Abstract / Introduction
Ryanodine receptors are ion channels that conduct calcium ions (Ca2+), a second messenger that transduce multiple cellular signalling pathways. They are located on the sarcoplasmic and endoplasmic reticulum membrane of the muscle and brain cells to release Ca2+ from the intracellular storage to the cytoplasm. Hypoxic-ischemic brain injury is caused by an inadequate level of blood and oxygen to the brain. An insufficient level of oxygen causes the influx of Ca2+ to the intracellular compartment. Previous studies have shown that an excess intracellular level of Ca2+ activates the calpain and caspase-3 pathways which eventually leads the cell to apoptosis. Ryanodine receptors, the intracellular Ca2+ "guard" can mediate neuronal cell death after hypoxic-ischemic brain injury. Therefore, dantrolene, a drug that prevents ryanodine receptors from releasing Ca2+, can potentially be a clinical treatment of the hypoxic-ischemic brain injury by inhibiting ryanodine receptor channels.
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Ryanodine Receptors: Structure and Functions
The ryanodine receptors (RyRs) are essential calcium (Ca2+) releasing channels on the sarcoplasmic and endoplasmic reticulum (SR & ER) membrane1 (Figure 1)12. The SR and ER are the main Ca2+ storage in myocytes and neurons2. RyR channels allow rapid Ca2+ release to the intracellular compartment which is an initial and essential step in many signalling pathways1, 2, 3. One interesting fact about RyRs is that they are the largest known ion channels in the mammalian system1. This feature makes them capable of conducting a large amount Ca2+ to the cytoplasm in a short time1.
Figure 1: ER and SR morphology and the arrangement of the common membrane receptors and pumps12, including RyR channels. Black arrows indicate the direction of Ca2+ flow.
RyR channels are found in three isoforms in the mammalian system: RyR1, RyR2, and RyR31, 2, 4. The RyR1 isoform is abundant in skeletal muscle; RyR2s are predominately found in cardiac muscle; RyR3, the least studied isoform, presents in the brain and other non-muscle cells and tissues at a low level of expression1, 2, 4.
Even though the size of RyR channels is massive, which makes them challenging to study, some studies elucidate many important structural features about the RyR channels and their multiple modulators. In general, all three RyR isoforms exist in the same structure1. They are homotetrameric channels with a molecular mass of more than 2.2 megadaltons1, 2. Electron cryomicroscopy (cryo-EM) and single-particle analysis have been used to study the RyR1 channel1, 2, 3, 4, 5. Under an average resolution of 6.1Å, RyR1 appears to have a huge cytoplasmic domain which takes up one-fifth of the overall structure1, 2, 4 (Figure 2)2. The transmembrane regions locate close to the carboxyl terminal and form the pore region of the channel1, 2. The cytoplasmic domain is divided into 15 subdomains and it is an important region that has multiple protein interaction motifs that are critical in regulating the channel activity and modulator interactions1, 3. One part of the cytoplasmic domain is called the "clamps"1 (Figure 2)2. The clamps are responsible for the closing and opening of the channel by undergoing conformational change during activation1, 2. The clamps also participate in interacting with neighbouring RyRs to exhibit their function properties1.
Figure 2: The Cryo-EM density map of RyR1 at an average resolution of 6.1Å (coloured by domains).2 This is a good illusion of the RyR1 structure, including its clamp domain.
In terms of the functional properties, RyR channels control the release of Ca2+ into the cytoplasm form the ER and SR1, 2, 3. This process is regulated by several factors, including ions, small molecules and multiple proteins2. One of the main contributors is the modulator complex on the cytoplasmic domain of the RyR channels1, 3. Many modulators interact with RyRs, such as voltage-dependent calcium channels (Cav1.1/Cav1.2) (Figure 3)1, protein kinase A (PKA), Calmodulin (CaM), and Ca2+/calmodulin-dependent protein kinase type II (CaMKII)1, 4, 5. Ca2+, magnesium ion (Mg2+) and adenosine triphosphate (ATP) are ion regulators that can also activate or inhibit RyR channel activity1, 3, 4. RyR channels are activated by low intracellular Ca2+ concentration (about 1 µM) and are inhibited by high intracellular Ca2+ concentration (about 1 mM)1. Mg2+ is known to be an inhibitor of RyR channels by reducing channel open probability and competing with Ca2+ at the activation binding site1, 3, 4. However, APT is a cytosolic agonist of RyR channels1. ATP allows even more release of Ca2+ from the ER and SR which is fatal for neurons after hypoxic-ischemic brain injury via the Ca2+ dependent programmed cell death1, 3, 4.
Figure 3: The interaction between RyR channels and various modulators.1 Black arrows indicate the direction of Ca2+ flow.
Interactions between Hypoxic-Ischemic Brain Injury and Ryanodine Receptors
Hypoxic-ischemic (HI) brain injury is due to a lack of blood flow and oxygen to the brain6. In neonatal HI brain injury, it is a severe health problem that causes developmental delay, cognitive and motor impairments, learning disabilities, and in the worst situation, neonatal death6,7.
During hypoxic-ischemia, blood flow to the brain region is stoped6. The anaerobic glycolysis quickly kicks in, which leads to a lack of energy supply and ATP production in the neurons7. Without sufficient ATP, glutamate, an excitatory neurotransmitter, that is released from firing neurons cannot be re-uptake7, 8. The high level of extracellular glutamate further activates postsynaptic neurons which spreads the effect of HI to the neighbouring neurons7, 8. Following HI, extensive Ca2+ enters the neuron and more Ca2+ is released from the ER and SR8, 9. Under a normal physiological condition, the intracellular Ca2+ concentration is about 100 nM9. There is a 10,000-fold difference in the concentration of Ca2+ between inside and outside of the cell9. Therefore, it is understandable that a small increase in intracellular Ca2+ concentration can trigger a whole series of cellular events. It is well known that an accumulation of intracellular Ca2+ directs many cellular signalling pathways including autophagy and apoptosis of the neurons5, 9, 10. Therefore, Ca2+ plays a determinative role in neuron survival after HI brain injury. Furthermore, when free intracellular Ca2+ binds to CaM, they together activate nitric oxide synthase and form nitric oxide in the cell8, 11. Free oxygen radicals can react with nitric oxide and form a highly toxic molecule that damages DNA and proteins8. The DNA repair system requires ATP for functioning8. Decreased ATP levels due to HI cause the formation of oxygen radicals in mitochondria and further increase cellular damage8.
The role of ER stress under HI condition is also highly important. ER stress is a complicated mechanism that determines the fate of the neuron responding to brain ischemia9.
The ER is not only the main Ca2+ storage but it is also important for protein synthesis that is extremely sensitive to stress9, 12, such as a drop in oxygen level due to ischemia. Studies have shown that when ER undergoes a stress state, it releases Ca2+ and accumulates defective proteins in the ER lumen9. Both the Ca2+ from influx due to spontaneously firing neurons and from the ER together contribute to an extremely high intracellular Ca2+ concentration. High levels of intracellular Ca2+ further activate RyR channels on the ER membrane, resulting in a depletion of Ca2+ in the ER and an even more exceeding increase of intracellular Ca2+ concentration. This process is termed as Ca2+-induced Ca2+ release (CICR)9, 11, 13. CICR can also be triggered by other voltage-gated calcium channels inducing Ca2+ influx into the cytoplasm.
Looking closely at the RyR's role in releasing Ca2+ from the ER, studies have shown that RyR channels are the main Ca2+ releasing channels on the ER membrane that are responsible for a high intracellular Ca2+ level after HI brain injury9, 11. The massive influx of Ca2+ into the cytoplasm may activate the calpain and caspase-3 pathway8, 9, 14, which eventually leads to neuronal dysfunction and neurodegeneration, a progressive loss of neurons due to cell death8, 10. Therefore, RyR channels are considered to be drug targets in treating HI brain injury.
Ryanodine Receptor Antagonists: Dantrolene and Azumolene
Dantrolene is a RyR channel antagonist and a muscle relaxant that has been clinically used as the primary drug to treat malignant hyperthermia (MH), a pharmacogenetic disorder of skeletal muscle3, 15, 16, 17. Under MH condition, an extensive amount of Ca2+ is released from the SR of the myocytes which causes severe muscle contraction in skeletal muscle15, 16. A study15 has shown that clinical use of dantrolene in treating MH has significantly reduced the mortality rate of patients from 80% to 5% today. Although there is no clinical use of dantrolene to treat HI brain injury, dantrolene, as a RyR channel antagonist, is considered to be a potential treatment and RyR3 in the brain can be its drug target18. Azumolene, although little it has been studied in detail, is an alternative antagonist that can potentially be used as well because of their similar chemical structure as compared in Figure 415.
Figure 4: A: The chemical structure of dantrolene B: The chemical structure of azumolene.15
Dantrolene is a chemical (structure shown in Figure 4) that is metabolized in the liver by oxidative and reductive pathways15. It has a beneficial effect on intracellular Ca2+ homeostasis and works through inhibiting abnormal CaM-dependent RyR activation and reducing RyR channel sensitivity. Thereby, reducing the release of Ca2+ to the cytoplasm from the ER and SR15,16.
Dantrolene also exerts neuroprotective properties in the brain after ischemia damage15, 18, 19, 20. Previous studies have shown that early dantrolene treatment after HI brain injury in gerbils can protect neurons from glutamate cytotoxicity and delayed neural death by reducing the activation of the ER stress-mediated apoptotic pathway18, 20. While dantrolene inhibits RyRs' function in skeletal muscle, there is no negative inotropic effect on the beating heart16. This finding suggests that dantrolene has selectivity on the isoforms of RyR channels: it works on RyR1 but not on RyR216. In addition, dantrolene can also exert its function in some non-muscle cells, such as neurons in the brain but the exact mechanism remains unclear16. Even though all three isoforms of the RyR channels have a similar structure1, why dantrolene is unaffected on RyR2 remains in question16.
Regardless, it is clear that dantrolene has a therapeutic effect on neural dysfunction after HI brain injury16. Therefore, in order to use dantrolene as a treatment for HI brain injury patients, it is valuable to understand the exact molecular mechanism of the effects of dantrolene on intracellular Ca2+ release via RyR3 channels. With RyR1 and RyR3 being the drug target, dantrolene is not only a lifesaving drug for MH3, 15, 16, 17, but it can also be a potential treatment for many other neurodegenerative diseases that are caused by an uncontrolled intracellular Ca2+ release from the ER, including HI brain injury16, 18.
Conclusion and Future Research Directions
RyR channels are major intracellular Ca2+ releasing channels on the ER and SR membranes. An over activation of the RyR channels is the cause of many skeletal and cardiac muscle diseases. Many RyR modulators regulate the activity of the channel and play a critical role in the physiopathology of RyR related diseases, including HI brain injury. HI brain injury is due to inadequate blood flow and oxygen to the brain, which causes a series of cellular event that finally leads neurons to apoptosis. The release of Ca2+ from the internal storage through RyR channels is the determinative step that decides the fate of the neuron. ER stress under HI condition also contributes to the uncontrolled release of Ca2+ into the cytoplasm through the CICR process. The calpain and caspase-3 pathways are then activated responding to the high intracellular Ca2+ concentration, which results in a progressive loss of neurons in the brain. The good news is that dantrolene, a RyR channel antagonist, has been described in detail and is used to treat RyR1 related skeletal muscle diseases.
While most of the studies focus on RyR1 and RyR2 isoforms, studies on RyR3 are very limited. Future studies may focus on the effect of dantrolene on RyR3 channels after HI brain injury since this type of study is still lacking. With RyR3 being the drug target, dantrolene has high hope of providing a new treatment in HI brain injury patients. Besides, azumolene may also be investigated more in detail of its similarities and differences with dantrolene. It is also one of the candidates that can be clinically used as a new treatment for HI brain injury patients.
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