The Potential Repurposing Effect 4‐Aminopyridine in Nerve and Muscle Injury‐induced Bone Loss

Muscle injuries are one of the most common injuries occurring in physical and sporting activities, and account for 10-55% of all injuries. Peripheral nerve injury (PNI) itself accounts for 3-5% of all injuries in trauma patients, and muscle injuries can be concurrent with PNI because of their close proximity. Importantly, the peripheral nervous system is critically involved in bone metabolism, bone mineralization, and bone remodeling, and PNI results in substantial bone loss. While first aid for muscle injuries follows the conservative RICE (Rest, Ice, Compression and Elevation) principle, there is no medical treatment available after the acute phase except anti-inflammatory medications and rehabilitation. The poor healing process in damaged muscle, prolonged muscle disuse, and the lack of therapeutic strategies affect the performance of daily activities and predispose to further risk of muscle injury and bone loss. Therefore, there is an unmet need to develop therapeutic strategies that can help the recovery of damaged muscle and nerve, and limit bone loss. Recently we demonstrated the potential repurposing effect of 4-aminopyridine (4-AP), a potassium channel blocker, on neurogenic muscle atrophy and functional recovery after PNI in mice. However, it is unknown whether these beneficial effects of 4-AP are present in traumatic muscle injury and muscle injury-induced bone loss. This study was thus designed to evaluate the potential therapeutic effect of 4-AP in traumatic muscle healing and PNI-induced bone loss. Standardized crush injury was performed on right sciatic nerve, biceps femoris (BF) muscle (innervated by sciatic nerve), and quadriceps femoris (QF) muscle (innervated by femoral nerve) of adult male mice (6 animals/group). Post-surgical animals were randomly assigned to normal saline and 4-AP. 4-AP (40 µg, intraperitoneal) was given daily for 21 days, and the functional recovery after injury was assessed by sciatic function index (SFI), sensation by von Frey test (VFT), and grip strength test. After 21 days, the muscles at the injury/peri-injury site were processed for histomorphometry and tibial bone density was analyzed using DEXA scanning. We found that 4-AP significantly enhanced SFI, VFT, and hind limb paw grip strength from post-injury day 7, improved BF muscle morphology, cross-sectional area, and minimum ferret diameter, and totally reversed trauma-induced muscle fiber type composition changes (type-1 vs. type-2). 4-AP-induced muscle effects were also associated with a significantly increased number of regenerating stem cells (Pax7+) and proliferating cells (Ki67+) compared with saline group. Of note, 4-AP-induced muscle effects were either less pronounced or nonsignificant in QF muscle. Most importantly, 4-AP treatment significantly reduced PNI-induced bone loss by enhancing bone mineral density and bone mineral content. In conclusion, these interesting findings, for the first time, demonstrate the potential therapeutic effect of 4-AP on the recovery of traumatic muscle and bone loss after mixed muscle and nerve injury.