Reactive oxygen species (ROS) are natural products of oxidative phosphorylation. With increased muscular activity (particularly in older individuals), ROS are elevated to the point where they overpower intracellular buffers and protein damage and muscle dysfunction occur. Diaphragmatic fatigue constitutes a life threatening condition for patients suffering from disorders that increase the work load of the diaphragm. The applicants propose to determine the role of ROS in diaphragmatic fatigue in mature and senescent rats, the cellular processes and proteins affected, and the protective role of heat shock proteins. Three hypotheses will be tested: 1) The response to and recovery from fatiguing stimulation are significantly influenced by ROS; 2) Diaphragms from senescent animals are more susceptible to damage by ROS than diaphragms from mature animals and that this damage is primarily to the excitation-contraction coupling (ECC) process; 3) The protein of the 70 kDa heat shock protein family protects the diaphragm from the damaging effects of fatiguing stimulation.
Four specific aims will test these hypotheses. The first three will study intact diaphragm to provide correlative evidence. The last one will utilize single skinned fibers to explicitly test the hypotheses.
The aims will determine: 1) the time course of isometric force and levels of superoxide (O2- and important EC regulatory proteins (FKBP12, triadin, sorcin and junctin)) in perfused mature and senescent rat diaphragms subjected to fatiguing stimulation in the presence and absence of intracellular ROS scavengers; 2) the effects of L(+)-lactate (a product of fatigue that is reported to stimulate ROS production) on the response to fatiguing stimulation; 3) how whole body heat shock affects the response to fatiguing stimulation; 4) how the contractile proteins and Ca++ uptake and release properties of the sarcoplasmic reticulum are affected by fatiguing stimulation in single skinned fast- and slow-twitch diaphragm muscle fibers.
