Human Muscle Fiber Function when Altering Phosphate, Hydrogen Ion, and Regulatory Light Chain Phosphorylation Status at Physiological Temperatures

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

Skeletal muscle single fiber force production declines early in juvenile male mice with chronic kidney disease

Abstract Children with chronic kidney disease (CKD) frequently exhibit delayed physical development and reduced physical performance, presumably due to skeletal muscle dysfunction. However, the cellular and molecular basis of skeletal muscle impairment in juvenile CKD remains poorly understood. Cellular (single fiber) and molecular (myosin‐actin interactions and myofilament properties) function was examined ex vivo in slow (soleus) and fast (extensor digitorum longus) contracting muscles of juvenile male (6 weeks old) CKD and control mice. CKD was induced by 0.2% adenine diet for 3 weeks starting at 3 weeks of age. Specific tension (maximal isometric force divided by cross‐sectional area) was reduced in larger myosin heavy chain (MHC) I and IIA fibers and in all IIB fibers in juvenile male mice with CKD due to fewer strongly bound myosin‐actin cross‐bridges. Fiber cross‐sectional area in juvenile CKD mice was unchanged in MHC I and IIB fibers and increased in MHC IIA fibers, compared to controls. CKD slowed cross‐bridge kinetics (slower rate of myosin force production and longer myosin attachment time, ton) in MHC IIA fibers, and accelerated kinetics (shorter ton) in MHC IIB fibers, which may indicate fiber type dependent shifts in contractile velocity in juvenile CKD. Overall, our findings show that single fiber myopathy is an early event during juvenile CKD, manifesting prior to the development of cellular atrophy as reduced force generation due to fewer strongly bound myosin heads. These results warrant clinical translation and call for early interventions to preserve physical function in children with CKD

Intradialytic exercise increases cardiac power index

Introduction: Mortality rates are high in end-stage renal disease due to cardiovascular complications. Perfusion of the myocardium declines during and after hemodialysis sessions with the potential for aerobic exercise to mitigate these during hemodialysis. Objectives: The purpose of this study was to investigate acute changes in hemodynamics in subjects with end-stage renal disease (ESRD) during exercise. Patients and Methods: Subjects (n = 10) were monitored for 1.5 hours during hemodialysis treatment during a control (CON) and an exercise (EX) session. Subjects cycled using an ergometer strapped to the reclining dialysis chair at an RPE of 11-13 for 30 minutes during the EX session beginning at 30 min into dialysis and ending at 60 minutes. Data for systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were collected using an automated blood pressure cuff attached to the hemodialysis machine. Data for cardiac output (Q̇ ), cardiac power index (CPI), stroke volume (SV), systemic vascular resistance (SVR), and heart rate (HR) were collected using the NICaS bioelectrical impedance device. Results: During the EX session, CPI, Q̇ , SV, and HR were significantly greater (P<0.05) than the CON session. Additionally, Q̇ was significantly (P< 0.05) greater at 45 minutes and 60 minutes compared to 15 minutes. HR was significantly (P<0.05) greater at 45 minutes compared to 90 minutes. No significant interactions were found for MAP, CPI, Q̇ , HR, SV, SBP, DBP, or SVR. Conclusion: In conclusion, exercise during dialysis may decrease the likelihood of experiencing ischemic or hypotensive events by enhancing myocardial perfusion through increasing CPI and Q̇