Electrical Coupling of Circulating Immune Cells to Peripheral Tissues

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Abstract: Peripheral injury responses require sophisticated interactions of target tissues, immune cells and primary sensory neurons. Crosstalk between these systems is essential for post-injury muscle repair and nociception. While a great deal is known about the role of the immune system in functional restoration of muscles and in pain development, it is not known if physical coupling of circulating immune cells to myofibers or neurons after injury directly modulates both of these unique biological processes. Information on novel interactions between muscles, the peripheral nervous system and immune cells could significantly advance understanding of myalgia and muscle repair. The goal of this study is to determine if infiltrating immune cells electrically couple to myofibers or neurons after injury to dually modulate functional muscle repair and nociception. Recent reports suggest that, after injury, connexin 43 (Cx43) gap junctions may form between macrophages and myofibers to modulate repair. It has also been shown that similar gap junctions form between adjacent neurons within the dorsal root ganglion (DRG) to regulate nociception. In the heart, electrical coupling between resident macrophages and cardiomyocytes is crucial for proper atrioventricular conduction. It is therefore reasonable to hypothesize that immune cells electrically couple to myofibers and nociceptors after skeletal muscle damage to coordinate responses to injury and dually modulate tissue repair and pain. Aim 1 (R61 Phase) will determine if electrical coupling of macrophages to nociceptors modulates incision-related hypersensitivity. This study will use novel transgenic strategies to specifically knockout Cx43 in macrophages in mice with hind paw muscle incision. This will be used in conjunction with chemogenetic or sono-genetic activation of macrophages. Impact of Cx43 knockout and macrophage activation will be assessed with our ex vivo muscle afferent recording preparations, muscle pain-related behavioral tests and calcium imaging (using GCaMP6 reporters) in co-cultures of macrophages and primary DRG cells. Aim 2 (R61 Phase) will use similar groups to determine if electrical coupling of infiltrating macrophages to myofibers facilitates repair of muscle tissue after incision. The impact of these manipulations will be determined using calcium imaging of hind paw muscle cells, electromyography/ compound muscle action potential recordings in vivo, and anatomical analyses of muscle membrane integrity. Aim 3 (R33 Phase) will further explore the functions of macrophage electrical coupling using different transgenic combinations, inhibitory chemogenetics and more severe models of muscle injury. Results will allow determination of novel means of communication between circulating immune factors and the peripheral structures they are affecting. Data will provide novel insights into muscle injury responses that will go well beyond the incremental expansion of current reports. These insights could identify a novel target for therapeutic intervention for pain or muscle repair in numerous muscle injury states.