Mark S. Miller

As human skeletal muscle cellular and molecular contractile properties are temperature‐sensitive, the ability to perform experiments at body temperature (∼37°C) may lead to a better understanding of their in vivo responses and, potentially, their effects on whole‐muscle and whole‐body performance. We utilized sinusoidal analysis to quantify molecular (myosin‐actin cross‐bridge mechanics and kinetics) and cellular (specific tension; force divided by cross‐sectional area) functions in slow‐contracting myosin heavy chain (MHC) I and fast‐contracting MHC IIA fibres from older adults ( N = 13, 8 female) at 37°C and compared these to results at 25°C. MHC I fibres were more temperature‐sensitive than MHC IIA fibres, showing greater increases in cross‐bridge kinetics (MHC I: 6.7–9.4‐fold; IIA: 4.7–4.8‐fold) and number or stiffness of strongly bound cross‐bridges (MHC I: 62%–77%; IIA: 31%–32%), leading to increased specific tension in MHC I (19%), but no change in MHC IIA fibres. The relationship between force and size (cross‐sectional area (CSA)) was stronger at 37°C in both fibre types, explaining 80%–82% of the variance compared to 51%–52% at 25°C. Specific tension was not associated with size at 37°C in both fibre types, indicating force increases proportionally with CSA, potentially due to the increased number or stiffness of strongly bound cross‐bridges at this temperature. At 25°C, specific tension decreased as size increased, in agreement with previous experiments. Overall MHC I and IIA fibres at body temperature and at near‐isometric conditions became more analogous, including similar specific tensions and closer cross‐bridge kinetics, and force production being more strongly correlated with fibre size compared to a subphysiological temperature. image Key points: Although skeletal muscle function is highly sensitive to temperature, human single‐fibre studies have been conducted only at ≤ 30°C. Small‐amplitude sinusoidal perturbations were utilized to elucidate mechanisms of single‐fibre force production at human physiological temperature (37°C). We found that functional differences in slow‐contracting myosin heavy chain (MHC I) and fast‐contracting MHC IIA fibres observed at 25°C were less apparent at 37°C, as force, cross‐bridge kinetics and strongly bound cross‐bridges increased more in MHC I fibres than MHC IIA fibres with the increase in temperature. These results indicate that, given the different sensitivity of each fibre type to changes in temperature, functional assessments of muscle should be conducted at 37°C to better translate to in vivo conditions.

Intracellular accumulation of hydrogen ions (H ⁺ ) and inorganic phosphate (P _i ) has temperature‐dependent effects on single‐fibre contractile function between 10°C and 30°C. In vivo, human skeletal muscle temperatures range between 35‐39°C, and although contractile function is highly dependent on temperature, the effects of fatigue‐inducing [H ⁺ ] and [P _i ] on contractile mechanics at 37°C are unknown. Using sinusoidal analysis, the independent and combined effects of these metabolites on cellular and molecular contractile function were determined at 37°C in slow‐contracting myosin heavy chain (MHC) I and fast‐contracting MHC IIA fibres from vastus lateralis muscle of 13 older adults (8 females), in four conditions: maximal calcium activation (‘control’; 5 mM P _i , pH 7.0), high P _i (30 mM), low pH (6.2) and fatigue (30 mM P _i and pH 6.2). Specific tension (force/cross‐sectional area, mN/mm ² ) in both fibre types was reduced only in the fatigue condition (20%–26%). MHC I fibres had slower cross‐bridge kinetics, with fewer or less stiff strongly bound myosin–actin cross‐bridges in high P _i , low pH and fatigue. The rate of myosin force production was slowest in low pH and fatigue conditions, whereas the myosin detachment rate was most altered by low pH alone. This indicates that during fatigue, high P _i moderates the slowing of cross‐bridge detachment owing to low pH. In contrast, fatigued MHC IIA fibres had faster cross‐bridge kinetics with increased myofilament viscosity. These findings quantify fibre type‐specific mechanical and kinetic mechanisms of fatigue in human skeletal muscle at 37°C, thus advancing our understanding of metabolite‐based muscle fatigue in vivo.

Intracellular accumulation of hydrogen ions (H ⁺ ) and inorganic phosphate (P _i ) have temperature-dependent effects on single fiber contractile function between 10-30°C. In vivo , human skeletal muscle temperatures range between 35-38°C, and although contractile function is highly dependent on temperature, the effects of fatigue-inducing [H ⁺ ] and [P _i ] on contractile mechanics at 37°C is unknown. Using sinusoidal analysis, the independent and combined effects of these metabolites on cellular and molecular contractile function were determined at 37°C in slow-contracting myosin heavy chain (MHC) I and fast-contracting MHC IIA fibers from vastus lateralis muscle of 13 older adults (8 females), under four conditions: maximal calcium activation (“control”; 5 mM P _i , pH 7.0), high P _i (30 mM), low pH (6.2), and fatigue (30 mM P _i and pH 6.2). Specific tension (force/cross-sectional area, mN/mm ² ) in both fiber types was reduced only under fatigue conditions (20-26%). MHC I fibers had slower cross-bridge kinetics with fewer or less stiff strongly-bound myosin-actin cross-bridges in high P _i , low pH, and fatigue. In contrast, fatigued MHC IIA fibers had faster cross-bridge kinetics with increased myofilament and/or cross-bridge viscosity. Single fiber oscillatory work was reduced in both fiber types when P _i or pH alone was altered. However, fatigue conditions returned oscillatory work values toward control through alterations to cross-bridge kinetics in MHC I fibers and changes to work absorption and production processes in MHC IIA fibers. These findings quantify fiber-type specific mechanical and kinetic mechanisms of fatigue in human skeletal muscle at 37°C, thus advancing our understanding of metabolite-based muscle fatigue in vivo . KEY POINTS SUMMARY: Working skeletal muscle increases intracellular concentrations of hydrogen ion and inorganic phosphate, leading to fatigue, or loss of force-generating capacity Temperature plays a well-established role in the muscle response to hydrogen ion and/or inorganic phosphate accumulation, but has not previously been studied at human body temperature (37°C) At 37°C, reduced force generation only occurs when high phosphate and hydrogen ions are combined, not when changed individually In slow-contracting fibers, fatigue slowed myosin-actin cross-bridge kinetics and reduced the number or stiffness of strongly-bound cross-bridges. In fast-contracting fibers, fatigue increased myosin-actin cross-bridge kinetics and increased myofilament viscosity. The distinct responses by fiber type to fatigue provides new insight into its mechanisms and advances our understanding of the whole muscle and body responses to fatigue Abstract Figure:

As human skeletal muscle cellular and molecular contractile properties are temperature-sensitive, the ability to perform experiments at body temperature (∼37°C) may lead to a better understanding of their in vivo responses and potentially their effects upon whole-muscle and whole-body performance. We quantified molecular (myosin-actin cross-bridge mechanics and kinetics) and cellular (specific tension; force divided by cross-sectional area) function in slow-contracting myosin heavy chain (MHC) I and fast-contracting MHC IIA fibers from older adults (n=13, 8 female) at 37°C and compared these to results at 25°C. MHC I fibers were more temperature-sensitive than MHC IIA fibers, showing greater increases in cross-bridge kinetics (MHC I: 4.9-8.7x; IIA: 4x) and number or stiffness of strongly-bound cross-bridges (MHC I: 86%; IIA: 34%), leading to increased specific tension in MHC I (19%), with no change in MHC IIA fibers. The expected relationship between fiber force and size (cross-sectional area, CSA) was stronger at 37°C in both fiber types, explaining 80-82% of the variance compared to 51-52% at 25°C. Specific tension was unchanged with size at 37°C in both fiber types, showing that force increases proportionally with CSA, which may be due to the increased number or stiffness of strongly-bound cross-bridges at this temperature. At 25°C, specific tension decreased with size in agreement with previous experiments. Overall, MHC I and IIA fibers at body temperature (37°C) became more analogous, including similar specific tension and closer cross-bridge kinetics, and force production was more strongly correlated with fiber size compared to a non-physiological temperature. Abstract Figure: KEY POINTS SUMMARY: Although skeletal muscle function is highly sensitive to temperature, human single fiber studies have only been conducted at ≤30°C. Small-amplitude sinusoidal perturbations were utilized to elucidate mechanisms of single fiber force production at human physiological temperature (37°C). We found that functional differences in slow-contracting myosin heavy chain (MHC I) and fast-contracting MHC IIA fibers observed at 25°C were less apparent at 37°C, as force, crossbridge kinetics, and strongly-bound crossbridges increased more in MHC I fibers than MHC IIA fibers at 37 vs. 25°C. These results indicate that, given the different sensitivity of each fiber type to changes in temperature, functional assessments of muscle should be conducted at 37°C to better translate to vivo conditions.

Stretch activation (SA) is the delayed increase in force following a rapid stretch and improves muscle performance during repetitive cyclical contractions in insect flight and cardiac muscles. Although historically considered too low to be physiologically relevant in skeletal muscle, our recent work showed that higher phosphate concentrations ([Pi]) increased SA in mouse soleus fibers. These results suggest SA has a role combating fatigue, which increases [Pi], lowers pH, and reduces active calcium concentration ([Ca2+]). To test this, we measured SA during Active, High [Ca2+] Fatigue and Low [Ca2+] Fatigue conditions in myosin heavy chain (MHC) I, IIA, IIX, and IIB fibers from mouse soleus and extensor digitorum longus muscles. In the fast-contracting MHC II fibers, calcium-activated isometric tension (F0) decreased from Active to High [Ca2+] Fatigue to Low [Ca2+] Fatigue, as expected. Remarkably, SA tension (FSA) was not decreased but remained unchanged or increased under High and Low [Ca2+] Fatigue, except for a small decrease in MHC IIB fibers in Low [Ca2+] Fatigue compared with Active. This results in SA’s percent contribution to total tension production (FSA/[F0 + FSA]) being much greater (58–114%) under fatiguing conditions in fast-contracting MHC II fibers. The SA tension peak for MHC I fibers was not visibly apparent under either fatigue condition, and the peak was about 20% of MHC II fibers’ peaks under active conditions. Our results show SA improves force production under fatiguing conditions in MHC II fibers, which could play an important role in increasing endurance for muscles that are lengthened prior to shortening.

Skeletal muscle fatigue is caused, in part, by increased production of phosphate and hydrogen ions, resulting in decreased force generation. We found that reduced force in fibers from older adults was due to altered function of myosin and actin, including slower protein interactions and reduced force per myosin head. Additionally, an ATP analog, dATP, partially reversed contractile dysfunction induced by increased phosphate and hydrogen, improving force production and altering myosin-actin interactions dependent upon fiber type.

Human studies examining the cellular mechanisms behind sarcopenia, or age-related loss of skeletal muscle mass and function, have produced inconsistent results. A systematic review and meta-analysis were performed to determine the aging effects on protein expression, size, and distribution of fibers with various myosin heavy chain (MyHC) isoforms. Study eligibility included MyHC comparisons between young (18–49 yr) and older (≥60 yr) adults, with 27 studies identified. Relative protein expression was higher with age for the slow-contracting MyHC I fibers, with correspondingly lower fast-contracting MyHC II and IIA values. Fiber sizes were similar with age for MyHC I, but smaller for MyHC II and IIA. Fiber distributions were similar with age. When separated by sex, the few studies that examined females showed atrophy of MyHC II and IIA fibers with age, but no change in MyHC protein expression. Additional analyses by measurement technique, physical activity, and muscle biopsies provided important insights. In summary, age-related atrophy in fast-contracting fibers lead to more of the slow-contracting, lower force-producing isoform in older male muscles, which helps explain their age-related loss in whole muscle force, velocity, and power. Exercise or pharmacological interventions that shift MyHC expression toward faster isoforms and/or increase fast-contracting fiber size should decrease the prevalence of sarcopenia. Our findings also indicate that future studies need to include or focus solely on females, measure MyHC IIA and IIX isoforms separately, examine fiber type distribution, sample additional muscles to the vastus lateralis (VL), and incorporate an objective measurement of physical activity.

INPLASY202460109 Effects of age on human skeletal muscle: A systematic review and meta-analysis of myosin heavy chain isoform protein expression, fiber size and distribution Keywords: aging, sarcop…

In males, skeletal muscle function may be altered by shifts in either circulating testosterone or estrogen. We examined the effect of acute (2 week) exposures to 17α-ethinyl estradiol (EE2), an estrogen receptor (ER) agonist, or flutamide, an androgen receptor (AR) antagonist, on the contractile function of individual skeletal muscle fibers from slow-contracting soleus and fast-contracting extensor digitorum longus muscles from adult male mice. Single fiber specific tension (force divided by cross-sectional area) was decreased with flutamide treatment in all myosin heavy chain fiber types examined (MHC I, IIA and IIB); similar effects were observed with EE2-treatment but only in the fastest-contracting MHC IIB fibers. The decreases in maximally Ca²⁺-activated specific tension were primarily a result of fewer strongly bound myosin-actin cross-bridges, with flutamide treatment also showing lower myofilament lattice stiffness. Myosin-actin cross-bridge kinetics were slower in MHC IIA fibers in flutamide-treated mice, but faster in EE2-treated mice, indicating that contractile velocity may be affected differently in this fiber type which is commonly expressed in human skeletal muscle. Importantly, these effects were observed in the absence of outcomes previously used to evaluate ER agonists or AR antagonists in rodents including weight of reproductive organs or mammary gland morphology. Our findings indicate that substantial shifts in skeletal muscle function occur in male mice following acute exposures to low doses of a pharmacological ER agonist and an AR antagonist. These results suggest that countermeasures to maintain physical function may be needed early in situations that induce similar ER agonist and AR antagonist conditions.

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, t_on) in MHC IIA fibers, and accelerated kinetics (shorter t_on) 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.