Potential molecular mechanisms underlying muscle fatigue mediated by reactive oxygen and nitrogen species

Intense contractile activity causes a dramatic decline in the force and velocity generating capacity of skeletal muscle within a few minutes, a phenomenon that characterizes fatigue. Much of the research effort has focused on how elevated levels of the metabolites of ATP hydrolysis might inhibit the function of the contractile proteins. However there is now growing evidence that elevated levels of reactive oxygen and nitrogen species (ROS/RNS), which also accumulate in the myoplasm during fatigue, also play a causative role in this type of fatigue. The most compelling evidence comes from observations demonstrating that pre-treatment of intact muscle with a ROS scavenger can significantly attenuate the development of fatigue. A clear advantage of this line of inquiry is that the molecular targets and protein modifications of some of the ROS scavengers are well-characterized enabling researchers to begin to identify potential regions and even specific amino acid residues modified during fatigue. Combining this knowledge with assessments of contractile properties from the whole muscle level down to the dynamic motions within specific contractile proteins enable the linking of the structural modifications to the functional impacts, using advanced chemical and biophysical techniques. Based on this approach at least two areas are beginning emerge as potentially important sites, the regulatory protein troponin and the actin binding region of myosin. This review highlights some of these recent efforts which have the potential to offer uniquely precise information on the underlying molecular basis of fatigue. This work may also have implications beyond muscle fatigue as ROS/RNS mediated protein modifications are also thought to play a role in the loss of muscle function with aging and in some acute pathologies like cardiac arrest and ischemia

Acidosis and Phosphate Directly Reduce Myosin\u27s Force-Generating Capacity Through Distinct Molecular Mechanism

Elevated levels of the metabolic by-products, including acidosis (i.e., high [H+]) and phosphate (Pi) are putative agents of muscle fatigue; however, the mechanism through which they affect myosin’s function remain unclear. To elucidate these mechanisms, we directly examined the effect of acidosis (pH 6.5 vs. 7.4), alone and in combination with elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin using a laser trap assay. Acidosis decreased myosin’s average force-generating capacity by 20% (p \u3c 0.05). The reduction was due to both a decrease in the force generated during each actomyosin interaction, as well as an increase in the number of binding events generating negative forces. Adding Pi to the acidic condition resulted in a quantitatively similar decrease in force but was solely due to an elimination of all high force-generating events (\u3e2 pN), resulting from an acceleration of the myosin’s rate of detachment from actin. Acidosis and Pi also had distinct effects on myosin’s steady state ATPase rate with acidosis slowing it by ∼90% (p \u3e 0.05), while the addition of Piunder acidic conditions caused a significant recovery in the ATPase rate. These data suggest that these two fatigue agents have distinct effects on myosin’s cross-bridge cycle that may underlie the synergistic effect that they have muscle force. Thus these data provide novel molecular insight into the mechanisms underlying the depressive effects of Pi and H+ on muscle contraction during fatigue

Acidosis and Phosphate Directly Reduce Myosin’s Force-Generating Capacity Through Distinct Molecular Mechanisms

Elevated levels of the metabolic by-products, including acidosis (i.e., high [H+]) and phosphate (Pi) are putative agents of muscle fatigue; however, the mechanism through which they affect myosin’s function remain unclear. To elucidate these mechanisms, we directly examined the effect of acidosis (pH 6.5 vs. 7.4), alone and in combination with elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin using a laser trap assay. Acidosis decreased myosin’s average force-generating capacity by 20% (p < 0.05). The reduction was due to both a decrease in the force generated during each actomyosin interaction, as well as an increase in the number of binding events generating negative forces. Adding Pi to the acidic condition resulted in a quantitatively similar decrease in force but was solely due to an elimination of all high force-generating events (>2 pN), resulting from an acceleration of the myosin’s rate of detachment from actin. Acidosis and Pi also had distinct effects on myosin’s steady state ATPase rate with acidosis slowing it by ∼90% (p > 0.05), while the addition of Pi under acidic conditions caused a significant recovery in the ATPase rate. These data suggest that these two fatigue agents have distinct effects on myosin’s cross-bridge cycle that may underlie the synergistic effect that they have muscle force. Thus these data provide novel molecular insight into the mechanisms underlying the depressive effects of Pi and H+ on muscle contraction during fatigue

Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules

AbstractElevated levels of phosphate (Pi) reduce isometric force, providing support for the notion that the release of Pi from myosin is closely associated with the generation of muscular force. Pi is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of Pi on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of Pi on the force-generating capacity of a small ensemble of myosin (∼12 myosin heads) using a three-bead laser trap assay. In the absence of Pi, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM Pi, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated Pi also caused a >65% reduction in binding-event lifetime, suggesting that Pi induces premature detachment from a strongly bound state. Definitive evidence of a Pi-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which Pi induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of Pi on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that Pi-induced inhibition of force may proceed along a kinetic pathway different from that of force generation

Active Self-Organization of Actin-Microtubule Composite Self-Propelled Rods

Spontaneous self-organization of active matter has been demonstrated in a number of biological systems including bacteria, cells, and cytoskeletal filaments. Cytoskeletal filaments act as active polar rods when they are propelled along a glass surface via motor proteins. Actin has previously been shown to display polar or nematic ordering, whereas microtubules have been shown to create large vortices. For the first time, we combine both the actin and microtubule gliding into a composite active system. In the absence of actin filaments, microtubule filament organization transitions from isotropic to nematic to polar as a function of filament density. We find that the presence of a crowder, methylcellulose, is essential for this transition. In the absence of microtubules, actin transitions from isotropic to nematic. In combination, microtubules are affected by the presence of actin and the overall density of the filaments, becoming entrained with the nematic alignment of actin. Actin filaments are not as affected by the presence of microtubules. These results serve as first step in exploring the rich emergent behavior that can result from composite active matter system with tunable particle properties, self-propulsion speeds, and interparticle interactions

The Role of Elevated Levels of Inorganic Phosphate and Hydrogen Ions in Muscular Fatigue: A Re-Examination Near Physiological Temperatures

The cause of muscular fatigue has been the quintessential question for exercise physiologists for over 100 years but despite intense efforts the etiology of the phenomenon remains elusive. Upon repeated high intensity stimulation muscular force and power output decline. A correlation between the build-up of metabolites and the decline in force and power output has been demonstrated. Additionally, elevating inorganic phosphate and hydrogen ions in skinned single muscle fibers depresses force and power output. These findings lead authors to conclude the build up of metabolites such as Pi and H+ were the major cause of fatigue. However, more recently authors have questioned the role of these ions based on the temperature-dependence of their depressive effects. The present investigation re-examined the role of elevations in Pi and H+ at low temperatures and at temperatures approaching mammalian physiological values. Efforts were also made to approximate the conditions experienced during fatigue. Chapter two examines the effect elevations in Pi on single fiber force, power and velocity. Chapter three examines the effect of elevated Pi on calcium sensitivity of single fibers. Finally Chapter four addresses the effect of simultaneous elevations in Pi and H+ on single fiber force, power and velocity. The results are interpreted in terms of the implication for muscular fatigue

Recent Insights into Muscle Fatigue at the Cross-Bridge Level

The depression in force and/or velocity associated with muscular fatigue can be the result of a failure at any level, from the initial events in the motor cortex of the brain to the formation of an actomyosin cross-bridge in the muscle cell. Since all the force and motion generated by muscle ultimately derives from the cyclical interaction of actin and myosin, researchers have focused heavily on the impact of the accumulation of intracellular metabolites [e.g., Pi, H+ and adenosine diphoshphate (ADP)] on the function these contractile proteins. At saturating Ca++ levels, elevated Pi appears to be the primary cause for the loss in maximal isometric force, while increased [H+] and possibly ADP act to slow unloaded shortening velocity in single muscle fibers, suggesting a causative role in muscular fatigue. However the precise mechanisms through which these metabolites might affect the individual function of the contractile proteins remain unclear because intact muscle is a highly complex structure. To simplify problem isolated actin and myosin have been studied in the in vitro motility assay and more recently the single molecule laser trap assay with the findings showing that both Pi and H+ alter single actomyosin function in unique ways. In addition to these new insights, we are also gaining important information about the roles played by the muscle regulatory proteins troponin (Tn) and tropomyosin (Tm) in the fatigue process. In vitro studies, suggest that both the acidosis and elevated levels of Pi can inhibit velocity and force at sub-saturating levels of Ca++ in the presence of Tn and Tm and that this inhibition can be greater than that observed in the absence of regulation. To understand the molecular basis of the role of regulatory proteins in the fatigue process researchers are taking advantage of modern molecular biological techniques to manipulate the structure and function of Tn/Tm. These efforts are beginning to reveal the relevant structures and how their functions might be altered during fatigue. Thus, it is a very exciting time to study muscle fatigue because the technological advances occurring in the fields of biophysics and molecular biology are providing researchers with the ability to directly test long held hypotheses and consequently reshaping our understanding of this age-old question

Image_1_Acidosis and Phosphate Directly Reduce Myosin’s Force-Generating Capacity Through Distinct Molecular Mechanisms.PDF

<p>Elevated levels of the metabolic by-products, including acidosis (i.e., high [H⁺]) and phosphate (P_i) are putative agents of muscle fatigue; however, the mechanism through which they affect myosin’s function remain unclear. To elucidate these mechanisms, we directly examined the effect of acidosis (pH 6.5 vs. 7.4), alone and in combination with elevated levels of P_i on the force-generating capacity of a mini-ensemble of myosin using a laser trap assay. Acidosis decreased myosin’s average force-generating capacity by 20% (p < 0.05). The reduction was due to both a decrease in the force generated during each actomyosin interaction, as well as an increase in the number of binding events generating negative forces. Adding P_i to the acidic condition resulted in a quantitatively similar decrease in force but was solely due to an elimination of all high force-generating events (>2 pN), resulting from an acceleration of the myosin’s rate of detachment from actin. Acidosis and P_i also had distinct effects on myosin’s steady state ATPase rate with acidosis slowing it by ∼90% (p > 0.05), while the addition of P_i under acidic conditions caused a significant recovery in the ATPase rate. These data suggest that these two fatigue agents have distinct effects on myosin’s cross-bridge cycle that may underlie the synergistic effect that they have muscle force. Thus these data provide novel molecular insight into the mechanisms underlying the depressive effects of P_i and H⁺ on muscle contraction during fatigue.</p