The role of acid-sensing ion channels (ASICs) in exercise-induced muscle pain

Tahsin Khataei

doi:10.25820/etd.007099

Pain is a universal experience. While acute pain can be protective against harm and injury, pain can also become persistent and chronic, causing tremendous suffering and financial burden to society. Despite the burden of chronic pain, efforts at treatment have fallen short. Opioids have generally been prescribed as the first-line therapy, but unfortunately, they frequently worsen chronic pain. Moreover, prescription opioids have been a major contributing factor to the growing opioid-addiction crisis. Additionally, the development of new non-opioid drug therapies has been hindered by a general failure to successfully translate basic science research into successful clinical trials. There is a clear need to identify alternative and complementary therapies to address the lack of successful pharmacological approaches to treat chronic pain. One such potential therapy for the treatment of chronic pain is exercise. Prescribed regular exercise is increasingly acknowledged as an effective strategy to treat a variety of chronic pain conditions, including chronic fatigue syndrome (CFS), fibromyalgia (FM), osteoarthritis, rheumatoid arthritis, and neuropathic pain, as well as a means to prevent the formation of chronic pain. Even in healthy humans, regular exercise can diminish pain perception and increase pain tolerance. Consistent with this idea, it has been observed that highly-trained athletes have a higher pain threshold than unconditioned people. On the other hand, exercise-induced pain can be a barrier to exercise. It is well understood that exercise, particularly those activities involving higher intensity, isometric, and eccentric contractions, can lead to sensations of fatigue and pain – often referred to as exercise-induced pain (EIP). These aversive sensations can prevent people from adhering to regular exercise programs, and act as barriers to achieving higher performance in competitive athletes. Athletes have long understood the benefit of training “through” EIP – hence, the concept of “no pain – no gain”. So why is an acute intensive exercise often painful, and yet persistent exercise training can diminish chronic pain? To understand this seeming paradox, we turned to our understanding of muscle pain associated with exercise. Skeletal muscle has the capacity for high metabolic activity and is susceptible to rapid drops in pH during ischemia, hypoxia, and/or intensive exercise. Consequently, lactic acid is generated, and along with ATP hydrolysis, reduces intracellular pH, which in turn leads to acidification of the muscle interstitium. With intense exercise, the extracellular pH in human skeletal muscle can drop to between 6.7 - 7.0. Skeletal muscle is richly innervated by sensory nerves (muscle afferents) that sense these pH changes, as well as other metabolites generated during exercise. Within these muscle afferents, increasing evidence suggests that ASICs and transient receptor potential cation channel subfamily V member 1 (TRPV1) are important sensors of these metabolic changes. ASICs are H+-gated channels of the DEG/ENaC family, expressed principally in the central nervous system and peripheral sensory neurons. Four genes encode at least six subunits (ASIC1a, -1b, -2a, -2b, -3, and -4; ASIC1 and -2 have alternate splice transcripts). Functional ASIC channels consist of a complex of three subunits; individual subunits form homotrimers, whereas two or more subunits can assemble to generate heterotrimers. In general, they seem to be highly expressed in organs that have high metabolic activity, including the brain, and sensory nerves that innervate the heart and skeletal muscle. Several observations highlight the potential importance of ASICs in skeletal muscle afferents during intense exercise. First, ASICs are highly expressed in muscle afferents (formed by ASIC1, -2, and -3 subunits), and are activated in the range of extracellular pH that occurs in the interstitium of exercising muscle. Second, besides being activated by protons, ASICs are potentiated by other chemicals released during muscle ischemia, hypoxia, and/or exercise including ATP, lactate, arachidonic acid, and nitric oxide. Third, ASICs are required for normal exercise-mediated reflexes. Activation of muscle afferents during exercise evokes reflex increases blood pressure, heart rate, and ventilation (termed the ‘exercise pressor reflex’). ASIC antagonists have been shown to attenuate the metabolic component, but not the mechanical component, of the exercise pressor reflex. Lastly, ASICs are required for the development of normal muscle pain. Inflammation-inducing insults, direct acid injection into the muscle, and electrically-induced muscle contraction all cause an increase in pain in mice that is dependent upon ASIC channels. Either genetic deletion of specific ASIC subunits, knockdown of ASICs by RNAi in muscle, or pharmacological inhibition of ASICs attenuates hyperalgesia in these mouse models of muscle pain. Other chemicals released during exercise and other stress conditions (including nerve growth factor (NGF), serotonin, interleukin-1β, and bradykinin) alter ASIC transcription. For example, ASIC3 protein is increased in rat muscle afferents after hindlimb muscle ischemia, which is dependent upon the upregulation of NGF within sensory neurons (Lu, Xing, and Li 2012). Similarly, ASIC2a and -3 mRNA increased 10-fold in dorsal root ganglion (DRG) in a mouse model of muscle inflammation. In humans, exercise is associated with altered mRNA levels of specific ASIC subunits and TRPV1 in blood cells. Together, these data suggest that muscle inflammation and ischemia, and possibly exercise, can selectively regulate the expression of sensory receptors. Given that ASICs are important sensors of muscle pain and fatigue, and contribute to the activation of exercise-mediated reflexes, it is becoming increasingly clear that ASICs might play an important role in exercise. Moreover, ASICs expression is regulated by many of the chemicals that are released during exercise. Therefore, we hypothesize that ASICs (and perhaps other sensory receptors) might play a role in immediate exercise-induced pain (IEIP) and delayed onset muscle soreness (DOMS). To test this, we used a genetic ASIC knockout model of mice (ASIC3 -/-) as well as pharmacological inhibition of ASICs in sensory neurons. Our studies found that exercise training reduced IEIP, and diminished ASICs mRNA levels in muscle afferents, and this downregulation of ASICs correlated with improved exercise capacity. While WT and ASIC3 -/- had the same exercise performance, they had different pain perceptions immediately after, but not a day after exercise. Unlike WT mice, ASIC3 -/- did not develop IEIP. At 24h after exercise, ASIC3 -/- developed similar DOMS features (hyperalgesia and lower strength) as WT mice. Interestingly, ASIC3 -/- mice had diminished locomotor movement and repeat exercise performance, and higher muscle injury (creatine kinase (CK) and lactate dehydrogenase (LDH)) at 24h after exercise. These results show ASICs are required for IEIP, but not DOMS, and they might play a protective role from muscle injury during metabolic perturbations like strenuous exercise. Our experiments discovered novel mechanisms underlying IEIP and DOMS, and define potential pharmacological targets and non-pharmacological (e.g., exercise) pain management strategies to treat certain diseases like chronic fatigue syndrome (CFS) and fibromyalgia (FM). To perform our study, we invented a new technique to run mice on a treadmill without using electrical shock, and we introduced a method to assess immediate exercise-induced muscle pain in mice. This preliminary study found that exercising mice without shock generated similar maximal exercise performance, however, these mice showed an increase in locomotor activity, less anxiety-like behavior, and altered muscle pain compared to mice that exercised with shock.

The role of acid-sensing ion channels (ASICs) in exercise-induced muscle pain

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