Optogenetic diaphragm muscle activation in mice

Breathing impairments are the leading cause of morbidity and mortality across all forms of neuromuscular disease. When voluntarily diaphragm activation is insufficient, clinical options include mechanical ventilation or electrical stimulation via intramuscular wires. Here we tested the hypothesis that an optogenetic approach would enable light to serve as an adequate stimulus for inducing diaphragm contraction to sustain breathing during neuromuscular impairments. Wild type, C57/bl6 mice (n=10) received an intrapleural injection of an adeno‐associated virus (AAV) construct containing the transgene for the light activated ion channel channelrhodopsin‐2 (34 µl, AAV9‐ACAGW‐ChR2‐mVenus, titer: 1.8x1013 vg/ml). After allowing 8‐10 weeks for transgene expression, electromyography (EMG) and pneumotachography were measured during spontaneous breathing under isoflurane anesthesia. To test optogenetic activation, a high‐power, 472 nm, micro‐LED was directed at the inferior diaphragm surface. Stimulus pulse durations (PD) ranging from 0.1‐1.0 ms and interpulse intervals (IPI) of 0.1, 0.5, 1.0, and 5.0 ms were tested. Directing light at the diaphragm evoked clearly discernable EMG activity in 10 of 10 animals treated with the AAV9 vector. The 5 ms IPI induced the largest EMG activation, occurring at a level comparable to endogenous diaphragm bursts. Stimulation with a 0.1 ms PD / 0.1 ms IPI, and 1 ms PD / 5 ms IPI both were able to generate robust increases in respiratory airflow. Optogenetic stimulation was repeated following an acute unilateral phrenic nerve section which abolished ipsilateral diaphragm EMG. The contralateral diaphragm inspiratory EMG signal was then used to “trigger” the light stimulation of the paralyzed hemi‐diaphragm. This procedure enabled a robust, bilateral diaphragm activation in all mice that received the unilateral phrenicotomy (n= 5). Histology confirmed robust expression of mVenus in diaphragm myofibers in mice treated with the vector. Directing light at the diaphragm of non‐AAV treated control mice caused no discernible impact on diaphragm EMG activity (n=2). We conclude that intrapleural AAV9‐ACAGW‐ChR2‐mVenus injection effectively drives transgene expression in the diaphragm, and this approach can be used to enable robust optogenetic activation of this critically important respiratory muscle. To our knowledge, this is the first demonstration of light activation of the diaphragm, and this success opens up new directions for therapeutic targeting of diaphragm paralysis and respiratory insufficiency.