Diaphragm Unloading via Controlled Mechanical Ventilation Alters the Gene Expression Profile

Abstract

Rationale: Prolonged controlled mechanical ventilation results in diaphragmatic inactivity and promotes oxidative injury, atrophy, and contractile dysfunction in this important inspiratory muscle. However, the impact of controlled mechanical ventilation on global mRNA alterations in the diaphragm remains unknown. Objectives: In these experiments, we used an Affymetrix oligonucleotide array to identify the temporal changes in diaphragmatic gene expression during controlled mechanical ventilation in the rat. Methods: Adult Sprague-Dawley rats were assigned to either control or mechanical ventilation groups (n ϭ 5/group). Mechanically ventilated animals were anesthetized, tracheostomized, and ventilated with room air for 6 or 18 h. Animals in the control group were acutely anesthetized but not exposed to mechanical ventilation. Measurements and Main Results: Compared with control diaphragms, microarray analysis identified 354 differentially expressed, unique gene products after 6 and 18 h of mechanical ventilation. In general, genes in the cell growth/cell maintenance, stress response, and nucleic acid metabolism categories showed predominant upregulation, whereas genes in the structural protein and energy metabolism categories were predominantly downregulated. Conclusions: We conclude that mechanical ventilation results in rapid changes in diaphragmatic gene expression, and subsequently, many of these changes may contribute to atrophy and muscle fiber remodeling associated with unloading this primary inspiratory muscle. Importantly, this study also provides new insights into why the diaphragm, after the onset of contractile inactivity, atrophies more rapidly than locomotor skeletal muscles and also highlights unique differences that exist between these muscles in the mRNA response to inactivity. Keywords: gene expression; microarray; muscle atrophy; respiratory muscle; weaning from mechanical ventilation Mechanical ventilation (MV) is used clinically to maintain blood gas homeostasis in patients who are incapable of maintaining adequate alveolar ventilation on their own. For many of these patients, an unfortunate consequence of prolonged MV is the ensuing development of respiratory muscle weakness (1). It is now clear that prolonged MV is associated with significant atrophy and contractile dysfunction of the diaphragm (2-6). Recent animal studies indicate that controlled MV leads to significantly greater respiratory muscle weakness than assisted MV (7) because controlled MV results in complete loss of neural activation and mechanical activity of the diaphragm (3,7,8). Therefore, controlled MV is a useful physiologic model to investigate the effects of prolonged inactivity of the diaphragm (2, 3).The diaphragm experiences remarkable physiologic and biochemical alterations during periods of unloading. The time frame of phenotypic changes that the diaphragm undergoes during MV occurs on the order of hours (3, 9), whereas diaphragmatic inactivity imposed by phrenic nerve denervation and cervical spinal cord hemisectio...

Calpain and caspase-3 play required roles in immobilization-induced limb muscle atrophy

“…Twenty-five microliters of reactions containing 1 l of cDNA were used to determine calpastatin expression (Entrez gene ID 25403) and expression of the calibrator sample [housekeeping gene, in this case ␤-glucuronidase (Entrez gene ID 244434)]. ␤-Glucuronidase, a lysosomal glycoside hydrolase, was chosen as the reference gene based on previous work showing unchanged expression in another model of disuse atrophy, mechanical ventilation (4,5).…”

Mechanical Ventilation-Induced Oxidative Stress in the Diaphragm

“…31 To determine whether this increase in messenger RNA results in a signifi cant increase in HO-1 protein in the diaphragm following 18 h of MV, we used Western blots to quantify the levels of HO-1 protein in the diaphragm. Compared with those of the control group, the diaphragmatic HO-1 protein levels in the other two groups were increased ~10-fold during the 18 h of MV ( Fig 1 ).…”

Metallothionein deficiency leads to soleus muscle contractile dysfunction following acute spinal cord injury in mice

“…In skeletal muscle, basal MT expression is low, but expression becomes largely upregulated in response to disuse (11,24,34,44). Consistent with the response of other cell types, it is generally thought that skeletal muscle MT upregulation represents a protective adaptation, although no studies have examined the functional role of MT in skeletal muscle.…”

“…Consistent with the response of other cell types, it is generally thought that skeletal muscle MT upregulation represents a protective adaptation, although no studies have examined the functional role of MT in skeletal muscle. To date, studies investigating skeletal muscle MT have been limited to reports of altered mRNA and protein expression levels (11,19,22,24,28,31,34,35,38,45). For example, significant elevations in MT protein levels have been observed in the soleus following limb immobilization (24).…”