Mechanical ventilation-induced oxidative stress in the diaphragm

Abstract

Prolonged mechanical ventilation (MV) results in oxidative damage in the diaphragm; however, it is unclear whether this MV-induced oxidative injury occurs rapidly or develops slowly over time. Furthermore, it is unknown whether both soluble (cytosolic) and insoluble (myofibrillar) proteins are equally susceptible to oxidation during MV. These experiments tested two hypotheses: 1) MV-induced oxidative injury in the diaphragm occurs within the first 6 h after the initiation of MV; and 2) MV is associated with oxidative modification of both soluble and insoluble proteins. Adult Sprague-Dawley rats were randomly divided into one of seven experimental groups: 1) control ( n = 8); 2) 3-h MV ( n = 8); 3) 6-h MV ( n = 6); 4) 18-h MV ( n = 8); 5) 3-h anesthesia-spontaneous breathing ( n = 8); 6) 6-h anesthesia-spontaneous breathing ( n = 6); and 7) 18-h anesthesia-spontaneous breathing ( n = 8). Markers of oxidative injury in the diaphragm included the measurement of reactive (protein) carbonyl derivatives (RCD) and total lipid hydroperoxides. Three hours of MV did not result in oxidative injury in the diaphragm. In contrast, both 6 and 18 h of MV promoted oxidative injury in the diaphragm, as indicated by increases in both protein RCD and lipid hydroperoxides. Electrophoretic separation of soluble and insoluble proteins indicated that the MV-induced accumulation of RCD was limited to insoluble proteins with molecular masses of ∼200, 120, 80, and 40 kDa. We conclude that MV results in a rapid onset of oxidative injury in the diaphragm and that insoluble proteins are primary targets of MV-induced protein oxidation.

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

“…Heart rate (365 Ϯ 9 beats/min) and mean blood pressure (90 Ϯ 4 mm Hg) homeostasis were maintained during MV across all treatment groups. Blood gas homeostasis and pH (data not shown) were maintained within physiologic levels during the experiment as reported previously (1,4,6,18). Because sepsis is associated with diaphragmatic contractile dysfunction, strict aseptic techniques were followed throughout the experiments (1,4,6,18).…”

“…This notion is supported by the observations that new nuclei are incorporated into locomotor skeletal muscle fibers during growth and that nuclei are lost during atrophy (12,14,16). Although it is known that controlled MV decreases protein synthesis and induces proteolysis and myofiber atrophy across all fiber types (6,17,18), it is unknown whether the myonuclear domain of diaphragm myofibers is altered during the fiber atrophy associated with MV.Regulation of myonuclear loss in skeletal muscle could occur through extrinsic death receptor and intrinsic (sarcoplasmic reticulum and mitochondrial) mediated pathways (19). Caspase (cysteine-dependent, aspartate-directed protease) activation results in protein cleavage and functions in intrinsic and extrinsic pathways of apoptosis.…”

“…Furthermore, we determined whether inhibition of caspase-3 activity at 6 h would alter the myonuclear response of the diaphragm to ventilation. We chose to investigate 6 h of MV because we have previously established that 6 h is the earliest time period that diaphragmatic oxidative injury can be detected after the initiation of MV (18). Six hours of MV with or without DEVD-CHO administration resulted in no diaphragmatic myofiber atrophy ( Figure 7A) and no alterations in myonuclear domain ( Figure 7B) of type I, type IIa, or type IIb/IIx diaphragm myofibers.…”

“…Numerous cellular mechanisms lead to caspase-3 activation, including mitochondrial-dependent (cytochrome c release) and mitochondrial-independent (e.g., Ca 2ϩ activation of calpain) pathways resulting in the stimulation of DNases responsible for cellular DNA fragmentation and protein degradation (19). Oxidative stress occurs rapidly in the diaphragm during mechanical ventilation (18), and oxidant stress can promote cytochrome c release and cellular Ca 2ϩ overload in cells (10,20). This fact suggests a potential role for caspase-3 activation in the regulation of myonuclear number during the rapid atrophy that occurs in the diaphragm after prolonged MV.…”

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

“…Studies demonstrated the following alterations in the diaphragm after controlled mechanical ventilation: reduced muscle mass 2,10,11 ; diminished type I, IIa, and IIx/b fiber dimensions 3,12,13 ; myofibrillar damage 4 ; enhanced proteolysis 12 ; increased protein oxidation and lipid peroxidation 14 ; reduced expression of the insulin-like growth factor (IGF-I) 3 ; decreased sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA-1a) expression 15 ; increased expression of muscle atrophy factor (MAF-box) 16 ; and a decrease in myonuclear content 17 . All of these factors seem to contribute to the development of VIDD, but the precise contribution of each factor and their apparition kinetics has yet to be defined.…”