Cumulative effects of H⁺ and P_i on force and power of skeletal muscle fibres from young and older adults

Abstract

Abstract

The cellular causes of the age‐related loss in power output and increased fatigability are unresolved. We previously observed that the depressive effects of hydrogen (H⁺) (pH 6.2) and inorganic phosphate (P_i) (30 mm) did not differ in muscle fibres from young and older men. However, the effects may have been saturated in the severe fatigue‐mimicking condition, potentially masking age differences in the sensitivity of the cross‐bridge to these metabolites. Thus, we compared the contractile mechanics of muscle fibres from the vastus lateralis of 13 young (20–32 years, seven women) and 12 older adults (70–90 years, six women) in conditions mimicking quiescent muscle and a range of elevated H⁺ (pH 6.8–6.6–6.2) and P_i (12–20–30 mm). The older adult knee extensor muscles showed hallmark signs of ageing, including 19% lower thigh lean mass, 60% lower power and a greater fatigability compared to young adult muscles. Progressively increasing concentrations of H⁺ and P_i in the chemically‐permeabilized fibre experiments caused a linear decrease in fibre force, velocity and power; however, the effects did not differ with age or sex. Fast fibre cross‐sectional area was 41% smaller in older compared to young adults, which corresponded with lower absolute power. Size‐specific power was greater in fibres from older compared to young adults, indicating the age‐related decline in absolute power was explained by differences in fibre size. These data suggest the age‐related loss in power is determined primarily by fast fibre atrophy in men and women, but the age‐related increase in fatigability cannot be explained by an increased sensitivity of the cross‐bridge to H⁺ and P_i.

Key points

• The causes of the age‐related loss in muscle power output and the increase in fatigability during dynamic exercise remain elusive.

• We show that progressively increasing concentrations of hydrogen (H⁺) and inorganic phosphate (P_i) causes a linear decrease in muscle fibre force, velocity and power, but the depressive effects of these metabolites on cross‐bridge function did not differ in fibres from older compared to young adults across a range of fatigue‐mimicking conditions.

• We also found peak absolute power did not differ in slow fibres from young and older adults but it was ∼33% lower in older adult fast fibres, which was explained entirely by age differences in fibre size.

• These data suggest that fast fibre atrophy is a major factor contributing to the loss in power of older men and women, but that the age‐related increase in fatigability cannot be explained by an increased sensitivity of the cross‐bridge to H⁺ and P_i.

Abstract figure legend Ageing of the knee extensor muscles is characterized by a loss of muscle mass that is vastly exceeded by the ability to generate power, and the problem is exacerbated by the increased fatigability when older adults perform dynamic exercise. Exposing isolated muscle fibres to progressively increasing concentrations of fatigue‐inducing metabolites (H⁺ and P_i) caused the force–power curves to shift down and to the left. However, the depressive effects of the metabolites did not differ in fibres from young and older men or women. Absolute force and power did not differ in the slow fibres with age but was markedly lower in older adult fast fibres, which was explained entirely by age differences in fibre size. These data suggest that the age‐related increase in fatigability cannot be explained by an increased sensitivity of the cross‐bridge to H⁺ and P_i, and that therapeutic interventions should target attenuating fast fibre atrophy to help offset some of the age‐related decrements in whole‐muscle power.

Introduction

Advanced ageing is accompanied by several impairments in the neuromuscular system that lead to a loss of mobility and functional independence. One essential factor contributing to the age‐related decrements in mobility and physical function is the decreased ability of older adults to generate power, which, after the fourth to fifth decade of life, is lost at a rate of ∼2–4% per year in muscles of the lower extremity (Alcazar et al., 2020, 2023; Bassey et al., 1992; Reid et al., 2014; Skelton et al., 1994). Another important factor is the fatigability of limb muscle, often referred to as fatigue or muscle fatigue, which is characterized by a contractile activity‐induced reduction in power that is reversible by periods of rest. This is particularly important to function in older adults (Foulis et al., 2017; Senefeld et al., 2017) because the age‐related loss in power is exacerbated by the increased fatigability that occurs when older adults perform moderate‐ to high‐velocity contractions (Callahan & Kent‐Braun, 2011; Dalton et al., 2012; McNeil & Rice, 2007; Petrella et al., 2005; Senefeld et al., 2017; Sundberg, Kuplic et al., 2018). Despite the growing recognition of the importance of power to mobility and physical function in older adults (Reid & Fielding, 2012), the causes of the loss in power output and the increase in fatigability with ageing remain poorly understood.

Several studies over the past 20 years have localized the primary site for the age‐related increase in fatigability during dynamic exercise to factors within the muscle rather than the nervous system (Baudry et al., 2007; Dalton et al., 2010, 2012; Sundberg, Hunter et al., 2018; Sundberg, Kuplic et al., 2018). Identifying the mechanisms, however, has proven considerably more challenging, in part, because of the complexity and number of mechanisms implicated in fatigue within the muscle (Allen et al., 2008; Fitts, 1994; Hostrup et al., 2021). Nevertheless, given the preponderance of evidence that a large portion of fatigue can be attributed to the effects of two metabolites, inorganic phosphate (P_i) and hydrogen (H⁺) (Debold et al., 2016; Sundberg & Fitts, 2019), we sought to determine whether age‐related changes in the muscle cause an increased accumulation of metabolites (Sundberg et al., 2019) and/or an increased sensitivity of the muscle fibres to a given concentration of metabolites (Sundberg, Hunter et al., 2018). To test the latter, we studied single fibre contractile mechanics from biopsies of the vastus lateralis and found that the decrements in single fibre force (P_o ), velocity (V _max and V_o ), and power elicited by a severe fatigue‐mimicking condition (pH 6.2 + 30 mm P_i) did not differ in fibres from young compared to older men (Sundberg, Hunter et al., 2018). The severe fatigue‐mimicking condition was used in that study because we hypothesized that the age differences in the sensitivity of the contractile proteins to these metabolites, if present, would be most obvious under this condition. However, the effects of elevated H⁺ and P_i on cross‐bridge function may have been saturated in the severe fatigue‐mimicking condition, potentially masking any age‐related differences in the sensitivity of the cross‐bridge to the metabolites. For example, numerous studies have observed a hyperbolic relationship between the concentration of P_i and peak isometric force, where any increase in [P_i] above ∼25–30 mm has minimal effect on peak force (Coupland et al., 2001; Fryer et al., 1995; Pathare et al., 2005; Tesi et al., 2002; Wang & Kawai, 1997). Whether a similar hyperbolic relationship occurs in peak power, velocity and/or force when H⁺ and P_i are elevated together in conditions that more closely mimic the fatigue environment in vivo is not known. It is also unknown whether the depressive effects of H⁺ and P_i on cross‐bridge function differs in muscle fibres from women compared to men.

Therefore, the present study aimed to test the force–velocity relationship and peak power of skeletal muscle fibres from young and older men and women across a range of elevated H⁺ and P_i that occur in vivo. We first hypothesize that the decrements in fibre force and power have a hyperbolic relationship with the increase in the concentrations of H⁺ and P_i. We also hypothesize that the decrements in power are more pronounced in fibres from older compared to young men and women at low to moderate levels of H⁺ and P_i, but that the age differences are not present in the severe fatigue‐mimicking condition (pH 6.2 + 30 mm P_i) because the effects of the metabolites are nearly saturated. Based on previous studies reporting that the mechanisms of fatigue do not appear to differ between men and women during most dynamic contraction tasks (Delgadillo et al., 2021; Hunter, 2016; Sundberg et al., 2017; Sundberg, Kuplic et al., 2018), we expect that the depressive effects of elevated H⁺ and P_i are similar in fibres isolated from women and men. Finally, because we incorporated measures of both whole‐muscle function and single fibre contractile mechanics, we used our integrated approach to explore the potential cellular mechanisms for the age‐related loss in power output of the knee extensor muscles.

Methods

Participants and ethical approval

Six young men (20–32 years), seven young women (20–29 years), six older men (70–90 years) and six older women (71–87 years) volunteered and provided their written informed consent to participate in this study. Participants underwent a general health screening and were excluded from the study if they were taking medications that affect the central nervous system, muscle mass or neuromuscular function (e.g. hormone‐replacement therapies, antidepressants, glucocorticoids, etc.). All participants were apparently healthy, community‐dwelling adults free of any known neurological, musculoskeletal, or cardiovascular diseases. All experimental procedures were approved by the Marquette University Institutional Review Board (Protocol Number HR‐2945) and conformed to the principles in the Declaration of Helsinki.

Experimental overview

Participants reported to the laboratory on two occasions, once for a muscle biopsy of the vastus lateralis muscle and the other to measure whole‐muscle function and fatigability of the knee extensor muscles. The whole‐muscle function and fatigability session was used to assess whether the older adults demonstrated conventional age‐related changes of the knee extensor muscles compared to the young adults, including (1) lower thigh lean mass, (2) lower absolute and mass specific mechanical power outputs and (3) an increased fatigability (reductions in mechanical power) when performing a moderate‐ to high‐velocity dynamic exercise (Dalton et al., 2012; Reid & Fielding, 2012; Sundberg, Hunter et al., 2018; Sundberg, Kuplic et al., 2018). Two young participants (one man and one woman) participated only in the muscle biopsy session and did not complete the whole‐muscle function and fatigability experiments.

Whole‐muscle knee extensor function and fatigability

The experimental setup to measure whole‐muscle function of the knee extensors was identical to the setup described previously (Sundberg, Kuplic et al., 2018). Briefly, testing was performed on the dominant leg of each participant (preferred kicking leg) and participants were seated upright in the high Fowler's position with the starting knee position set at 90° flexion in a Biodex System 4 Dynamometer (Biodex Medical, Shirley, NY, USA). Extraneous movements and changes in hip angle were minimized by securing the participants to the seat with the dynamometer's four‐point restraint system. To ensure the measured torques and velocities were generated primarily by the knee extensor muscles, participants were prohibited from grasping the dynamometer with their hands.

Maximal voluntary contraction (MVC) torque

Prior to the maximal voluntary isometric strength assessment, participants performed a standardized warmup consisting of dynamic knee extension exercise lifting a light load (1 Nm). Following the warmup, participants performed a minimum of three brief (2–3 s) knee extension MVCs with at least 60 s rest between contractions. Participants were provided strong verbal encouragement and visual feedback on their performance with a 56 cm monitor mounted 1–1.5 m directly in front of their line of vision. The torque during each MVC was quantified as the average over an interval of 0.5 s centered on the peak torque, and MVC attempts were continued until the two highest values were within 5% of each other.

Dynamic fatiguing exercise

Following the isometric MVC measurements, participants were habituated to performing maximal velocity knee extensions against a 20% MVC load applied by the dynamometer. To minimize the effect of the additional braking force applied by the dynamometer at the end of the range of motion, the maximum total displacement was set to 95° with the starting position at 90° knee flexion. For the dynamic fatiguing exercise, participants were provided strong verbal encouragement to kick as fast as possible once every 3 s for a total of 4 min (80 contractions). Contraction‐by‐contraction power outputs (W) were calculated as the product of the measured torque (Nm) and angular velocity (rad s⁻¹) and averaged over the entire shortening phase of the knee extension. Because power output increased over the first few contractions in some participants as observed previously (Sundberg, Hunter et al., 2018; Sundberg, Kuplic et al., 2018; Sundberg et al., 2019), the baseline power output for each participant was the highest average obtained from five sequential contractions within the first 10 contractions. Fatigability was quantified by expressing the average power output from the last five contractions as a percentage of the individual‐specific baseline power output. The fatigability data from one older male was excluded from the analysis, because their power output increased over the 4 min exercise indicating the participant did not follow the instructions to perform a maximal effort exercise.

Thigh lean mass

Body composition and thigh lean mass was assessed with dual X‐ray absorptiometry (Lunar iDXA; GE, Madison, WI, USA). Thigh lean mass was quantified for the region of interest from the manufacturer's software (enCORE 14.10.022; GE), with the distal demarcation set at the tibiofemoral joint and the proximal demarcation set as a diagonal bifurcation through the femoral neck. DXA measures of thigh lean mass with these landmarks are strongly correlated with measures from magnetic resonance imaging but underestimate the age‐related loss in thigh muscle mass (Maden‐Wilkinson et al., 2013).

Muscle biopsy

A muscle biopsy from the vastus lateralis was obtained from each participant as described previously (Bergstrom, 1962; Sundberg, Hunter et al., 2018). Participants were instructed to refrain from strenuous exercise of the lower limbs for 48 h prior to the biopsy and arrive at the laboratory fasted and without consumption of caffeine for ≥8 h. The biopsy location was cleaned with 70% ethanol, sterilized with 10% povidone–iodine and anaesthetized with 1% lidocaine HCl. A small ∼1 cm incision was made overlying the distal 1/3 of the muscle belly, and the biopsy needle inserted under local suction to obtain the tissue sample. Two longitudinal bundles from the biopsy sample were immediately submerged in cold glycerol skinning solution (see below) and stored at −20°C. All single fibre contractile experiments were completed within 4 weeks of the biopsy.

Single fibre morphology and contractile mechanics experiments

Solutions

The composition of the relaxing (pCa 9.0, where pCa = −log[Ca²⁺]) and activating (pCa 4.5) solutions were derived from an iterative program using the stability constants adjusted for temperature, pH and ionic strength (Fabiato, 1988; Fabiato & Fabiato, 1979). All solutions contained (in mm): 20 imidazole, 7 EGTA, 4 MgATP and 14.5 creatine phosphate. Inorganic phosphate (P_i) was added as K₂HPO₄ to yield a concentration of 4, 12, 20 or 30 mm. Although no P_i was added to the 0 mm P_i solution, there is evidence that the actual concentration of P_i is between 0.4 and 0.7 mm as a result of the hydrolysis and resynthesis of ATP and to impurities in stock reagents (Pate & Cooke, 1989). Mg²⁺ was added as MgCl₂ with a specified free concentration of 1 mm, and Ca²⁺ was added as CaCl₂. The ionic strength was adjusted to 180 mm with KCl, and the pH was adjusted to 7.0, 6.8, 6.6 or 6.2 with KOH or HCl. The skinning solution was composed of 50% relaxing solution and 50% glycerol (v:v).

Experimental setup

Contractile function experiments were performed on ∼2–3 mm long single fibre segments isolated from the muscle biopsy sample as described previously (Sundberg, Hunter et al., 2018). On the day of experimentation, a fibre was isolated from the biopsy, and the ends secured with 4.0 monofilament posts tied with 10.0 nylon sutures to a force transducer (400A, Aurora Scientific, Aurora, Ontario, CA) and high‐speed servomotor (controller 312C; Aurora Scientific) in a plastic chamber containing cold relaxing solution (Moss, 1979). Once the fibre was attached, the position of the force transducer and servomotor were adjusted so the fibre could be suspended in 100–120 µL of relaxing solution cooled to 12°C by a temperature‐controlled Peltier unit. The fibre remained in the 12°C relaxing solution, except when transferred either into air for imaging or to a second Peltier for activation at 15°C.

To view the fibre at 800× magnification, the microsystem was transferred to the stage of an inverted microscope. The sarcomere length was adjusted to 2.5 µm using a calibrated eyepiece micrometer and the fibre length measured as the distance between the two points of attachment via a mechanical micrometer (Starrett; Athol, MA, USA). Fibre width was determined from a digital image (CoolSNAP ES; Roper Scientific Photometrics, Tucson, AZ, USA) taken when the fibre was briefly suspended in air (<5 s). The fibre width was measured at three locations along the segment length, and the average fibre diameter and cross‐sectional area (CSA) were calculated assuming the fibre forms a cylinder when in the air.

Experimental design

All single fibre contractile experiments began with a sequence of four or five contractions (activating solution – pH 7.0 + 0 mm P_i) to determine the maximal Ca²⁺‐activated isometric force (P_o ) and unloaded shortening velocity (V_o ). Each fibre then underwent a series of force–velocity experiments at 15°C in a condition that mimics quiescent human skeletal muscle (pH 7.0 + 4 mm P_i) and in conditions that mimic mild (pH 6.8 + 12 mm P_i), moderate (pH 6.6 + 20 mm P_i) and severe metabolite accumulation (pH 6.2 + 30 mm P_i). The pH 7.0 + 0 mm P_i activating condition was used for comparison with other single fibre experiments in animals (Knuth et al., 2006; Metzger & Moss, 1990; Nelson et al., 2014) and humans (D'Antona et al., 2003; Frontera et al., 2008; Lamboley et al., 2015; Trappe et al., 2003), whereas the pH 7.0 + 4 mm P_i activating condition was used to more closely mimic the [P_i] in the quiescent human quadriceps muscle (Kemp et al., 2007). The order of the experimental conditions was pseudo‐randomized for each fibre to alleviate the potential of an order effect. Fibres with visible tears or that had a decrease in the maximal Ca²⁺‐activated isometric tension to <90% of the initial P_o within a condition were excluded from further analysis. This occurred in 25 out of the 327 fibres studied [3 myosin heavy chain (MyHC) I, 16 MyHC IIa and 6 MyHC IIa/Iix] resulting in 302 fibres completing all 5 experimental conditions (134 MyHC I, 2 MyHC I/IIa, 150 MyHC IIa, 15 MyHC IIa/IIx and 1 MyHC IIx).

Unloaded shortening velocity (V_o ) experiment

Unloaded shortening velocity (V_o ) was determined using the slack test (Edman, 1979) as described previously (Sundberg, Hunter et al., 2018; Widrick et al., 1996). Fibres were maximally activated in saturating Ca²⁺ (pCa 4.5), allowed to generate peak isometric force (P_o ), and then rapidly shortened with the servomotor to a predetermined distance so that force was momentarily reduced to zero. The fibre remained activated until the redevelopment of force, after which, the fibre was returned to relaxing solution and re‐extended to its original position. Fibres were activated four or five times in pH 7.0 + 0 mm P_i and slacked at varying distances (100–450 µm) that never exceeded a distance >20% fibre length. The V_o was the slope of the least squares regression line between the slack distance and the time required to begin the redevelopment of force.

Force–velocity experiments

In the second set of experiments, force–velocity and force–power curves were obtained as described previously (Sundberg, Hunter et al., 2018). Fibres were maximally activated in saturating Ca²⁺ (pCa 4.5), allowed to generate peak isometric force (P_o ), and then subjected to three predetermined submaximal isotonic loads (300‐FC1 Force Controller; Positron Development, Inglewood, CA, USA). Fibres were activated four to six times under each condition to obtain 12–18 different isotonic loads. Each force–velocity curve was fit with an iterative non‐linear curve fitting procedure (Levenberg–Marquardt algorithm) using the hyperbolic Hill equation (Hill, 1938) (Fig. 1) in accordance with:

$$\left(P+a\right)\left(V+b\right)=\frac{\left(P_o+a\right)}{b}$$ (1)

where P is force, V is velocity, P_o is peak isometric force, and a and b are constants with dimensions of force and velocity, respectively (Widrick et al., 1996). Absolute (µN fl s⁻¹) and size specific power (W L⁻¹) were calculated as the product of shortening velocity (fl s⁻¹) and absolute (µN) and size specific force (kN m⁻²), respectively, and the peak fibre power determined using the fitted parameters from the force–velocity curve (Widrick et al., 1996). The reported absolute and size specific P_o was the average from all the contractions within each condition.

Figure 1. Representative force–velocity and force–power curves from a fast MyHC IIa fibre at 15°C.

Muscle fibres were activated four to six times in each condition to obtain the fibre shortening velocities from at least 12 isotonic loads in all four activating conditions. The activating conditions included one mimicking quiescent human skeletal muscle (pH 7.0 + 4 mm P_i) and three with a range of metabolite concentrations to mimic mild (pH 6.8 + 12 mm P_i), moderate (pH 6.6 + 20 mm P_i) and severe fatigue (pH 6.2 + 30 mm P_i). Presented in (A) are data traces of the fibre shortening (top) at three isotonic loads (bottom) during maximal Ca²⁺ activations in both the quiescent and severe fatigue conditions. Traces are overlaid for visual comparison between the two conditions. Force–velocity plots were fit with the hyperbolic Hill equation for each condition (B) and the force–power curves (C) were constructed by calculating the power values for each force level from the force–velocity curves. The labelled points in (B) correspond with the data traces presented in (A). Data are from a fast MyHC IIa fibre extracted from a biopsy of a 32‐year‐old man. The contractile mechanics from this fibre were P_{o  =} 1.99 mN, V _max = 3.36 fl s⁻¹, PPw = 252 µN fl s⁻¹ in pH 7.0 + 4 mm P_i (Quiescent), P_o  = 1.76 mN, V _max = 3.35 fl s⁻¹, PPw = 215 µN fl s⁻¹ in pH 6.8 + 12 mm P_i (Mild), P_{o  =} 1.52 mN, V _max = 3.08 fl s⁻¹, PPw = 173 µN fl s⁻¹ in pH 6.6 + 20 mm P_i (Moderate) and P_{o  =} 1.26 mN, V _max = 2.75 fl s⁻¹, PPw = 114 µN fl s⁻¹ in pH 6.2 + 30 mm P_i (Severe).

Myosin heavy chain (MyHC) composition

MyHC composition of the isolated fibres were determined by SDS‐PAGE and silver staining as described previously (Giulian et al., 1983). Briefly, following the contractile experiments each fibre was solubilized in 80 µL of SDS sample buffer and loaded on a gel made up of a 3% acrylamide/bis (19:1) stacking layer and 5% separating layer. Gels were run 20–24 h at 4°C (SE600; Hoefer, Holliston, MA, USA), stained, imaged and visually inspected to classify the MyHC isoform composition (I, I/IIa, I/IIa/IIx, IIa, IIa/IIx and IIx) of each fibre.

The MyHC distribution of the vastus lateralis muscle for each participant was determined by homogenizing a portion of the biopsy sample (>10 mg) in 30X (vol:weight) RIPA buffer with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The homogenized samples were run in quadruplicate for each participant with SDS‐PAGE, and the relative abundance of each MyHC isoform (I, IIa, and IIx) was quantified using densitometry and averaged over the four runs for each participant. Because of the low abundance of MyHC IIx in most participants, we combined the IIa and IIx isoforms for each participant and report the values as MyHC II.

Statistical analyses

Anthropometrics, whole‐muscle knee extensor function and MyHC distribution were compared between age groups (young or old) and sex (men or women) with individual univariate ANOVA. Statistical analyses for the anthropometrics, whole‐muscle knee extensor function and MyHC distribution were performed using SPSS, version 27.0 (IBM Corp., Armonk, NY, USA).

To test for differences in single fibre morphology and contractile mechanics between young and old men and women, a nested ANOVA was used with age (young or old), sex (men or women) and fibre type (I or IIa) as the fixed factors. Of the 327 fibres studied, 303 fibres were either pure MyHC IIa (166 fibres) or I (137 fibres) and only 1 pure IIx, 2 hybrid I/IIa and 21 hybrid IIa/IIx fibres were observed. Because of the small sample sizes of the pure IIx and hybrid I/IIa and IIa/IIx fibres, only pure I and IIa fibres could be included in all the statistical comparisons to test for differences in age and sex, whereas the IIa/IIx fibres were included to test for fibre type differences in the pH 7.0 + 0 mm P_i activating condition irrespective of age or sex. Repeated‐measures nested ANOVAs were employed to test the effect of the activating condition (pH 7.0 + 0 mm P_i, pH 7.0 + 4 mm P_i, pH 6.8 + 12 mm P_i, pH 6.6 + 12 mm P_i and pH 6.2 + 30 mm P_i) on P_o and the force–velocity parameters of the pure MyHC I and IIa fibres from the young and old men and women, with pairwise post hoc comparisons performed using Tukey's method. Statistical analyses for the single fibre data were performed using Minitab, version 21.0 (Minitab Inc., State College, PA, USA). P < 0.05 was considered statistically significant. Data are presented as the mean ± SD in the text and tables and the mean ± SEM in the figures.

Results

Whole‐muscle knee extensor function, fatigability and MyHC distribution

Anthropometrics and whole‐muscle function of the knee extensors are presented in Table 1. As expected, the thigh lean mass was ∼24% greater in young (6.7 ± 1.8 kg) compared to old (5.4 ± 1.0 kg; P = 0.008) and ∼35% greater in men (6.9 ± 1.7 kg) compared to women (5.1 ± 0.8 kg; P = 0.001). Absolute mechanical power output of the knee extensors was 147% greater in young (270.7 ± 123.5 W) compared to old (109.5 ± 38.7 W; P < 0.001) and 60% greater in men (231.7 ± 143.3 W) compared to women (145.2 ± 79.7 W; P = 0.007). After normalizing for the differences in thigh lean mass, mass‐specific power output remained 98% higher in young (39.4 ± 7.5 W kg⁻¹) compared to old (19.9 ± 3.9 W kg⁻¹; P < 0.001) and 16% higher in men (31.5 ± 11.5 W kg⁻¹) compared to women (27.1 ± 11.5 W kg⁻¹; P = 0.032) (Fig. 2). Fatigability of the knee extensor muscles was greater with ageing as indicated by the average relative reduction in power of 37% ± 18% in the old compared to 21% ± 15% in young (P = 0.045), with no differences between men (31% ± 18%) and women (28% ± 18%; P = 0.615). The MyHC distribution of the vastus lateralis did not differ between young (52% ± 18% MyHC I) and old (58% ± 11% MyHC I; P = 0.177) or between men (52% ± 19% MyHC I) and women (57% ± 11% MyHC I; P = 0.411).

Table 1.

Anthropometrics and neuromuscular function of the knee extensor muscles

			Young		Old		P value
Variable		Units	Men (6)	Women (7)	Men (6)	Women (6)	Age	Sex	Age × Sex
	Age	years	25.4 ± 4.2	23.5 ± 3.3	76.1 ± 7.4	72.6 ± 5.9	<0.001	0.709	0.204
	Height	cm	178.3 ± 10.2	168.6 ± 3.2	174.5 ± 6.9	163.5 ± 4.4	0.107	<0.001	0.817
	Weight	kg	81.4 ± 18.3	63.1 ± 7.6	77.5 ± 8.2	69.1 ± 10.3	0.831	0.010	0.305
	BMI	kg m−2	25.3 ± 3.0	22.2 ± 2.4	25.5 ± 3.3	25.9 ± 4.0	0.150	0.304	0.187
	Body fat	%	17.2 ± 3.5 (5)	26.0 ± 6.2 (6)	26.7 ± 7.3	38.7 ± 8.3	<0.001	0.001	0.576
	Thigh lean mass	kg	7.8 ± 2.1 (5)	5.7 ± 0.6 (6)	6.2 ± 0.8	4.6 ± 0.5	0.008	0.001	0.524
Knee extensor function
	MVC torque – Absolute	Nm	296.6 ± 62.0 (5)	164.3 ± 30.3 (6)	191.4 ± 44.6	100.8 ± 16.6	<0.001	<0.001	0.234
	MVC torque – Mass specific	Nm kg−1	38.4 ± 5.9 (5)	28.8 ± 3.6 (6)	31.0 ± 5.7	21.9 ± 2.4	0.001	<0.001	0.895
	Power – Absolute	W	342.2 ± 149.0 (5)	211.1 ± 58.0 (6)	139.6 ± 30.2	79.3 ± 14.2	<0.001	0.007	0.283
	Power – Mass specific	W kg−1	42.3 ± 7.3 (5)	36.9 ± 7.3 (6)	22.5 ± 3.0	17.3 ± 2.9	<0.001	0.032	0.968
	Fatigability – Power	% ∆	−26 ± 14 (5)	−17 ± 16 (6)	−36 ± 22 (5)	−38 ± 15	0.045	0.615	0.486

Body fat percentage and lean mass were measured via dual X‐ray absorptiometry. MVC torque was the highest torque output recorded from the MVC attempts in the whole‐muscle function testing session. Mechanical power was the highest average obtained from five sequential contractions of the first 10 contractions performed during the dynamic fatiguing exercise. Mass‐specific torque and power were calculated with the thigh lean mass. The sample size (n) for each cohort is reported in parentheses. A significant age or sex difference is indicated in bold when P < 0.05. Values are the mean ± SD.

Figure 2. Whole‐muscle mechanical power output and fatigability of the knee extensors.

Mean absolute mechanical power outputs of the knee extensors were ∼60% lower in the old compared to young and ∼37% lower in women compared to men (A). After normalizing for the age and sex differences in thigh lean mass, mass‐specific power remained ∼49% lower in the old compared to young and ∼14% lower in women compared to men (B). The fatigability of the knee extensors did not differ between men and women but was greater with ageing as indicated by an average relative reduction in power of 37% in the old compared to 21% in the young (C). The number of participants (n) is displayed within the bars, and each dot in (A) and (B) is the data from an individual participant. Values are displayed as the mean ± SEM. The asterisk (*) in (C) indicates a main effect of age at P = 0.045.

Single fibre morphology and contractile mechanics

Presented in Table 2 are the fibre diameter and CSA for the pure MyHC I and IIa fibres, along with the peak isometric force (P_o ) and unloaded shortening velocity (V_o ) from the pH 7.0 + 0 mm P_i activating condition. The CSA of MyHC I fibres did not differ between young (6037 ± 1811 µm²) and old (5303 ± 2159 µm², P = 0.205) or between men (5926 ± 2477 µm²) and women (5402 ± 1432 µm², P = 0.408). Similarly, absolute P_o of MyHC I fibres did not differ between young (0.98 ± 0.23 mN) and old (0.96 ± 0.33 mN, P = 0.718) or between men (0.98 ± 0.33 mN) and women (0.95 ± 0.24 mN, P = 0.549), nor did size‐specific P_o differ between young (165 ± 24 kN m⁻²) and old (186 ± 34 kN m⁻², P = 0.060) or between men (172 ± 29 kN m⁻²) and women (179 ± 33 kN m⁻², P = 0.789). By contrast, the CSA of MyHC IIa fibres was 41% smaller in fibres from old (3639 ± 1821 µm²) compared to young (6193 ± 2527 µm², P < 0.001) and 42% smaller in fibres from women (3500 ± 1274 µm²) compared to men (6084 ± 2750 µm², P < 0.001). Accordingly, the absolute P_o was 36% lower for the MyHC IIa fibres from old (0.79 ± 0.32 mN) compared to young (1.23 ± 0.49 mN, P < 0.001) and 38% lower in fibres from women (0.76 ± 0.25 mN) compared to men (1.22 ± 0.51 mN, P < 0.001). The age and sex differences in absolute P_o were explained entirely by the differences in the MyHC IIa fibre CSA as indicated by the greater size‐specific P_o in old (227 ± 37 kN m⁻²) compared to young (202 ± 30 kN m⁻², P = 0.002) and no differences between women (221 ± 32 kN m⁻²) and men (210 ± 38 kN m⁻², P = 0.115). Independent of age and sex, the size‐specific P_o of MyHC I fibres (176 ± 31 kN m⁻²) was 18% lower than MyHC IIa fibres (215 ± 36 kN m⁻², P < 0.001) and 24% lower than MyHC IIa/IIx fibres (233 ± 40 kN m⁻², P < 0.001), with no differences between IIa and IIa/IIx fibres (P = 0.241).

Table 2.

Peak fibre force (P_o ) and unloaded shortening velocity (V_o ) in pH 7.0 + 0 mm P_i

			Men			Women			P value
			Young	Old	Diff.	Young	Old	Diff.	Age	Sex	Age × Sex
Slow MyHC I		n	32	36		35	34
	Diameter	µm	87.1 ± 15.6	83.5 ± 18.5	↔	86.5 ± 9.5	77.8 ± 11.1	↔	0.144	0.484	0.500
	CSA	µm2	6,141 ± 2,253	5,735 ± 2,659	↔	5,942 ± 1,311	4,846 ± 1,352	↔	0.205	0.408	0.530
	Absolute Po	mN	0.96 ± 0.27	1.00 ± 0.38	↔	0.99 ± 0.20	0.91 ± 0.27	↔	0.718	0.549	0.293
	Size‐specific Po	kN m−2	161.1 ± 21.5	181.9 ± 31.3	↔	169.3 ± 25.4	189.7 ± 36.6	↔	0.060	0.789	0.667
	Vo	fl s−1	1.36 ± 0.32	1.40 ± 0.29	↔	1.36 ± 0.30	1.20 ± 0.21	↔	0.355	0.230	0.155
Fast MyHC IIa		n	41	46		38	41
	Diameter	µm	100.4 ± 12.5	72.3 ± 17.5	↓ 28%	72.6 ± 8.8	59.0 ± 12.7	↓ 19%	<0.001	<0.001	0.192
	CSA	µm2	8,045 ± 2,038	4,337 ± 2,033	↓ 46%	4,195 ± 1,036	2,857 ± 1,135	↓ 32%	<0.001	<0.001	0.087
	Absolute Po	mN	1.57 ± 0.43	0.91 ± 0.34	↓ 42%	0.86 ± 0.22	0.66 ± 0.25	↓ 23%	<0.001	<0.001	0.111
	Size‐specific Po	kN m−2	196.5 ± 31.5	221.3 ± 40.5	↑ 13%	207.7 ± 26.7	233.6 ± 31.9	↑ 12%	0.002	0.115	0.860
	Vo	fl s−1	3.81 ± 0.84	3.96 ± 0.86	↔	3.58 ± 0.72	3.29 ± 0.85	↔	0.765	0.019	0.473

Fibre diameter and cross‐sectional area (CSA) were calculated from a digital image taken when the fibre was briefly suspended in air (<5 s). P_o and V_o were measured from the slack test. The number of fibres (n) is reported for each fibre type from the young and older men and women. A significant age or sex difference is indicated in bold when P < 0.05. To compare these data with the prevailing ageing single fibre literature, which are primarily single sex studies (Grosicki et al. 2022), the percentage difference (Diff.) between young and old for men and women is also reported separately when P < 0.05. Values are the mean ± SD.

The unloaded shortening velocity (V_o ) of the slow MyHC I fibres did not differ between young (1.36 ± 0.30 fl s⁻¹) and old (1.30 ± 0.27 fl s⁻¹; P = 0.355) or between men (1.38 ± 0.30 fl s⁻¹) and women (1.28 ± 0.27 fl s⁻¹; P = 0.230). Similarly, the V_o of the fast MyHC IIa fibres did not differ between young (3.70 ± 0.79 fl s⁻¹) and old (3.64 ± 0.92 fl s⁻¹; P = 0.765) but was 13% greater in men (3.89 ± 0.85 fl s⁻¹) compared to women (3.43 ± 0.80 fl s⁻¹; P = 0.019). Independent of age and sex, V_o was 64% lower in MyHC I (1.33 ± 0.29 fl s⁻¹) compared to IIa fibres (3.67 ± 0.86 fl s⁻¹; P < 0.001) and 23% lower in MyHC IIa compared to IIa/IIx fibres (4.77 ± 0.95 fl s⁻¹; P < 0.001).

Cumulative effects of H⁺ and P_i on the force–velocity relationship and peak power

Slow MyHC I fibres

The mean force–velocity curves, absolute P_o and maximal shortening velocity (V _max) from the MyHC I fibres are shown in Fig. 3, with the force–velocity parameters reported in the Appendix (Table A1). Elevated levels of H⁺ and P_i caused the force–velocity relationship to shift down and to the left (Fig. 3), with P_o and V _max both progressively decreasing with increasing concentrations of these metabolites (P < 0.001). However, the relative reduction in P_o and V _max induced by H⁺ and P_i did not differ between the cohorts in any of the conditions (P > 0.05). Independent of age and sex, the P_o of slow MyHC I fibres was reduced by 17% ± 5%, 25% ± 8% and 40% ± 9% and V _max reduced by 6% ± 6%, 12% ± 7% and 21% ± 8% in the mild‐, moderate‐ and severe‐fatigue conditions compared to the 4 mm P_i condition, respectively (P < 0.001).

Figure 3. Force–velocity curves, P_o and V _max of slow MyHC I fibres.

Increasing the concentrations of H⁺ and P_i shifted the force–velocity curves of the MyHC I fibres down and to the left (A). Peak isometric force (B) and maximal shortening velocity (C) both progressively decreased with increasing concentrations of H⁺ and P_i in the MyHC I fibres from all four cohorts. However, the combined depressive effects of H⁺ and P_i, which is depicted as the [H₂PO₄ ⁻], did not differ between any of the cohorts. The [H₂PO₄ ⁻] was calculated with the equation $[{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_{4^{-}}]=\frac{[{\mathrm{P}}_{\mathrm{i}}]}{1+{10}^{\mathrm{pH}-6.838}}$, where 6.838 is the calculated pK _a of [H₂PO₄ ⁻] at 15°C and an ionic strength of 0.18 m using equation 12 from Mauger (2017). The number of fibres (n) is displayed within the bars, and each dot is the data from an individual fibre. Values are displayed as the mean ± SEM. The variances around the mean curves in (A) have been omitted for clarity, and the error bars in (B) and (C) are obscured by the symbols.

The mean force–power curves and absolute peak power from the slow MyHC I fibres are shown in Fig. 4, with peak power values reported in the Appendix (Table A1). There were no age or sex differences in absolute peak power for any of the activating conditions (P > 0.05). There were no sex differences in size‐specific peak power for any of the activating conditions (P > 0.05), and no age differences in the pH 7.0 + 4 mm P_i (P = 0.056). However, the size‐specific peak power was greater in the older compared to the young in the pH 6.8 + 12 mm P_i (P = 0.049), the pH 6.6 + 20 mm P_i (P = 0.013), and pH 6.2 + 30 mm P_i (P = 0.031) conditions. Similar to the findings on P_o and V _max, peak power progressively decreased with increasing concentrations of H⁺ and P_i (P < 0.001), but the relative reduction in power induced by H⁺ and P_i did not differ between the cohorts in any of the conditions (P > 0.05). Independent of age and sex, the peak power of slow MyHC I fibres was reduced by 10 ± 5%, 25 ± 9 and 51 ± 6% in the mild‐, moderate‐ and severe‐fatigue conditions compared to the 4 mm P_i condition, respectively (P < 0.001).

Figure 4. Force–power curves and peak power of slow MyHC I fibres.

Increasing the concentrations of H⁺ and P_i shifted the force‐power curves of the MyHC I fibres down and to the left (A). Peak power (B) progressively decreased with increasing concentrations of H⁺ and P_i in the MyHC I fibres from all four cohorts. However, the combined depressive effects of H⁺ and P_i, which is depicted as the [H₂PO₄ ⁻], did not differ between any of the cohorts. The [H₂PO₄ ⁻] was calculated with the equation $[{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_{4^{-}}]=\frac{[{\mathrm{P}}_{\mathrm{i}}]}{1+{10}^{\mathrm{pH}-6.838}}$, where 6.838 is the calculated pK _a of [H₂PO₄ ⁻] at 15°C and an ionic strength of 0.18 m using equation 12 from Mauger (2017). The number of fibres (n) is displayed within the bars, and each dot is the data from an individual fibre. Values are displayed as the mean ± SEM. The variances around the mean curves in (A) have been omitted for clarity, and the error bars in (B) are obscured by the symbols.

Fast MyHC IIa fibres

The mean force–velocity curves, absolute P_o and V _max from the MyHC IIa fibres are shown in Fig. 5, with key force–velocity parameters reported in the Appendix (Table A2). Similar to the MyHC I fibres, elevated levels of H⁺ and P_i caused the force–velocity relationship to shift down and to the left (Fig. 5), with P_o and V_max both progressively decreasing with the increasing concentrations of these metabolites (P < 0.001). Also similar to the MyHC I fibres, the relative reduction in P_o and V_max induced by H⁺ and P_i did not differ between the cohorts in any of the conditions (P > 0.05). Independent of age and sex, the P_o of fast MyHC IIa fibres was reduced by 13 ± 4%, 24 ± 5% and 40 ± 5% and V_max reduced by 7 ± 6%, 13 ± 8% and 24 ± 10% in the mild‐, moderate‐ and severe‐fatigue conditions compared to the 4 mm P_i condition, respectively (P < 0.001).

Figure 5. Force–velocity curves, P_o and V _max of fast MyHC IIa fibres.

Increasing the concentrations of H⁺ and P_i shifted the force–velocity curves of the MyHC IIa fibres down and to the left (A). Peak isometric force (B) and maximal shortening velocity (C) both progressively decreased with increasing concentrations of H⁺ and P_i in the MyHC IIa fibres from all four cohorts. However, the combined depressive effects of H⁺ and P_i, which is depicted as the [H₂PO₄ ⁻], did not differ between any of the cohorts. The [H₂PO₄ ⁻] was calculated with the equation $[{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_{4^{-}}]=\frac{[{\mathrm{P}}_{\mathrm{i}}]}{1+{10}^{\mathrm{pH}-6.838}}$, where 6.838 is the calculated pK _a of [H₂PO₄ ⁻] at 15°C and an ionic strength of 0.18 m using equation 12 from Mauger (2017). The number of fibres (n) is displayed within the bars, and each dot is the data from an individual fibre. Values are displayed as the mean ± SEM. The variances around the mean curves in (A) have been omitted for clarity, and the error bars in (B) and (C) are obscured by the symbols.

The mean force–power curves and absolute and size‐specific peak power from the fast MyHC IIa fibres are shown in Fig. 6, with peak power values reported in the Appendix (Table A2). Peak absolute power outputs were 31–33% lower in old compared to young adults (P < 0.01) and 40–43% lower in women compared to men in all four activating conditions (P < 0.001). There was no sex difference in size‐specific peak power; however, older adults had a greater size‐specific peak power compared to young adults in all conditions (P < 0.05). Similar to the findings on P_o and V_max , peak power progressively decreased with increasing concentrations of H⁺ and P_i (P < 0.001), but the relative reduction in peak power induced by H⁺ and P_i did not differ between the cohorts in any of the conditions (P > 0.05). Independent of age and sex, the peak power of fast MyHC IIa fibres was reduced by 12 ± 6%, 29 ± 7% and 55 ± 6% in the mild‐, moderate‐ and severe‐fatigue conditions compared to the 4 mm P_i condition, respectively (P < 0.001).

Figure 6. Force–power curves and peak power of fast MyHC IIa fibres.

Increasing the concentrations of H⁺ and P_i shifted the force‐power curves of the MyHC IIa fibres down and to the left (A). Peak power (B) progressively decreased with increasing concentrations of H⁺ and P_i in the MyHC IIa fibres from all four cohorts. However, the combined depressive effects of H⁺ and P_i, which is depicted as the [H₂PO₄ ⁻], did not differ between any of the cohorts. The [H₂PO₄ ⁻] was calculated with the equation $[{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_{4^{-}}]=\frac{[{\mathrm{P}}_{\mathrm{i}}]}{1+{10}^{\mathrm{pH}-6.838}}$, where 6.838 is the calculated pK _a of [H₂PO₄ ⁻] at 15°C and an ionic strength of 0.18 m using equation 12 from Mauger (2017). The age and sex differences in absolute peak power were explained entirely by differences in the fibre size, as indicated by the lack of a sex difference in size‐specific peak power and the higher size‐specific peak power in the old compared to young adults (C). The number of fibres (n) is displayed within the bars, and each dot is the data from an individual fibre. Values are displayed as the mean ± SEM. The variances around the mean curves in (A) have been omitted for clarity, and the error bars in (B) are obscured by the symbols.

Fibre type differences in the detrimental effects of H⁺ and P_i in human skeletal muscle independent of age and sex

Peak isometric force (P_o )

The fibre type differences in size‐specific P_o more than doubled when [P_i] was increased from ∼0 to 4 mm (Fig. 7A ), whereby the size‐specific P_o was 39% lower in MyHC I (105 ± 22 kN m⁻²) compared to MyHC IIa fibres in the pH 7.0 + 4 mm P_i condition (171 ± 31 kN m⁻²) (P < 0.001). The increase in the fibre type difference in size‐specific P_o with the small increase in [P_i] was the result of a greater sensitivity of MyHC I fibres to P_i (P < 0.001). The H⁺‐ and P_i‐induced decrements in P_o in the moderate‐ and severe‐fatigue mimicking conditions did not differ between the fibre types when expressed as a percentage of P_o in the 4 mm P_i condition (P > 0.05) (Fig. 7A ), but the depressive effects in the mild‐fatigue mimicking condition was 4% greater in the MyHC I compared to the MyHC IIa fibres (P < 0.001).

Figure 7. Cumulative effects of H⁺ and P_i on force, velocity and power in fast MyHC IIa and slow MyHC I fibres.

Peak isometric force (P_o ), maximal shortening velocity (V _max), and peak power progressively decreased with increasing concentrations of H⁺ and P_i in both MyHC IIa and MyHC I fibres. Decreasing the [P_i] from 4 to ∼0 mm increased P_o more in slow compared to fast fibres (A). The reductions in V _max were greater in MyHC IIa compared to MyHC I fibres in the severe fatigue condition (B), whereas the reductions in peak power were greater in MyHC IIa compared to MyHC I fibres in all three fatigue‐mimicking conditions (C). The combined depressive effects of H⁺ and P_i are depicted as [H₂PO₄ ⁻], which was calculated with the equation $[{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_{4^{-}}]=\frac{[{\mathrm{P}}_{\mathrm{i}}]}{1+{10}^{\mathrm{pH}-6.838}}$, where 6.838 is the calculated pK _a of [H₂PO₄ ⁻] at 15°C and an ionic strength of 0.18 m using equation 12 from Mauger (2017). Note that the P_o has data above 100% because we measured P_o in the pH 7.0 + 0 mm P_i condition, which is the most commonly used activating condition in the single fibre literature (for a review, see Grosicki et al., 2022). However, this metabolic environment does not exist in vivo, which suggests that these values are supraphysiological. We did not conduct the force–velocity experiments in the pH 7.0 + 0 mm P_i condition, which is why the data points are absent in (B) and (C). Values are the mean ± SEM (Error bars are obscured by the symbols. The asterisk (*) denotes a significant difference between slow and fast fibres at P < 0.05.

Maximal shortening velocity (V_max )

The V_max in all four activating conditions was ∼140% greater in fast MyHC IIa compared to slow MyHC I fibres (P < 0.001) (Fig. 7B ). The V_max decreased linearly in both fibre types with the increase in the concentrations of H⁺ and P_i. However, the reduction in V_max was greater in MyHC IIa compared to MyHC I fibres in the severe‐fatigue mimicking condition (P < 0.001), with no fibre type differences for the reductions in V_max in the mild‐ and moderate‐fatigue mimicking conditions (P > 0.05).

Peak power

The size‐specific peak power in all 4 activating conditions was ∼5 times greater in fast MyHC IIa compared to slow MyHC I fibres (P < 0.001) (Fig. 7C ). The higher peak power in MyHC IIa fibres was primarily explained by the ∼60% greater size‐specific P_o and ∼140% greater V_max in MyHC IIa compared to MyHC I fibres but was also partially a result of the lesser curvature (i.e. higher a/P_o ratio) of the force–velocity relationship in MyHC IIa compared to MyHC I fibres (data reported in the Appendix in Tables A1 and A2). Consistent with the effects of H⁺ and P_i on V_max , peak power decreased linearly in both fibre types with the increase in the concentrations of H⁺ and P_i. However, the H⁺‐ and P_i‐induced decrements in power were 2–4% greater in MyHC IIa compared to MyHC I fibres for all three fatigue‐mimicking conditions (P < 0.05) (Fig. 7C ).

Discussion

The primary aim of the present study was to determine the effects of H⁺ and P_i on the force–velocity relationship and peak power of skeletal muscle fibres from young and older adults across a range of metabolite concentrations that occur in vivo. In agreement with our first hypothesis, there was a hyperbolic relationship between the decrements in peak isometric force and the increase in concentrations of H⁺ and P_i for both the slow MyHC I and fast MyHC IIa fibres (Figs 3, 5 and 7). However, when the data from the ∼0 mm P_i condition were removed to only include the concentrations of metabolites observed in vivo (Kemp et al., 2007), the relationship between the decrements in isometric force and the increased concentration of metabolites was linear. Similarly, the shortening velocity and peak power decreased linearly with increasing concentrations of H⁺ and P_i (Figs 3, 4, 5, 6, 7). Contrary to our second hypothesis, the depressive effects of the metabolites were similar in fibres from young and older adults across all conditions. Although the decrements in cross‐bridge function from H⁺ and P_i did not differ with age or sex, the absolute force and power of the fast MyHC IIa fibres from older adults (Figs 5 and 6), particularly the older women, may have reached critically low levels in the fatigue‐mimicking conditions where the power necessary to maintain mobility and balance might be compromised. The markedly lower absolute force and power of the fast MyHC IIa fibres from the older men and women was explained entirely by the age differences in fibre size (Figs 5 and 6 and Table 2; see also the Appendix, Table A2). These data suggest the age‐related loss in power is determined primarily by fast fibre atrophy in men and women, but the age‐related increase in fatigability cannot be explained by an increased sensitivity of the cross‐bridge to H⁺ and P_i.

Cumulative effects of H⁺ and P_i on cross‐bridge function: important mediators of human muscle fatigue

Most studies on the effects of H⁺ and P_i on cross‐bridge function have focused on either the individual effects of these ions or tested their effects under extreme concentrations (Chase & Kushmerick, 1988; Debold et al., 2004, 2006; Fryer et al., 1995; Knuth et al., 2006; Metzger & Moss, 1987, 1990; Nelson & Fitts, 2014; Nelson et al., 2014; Pate & Cooke, 1989; Pate et al., 1995; Potma et al., 1995). In addition, only one study has tested the effects of H⁺ and P_i on human skeletal muscle (Sundberg, Hunter et al., 2018), which differs markedly in contractile kinetics, fibre type distribution and metabolic properties compared to muscles from smaller mammalian or amphibious species (Edman, 2005; Schiaffino, 2010; Schiaffino & Reggiani, 2011; Shirokova et al., 1996). In the present study, we observed that isometric force, shortening velocity and peak power of human skeletal muscle fibres all progressively decreased with the increase in the concentrations of H⁺ and P_i, and that the detrimental effects of these metabolites on cross‐bridge function showed no signs of saturating at the higher concentrations. These novel findings have important implications for human skeletal muscle performance, because changes in the concentrations of H⁺ and P_i within the myoplasm occur at the onset of high‐intensity contractile activity and can continue to accumulate until the individual can no longer perform the task (Broxterman et al., 2017; Chidnok et al., 2013; Hogan et al., 1999; Jones et al., 2008). The detrimental effects of these metabolites may also be particularly important to the physical function of older adults because (1) they already have impaired force and power generating capacity compared to younger adults (Alcazar et al., 2020; Wrucke et al., 2024) and (2) older adults have a greater accumulation of metabolites within the muscle during dynamic fatiguing contractions compared to younger adults (Sundberg et al., 2019).

Similar to previous findings on the effects of P_i on isometric force (Coupland et al., 2001; Fryer et al., 1995; Pathare et al., 2005; Tesi et al., 2002; Wang & Kawai, 1997), the relationship between the decrements in isometric force and the increase in the concentrations of both H⁺ and P_i was hyperbolic (Figs 3, 5 and 7). However, the hyperbolic relationship was only observed when the data from the pH 7.0 + 0 mm P_i condition were included, which is a condition that does not occur in human skeletal muscle in vivo (Kemp et al., 2007). By contrast, when the data from the ∼0 mm P_i condition were excluded, there was a linear relationship between the decrements in isometric force and the increase in the concentrations of H⁺ and P_i that occurred similarly in fibres from young and older men and women. This finding is in close agreement with the linear relationship observed between the concentrations of dihydrogen phosphate (H₂PO₄ ⁻) and the reductions in force in young and older adults during a voluntary fatiguing isometric exercise (Kent‐Braun et al., 2002; Lanza et al., 2007). Collectively, these results provide strong evidence that H⁺ and P_i are important mediators of human muscle fatigue by directly impairing the ability of the cross‐bridge to generate force. It should be noted that the role H⁺ and P_i each individually have on force depression remains an area of intense debate (Fitts, 2016; Westerblad, 2016); however, there is compelling evidence in skinned fibres from rodent muscle that when H⁺ and P_i are elevated together, there is a much more pronounced effect on force than when studying either P_i or H⁺ in isolation (Karatzaferi et al., 2008; Nelson & Fitts, 2014). Additionally, whether H₂PO₄ ⁻ is the primary phosphate species to inhibit isometric force is unknown (Fitts, 1994; Nosek et al., 1987), but it does provide an accurate marker to quantify the changes occurring in both H⁺ and P_i because H₂PO₄ ⁻ becomes the predominant phosphate species with the decrease in pH (H₂PO₄ ⁻ pK _a = 6.75 at 38°C) (Lawson & Veech, 1979).

Surprisingly, the decrements in the maximal shortening velocity also showed a linear relationship with the increase in the concentrations of H⁺ and P_i for the fast MyHC IIa and slow MyHC I fibres from young and older men and women (Figs 3, 5 and 7). There are several non‐human, animal studies demonstrating that P_i has little‐to‐no effect on shortening velocity, and that H⁺ is the primary metabolite that inhibits velocity (Chase & Kushmerick, 1988; Cooke et al., 1988; Debold et al., 2004, 2016; Karatzaferi et al., 2008; Metzger & Moss, 1987; Nelson et al., 2014; Widrick, 2002). However, controversy still exists as to the relative importance of decreased pH in inhibiting velocity (Fitts, 2016; Karatzaferi et al., 2008; Knuth et al., 2006; Pate et al., 1995; Westerblad, 2016; Westerblad et al., 1997), which led to the hypothesis that H⁺ does not inhibit shortening velocity until pH drops below ∼6.7 (Fitts, 2008). By contrast, we observed that the maximal shortening velocity measured by extrapolation of the force–velocity relationship (V _max) was already significantly decreased in the pH 6.8 + 12 mm P_i condition, albeit only by ∼6%, and continued to decrease with the increase in the concentrations of H⁺ and P_i. The explanation for the discrepancies between these findings compared to findings from animal studies is unclear, but perhaps is a result of differences in the contractile kinetics between mammalian species or that the pH 6.8 condition was studied in combination with 12 mm P_i. Future studies should provide a more comprehensive assessment of the individual and combined effects of elevated H⁺ and P_i to assess how these metabolites affect shortening velocity in human skeletal muscle.

A noteworthy observation is that the fatigue‐induced reductions in the maximal shortening velocity of the human adductor pollicis muscle in vivo (Jones et al., 2006) has a time course that is generally similar to the changes in intracellular pH during fatiguing exercise (Broxterman et al., 2017; Fiedler et al., 2016; Sundberg et al., 2019). Specifically, during high‐intensity exercise, intracellular pH initially becomes more alkaline because of the predominance of ATP synthesized by the creatine kinase reaction (Adams et al., 1990) followed by the precipitous decline in pH from the high rates of ATP hydrolysis and increased glycolytic flux (Robergs et al., 2004). The delay in H⁺ accumulation mirrors the delay in the fatigue‐induced reduction in shortening velocity observed in the human adductor pollicis muscle in vivo (Jones et al., 2006). These results, interpreted together with the findings presented in this study, suggest that H⁺ is an important factor for the fatigue‐induced reduction in shortening velocity in human skeletal muscle.

Although valuable mechanistic insight is gleaned from examining the effects of H⁺ and P_i on peak isometric force and maximal shortening velocity, these parameters represent only two extremes of the force–velocity relationship. Thus, it is important from both a human performance and an ageing perspective to examine how these ions influence the ability of skeletal muscle to shorten under submaximal loads and generate power. The combination of the H⁺‐ and P_i‐induced reductions of force and shortening velocity resulted in marked decrements in peak power in both MyHC I and IIa fibres (Figs 4, 6 and 7). By contrast to the findings on isometric force and shortening velocity, the H⁺‐ and P_i‐induced reductions in peak power were 2%–4% greater in MyHC IIa compared to MyHC I fibres in all three fatigue‐mimicking conditions (Fig. 7). The explanations for the fibre type dependence in the decrements in power are unclear, but may partly be a result of the modest differences in the H⁺‐ and P_i‐induced changes in the curvature of the force–velocity relationship (a/P_o ) in the fatigue‐mimicking conditions (see Appendix, Tables A1 and A2). Irrespective of the potential explanation for the fibre type differences, there was a linear relationship between the decrements in peak power and the increase in concentrations of H⁺ and P_i that occurred similarly in both fibre types from young and older men and women. This finding is in close agreement with the linear relationship observed between the concentrations of H₂PO₄ ⁻ and the reductions in power output in young and older adults during a voluntary fatiguing dynamic exercise (Sundberg et al. 2019). Collectively, these data provide compelling evidence that H⁺ and P_i are important mediators of human muscle fatigue by directly impairing the power generating capacity of the cross‐bridge. By contrast to our hypothesis, we found no evidence in any of our contractile mechanics measurements that fibres from older men and women are more sensitive to the depressive effects of elevated H⁺ and P_i compared to fibres from younger men and women, at least not in saturating Ca²⁺ conditions. Future studies should examine the effects of elevated H⁺ and P_i under submaximal Ca²⁺ conditions in fibres from young and older men and women, because fatigue during high‐intensity contractions is thought to also involve a decrease in myoplasmic free Ca²⁺ (Allen et al. 2011; Lee et al. 1991).

Fast fibre atrophy is an important factor contributing to power loss with ageing

A secondary aim of the present study was to gain insight into the potential mechanisms for the age‐related loss in mechanical power output by integrating measures of whole‐muscle function with single fibre contractile mechanics. As expected, the older men and women demonstrated hallmark signs of ageing in the knee extensor muscles, including a 19% lower thigh lean mass, 60% lower absolute mechanical power and 49% lower mass‐specific power compared to the younger men and women (Fig. 1 and Table 1). The lower mass‐specific power is evidence for the accelerated loss in power output relative to muscle mass that is commonly observed with ageing. However, despite the widespread recognition, the mechanisms for this ageing phenomenon remains elusive. Our data suggests that the age‐related loss in whole‐muscle power is determined, in large part, by the atrophy of the fibres expressing the fast MyHC isoforms in both men and women (Fig. 6).

Several mechanisms have been proposed to contribute to the greater loss in power relative to muscle mass with ageing, including a decreased ability of the nervous system to voluntarily activate the muscle (Harridge et al., 1999; Russ et al., 2012), infiltration of intermuscular adipose and fibrotic tissue (Beavers et al., 2013; Kent‐Braun et al., 2000; Lexell, 1995; Straight et al., 2019), motor unit remodeling and instability of the neuromuscular junction (Hepple & Rice, 2016), impaired cross‐bridge mechanics and Ca²⁺ handling (Lamboley et al., 2015; Larsson et al., 1997; Miller & Toth, 2013), and/or the selective atrophy of fibres expressing the fast MyHC II isoforms (Grosicki et al., 2022; Sundberg, Hunter et al., 2018; Teigen et al., 2020). Although we did not measure voluntary activation of the participants in the present study, there is compelling evidence that most community‐dwelling older adults are able to voluntarily activate their muscles to a similar level as younger adults (Rozand et al., 2020) and that the age‐related loss of whole‐muscle power is determined primarily by factors disrupting the contractile properties with the muscle (Wrucke et al., 2024). In our study, we found that the only contractile properties depressed in the muscle fibres from older men and women were the absolute P_o and peak power of the fast MyHC IIa fibres (Figs 3, 4, 5, 6). However, when the differences in absolute P_o and peak power were normalized to the differences in fibre size, the older men and women generated either similar or higher size‐specific P_o and power in all the activating conditions compared to younger men and women. These data are in agreement with a large body of literature observing similar or higher size‐specific fibre force and power in older compared to younger adults (Gries et al., 2019; Grosicki et al., 2021; Miller et al., 2013; Sundberg, Hunter et al., 2018; Teigen et al., 2020; Trappe et al., 2003; Venturelli et al., 2015) but in contrast to others (Brocca et al., 2017; Lamboley et al., 2015; Larsson et al., 1997; Yu et al., 2007). The explanation for the disparate findings between studies is unclear, but, as suggested previously (Grosicki et al. 2022), probably involves methodological challenges with single fibre experiments, such as the low fibre n in most studies and/or the difficulty in accurately estimating fibre CSA with 2‐D images. Notably, we found that V_o , which is a measure of cross‐bridge kinetics that is independent of fibre CSA, also did not differ between the young and older adults (Table 2). These data provide additional support that the lower absolute P_o and peak power in the fast MyHC IIa fibres from the older men and women were primarily a result of fibre atrophy rather than age‐related alterations in the intrinsic contractile properties. Given the fast fibres generate markedly greater power than slow fibres (Fig. 7; see also the Appendix, Tables A1 and A2), these data implicate fast fibre atrophy as a potential important therapeutic target for preserving whole‐muscle power with ageing in both men and women.

Concluding remarks

The data in the present study provide novel evidence that progressively increasing the concentrations of H⁺ and P_i elicits a linear decrease in force, velocity and power of human skeletal muscle fibres and that the detrimental effects of these metabolites on cross‐bridge function show no signs of saturating at higher concentrations. Importantly, the depressive effects of H⁺ and P_i did not differ in any of the fatigue‐mimicking conditions in fibres from young and older men and women, suggesting that the age‐related increase in fatigability cannot be attributed to a greater sensitivity of the cross‐bridge to these metabolites. In agreement with our previous findings (Sundberg, Hunter et al., 2018; Teigen et al., 2020), we observed that the markedly lower absolute force and power of the fast fibres in older men and women was as a result of differences in the size of the fibres, not impaired intrinsic contractile function. These findings have important applied implications because the power of the fast fibres from the older adults, particularly the older women, may reach critically low levels in the fatigue conditions where generating the power necessary for mobility may become compromised. We conclude that H⁺ and P_i are both important mediators of human muscle fatigue by directly impairing cross‐bridge function and that therapeutic interventions should target attenuating fast fibre atrophy to help offset some of the age‐related decrements in whole‐muscle power.

Competing interests

The authors declare that they have no competing interests.

Author contributions

C.W.S. conceived, designed and conducted the experiments. C.W.S. and L.E.T. analyzed and interpreted the data. C.W.S. and L.E.T. drafted the manuscript. C.W.S., L.E.T., S.K.H. and R.H.F. revised the manuscript for important intellectual content. All authors approved the final version of the manuscript submitted for publication and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was supported by an American Heart Association postdoctoral fellowship (19POST34380411) to CWS and a National Institute of Aging grant (R01AG048262) to CWS, SKH and RHF.

Supporting information

Peer Review History

Biography

Christopher Sundberg is an Assistant Professor in the Department of Physical Therapy and director of the Integrative Muscle Physiology and Energetics laboratory at Marquette University. The goal of his laboratory is to identify the aetiologies of muscle fatigue and the physiological processes that limit human neuromuscular function in health, ageing and disease. A major focus of his research aims to develop a comprehensive understanding of the mechanisms responsible for the age‐related loss in muscle power output and increased fatigability by integrating techniques to study these phenomena in the intact neuromuscular system down to the cellular and molecular levels.

Table A1.

Force–velocity parameters and peak power of slow MyHC I fibers at 15°C

		Men		Women		Combined		P value
		Young (31)	Old (34)	Young (35)	Old (34)	Young (66)	Old (68)	Age	Sex	Age × Sex
Po (mN)
	pH 7.0 + 4 mm Pi	0.56 ± 0.15	0.60 ± 0.22	0.59 ± 0.14	0.54 ± 0.17	0.57 ± 0.14	0.57 ± 0.20	0.890	0.572	0.174
	pH 6.8 + 12 mm Pi	0.45 ± 0.12	0.51 ± 0.18	0.48 ± 0.12	0.45 ± 0.15	0.47 ± 0.12	0.48 ± 0.17	0.734	0.459	0.106
	pH 6.6 + 20 mm Pi	0.41 ± 0.12	0.47 ± 0.17	0.44 ± 0.13	0.41 ± 0.15	0.42 ± 0.13	0.44 ± 0.16	0.887	0.466	0.160
	pH 6.2 + 30 mm Pi	0.33 ± 0.09	0.36 ± 0.13	0.34 ± 0.11	0.34 ± 0.13	0.34 ± 0.10	0.35 ± 0.13	0.800	0.515	0.382
Po (kN m−2)
	pH 7.0 + 4 mm Pi	94 ± 17	113 ± 20	101 ± 18	113 ± 26	98 ± 18	113 ± 23	0.054	0.972	0.337
	pH 6.8 + 12 mm Pi	76 ± 17	98 ± 20	83 ± 17	93 ± 25	80 ± 17	96 ± 21	0.045	0.675	0.269
	pH 6.6 + 20 mm Pi	70 ± 18	89 ± 18	74 ± 18	86 ± 24	72 ± 18	87 ± 22	0.041	0.822	0.485
	pH 6.2 + 30 mm Pi	57 ± 17	70 ± 16	60 ± 14	71 ± 22	58 ± 15	70 ± 19	0.068	0.812	0.879
V max (fl s−1)
	pH 7.0 + 4 mm Pi	1.54 ± 0.19	1.57 ± 0.18	1.56 ± 0.18	1.44 ± 0.20	1.55 ± 0.19	1.51 ± 0.20	0.369	0.372	0.126
	pH 6.8 + 12 mm Pi	1.45 ± 0.18	1.48 ± 0.16	1.46 ± 0.16	1.34 ± 0.17	1.46 ± 0.17	1.41 ± 0.18	0.256	0.122	0.092
	pH 6.6 + 20 mm Pi	1.36 ± 0.18	1.39 ± 0.14	1.35 ± 0.16	1.25 ± 0.18	1.35 ± 0.17	1.32 ± 0.17	0.434	0.139	0.105
	pH 6.2 + 30 mm Pi	1.20 ± 0.14	1.25 ± 0.13	1.22 ± 0.18	1.14 ± 0.16	1.22 ± 0.16	1.20 ± 0.15	0.600	0.497	0.108
Peak power (µN fl s−1)
	pH 7.0 + 4 mm Pi	22.4 ± 6.9	24.6 ± 8.9	23.5 ± 7.3	20.7 ± 6.0	23.0 ± 7.1	22.6 ± 7.8	0.660	0.476	0.115
	pH 6.8 + 12 mm Pi	20.2 ± 6.6	22.2 ± 8.2	21.2 ± 6.8	18.7 ± 5.6	20.7 ± 6.7	20.5 ± 7.2	0.720	0.471	0.120
	pH 6.6 + 20 mm Pi	16.9 ± 5.8	18.5 ± 6.7	16.7 ± 4.9	15.6 ± 5.1	16.8 ± 5.3	17.1 ± 6.1	0.980	0.335	0.224
	pH 6.2 + 30 mm Pi	11.0 ± 3.4	12.0 ± 4.5	11.1 ± 3.5	10.2 ± 3.4	11.1 ± 3.4	11.1 ± 4.0	0.954	0.400	0.285
Peak power (W L−1)
	pH 7.0 + 4 mm Pi	3.8 ± 0.8	4.7 ± 1.2	4.0 ± 1.0	4.3 ± 0.9	3.9 ± 0.9	4.5 ± 1.0	0.056	0.786	0.319
	pH 6.8 + 12 mm Pi	3.4 ± 0.7	4.2 ± 1.0	3.6 ± 1.0	3.9 ± 0.8	3.5 ± 0.9	4.1 ± 0.9	0.049	0.849	0.314
	pH 6.6 + 20 mm Pi	2.8 ± 0.6	3.5 ± 0.8	2.9 ± 0.7	3.3 ± 0.7	2.8 ± 0.7	3.4 ± 0.8	0.013	0.589	0.523
	pH 6.2 + 30 mm Pi	1.9 ± 0.4	2.3 ± 0.6	1.9 ± 0.5	2.1 ± 0.5	1.9 ± 0.5	2.2 ± 0.5	0.031	0.650	0.697
a/Po
	pH 7.0 + 4 mm Pi	0.039 ± 0.007	0.039 ± 0.005	0.037 ± 0.007	0.040 ± 0.007	0.038 ± 0.007	0.039 ± 0.006	0.498	0.802	0.325
	pH 6.8 + 12 mm Pi	0.048 ± 0.010	0.044 ± 0.005	0.047 ± 0.012	0.050 ± 0.011	0.047 ± 0.011	0.047 ± 0.009	0.439	0.573	0.289
	pH 6.6 + 20 mm Pi	0.047 ± 0.011	0.043 ± 0.005	0.044 ± 0.013	0.049 ± 0.011	0.046 ± 0.012	0.046 ± 0.009	0.991	0.426	0.152
	pH 6.2 + 30 mm Pi	0.045 ± 0.014	0.039 ± 0.007	0.040 ± 0.010	0.042 ± 0.013	0.042 ± 0.012	0.040 ± 0.010	0.714	0.894	0.174

Absolute (µN fl s⁻¹) and size‐specific (W L⁻¹) peak power were calculated with the fitted‐parameters from the force–velocity curves. The absolute (mN) and size‐specific (kN m⁻²) P_o for each fibre was the average from all the contractions within each condition. The maximal shortening velocity (V _max) was calculated using the Hill equation, and the a/P_o ratio is a unitless parameter describing the curvature of the force–velocity relationship. The number of fibres (n) for each cohort is reported in parentheses. Values are the mean ± SD.

Table A2.

Force–velocity parameters and peak power of fast MyHC IIa fibres at 15°C

		Men		Women		Combined		P value
		Young (39)	Old (38)	Young (36)	Old (37)	Young (75)	Old (75)	Age	Sex	Age × Sex
Po (mN)
	pH 7.0 + 4 mm Pi	1.25 ± 0.37	0.75 ± 0.30	0.69 ± 0.17	0.53 ± 0.20	0.98 ± 0.41	0.64 ± 0.28	0.004	0.001	0.155
	pH 6.8 + 12 mm Pi	1.09 ± 0.37	0.66 ± 0.26	0.59 ± 0.14	0.45 ± 0.18	0.85 ± 0.36	0.56 ± 0.25	0.004	0.001	0.160
	pH 6.6 + 20 mm Pi	0.95 ± 0.27	0.57 ± 0.22	0.51 ± 0.13	0.40 ± 0.17	0.74 ± 0.31	0.49 ± 0.21	0.003	0.001	0.106
	pH 6.2 + 30 mm Pi	0.75 ± 0.23	0.44 ± 0.16	0.40 ± 0.11	0.33 ± 0.13	0.58 ± 0.26	0.38 ± 0.16	0.003	0.001	0.175
Po (kN m−2)
	pH 7.0 + 4 mm Pi	156 ± 28	180 ± 33	162 ± 20	185 ± 32	161 ± 24	182 ± 33	0.003	0.383	0.939
	pH 6.8 + 12 mm Pi	136 ± 25	158 ± 29	141 ± 18	158 ± 33	139 ± 22	158 ± 31	0.010	0.757	0.702
	pH 6.6 + 20 mm Pi	120 ± 22	138 ± 29	122 ± 17	141 ± 31	121 ± 20	139 ± 30	0.011	0.708	0.884
	pH 6.2 + 30 mm Pi	94 ± 17	107 ± 23	94 ± 15	114 ± 26	94 ± 16	111 ± 25	0.006	0.553	0.537
V max (fl s−1)
	pH 7.0 + 4 mm Pi	3.98 ± 0.51	3.91 ± 0.58	3.75 ± 0.51	3.25 ± 0.67	3.87 ± 0.52	3.58 ± 0.71	0.070	0.009	0.175
	pH 6.8 + 12 mm Pi	3.72 ± 0.50	3.58 ± 0.45	3.48 ± 0.45	2.99 ± 0.58	3.60 ± 0.55	3.29 ± 0.59	0.025	0.005	0.199
	pH 6.6 + 20 mm Pi	3.46 ± 0.49	3.37 ± 0.38	3.25 ± 0.41	2.83 ± 0.62	3.36 ± 0.47	3.10 ± 0.58	0.042	0.003	0.227
	pH 6.2 + 30 mm Pi	3.05 ± 0.50	2.86 ± 0.42	2.86 ± 0.35	2.46 ± 0.60	2.96 ± 0.44	2.66 ± 0.55	0.008	0.009	0.476
Peak power (µN fl s−1)
	pH 7.0 + 4 mm Pi	153.0 ± 46.1	99.1 ± 37.1	87.5 ± 23.6	64.1 ± 23.5	121.6 ± 49.4	81.8 ± 35.6	0.003	0.000	0.255
	pH 6.8 + 12 mm Pi	134.1 ± 42.9	87.8 ± 33.4	77.3 ± 21.8	55.9 ± 22.2	106.9 ± 44.6	72.1 ± 32.5	0.005	0.000	0.288
	pH 6.6 + 20 mm Pi	110.8 ± 34.7	70.4 ± 24.9	62.0 ± 18.3	45.6 ± 18.6	87.3 ± 37.2	58.2 ± 25.2	0.004	0.000	0.170
	pH 6.2 + 30 mm Pi	69.4 ± 21.0	45.3 ± 16.2	37.5 ± 11.3	28.6 ± 12.5	54.1 ± 23.3	37.1 ± 16.7	0.006	0.000	0.170
Peak power (W L−1)
	pH 7.0 + 4 mm Pi	19.4 ± 4.6	24.5 ± 6.2	20.8 ± 3.2	23.3 ± 6.9	20.0 ± 4.0	23.9 ± 6.5	0.005	0.673	0.782
	pH 6.8 + 12 mm Pi	17.0 ± 4.2	21.7 ± 5.7	18.3 ± 2.9	20.2 ± 6.1	17.6 ± 3.7	21.0 ± 5.9	0.007	0.779	0.874
	pH 6.6 + 20 mm Pi	14.1 ± 3.7	17.7 ± 5.0	14.7 ± 2.6	16.5 ± 5.6	14.4 ± 3.2	17.1 ± 5.3	0.012	0.902	0.793
	pH 6.2 + 30 mm Pi	8.8 ± 2.3	11.3 ± 3.1	8.9 ± 1.6	10.4 ± 4.1	8.9 ± 2.0	10.9 ± 3.6	0.007	0.967	0.762
a/Po
	pH 7.0 + 4 mm Pi	0.048 ± 0.010	0.057 ± 0.014	0.055 ± 0.009	0.065 ± 0.020	0.051 ± 0.010	0.061 ± 0.018	0.014	0.059	0.659
	pH 6.8 + 12 mm Pi	0.053 ± 0.011	0.064 ± 0.018	0.062 ± 0.012	0.076 ± 0.057	0.057 ± 0.012	0.070 ± 0.028	0.009	0.072	0.703
	pH 6.6 + 20 mm Pi	0.054 ± 0.013	0.063 ± 0.017	0.061 ± 0.012	0.073 ± 0.034	0.057 ± 0.013	0.068 ± 0.027	0.046	0.097	0.806
	pH 6.2 + 30 mm Pi	0.048 ± 0.012	0.061 ± 0.018	0.052 ± 0.011	0.062 ± 0.031	0.050 ± 0.012	0.061 ± 0.025	0.021	0.414	0.773

Absolute (µN fl s⁻¹) and size‐specific (W l⁻¹) peak power were calculated with the fitted‐parameters from the force–velocity curves. The absolute (mN) and size‐specific (kN m⁻²) P_o for each fibre was the average from all the contractions within each condition. The maximal shortening velocity (V _max) was calculated using the Hill equation (1938), and the a/P_o ratio is a unitless parameter describing the curvature of the force–velocity relationship. The number of fibres (n) for each cohort is reported in parentheses. Values are the mean ± SD.

Data availability statement

The data in the present study are available from the corresponding author upon reasonable request.