Introduction: The impact of skeletal muscle fatigue, which is the contraction-induced decline in muscle force or power, is associated with elements that are temperature-sensitive, such as myosin regulatory light chain (RLC) phosphorylation, increased phosphate (Pi) and hydrogen (H+) ion accumulation. However, to maintain protein stability, molecular and cellular experiments that illuminate the underlying mechanisms of muscle fatigue are typically examined at temperatures from 15-30°C, which is lower than in vivo (37°C). This study sought to characterize the molecular and cellular effects of fatiguing conditions, including the rarely-studied effect of RLC phosphorylation, at physiological temperatures.
Methods: Biopsies of the vastus lateralis muscle of females (n = 8) aged 71.8 + 1.3 years and males (n = 5) aged 69.4 + 1.7 years in ongoing studies (Cultivating Healthy Aging in Older Adults and Understanding Fatigue in Older Adults) were completed and single fibers used for mechanical testing. Maximal calcium-activated cellular force production and molecular-level interactions (myosin-actin cross-bridge kinetics and mechanical myofilament properties) in slow-contracting myosin heavy chain (MHC) I and fast-contracting MHC IIA fibers were tested at physiological temperatures (37°) to examine the effects of elevated Pi, H+, and RLC phosphorylation. To explore the effects of Pi and H+, repeated measures were performed on single fibers under control (pH = 7, Pi = 5 mM), high phosphate (pH = 7, Pi = 30 mM), high hydrogen ion (pH = 6.2, Pi = 5 mM) and fatigue (pH = 6.2, Pi = 30 mM) conditions. To explore the effects of RLC phosphorylation, repeated measures were performed on single fibers under control and fatigue, then control with RLC phosphorylation and fatigue with RLC phosphorylation.
Results: At 37°C, specific tension (force normalized to cross-sectional area) was greater than 25°C, apparently due to greater numbers of strongly-bound cross-bridges, which in turn were established by quicker cross-bridge kinetics. With fatigue, specific tension was lower at 37° and 25°C, presumably due to fewer strongly-bound cross-bridges and slowed cross-bridge kinetics observed compared to control conditions in MHC I fibers. In MHC IIA fibers at 25°C, fatigue was accompanied by a reduction in strongly-bound cross-bridges and slower cross-bridge kinetics, but at 37°C due to increases in the work-absorbing properties of single fibers, and faster cross-bridge kinetics compared to control. When examining Pi and pH independently at 37°C, no change in specific tension was found due to increased myofilament stiffness and decreased strongly-bound cross-bridges in both MHC I and IIA fibers. In both cases for MHC I and IIA fibers, oscillatory work and power were depressed with alterations to Pi and pH, but recovered completely for MHC I fibers with fatigue due to alterations in the cross-bridge kinetic ratio, and slightly in MHC IIA due to maintenance of strongly-bound cross-bridges and faster cross-bridge kinetics. With RLC phosphorylation, specific tension was reduced compared to control conditions due to a loss of strongly-bound cross-bridges in both MHC I and IIA fibers, with an additional drop in myofilament stiffness in MHC IIA fibers. However, the relative drop in specific tension from control to fatigue was lower with RLC phosphorylation in both MHC I and IIA fibers. Shifts in the cross-bridge kinetic ratios lead to differing results for oscillatory work and power, such that MHC I fibers had dramatically increased work and power under fatigue with RLC, and in MHC IIA fibers work and power were dramatically increased under control with RLC phosphorylation.
Summary: Fiber type specific changes occurred with alterations in temperature in fatiguing conditions indicate the need to conduct experiments at physiological temperatures when attempting to extrapolate to in vivo conditions. While the alterations in specific tension may not occur with changes in phosphate or hydrogen ion concentration, the change in oscillatory work and power production, i.e. force transmission or generation, may be substantial. Additionally, RLC phosphorylation brought about differing effects dependent upon the fiber type examined and fatigue status, thus the relevance of phosphorylation combined with other fatiguing metabolites, should be further questioned and quantified.Doctor of Philosophy (PhD
