Selective targeting of ASIC3 using artificial miRNAs inhibits primary and secondary hyperalgesia after muscle inflammation

Abstract

Acid-sensing ion channels (ASICs) are activated by acidic pH and may play a significant role in the development of hyperalgesia. Earlier studies show ASIC3 is important for induction of hyperalgesia after muscle insult using ASIC3−/− mice. ASIC3−/− mice lack ASIC3 throughout the body, and the distribution and composition of ASICs could be different from wild-type mice. We therefore tested whether knockdown of ASIC3 in primary afferents innervating muscle of adult wild-type mice prevented development of hyper-algesia to muscle inflammation. We cloned and characterized artificial miRNAs (miR-ASIC3) directed against mouse ASIC3 (mASIC3) to downregulate ASIC3 expression in vitro and in vivo. In CHO-K1 cells transfected with mASIC3 cDNA in culture, the miR-ASIC3 constructs inhibited protein expression of mASIC3 and acidic pH-evoked currents and had no effect on protein expression or acidic pH-evoked currents of ASIC1a. When miR-ASIC3 was used in vivo, delivered into the muscle of mice using a herpes simplex viral vector, both muscle and paw mechanical hyperalgesia were reduced after carrageenan-induced muscle inflammation. ASIC3 mRNA in DRG and protein levels in muscle were decreased in vivo by miRASIC3. In CHO-K1 cells co-transfected with ASIC1a and ASIC3, miR-ASIC3 reduced the amplitude of acidic pH-evoked currents, suggesting an overall inhibition in the surface expression of heteromeric ASIC3-containing channels. Our results show, for the first time, that reducing ASIC3 in vivo in primary afferent fibers innervating muscle prevents the development of inflammatory hyperalgesia in wild-type mice, and thus, may have applications in the treatment of musculoskeletal pain in humans.

ENaCs and ASICs as therapeutic targets

“…ASIC3 knockout mice (ASIC3 Ϫ/Ϫ ) did not develop hyperalgesia of the paw consequent on carrageenan-induced inflammation of the gastrocnemius muscle, although the response to heat was maintained (354,355), most likely due to the role of TRPV channels in mediating this pain modality. Similar results have been obtained in a localized in vivo knockout mouse model (402). Furthermore, ASIC3 Ϫ/Ϫ mice did not exhibit the hyperalgesia in surrounding tissues associated with joint inflammation (secondary hyperalgesia) (191).…”

Expression of acid-sensing ion channels in nucleus pulposus cells of the human intervertebral disk is regulated by non-steroid anti-inflammatory drugs

“…Recently, Cuesta et al [17] showed the presence and localization of different ASICs in the human healthy intervertebral disk, and an increased expression of ASICs in the degenerated intervertebral disk, indicating that ASICs may be involved in intervertebral disk degeneration. In this study, we investigated two ASIC subunits (ASIC1 and ASIC3) in nucleus pulposus cells of the human intervertebral disk, which were reported to be relevant for the function of connective tissues including bone and cartilage [18][19][20]. Uchiyama et al [21] confirmed the presence of ASIC3 subunit in nucleus cells of the intervertebral disk.…”

The dichotomized role for acid sensing ion channels in musculoskeletal pain and inflammation

“…In direct contrast, ASIC1−/− mice still develop decreases in withdrawal thresholds of the paw but do not develop decreases in withdrawal thresholds of the muscle or knee induced by muscle or joint inflammation, respectively [8,31]. In adult mice, knockdown of ASIC3 in DRG innervating muscle with a miRNA prevents the development of both paw and muscle hyperalgesia in mice with muscle inflammation [32]. After development of muscle inflammation, blockade of ASICs with the non-selective blockers or a selective ASIC3 antagonist reverses both the muscle and paw hyperalgesia [28,31,33].…”