Caspase-3 Regulation of Diaphragm Myonuclear Domain during Mechanical Ventilation–induced Atrophy

Abstract

Rationale: Unloading the diaphragm via mechanical ventilation (MV) results in rapid diaphragmatic fiber atrophy. It is unknown whether the myonuclear domain (cytoplasmic myofiber volume/ myonucleus) of diaphragm myofibers is altered during MV. Objective: We tested the hypothesis that MV-induced diaphragmatic atrophy is associated with a loss of myonuclei via a caspase-3-mediated, apoptotic-like mechanism resulting in a constant myonuclear domain. Methods: To test this postulate, Sprague-Dawley rats were randomly assigned to a control group or to experimental groups exposed to 6 or 12 h of MV with or without administration of a caspase-3 inhibitor. Measurements and Main Results:After 12 h of MV, type I and type IIa diaphragm myofiber areas were decreased by 17 and 23%, respectively, and caspase-3 inhibition attenuated this decrease. Diaphragmatic myonuclear content decreased after 12 h of MV and resulted in the maintenance of a constant myonuclear domain in all fiber types. Both 6 and 12 h of MV resulted in caspase-3-dependent increases in apoptotic markers in the diaphragm (e.g., number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive nuclei and DNA fragmentation). Caspase-3-dependent increases in apoptotic markers occurred after 6 h of MV, before the onset of myofiber atrophy. Conclusions: Collectively, these data support the hypothesis that the myonuclear domain of diaphragm myofibers is maintained during prolonged MV and that caspase-3-mediated myonuclear apoptosis contributes to this process. Keywords: muscle atrophy; respiratory muscle; apoptosis; ventilatory weaning Mechanical ventilation (MV) is a clinical intervention for patients who are unable to maintain adequate alveolar ventilation. Recent evidence reveals that controlled MV results in a swift progression of diaphragmatic atrophy and weakness (1-6). It seems that this diaphragmatic atrophy and weakness contributes to difficulty in weaning patients from the ventilator (7). The mechanism(s) responsible for the rapid onset of diaphragmatic atrophy and weakness are not fully understood. Therefore, delineating these mechanisms is a prerequisite for the development of therapeutic strategies to circumvent weaning difficulties. Although mechanical ventilation-induced diaphragm inactivity results in fiber atrophy, it is unknown if prolonged mechanical ventilation is associated with alterations in myonuclear domain via apoptotic mechanisms. What This Study Adds to the FieldOur results reveal that inhibiting caspase-3 activation and myonuclear loss during mechanical ventilation attenuates diaphragmatic muscle atrophy.Mechanical ventilation-induced diaphragmatic atrophy and contractile dysfunction is characterized by oxidative stress and stress-related gene expression in myofibers that occurs within a matter of hours (7,8). In addition to myofibrillar protein loss, extracellular matrix expansion, and metabolic enzyme alterations (9-11), prolonged disuse of skeletal muscle results in the selective loss of myonuclei (12-16). Myonu...

Effects of controlled and pressure support mechanical ventilation on rat diaphragm muscle

“…All mammalian cells proteases systems (lysossomal proteases, calpains, caspases and proteasome system 43 ) are activated after the onset of CMV 7,12,44 , which is one more cause of the muscle fibers atrophy in PCV-C group observed in our study.…”

“…Skeletal muscle cells are multinucleated and, theoretically, a single myonucleus can supply the necessary gene expression for a determined area of cytoplasm, a relationship called myonuclear domain 17 . Literature demonstrates a decreased volume of the cytoplasm (atrophy), which is accompanied by a decreased number of myonuclei, not analyzed in our study, but resulting in constant myonuclear domain 7 .That decrease in myonuclear content is mediated by an increase in caspase-3-dependent apoptosis, which was evident after only 6 hours of CMV 7 . Both apoptosis and atrophy were attenuated with caspase-3 inhibition.…”

“…This ventilatorinduced diaphragmatic dysfunction (VIDD), analyzed in many studies, may be due to: muscle disuse atrophy, reduced protein synthesis, increased proteolytic activity, oxidative stress, intrinsic abnormalities (ultrastructural level) of muscle fibers, problems with the excitation-contraction coupling and myonuclear loss 2,7,8,12,16,23,24 .…”

“…The decreased volume of the cytoplasm (atrophy) was observed in the presence of decreased number of myonuclei, although the myonuclear domain (the concept that each myonucleus controls the gene products in a finite volume of a muscle fiber 17 ) remained constant. This decrease in myonuclear content was mediated by caspase-3-dependent increased apoptosis, which was evident as early as 6 hours after the onset of CMV 7 . Both the apoptosis and the atrophy were attenuated by the inhibition of caspase-3 7 .…”

“…This decrease in myonuclear content was mediated by caspase-3-dependent increased apoptosis, which was evident as early as 6 hours after the onset of CMV 7 . Both the apoptosis and the atrophy were attenuated by the inhibition of caspase-3 7 .…”