Calpain activation is required for ventilator‐induced diaphragmatic atrophy
Authors: Hyatt Hayden William , Ozdemir Mustafa , Nguyen Branden , Deminice Rafael , Yoshihara Toshinori , Powers Scott K. 
Published: April 2020

BACKGROUNDMechanical ventilation (MV) is a life‐saving intervention for many critically ill patients. However, prolonged MV is often associated with complications including difficulty in “weaning” patients from the ventilator. Although the cause of difficult weaning can be multifactorial, weak inspiratory muscles (i.e., diaphragm) are a major contributor. Unfortunately, an unintended consequence of MV is that ventilator support results in inactivity of the diaphragm and the rapid development (i.e., within 12 hours) of both diaphragmatic atrophy and contractile dysfunction. While it is established that MV‐induced diaphragmatic atrophy is largely due to accelerated proteolysis that is preceded by increased mitochondrial emission of reactive oxygen species (ROS), the dominant proteolytic system involved remains unknown.PURPOSEWe tested the hypothesis that active calpains play an essential role in MV‐induced diaphragmatic atrophy.METHODSTo test our hypothesis, we utilized a targeted approach of directly inhibiting calpain activation in the diaphragm via rAAV9‐mediated transgene overexpression of calpastatin (CAST). Notably, the only known function of calpastatin is the inhibition of calpain activity. Adult Sprague‐Dawley rats underwent survival surgery for either sham (SALINE injections) or CAST overexpression (AAV9‐CAST injections) in the diaphragm. Following four weeks of post‐surgery recovery, the diaphragm was harvested from animals that were either allowed to breathe spontaneously for 12 hours (CONTROL‐SALINE and CONTROL‐CAST animals) or were exposed to 12 hours of MV (MV‐SALINE and MV‐CAST). Diaphragm muscle was used for measurements of mitochondrial respiration, mitochondrial reactive oxygen species (ROS) emission, muscle specific force production, fiber cross sectional area, calpain activity, and calpastatin abundance.RESULTSCompared to CONTROL‐SALINE, AAV9‐mediated transgene overexpression increased CAST protein abundance in the diaphragm of CONTROL‐CAST and MV‐CAST animals by >80%. In MV‐SALINE animals, prolonged MV increased calpain activity in the diaphragm by ~83%. In contrast, overexpression of CAST in the diaphragm of MV‐CAST animals prevented MV‐induced increases in calpain activity. Importantly, prevention of MV‐induced calpain activation protected against MV‐induced reductions in both diaphragm fiber cross‐sectional area and maximal specific force production. Moreover, CAST overexpression in the diaphragm prevented MV‐induced decreases in the mitochondrial respiratory control ratio (RCR) and ameliorated MV‐induced increases in mitochondrial ROS emission.CONCLUSIONSThese results support the hypothesis that calpain activation is essential for MV‐induced diaphragmatic atrophy. Importantly, our findings confirm that calpain activation is required for MV‐induced mitochondrial dysfunction in the diaphragm and challenge the dogma that the classical calpains (i.e., calpain 1 and 2) play a limited role in disuse skeletal muscle atrophy.Support or Funding Information1R21AR073956‐01A1