Although the development of respiratory muscle fatigue has been well documented, its molecular basis is poorly understood. We wished to characterize the development of fatigue in rats subjected to severe inspiratory resistive loading (IRL) and identify IRL-induced changes to the diaphragmatic myofilament proteome. We hypothesized that inspiratory resistive loading (IRL) would elicit diaphragmatic fatigue (a decrease in the ratio of transdiaphragmatic pressure to integrated phrenic; Pdi/|MiPhr) due, in part, to modifications to myofilament proteins.
We subjected 14 spontaneously breathing anesthetized rats to IRL until pump failure (decreased pressure generation) at which point we terminated the load and harvested diaphragmatic tissue for proteomic analysis.
IRL elicited a rapid (∼ 2 min) decrease in Pdi/|MiPhr which plateaued until a later decrease at ∼ 42 min; this was followed by central failure (decreased minute phrenic activity) and pump failure at ∼ 44 min. One-dimensional western blot analysis of myofilament proteins indicated changes only to the fast isoforms of troponin T (TnT), particularly a loss of the dominant isoforms of type IIB fibers. In western blots of serum taken before and during IRL, we detected the presence of only the fast, not slow, isoform of troponin I, confirming damage to fast-twitch fibers. In addition, differential antibody immunoreactivity revealed an altered affinity to the other fast TnT isoforms, indicating the presence of a post-translational modification.
These results demonstrate for the first time that type IIB fibers are preferentially injured during inspiratory resistive loading and that TnT, a key contractile protein, is modified in the diaphragms of rats subjected to IRL. The exact nature of this modification remains to be determined but likely plays an important role in the development of fatigue.
Our results suggest that fatigue and injury to fast fatiguable fibers alone is sufficient to cause respiratory pump failure.
Jeremy Simpson, University grant monies Wm. M. Spear Foundation (funds bequested to Queen’s University for respiratory-related research); Block Term Grant (funds awarded to Queen’s University from the Ontario Thoracic Society for respiratory-related research); Grant monies (from sources other than industry) Canadian Institutes for Health Research and Ontario Thoracic Society.