the best ways to reduce exposure is to avoid contaminated foods and herbs as well as personal care products and make-up that can often be loaded with these sorts of substances. Bisphenols and phthalates are found in plastics, some food packaging and on most shop receipts.
  
• Reduce exposure to EMFs: EMFs have increased exponentially over the last few decades, and although regulatory bodies focus on reducing damaging thermal effects, the non thermal effects are often ignored, and perhaps the most well studied of this is the effect on Voltage Gated Calcium Channels causing massive calcium ion influx into the cell. Reducing exposure includes plugin in computers to an ethernet connection and turning off WiFi, especially at night, reducing smartphone use and checking your building for other sources including low frequency EMFs from transformers, cables and sockets and strong magnetic fields coming from electro domestic appliances and faulty home wiring.

• Reduce antigen exposure: where possible, we should reduce exposure to things which directly stimulate calcium influx including viruses such as covid.
  
• Avoid MSG and other excitotoxins: many Asian restaurants use MSG to flavour their food, and MSG may also be high in packaged foods such as stock cubes, so always read the labels and check if your favourite restaurants are MSG free.
  
• Increase NRF2 signalling: NRF2 is a pathway activated by oxidative stress that switches on our natural antioxidant and antiinflammatory systems, helps with xenobiotic detoxification, aids lipid and glucose metabolism and stimulates DNA and tissue repair. An animal study found a reduction in calpain overactivation by stimulating NRF2 with a drug [R], so as well as the benefits listed above, NRF2 might also help with excessive calpains. Natural activators include exercise, sulforaphane found in broccoli sprouts, olive oil and olives, moringa, curcumin, quercetin and NAC.
   
• Follow a good circadian rhythm and sleep in the dark: melatonin has been found to inhibit calpain [R] and increase calpastatin [R], but artificial light at night has been shown to significantly reduce its release. Melatonin production is also stimulated by sunshine during the day
  

Other things that may inhibit excessive calpains: Taurine [R]. Rosemary (in acrylamide toxicity) [R], melatonin [R], moringa [R], turmeric [R][R][R], olive [R], pomegranate seed oil (CLA) [R], lithium (GSK-3b inhibition) [R][R][R], Astaxanthin [R], Grape seed polyphenols [R]

With thanks to Dr Stephanie Seneff for her input regarding glyphosate