Hypoxia and fatigue-induced modification of function and proteins in intact and skinned murine diaphragm muscle

Abstract

Fatigue studies of isolated, intact muscles typically utilize solutions saturated with O2. However, under in vivo fatiguing conditions, less oxygen is delivered to the muscles and they actually experience hypoxia. No studies to date have correlated the effects of acute hypoxia on the isometric contractile properties of intact muscles, skinned fibers isolated from the same muscles, and the cellular content of specific muscle proteins. Therefore, we have studied the effects of in vitro acute hypoxia on the fatigability of intact diaphragm muscle strips and on the isometric contractile properties of single Triton-skinned fibers isolated from control and hypoxic diaphragm muscles. We found that hypoxia and fatiguing stimulation per se affect the tetanic force of intact muscle strips without exhibiting any significant deleterious effects on the calcium-activated force of skinned muscle fibers dissected from the intact muscles. In contrast, fatiguing stimulation under hypoxic conditions decreased both the tetanic force of muscle strips and the calcium-activated force of skinned muscle fibers. Gel electrophoresis of muscles subjected to hypoxia and hypoxic-fatigue revealed that there is a significant reduction in three protein bands when compared to control muscles. Protein modification may be the underlying mechanism of muscle fatigue under physiologic conditions.

Hypoxia/fatigue-induced degradation of troponin I and troponin C: new insights into physiologic muscle fatigue

“…After muscles were mounted, the resting tension and the stimulatory voltage (provided by a Grass digital stimulator) were adjusted to produce T max . In our recently published study [27], we found that the stimulatory frequency necessary to produce T max was 100 Hz. The stimulatory pulses for the experiment were computer-controlled by a PC (Pentium II) running a customized LABView 4.1 program that controlled the Grass stimulator, collected the digitized force, and analyzed all the data collected.…”

“…Intact muscle preparation All experimental protocols were performed at room temperature (22±2°C) and followed protocols previously described by our research group [27]. Mice were killed by CO 2 inhalation and the intact diaphragm muscles were removed and placed in a dissecting dish containing a modified Ringer solution with the following composition (in mM): 142.0 NaCl, 5.0 KCl, 2.5 CaCl 2 , 2.0 MgCl 2 , 5.0 HEPES, 11 glucose, pH 7.35 (adjusted with NaOH), continuously bubbled with 100% O 2 .…”

“…We used physiological intermittent stimulation (muscles were never stimulated on a continuous basis) in which the frequency of stimulation was that producing maximal tetanic force (T max ). Our research group has recently demonstrated that fatiguing stimulation under hypoxia adversely affects the T max of intact muscle strips as well as the maximum Ca 2+ -activated force (F max ) and the Ca 2+ sensitivity of skinned muscle fibers isolated from these muscles [27]. We have reported significant changes in the protein content of three protein bands when diaphragm muscle strips were subjected to hypoxic-fatigue and then analyzed by SDS-PAGE [27].…”

“…Fatiguing stimulation protocols typically use solutions to superfuse isolated muscles that are constantly bubbled with 95-100% O 2 [24,25,26,27]. Such in vitro experiments fail to adequately mimic in vivo conditions.…”

“…Our research group has recently demonstrated that fatiguing stimulation under hypoxia adversely affects the T max of intact muscle strips as well as the maximum Ca 2+ -activated force (F max ) and the Ca 2+ sensitivity of skinned muscle fibers isolated from these muscles [27]. We have reported significant changes in the protein content of three protein bands when diaphragm muscle strips were subjected to hypoxic-fatigue and then analyzed by SDS-PAGE [27]. These protein bands are at a molecular weight coinciding with that of the troponins responsible for modulating force generation; therefore, we hypothesize that the longlasting and sustained effects of hypoxia-fatigue on the T max of intact muscles and the contractile properties of skinned fibers isolated from hypoxic-fatigued muscles are due to modification/degradation of these proteins.…”