Po-01-170 pharmacological and gene-therapy based inhibition of nox2-generated ros attenuates disruption of microtubule network and emergence of triggered ca waves in atrial myocytes and in chronic af canine model

Recent research indicate an important role for derailed proteostasis in atrial fibrillation (AF). Major pathways of derailed proteostasis include disruption of microtubule network (MTN). Experimental and clinical studies have documented a significant contribution of histone deactylase 6 (HDAC6) mediated disruption of MTN. Another major causative factor for AF is excessive oxidative stress (OS). However, a clear connection of disruption of MTN with OS is not known in atrial myocytes and chronic model of AF. In particular, it remains elusive whether disruption of MTN by NOX2-generated reactive oxygen species (ROS) contributes to the emergence of triggered Ca2+ waves (TCWs), which can cause membrane depolarization and promote early-afterdepolarization (EAD). To establish the connection between OS and disruption of MTN in isolated canine atrial myocytes and in atrial myocardium of a canine model of chronic AF Normal atrial myocytes were isolated from 4 dogs and pre-incubated in Colchicine (a microtubule disruptor) and H2O2. While myocytes, loaded with Ca2+ sensitive dye, were paced at different cycle lengths (1000ms to 200ms), line-scan confocal Ca2+ imaging were performed in the presence of gp91-tat (a NOX2 inhibitor) and TSA (a HDAC6 inhibitor). In the same cells, MTN was examined by Immunofluorescence. To determine the MTN organization in chronic AF, immunohischemistry was performed in atrial tissue sections from 3 normal animals, 4 control rapid atrial pacing (RAP) animals, and 4 NOX2-shRNA injected RAP animals. In Colchicine treated- isolated atrial myocytes, acute application of gp91-tat and TSA attenuated the disruption of MTN (a) and the generation of TCWs (b). Similarly, gp91-tat and TSA attenuated the disruption of MTN in H2O2 pretreated atrial myocytes (data not shown). Notably, while distinctive MTN was abolished in RAP induced chronic AF atrial sections, there was an attenuation of expected MTN decrease in atrial sections from NOX2-shRNA injected RAP dogs (c). Inhibition of NOX2 by either NOX2 shRNA injection in the chronic AF model or acute application of NOX2 inhibitor as well as HDAC6 inhibitor in atrial myocytes is associated with attenuation of the disruption of MTN and generation of TCWs. Identifying the connection between OS and derailed proteostasis not only sheds light on the molecular mechanisms underlying AF, but also may allow to develop novel therapeutic approach for AF.