Researchers from Maikop State Technological University, Republic of Adygeya-Russia and Adyghe State University-Russia in a new study have discovered that SARS-CoV-2 infections can cause disruptions in the signaling of the acetylcholine system (AChS) in the human host, causing cholinergic deficiency that can lead to various serious symptoms and health conditions arising especially cardiovascular issues.
The study findings were published in the peer reviewed Chinese Journal of Physiology.
https://www.cjphysiology.org/article.asp?issn=0304-4920;year=2023;volume=66;issue=1;spage=1;epage=13;aulast=Lysenkov
Suppression of the SARS-CoV-2 Acetylcholine System
It is known that ACh and its nicotinic receptors (nAChR) are one of the key components of the CNS, and the cholinergic pathway plays an important role in modulating the inflammatory response.[74],[75] It has been established that stimulation of the homopentameric α7 nAChR present on the surface of tissue macrophages blocks the expression of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6.[76],[77] Initiation of vagus nerve activity and its anti-inflammatory mechanism before viremia with a nicotinic receptor activator (nicotine) may have a modulating effect on macrophage activity.
However, dysfunction of the cholinergic anti-inflammatory mechanism can lead to a cytokine storm and failure of the immune response to return to homeostasis.[78] This fact is confirmed in the work of Alexandris et al., who conducted molecular modeling of the interaction of SARS-CoV-2 with nAChRs, as well as using a series of complexes with cholinergic agonists.[79]
Based on these results, the authors suggested that one of the causes for the disruption of the AChS is that the SARS-CoV-2 glycoprotein spike, which carries a “toxin-like” sequence in its receptor-binding domain, can bind using the α-subunits of nicotinic receptors (nAChRs), in particular α7 nAChR, by inhibiting AChS activity.
Perhaps this is due precisely to the competition for nAChRs and their moderate stimulation. The interaction of nicotine entering the body through smoking blocks these receptors and prevents SARS-CoV-2 from binding to them, acting, in its own way, as a protective mechanism, but this fact requires further study.[81],[82]
Change in Acetylcholine System Receptor Activity in COVID-19
According to recent studies, the number of ACh receptors is maintained both by the insertion of newly synthesized AChRs and by their reuse through the so-called AChR recycling.[84],[85]
Thus, the rate and balance between synthesis and destruction of receptors play an important role in maintaining the normal functioning of the synapse. However, this process can be disturbed during the development of pathological diseases and cause negative consequences. For example, in “myasthenia gravis” it was found that the formed antibodies to nAChRs on the surface of muscle cells cause the internalization and degradation of nAChR. Internalized and degraded receptors are not replaced by increased synthesis of new nAChRs, so an overall decrease in nAChRs occurs at neuromuscular junctions, with subsequent loss of synaptic efficiency.[86],[87]
On the other hand, Bruneau and Akaaboune showed that the rate of removal of reducible receptors increases with muscle denervation and pharmacological nerve blockade in a rat neuromuscular preparation, and the rate of formation of new receptors decreases.[88] It was shown as a result experiment on a rat neuromuscular preparation that the half-life of recycled receptors was 28 h in innervated synapses, and the half-life of pre-existing receptors was 102 h.
However, the mechanism of synaptic transmission disorders, that occurs with COVID-19, is likely to differ from those described above.[84],[85],[86],[87],[88] We believe that one of the causes of the disorders may be associated with the hyperproduction of nitric oxide and the formation of toxic compounds in synapses, such as peroxynitrite.
In our opinion, the toxic effect of peroxynitrite during a cytokine storm can be one of the main causes for the development of failures in signal transmission due to the fact that it can oxidize receptors and other synapse structures, as well as disrupt the formation of new ones. As a result, we can get a similar effect observed with surgical or pharmacological nerve denervation.
Hyperproduction of Nitric Oxide as a Factor Contributing to the Disruption of the Normal Functioning of the Synapse in COVID-19
Reactions that disrupt normal physiological processes are possible with a viral infection. Currently, the role of nitric oxide in the formation of the inflammatory response in COVID infection is shown here. On this issue, we have already published an article,[89] in which we tried to substantiate this thesis.
Acting as an element of antiviral protection, nitric oxide translates the inflammatory response into the pathological nature of inflammation. An increase in NO synthesis during viremia and excessive release of pro-inflammatory cytokines (IL-1, IL-2, IL-6; TNF-γ, etc.) disrupts NO utilization processes. Calcium channels on the presynaptic membrane, which promote the release of ACh from vesicles into the synaptic cleft, are simultaneously activated.
It is known that NO inhibits the synthesis and action of AChE, which manifests itself in a longer activation of ACh receptors, opening of sodium and calcium channels and slowing down their inactivation. It was determined by analogy with this process that changes in the amplitude-time parameters, distinguishing for AChE inhibition, are observed on the activation of NMDARs by glutamate and glycine. Conductivity in the synapse is disturbed; muscle activity is inhibited.[67],[90],[91]
Therefore, it can be assumed that similar mechanisms of AChE regulation may also lie in the central synapses. Evidence for this was obtained in the works of Udayabanu et al., Hua et al. using the nitric oxide donor Spermine NONOate.[92],[93] Hyperproduction of nitric oxide is accompanied by the formation of a very aggressive oxidizing agent – peroxynitrite (and a number of aggressive nitro compounds).
Peroxynitrite reacts with lipids of presynaptic and postsynaptic membranes, disrupts the processes of endo- and exocytosis of ACh. In addition, oxidation and denaturation of protein-lipid complexes of ACh, Na+, Ca2+ channels occur.[89],[94] All these compounds begin to acquire the properties of antigens. An autoimmune reaction is formed with the production of appropriate antibodies that disrupt the functioning of ACh synapses (and possibly other synapses).
Indeed, antibodies to AChR have been detected in a number of studies in the post-COVID period. In this regard, in the second phase of the infectious process, ACh receptors can be partially blocked by self-antibodies after a cytokine storm. There is one of the variants of myasthenia gravis.[95],[96] Its symptoms can appear for a long time, and their severity depends on the concentration of antibodies to AChR. The produced antibodies activate the complement system, which is accompanied by damage to the postsynaptic membrane.[67],[75],[76],[77],[79],[83],[84],[85]
In addition, antibodies to muscle-specific tyrosine kinase (MuSK) can be produced, but they form a reaction without the participation of the complement system but block the interaction of MuSK with a protein similar to the low-density lipoprotein receptor 4. This leads to a reduction in the AChR clustering process.[97]
We have already drawn attention to the fact that endothelial NO synthase is contained in caveolae,[50],[51] which is activated by pro-inflammatory cytokines with the formation of excess amounts of nitric oxide and peroxynitrite.[89] Activation of the process of lipid peroxidation plays a role in maintaining viral inflammation.
A decrease in the concentration of cholesterol as a result of the action of peroxynitrite leads to a flattening of the structure of caveolae or their disappearance. It should be assumed that under the influence of an active inflammatory process, the processes with the activation of AP generation on the postsynaptic membrane and the operation of the K+/Ca2+ pump are disrupted.
Thus, partial removal of cholesterol in an experiment on a frog neuromuscular preparation leads to disruption of the process of endocytosis, recycling of synaptic vesicles with ACh, and depletion of the vesicle population during synaptic activity. Cholesterol depletion enhances the production of ROS, causing the activation of lipid peroxidation in the synaptic region.[98],[99]
The resulting oxysterols are able to interact with oxysterol-binding proteins and receptors.[99],[100] In addition, oxysterol destroys lipid rafts in the synaptic zone and inhibits the release of the ACh mediator in the neuromuscular preparation during low- and high-frequency stimulation.[98]
Moreover, it was shown that cholesterol oxidation products 5L-cholestan-3-OH, without affecting the spontaneous release of ACh, inhibited ACh secretion during low- and high-frequency stimulation and disrupted short-term synaptic plasticity.[101],[102] In turn, the activity of 5L-cholestan-3-OH depended on the content of membrane cholesterol.
In an experiment on mice by Grajales-Reyes et al., it was found that exposure to statins leads to a decrease in cholesterol synthesis and disruption of the AChR, which leads to the development of myasthenia gravis.[103] These processes are aggravated by tissue hypoxia. In view of the above, we believe that peroxidation is one of the mechanisms for the development of post-COVID myasthenia gravis, but not the only one.
Similar changes occur in the synapses of the CNS, but clinically, this manifests itself in impaired functioning of the AChS of the brain: memory disorders and sensory aphasia. Symptoms such as “fog in the head,” reduced attention, impaired short-term memory, impaired orientation in space, impaired memory for names, and names of objects are characteristic of CNS synapses. In the post-COVID period, partial blockade of AChR significantly weakens the anti-inflammatory function of the parasympathetic system, and the main representative of this system – n. vagus.
Deterioration of Calcium Transport in the Synapse in COVID-19
It must be assumed that a huge number of modulating influences and especially biologically active compounds, that act as neurotransmitters and neuromodulators (norepinephrine, somatostatin, calcitonin, and serotonin), have an effect on the acetylcholine system. One of the important participants in the mechanisms of synaptic transmission is calcium ions. For example, the role of calcium ions in the progression of a viral infection is confirmed by the use of calcium channel blockers (amlodipine) as a therapeutic agent that improves the course of the process in COVID-19.[104],[105],[106]
Moreover, it turned out that the process of cooperation of the S-protein of the virus with the host ACE2 receptors occurs with the obligatory participation of the activation (or formation) of the calcium channel. Coronavirus produces a special protein that can assemble as Ca2+ conductor viroporins, which facilitates the entry of the virus into the cell. The accumulation of calcium ions in the cell leads to a deviation of the concentration balance and, as a result, can cause disturbances in the work of mitochondria.
According to the results presented by Ramadan et al., mitochondrial metabolism and cytosolic calcium have a dynamic relationship.[106] Increasing Ca2+ inside the cell stimulates its absorption by mitochondria, in this case, they act as a kind of buffer system of the cell; however, calcium overload in mitochondria can lead to activation of cell death pathways. Active absorption of calcium ions by mitochondria through the pores in the membrane causes osmotic changes that lead to the swelling of mitochondria and an increase in the production of ROS.[107],[108],[109]
If we turn to the neuromuscular synapse, then the accumulation of Ca2+ in mitochondria and ATPase deficiency in the sarcoplasmic reticulum form the syndrome of mitochondrial myopathy. On the other hand, as we noted above, a change in synaptic transmission due to CNS viral invasion can also lead to disruption of muscle work throughout the body. In severe cases, the process of necrosis can cause an increase in the level of K+, destruction of the sarcolemma, and myoglobin, followed by heart rhythm disorder and the formation of multiple organ failure.
It is known that for adequate muscle contraction it is necessary that the calcium concentration between myofibrils increases 500 times (up to 2 × 10-4 mmol/l). After actin-myosin interaction, calcium is “pumped out” into the sarcoplasmic reticulum. It should be noted that the process of “pumping out” of Ca2+ into the sarcoplasmic reticulum is energy-consuming and is carried out with the participation of ATPase. If there is a deficiency in the synthesis of ATPase, then this process slows down. In advanced cases, this can cause prolonged contraction (spasm) of the muscles.[110],[111],[112],[113]
The appearance of a whole group of trigger activators of Ca2+ channels in COVID infection is accompanied by a sharp increase in Ca2+ in the sarcoplasm and active hydrolysis of ATPase. With the consumption of ATPase, there is a slowdown in the work of Ca-ATPase, which causes a slowdown in the relaxation of skeletal muscles and, at the same time, the start of anaerobic respiration in the muscles.[112],[113]
This may be accompanied by an increase in nonshivering (and partially contractile) thermogenesis, an increase in oxygen debt and an accumulation of lactic acid. It is possible that thermogenesis in muscles during COVID infection involving RyR1s is parasympathetically controlled through AChR,[114],[115],[116] as is the case in fish in the heating organ.[117]
Clinically, this is manifested by rapid muscle fatigue. Attempts to make voluntary movements are accompanied by the depletion of the recirculating ACh, and, subsequently, the reserve pool. In addition, the formation of vesicles is impaired in parallel with a decrease in the concentration of membrane cholesterol. The described situation is dangerous for sarcomeres, which can partially go into the stage of apoptosis and necrosis due to hypoxia and calcium overload.
Systemic endotheliitis in the “cytokine storm” causes disturbances in blood flow in muscle tissue, the development of circulatory and tissue hypoxia, metabolic acidosis and impaired resynthesis of ATPase. All these factors are components of neuromuscular transmission disorders.[118] The concentration of ions in the blood has a significant impact on the activity of the calcium channel. There is still no clarity on this issue, although hypocalcemia has been recorded in 60% of patients admitted to the clinic.[119],[120],[121]
A nonspecific increase in the level of procalcitonin in the blood during a viral infection may indirectly indicate an increase in synthesis not only in nonspecialized cells but also in specialized C-cells of the thyroid gland. The result of the action of calcitonin is a decrease in ionized calcium in the blood.[120],[121] Given the ability of Ca2+ to induce the penetration of the virus into the cell, hypocalcemia can be regarded as the host’s evolutionary defense against the virus.
This reaction turned out to be universal since the mechanism of virus penetration into the cell turned out to be common for such viruses as swine coronavirus, recovirus, hepatitis B virus, West Nile virus, herpes virus, influenza A, rotovirus, cytomegalovirus, and coronavirus COVID-19.[106] The concentration of calcium in most cells is 10−8–10−7 mol/L. As soon as the level of 10−6–10−5 mol/L is reached, calmodulin shows its effects.[118],[121],[122]
However, the correction of hypocalcemia is recognized as an unreasonable tactic in clinical practice today.[106] The issue of whether such a reaction is biologically appropriate for the host has not been resolved, although there are more arguments in favor of conditional host protection from the virus.
The issue of participation of calcium ions, calcium channels in the genesis of virus replication, modulation of the inflammatory and anti-inflammatory mechanism of the AChS, disruption of the mechanisms of synaptic transmission of information, both in the acute period of viral infection and in the long-term, deserves further study.
Acetylcholine System Disorders and Effects on the Cardiovascular System
Many post-COVID patients often complain about pathologies of the cardiovascular system: cardiac arrhythmias and orthostatic collapses. It is noted in many studies, that 1/3 or more of those with COVID have a lesion of the cardiovascular system. However, as the authors of the studies themselves note, damage to the cardiovascular system in most cases is not directly related to viral invasion, and the nature of the pathologies is not entirely clear.[70],[71],[123],[124]
We believe that after suffering from COVID-19 disease, disturbances in the autonomic nervous system can significantly affect the functions of the cardiovascular system, expressed in the inadequacy of orthostatic,[125] due to impaired sympathetic control of tone, vessels during the redistribution of blood volume under the influence of gravity.[126],[127] The innervation of resistive vessels is provided through the sympathetic ganglia, in which ACh acts as a mediator.
The loss of sympathetic tone is probably due to a deterioration of neuromuscular transmission in adrenergic synapses of vascular smooth muscles.[128],[129] The events that we described for the ACh synapse could also occur in this region. The basis for this statement is the data that one of the leading factors in the progression of the disease is inflammation of the endothelium (systemic endotheliitis) in COVID infection. Under these conditions, energy processes are disrupted in all underlying subendothelial areas, including vascular muscles and synapses.
On the other hand, despite the strong influence of the central sections of the CNS on the work of the heart through the parasympathetic and sympathetic sections, it is necessary to take into account the fact that there are intracardiac reflex arcs, which include Dogel cells (1, 2, 3 orders) with the mediator ACh. These reflexes normally provide a rapid restructuring of the heart, after which the mechanisms of systemic regulation are activated through the vasomotor and cardioinhibitory centers.[122]
Loss of control from the vagus nerve (its afferent and efferent links) may be accompanied by attacks of tachycardia and the appearance of ectopic foci in the myocardium. Similar reactions were noted in the elderly. It has been shown that with age the efficiency of cardiovagal baroreceptor regulation decreases due to modification of the receptors of the aortic arch, carotid sinus, cardiopulmonary, and other reflexogenic zones.[130],[131],[132]
Under physiological conditions, lowering blood pressure (BP) includes baroreflex regulation of cardiac activity and blood vessels.[131],[132] It consists of a narrowing of resistive vessels, tachycardia, and an increase in BP, as well as due to the release of adrenaline from the adrenal medulla in response to a decrease in BP. However, it can be assumed that reflex responses in some cases after exposure to the virus will be inadequate and delayed in some patients due to a malfunction of the ACh synapse that innervates this area.
The classic work of Nemecek showed that postganglionic sympathetic fibers innervating sweat glands, skin vessels, and striated muscles use ACh as a mediator.[133]
A more significant evidence of the failure of nervous regulation is orthostatic hypotension, accompanied by a decrease in venous return to the heart at the time of taking a vertical position, and in some cases, the inclusion of the so-called “reverse” Bainbridge reflex with the development of bradycardia and hypotension. The physiological expediency of this reflex is not entirely clear, and, most likely, this is a manifestation of pathology.[131],[133]
In rare cases, the manifestation of the Bezold-Jarisch reflex with the development of bradycardia, hypotension, and apnea is possible. The starting point of this reflex is the increased activity of the afferent link (atrial stretch receptors) of the vagus nerve with excessive contraction of the “empty” ventricles. The reflex is consonant with the “reverse” Bainbridge reflex. In all cases (except others), there is a disruption in the functioning of the AChS and its interaction with other regulatory systems.[130],[134],[135]
