Effects of acidosis and inorganic phosphate on Ca²⁺ sensitivity of young and older adult skeletal muscle fibers

Abstract

The cellular mechanisms for the age-related loss in skeletal muscle contractile function and increased fatigability are unresolved. We previously observed that the depressive effects of fatiguing levels of hydrogen (H⁺)(pH6.8–6.6–6.2) and inorganic phosphate (P_i)(12-20-30mM) did not differ in myofibers from young compared with older adults. However, these studies used saturating Ca²⁺, when fatigue during high-intensity contractions in vivo also likely involves a decrease in myoplasmic free Ca²⁺. Thus, we compared the Ca²⁺ sensitivity of myofibers from 10 young (22.1±3.6; 5women) and 13 older (71.7±5.5; 7women) adults in conditions mimicking quiescent (pH7+4mM P_i) and fatigued (pH6.2+30mM P_i) muscle. Fast fiber cross-sectional area was ~35% smaller in older (4,859±2,116µm²) compared with young (7,446±2,399µm², P=0.002), which corresponded with lower maximal absolute force (P_o) in both quiescent (old=0.75±0.30mN; young=1.13±0.32 mN, P=0.002) and fatigue conditions (old=0.35±0.14mN; young=0.52±0.16mN, P=0.002). There were no differences in fast fiber size-specific P_o, indicating the age-related decline in force was due to differences in fiber size. Elevated H⁺ and P_i shifted the force-pCa relationship to the right, confirming non-human studies that these metabolites contribute to fatigue by depressing the sensitivity of the myofilaments to Ca²⁺. However, Ca²⁺ sensitivity was not different with age or sex in either condition, and the metabolite-induced shift in the force-pCa relationship did not differ with age in either the slow (P=0.507) or fast (P=0.115) fibers. These data suggest the age-related increase in fatigability of limb muscles cannot be explained by an increased sensitivity of the myofibers to elevated H⁺ and P_i in maximal or submaximal Ca²⁺.

Graphical Abstract

NEW & NOTEWORTHY

This study reports the effects of elevated H⁺ and P_i on Ca²⁺ sensitivity of human skeletal muscle fibers and determines whether the effects of these metabolites are altered by aging in submaximal Ca²⁺. The metabolites markedly depressed Ca²⁺ sensitivity in human muscle fibers, but there was no effect of age or sex. These data suggest that Ca²⁺ sensitivity is preserved with age in conditions that mimic both quiescent and fatigued muscle.

INTRODUCTION

The ability of older adults to generate the force and power necessary to maintain functional independence and mobility is compromised by several factors, including a loss of muscle mass and an increased fatigability during dynamic contractions (1–3). For example, muscle mass can decline by as much as 30% in individuals aged 60 and older (1). However, it is well-established that the age-related loss of strength and power occurs at a much greater rate than the loss of muscle mass (4–7), suggesting that other factors – such as changes in the nervous system’s ability to activate the muscle and/or alterations in the muscle’s intrinsic contractile function – must also play an important role for the decrements in function. Several studies on community-dwelling, healthy older adults have localized the primary site for the accelerated age-related loss in force and power relative to muscle mass and the increased fatigability during dynamic exercise to factors within the muscle, rather than the nervous system (8–15). Identifying the cellular and molecular mechanisms, however, has proven considerably more challenging, in part, due to the complexity and number of sites within the muscle that may contribute to fatigue and muscle weakness (16–19).

One potential factor contributing to the more precipitous loss in force and power relative to muscle mass with aging is that the structure of the troponin-tropomyosin complex may be altered, resulting in a decreased sensitivity of the myofilaments to Ca²⁺ in fibers from older adults (20). Indeed, several studies have investigated the effects of aging on Ca²⁺ sensitivity in human skeletal muscle fibers, however the findings are equivocal, with some studies reporting decreased Ca²⁺ sensitivity in older compared with young adults, particularly in the fast fibers (21–23), and others observing no age differences (24–26). The explanation for the discrepancies between studies is unknown, but one major limitation of all previous studies is they were conducted in an activating condition mimicking near quiescent skeletal muscle (pH 7.0 + 0 mM P_i), which is a metabolic environment that never exists in human skeletal muscle in vivo. Specifically, the resting concentration of P_i in quiescent human skeletal muscle is ~3–5 mM (27), and both H⁺ and P_i rapidly accumulate in the muscle during high-intensity contractile activity in young and older adults (28). This is critically important because H⁺ and P_i individually and collectively disrupt cross-bridge function in saturating Ca²⁺, while also decreasing the sensitivity of the myofilaments to Ca²⁺ (16, 17). Thus, the first aim of the present study was to compare the Ca²⁺ sensitivity of limb skeletal muscle fibers from young and older men and women under a condition that more closely mimics human quiescent skeletal muscle in vivo (pH 7.0 + 4 mM P_i).

Energetic demands from high-intensity contractile activity can easily exceed the ATP synthesis rates of the mitochondria, resulting in a net reliance on anaerobic metabolic pathways and a buildup of the ATP hydrolysis byproducts, H⁺ and P_i. Given the central role of H⁺ and P_i in the fatigue process (16, 17), we have sought to determine whether the age-related increase in fatigability during dynamic exercise is the result of an increased accumulation of metabolites in the working muscle of older adults (28) and/or an increased sensitivity of the muscle fibers to a given concentration of metabolites (15, 29). To examine the latter, we studied single fiber contractile mechanics from biopsies of the vastus lateralis and found that the decrements in fiber force (P_o), velocity (V_max and V_o), and power elicited by metabolite conditions mimicking 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) did not differ in fibers from young compared to older adults (15, 29). However, these studies were conducted in saturating Ca²⁺ conditions, while fatigue during high-intensity contractions is thought to also involve a decrease in myoplasmic free Ca²⁺ to submaximal levels (30–33). Importantly, studies on muscle fibers from rats, rabbits, and chickens have found that H⁺ and P_i both reduce the sensitivity of the myofilament to Ca²⁺, but through different mechanisms, and that when the metabolites are elevated together the decrements in Ca²⁺ sensitivity are much greater than when elevated in isolation (34–38). To our knowledge, only a single study has measured the effects of H⁺ on Ca²⁺ sensitivity in human muscle fibers and found that reducing the pH from 7.1 to 6.6 decreased the Ca²⁺ sensitivity of fibers from young men, however, no P_i was added in either pH condition (39). Thus, the second aim of the present study was to examine the Ca²⁺ sensitivity of human muscle fibers in a fatigue-mimicking condition (pH 6.2 + 30 mM P_i) and compare the effects of the metabolites on force production in submaximal Ca²⁺ in fibers from young and older men and women. We hypothesized that 1) fibers from older men and women, particularly fast fibers, would exhibit lower Ca²⁺ sensitivity compared to fibers from young adults, 2) elevated levels of H⁺ and P_i would decrease the sensitivity of the myofilaments to Ca²⁺ in human skeletal muscle fibers, and 3) the depressive effects of the metabolites on Ca²⁺ sensitivity and force production at submaximal Ca²⁺ levels would be more pronounced in muscle fibers from older men and women.

MATERIALS AND METHODS

Subjects.

Ten young adults (5 men and 5 women; 19–31 years) and 13 older adults (6 men and 7 women; 64–81 years) volunteered to participate in this study. Participants were given a general health screening that included an assessment of body composition and thigh lean mass with dual X-ray absorptiometry (Lunar iDXA; GE, Madison, WI). Participants were healthy, community dwelling adults free of any known neurological, musculoskeletal, and cardiovascular diseases, and were excluded from participation if they had any major health concerns. All subjects provided written informed consent, and procedures were approved by the Marquette University Institutional Review Board 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 and once to assess whole-muscle function and fatigability of the knee extensor muscles. The whole-muscle function and fatigability session was conducted using a Biodex System 4 Dynamometer (Biodex Medical, Shirley, NY, USA), as described in detail previously (9, 15). Briefly, after a warmup, participants performed a minimum of three brief (2–3 s) maximal voluntary isometric contractions (MVCs) of the knee extensors, with at least 60 s rest between contractions. Following MVC measurements, a dynamic fatiguing exercise was performed at a load equivalent to 20% MVC, where participants were instructed to kick as fast as possible once every 3 s for a total of 4 min (80 contractions). Fatigability was quantified by expressing the mean power output from the last five contractions as a percentage of the individual-specific baseline power output measured at the beginning of the exercise. The whole-muscle session aimed to determine whether older adults exhibited conventional age-related differences in knee extensor muscle performance compared with young adults, including 1) lower absolute and mass-specific mechanical force and power outputs (4, 5, 8), and 2) greater fatigability (reduction in mechanical power) during moderate- to high-velocity dynamic exercise (9, 14, 40).

Physical activity assessment.

Physical activity was quantified for each participant with a triaxial accelerometer (GT3X; ActiGraph, Pensacola, FL, USA) worn around the waist for at least 4 days (2 weekdays and 2 weekend days) as reported previously (15, 41). The data were recorded for each participant if the accelerometer was worn for a minimum of 8 hours on at least 3 days (42).

Muscle biopsy.

Muscle biopsies of the vastus lateralis were performed using the modified Bergström technique as previously described (15). A longitudinal bundle of each biopsy for the single fiber contractile experiments was immediately placed in ice cold glycerol skinning solution (see below) and stored at −20 °C for up to 2 weeks. The remaining portions of the biopsy sample were flash frozen in liquid nitrogen-cooled isopentane and stored at −80 °C until sectioned for immunohistochemistry (IHC).

Solutions.

The solutions were designed to specifically isolate the effects of elevated H⁺ and P_i on single fiber force and Ca²⁺ sensitivity, independent of other metabolite changes that typically occur during fatiguing exercise in vivo (e.g., elevated creatine, lactate, ADP, AMP, IMP, and Mg²⁺, or reduced creatine phosphate). Solutions were derived from an iterative program using the stability constants adjusted for temperature, pH and ionic strength (43, 44) and contained (in mM): 20 imidazole, 7 EGTA, 4 free MgATP, 1 free Mg²⁺, and 14.5 creatine phosphate. ATP was added as Na₂ATP, Mg as MgCl₂, creatine phosphate as Na₂ phosphocreatine, and Ca²⁺ as CaCl₂. Inorganic phosphate (P_i) was added as K₂HPO₄ to yield a concentration of 4 mM (pH 7.0) or 30 mM (pH 6.2). The ionic strength of both the activating and relaxing solutions were adjusted to 180 mM with KCl. The pH was adjusted to 7.0 with KOH in the quiescent condition and to 6.2 with HCl in the fatigue condition. The relaxing solution contained negligible amounts of Ca²⁺ (pCa 9.0, where pCa = −log₁₀[Ca²⁺]), while the activating solution contained saturating levels of Ca²⁺ (pCa 4.5). A range of activating solutions from pCa 6.6 to 4.8 were made by mixing appropriate volumes of pCa 9.0 and 4.5 solutions (45). Glycerol skinning solution was composed of 50% relaxing solution and 50% glycerol (vol/vol).

Single fiber preparation.

Fibers were prepared as described previously (15, 26). Briefly, single fiber segments between 5 and 10 mm in length were isolated from the biopsy sample and tied to a force transducer (400A; Aurora Scientific) and a high-speed servomotor (controller 312B; Aurora Scientific) with ~2–3 mm of the fiber suspended between the two attachment points. Fibers were kept at 16 °C with a temperature-controlled Peltier unit in 120 µL relaxing solution for the duration of the experiment, except when transferred briefly to a second Peltier unit containing 120 µL of activating solution set at 20 °C (46). Sarcomere length was adjusted to 2.5 µm, and fiber length and diameter were determined as described previously (26). CSA was calculated from the mean fiber diameter, assuming the fiber forms a cylinder when suspended briefly in air (<2 s). Fibers were used for multiple conditions if the force remained at >90% of the initial force measured in the first experimental condition. After completing experiments, the MyHC composition of each fiber was determined by SDS-PAGE and silver staining as previously described (15).

Force-pCa relationship.

The force-pCa relationship was determined as described previously (26, 37, 45) using solutions to mimic both quiescent (pH 7.0 + 4 mM P_i) and fatigued (pH 6.2 + 30 mM P_i) muscle (Figure 1). Single fibers were activated a total of 25 times in a series of solutions with [Ca²⁺] ranging from pCa 6.6 to 5.4 (quiescent) and from pCa 5.8 to 4.8 (fatigued). Each fiber was activated until it reached peak force, which was determined visually by monitoring the force traces on a digital oscilloscope (PicoScope 4262, Pico Technologies). At lower forces, where small changes in force were more difficult to detect visually, contractions were allowed to proceed until forces appeared stable for several seconds (Figure 1A). At higher forces, where peak force was reached more rapidly, contractions were terminated once it was evident that little to no further increase in force would occur with continued activation. This approach was used to preserve fiber integrity, as human single muscle fibers, particularly fast fibers, are more susceptible to damage and tearing than rodent fibers (unpublished observations). The force-pCa relationship of both conditions were fit with Hill plots to determine the [Ca²⁺] eliciting half-maximal force (pCa₅₀), the lowest [Ca²⁺] that elicits force (activation threshold), and the slope of the force-pCa relationship above (n₁) and below (n₂) pCa₅₀ (see discussion for a more thorough description of the Hill slope). The first and last contractions in each condition were performed at pCa 4.5 to assess fiber quality and ensure force remained >90% of the initial force. The quiescent and fatigue conditions were randomized to prevent an order effect and only fibers that successfully completed the force-pCa curves for both conditions were included.

Fig. 1.

Representative force traces and force-pCa curves from fast myosin heavy chain (MyHC) IIa fibers from a 69-year-old woman. A: Force traces at different free [Ca²⁺] (pCa) from a single MyHC IIa fiber in both quiescent (pH 7 + 4 mM P_i, black) and fatigue (pH 6.2 + 30 mM P_i, grey) conditions. The fiber was moved from relaxing solution (pCa 9.0) to activating solution with the indicated pCa, allowed to reach peak, and then slacked 20% of the fiber length before being moved back into relaxing solution. B and C: Representative force-pCa curves from a single MyHC IIa fiber in both quiescent and fatigue conditions in absolute force (B) and expressed relative to the condition specific peak force (P_o) produced in pCa 4.5 (C).

Immunohistochemistry.

To complement the single fiber CSA data from the contractile mechanics experiments, which are known to be limited by sample size and unintentional selection bias against the smaller fibers (47), we performed immunohistochemistry (IHC) to assess the CSA, percent distribution, and proportional area of each fiber type. Muscle tissue was equilibrated to the cryostat (HM525NX, ThermoScientific) temperature of −21 °C for ≥30 min, oriented upright and exposed in Neg-50 (Ref. 6502, Epredia), and then cross-sectioned at 7 µm with a low-profile disposable microtome blade (Ref. 3053835, Epredia) at a 10° clearance angle. Muscle sections were picked up with Superfrost Plus slides (Ref. 6776214, Epredia), air-dried at room temperature (RT) for ≥1 hr, and stored at −80 °C until staining for myosin heavy chain (MyHC) distribution. On the day of staining, slides were taken from −80 °C, air-dried for ≥30 min at RT, and sections circled with an ImmEdge pen (H-4000, Vector Laboratories), allowing ≥15 min to air-dry and establish a hydrophobic barrier. After rehydration in a coplin staining jar with 3x 5 min washes of 1X phosphate-buffered saline (PBS) at pH 7.4, muscle sections were incubated in a humidified slide stain tray (M918–2, Simport Scientific) with a primary antibody (1°Ab) cocktail consisting of rabbit (Rb) α-laminin IgG (1:100, L9393, Sigma-Aldrich, RRID:AB_477163), mouse (Ms) α-MyHC I IgG2b (1:100, BA-D5-c; DSHB, RRID:AB_2235587), Ms α-MyHC IIa IgG1 (1:500, SC-71-s; DSHB, RRID:AB_2147165), Ms α-MyHC IIx IgM (1:50, 6H1-s; DSHB, RRID:AB_1157897), and 1X PBS for 90 min at RT. Muscle sections were then washed in a coplin staining jar 3x for 5 min each with fresh 1X PBS and subsequently incubated in a secondary antibody (2°Ab) cocktail of Goat (Gt) α-Rb IgG (H+L), AMCA (1:50, CI-1000-1.5, Vector Laboratories, RRID:AB_2336195), Gt α-Ms IgG2b Alexa Fluor (AF) 647 (1:250, A21242, Invitrogen, RRID:AB_2535811), Gt α-Ms IgG1 AF488 (1:250, A21121, Invitrogen, RRID:AB_2535764), Gt α-Ms IgM AF555 (1:500, A21426, Invitrogen, RRID:AB_2535847), and 1X PBS for 60 min at RT. Sections were again washed 3x for 5 min with fresh 1X PBS, mounted with 1:1 1X PBS/glycerol (5092-02, Macron Fine Chemicals), and coverslipped with a micro cover glass (48393-221, VWR). Coverslips were sealed onto the slides with clear nail polish and then stored at 4 °C until imaging.

Muscle section images were acquired (Fig. S1) using a high-resolution fluorescence microscope (BZ-X810, Keyence) with a Plan Apochromat 20x mounted objective (BZ-PA20, Keyence) and filters DAPI (49000, Chroma Technology), Cy5 (49006, Chroma Technology), FITC/Alexa Fluor 488/Fluo3/Oregon Green (49011, Chroma Technology), and CY3/TRITC (49004, Chroma Technology). Automated detection software, MyoVision (48), was used for fiber type specific CSA and distribution analysis with manual verification of all fibers. Proportional area of each fiber type was then quantified as the fiber type specific area (µm²) divided by total fiber area (µm²) multiplied by 100.

Statistical analysis.

For IHC, MyHC I, IIa, IIa/IIx, and II (IIa, IIa/IIx, and IIx combined) were included in the analysis, whereas the MyHC I/IIa hybrids and pure IIx were excluded due to their low prevalence in most participants. Similarly, there was a small number of hybrids (MyHC I/IIa and IIa/IIx) in the single fiber mechanics experiments, and thus, only pure MyHC I and IIa fibers were included in the analysis. Additionally, some fibers were excluded from the contractile mechanics analysis if they did not meet quality criteria (see above). Accordingly, a total of 404 fibers were attempted, 16 of which did not meet quality criteria and were discarded. Of the remaining 388 fibers, 8 were MyHC I/IIa hybrids, 59 were MyHC IIa/IIx hybrids, and 1 was MyHC IIx, leaving 320 fibers for the analysis (158 MyHC I and 162 MyHC IIa). To account for the hierarchical structure of the single fiber experiments that is caused by studying multiple fibers from each participant, we used a mixed effects ANOVA with the fiber size and contractile mechanics as the dependent variables, and age and sex as the independent variables. In this ANOVA design, testing for a main effect of age requires combining data from men and women, while testing for a main effect of sex requires combining data from young and old participants. To test for an effect of fatigue on contractile mechanics, a repeated measure mixed effects ANOVA was used. MyHC content from IHC, physical activity, whole muscle function and anthropometric data were assessed using a two-way univariate ANOVA. Post-hoc Tukey’s tests were conducted to assess differences between cohorts. When necessary, data were transformed to meet assumptions of normality and homogeneity of variance. Statistical analyses for anthropometrics and whole-muscle knee extensor function were performed with SPSS, version 31.0 (IMB Corp, Armonk, NY, USA). Statistical analyses for single fiber and IHC data were performed using Minitab, version 21.1 (Minitab Inc., State College, PA, USA). Statistical significance was set at P < 0.05. Data are presented as mean ± SD in the text and tables.

RESULTS

Whole-muscle knee extensor function

Anthropometrics, physical activity levels, and whole-muscle knee extensor function of the participants are reported in Table 1. As expected, absolute maximal voluntary isometric contraction (MVC) torque of the knee extensors was ~38% lower in old compared with young (P < 0.001; η²_p = 0.58) and ~43% lower in women (143.7 ± 51.0 N∙m) compared with men (251.5 ± 82.2 N∙m, P < 0.001; η²_p = 0.66). After normalizing the MVC torque to thigh lean mass, mass specific MVC torque remained ~28% lower in old compared with young (P < 0.001; η²_p = 0.55) but did not differ between men (32.7 ± 7.5 N∙m∙kg⁻¹) and women (29.1 ± 6.5 N∙m∙kg⁻¹, P = 0.126; η²_p = 0.12). Absolute mechanical power output was ~48% lower in old compared with young (P < 0.001; η²_p = 0.63) and ~41% lower in women (137.4 ± 63.0 W) than men (234.4 ± 92.5 W, P < 0.001; η²_p = 0.51). Mass specific power remained ~40% lower in old compared with young (P < 0.001; η²_p = 0.76) but did not differ between men (30.2 ± 8.9 W∙kg⁻¹) and women (27.9 ± 8.9 W∙kg⁻¹, P = 0.381; η²_p = 0.04). Fatigability (relative reduction in power during the 4-min exercise) of the knee extensors did not differ between old and young (P = 0.055; η²_p = 0.18), or between the men (23 ± 10%) and women (27 ± 8%, P = 0.311; η²_p = 0.05).

Table 1.

Anthropometrics, knee extensor function, and physical activity of the young and older adults

		Men		Women		Combined		P-value
Variable	Units	Young (5)	Old (6)	Young (5)	Old (7)	Young (10)	Old (13)	Age	Sex	Age x Sex
Age	years	23.3 ± 4.9	70.2 ± 6.4	20.9 ± 1.2	73.0 ± 4.6	22.1 ± 3.6	71.7 ± 5.5	<0.001	0.936	0.205
Height	cm	179.0 ± 6.0	183.2 ± 11.1	161.7 ± 6.1	161.0 ± 7.5	170.3 ± 10.8	171.4 ± 14.7	0.605	<0.001	0.474
Weight	kg	82.0 ± 16.1	88.2 ± 9.7	60.6 ± 10.6	67.6 ± 13.9	71.3 ± 17.1	77.1 ± 15.8	0.239	0.001	0.942
Body mass index	kg m−2	25.5 ± 4.3	26.4 ± 3.6	23.2 ± 3.9	25.9 ± 3.6	24.4 ± 4.1	26.1 ± 3.5	0.282	0.378	0.584
Whole-body fat	%	20.0 ± 4.9	29.0 ± 5.5	29.2 ± 7.1	38.3 ± 6.4	24.6 ± 7.5	34.0 ± 7.5	0.002	0.002	0.980
Whole-body lean mass	kg	62.5 ± 10.5	59.9 ± 6.2	40.5 ± 3.8	39.5 ± 5.1	51.5 ± 13.8	48.9 ± 11.9	0.530	<0.001	0.783
Thigh lean mass	kg	8.0 ± 1.9	7.4 ± 1.2	5.4 ± 0.8	4.5 ± 0.6	6.7 ± 2.0	5.8 ± 1.7	0.129	<0.001	0.841
Physical activity	Steps day−1	9,444 ± 4,353	7,261 ± 1,953	9,664 ± 1,540	6,675 ± 1,985	9,554 ± 3,080	6,945 ± 1,911	0.029	0.869	0.716
Knee extensor function
MVC Torque - Absolute	Nm	310.6 ± 88.8	202.2 ± 28.6	189.2 ± 47.6	111.2 ± 17.2	249.9 ± 92.7	153.2 ± 52.2	<0.001	<0.001	0.592
MVC Torque - Mass Specific	Nm kg−1	38.4 ± 5.6	28.0 ± 5.3	34.7 ± 3.9	25.1 ± 4.8	36.6 ± 4.9	26.4 ± 5.0	<0.001	0.126	0.852
Power - Absolute	W	308.3 ± 73.4	172.8 ± 52.9	198.3 ± 47.4	96.4 ± 21.5	253.3 ± 82.2	131.7 ± 54.5	<0.001	<0.001	0.435
Power - Mass specific	W kg−1	38.4 ± 3.8	23.3 ± 4.7	36.6 ± 4.3	21.7 ± 5.1	37.5 ± 4.0	22.4 ± 4.8	<0.001	0.381	0.942
Fatigability - Power	% Δ	−17 ± 10	−29 ± 8	−26 ± 9	−28 ± 7	−21 ± 10	−28 ± 7	0.055	0.311	0.161

Data are presented as mean ± SD. Whole-body fat, whole-body lean mass, and thigh lean mass were measured via dual X-ray absorptiometry (Lunar iDXA, GE Healthcare) and physical activity was measured via triaxial accelerometery (GT3X; ActiGraph). The sample size (N) for each cohort is reported in parentheses. Boldfaced P-values highlight statistical significance at P < 0.05.

Single fiber morphology and peak isometric force (P_o)

Slow MyHC I Fibers.

The MyHC I fiber CSA measured during the contractile experiments did not differ between young and old (P = 0.468) but was ~21% smaller in women (5585 ± 2120 µm²) compared with men (7073 ± 2620 µm², P = 0.042) (Figure 2 and Table 2). Accordingly, P_o of the MyHC I fibers did not differ between young and old for either the quiescent (P = 0.440) or fatigue-mimicking conditions (P = 0.419), while P_o was ~20% lower in women (0.61 ± 0.22 mN) compared with men (0.76 ± 0.27 mN, P = 0.039) in the quiescent condition but did not differ in the fatigue-mimicking condition (women: 0.27 ± 0.11 mN, men: 0.34 ± 0.14 mN, P = 0.071). There were no effects of age or sex on 1) the relative reduction in P_o elicited by the fatigue-mimicking condition in saturating Ca²⁺ (pCa 4.5) or 2) the size-specific P_o in the quiescent or fatigue-mimicking conditions (Figure 2 and Table 2).

Fig. 2.

Single fiber cross-sectional area (CSA), peak force (P_o), and size-specific P_o of myosin heavy chain (MyHC) I and IIa fibers. A: CSA. Men had larger CSA compared with women for both MyHC I and IIa fibers, but only MyHC IIa fibers were smaller in older compared to young adults. B: Absolute P_o in maximal Ca²⁺ (pCa 4.5). Men had greater P_o in the quiescent condition compared with women in both MyHC I and IIa fibers, but in the fatigue condition P_o was greater in men compared to women only for MyHC IIa fibers. There was no effect of age on P_o for MyHC I fibers, however P_o was greater in young compared with older adults in MyHC IIa fibers. C: Size-specific P_o in maximal Ca²⁺ (pCa 4.5). There were no age or sex differences in size-specific P_o in either condition indicating that any age and/or sex differences in absolute P_o were explained by fiber size. Significant main effect of *sex and †age with a significance level of P < 0.05. The horizontal line in each box plot indicates the median, while the whiskers represent 1.5 times the upper- and lower-interquartile range. Each open circle is an individual fiber, with the number of fibers (n) displayed below the box plots, from 5–7 subjects. n is the same in A, B, and C. Quiescent condition (pH 7 + 4 mM P_i) is in black and fatigue condition (pH 6.2 + 30 mM P_i) is in grey with diagonal lines. Data were analyzed using a mixed effects ANOVA.

Table 2.

Cross-sectional area and peak force (P_o) of single muscle fibers

			Men		Women		Combined			P-value
			Young	Old	Young	Old	Young	Old	Diff.	Age	Sex	Age*Sex
MyHC I		n(N)	37 (5)	39 (6)	35 (5)	47 (7)	72 (10)	86 (13)
CSA		µm2	6,782 ± 3,163	7,350 ± 1,978	5,317 ± 2,167	5,784 ± 2,084	6,070 ± 2,804	6,494 ± 2,171	↔	0.468	0.042	0.920
pH 7 + 4 mM Pi
Absolute Po	mN		0.74 ± 0.33	0.78 ± 0.21	0.59 ± 0.23	0.63 ± 0.21	0.67 ± 0.30	0.70 ± 0.22	↔	0.440	0.039	0.985
Size-Specific Po	kN∙m−2		110.8 ± 15.1	107.4 ± 12.2	111.6 ± 15.3	111.3 ± 17.5	111.2 ± 15.1	109.5 ± 15.4	↔	0.556	0.617	0.644
pH 6.2 + 30 mM Pi
Absolute Po	mN		0.32 ± 0.16	0.36 ± 0.12	0.27 ± 0.11	0.28 ± 0.10	0.30 ± 0.14	0.32 ± 0.12	↔	0.419	0.071	0.713
Size-Specific Po	kN∙m−2		47.9 ± 10.6	49.2 ± 9.0	50.9 ± 8.8	49.2 ± 11.1	49.3 ± 9.8	49.2 ± 10.2	↔	0.850	0.521	0.647
↓ with Fatigue	%		−57 ± 5	−54 ± 5	−55 ± 3	−56 ± 5	−56 ± 4	−55 ± 5	↔	0.620	0.892	0.170
MyHC IIa		n(N)	34 (5)	38 (6)	34 (5)	56 (7)	68 (10)	94 (13)
CSA		µm2	7,747 ± 2,348	6,302 ± 2,208	7,144 ± 2,447	3,880 ± 1,370	7,446 ± 2,399	4,859 ± 2,116	↓ 35%	0.002	0.006	0.079
pH 7 + 4 mM Pi
Absolute Po	mN		1.20 ± 0.31	0.98 ± 0.27	1.06 ± 0.31	0.60 ± 0.21	1.13 ± 0.32	0.75 ± 0.30	↓ 34%	0.002	0.002	0.094
Size-Specific Po	kN∙m−2		157.6 ± 19.8	158.9 ± 18.7	151.6 ± 20.5	155.3 ± 18.3	154.6 ± 20.2	156.7 ± 18.4	↔	0.437	0.388	0.703
pH 6.2 + 30 mM Pi
Absolute Po	mN		0.55 ± 0.15	0.45 ± 0.12	0.50 ± 0.17	0.28 ± 0.09	0.52 ± 0.16	0.35 ± 0.14	↓ 33%	0.002	0.003	0.082
Size-Specific Po	kN∙m−2		72.1 ± 10.0	74.0 ± 9.2	70.6 ± 11.3	73.4 ± 10.4	71.3 ± 10.6	73.6 ± 9.9	↔	0.228	0.858	0.765
↓ with Fatigue	%		−54 ± 2	−53 ± 2	−54 ± 3	−53 ± 3	−54 ± 3	−53 ± 3	↔	0.135	0.228	0.889

Data are presented as mean ± SD. N: number of subjects; n: number of fibers; CSA: cross-sectional area; ↓ with Fatigue: decrease in absolute force with fatigue-mimicking solution compared to quiescent condition. Force was measured in maximal Ca²⁺ activating solution (pCa 4.5). Boldfaced P-values highlight statistical significance at P < 0.05.

Fast MyHC IIa Fibers.

The CSA measured during the contractile experiments was ~35% smaller in MyHC IIa fibers from older compared with young (P < 0.002) and ~27% smaller in women (5113 ± 2432 µm²) compared with men (6985 ± 2373 µm², P < 0.006) (Figure 2 and Table 2). Accordingly, P_o of the MyHC IIa fibers were ~34% and 33% lower in older compared with young in the quiescent (P = 0.002) and fatigue-mimicking conditions (P = 0.002). In addition, P_o was ~29% lower in women (0.77 ± 0.34 mN) compared with men (1.08 ± 0.31 mN, P = 0.002) in the quiescent condition, and ~28% lower in the fatigue-mimicking condition (women: 0.36 ± 0.16 mN, men: 0.50 ± 0.14 mN, P = 0.003). Similar to the findings from the slow MyHC I fibers, there were no effects of age or sex on 1) the relative reduction in P_o elicited by the fatigue-mimicking condition in saturating Ca²⁺ (pCa 4.5) or 2) the size-specific P_o in the quiescent or fatigue-mimicking conditions for the fast MyHC IIa fibers (Figure 2 and Table 2).

Fiber type differences.

Irrespective of age or sex, MyHC I fibers generated ~25% and 26% lower absolute P_o compared with IIa fibers in both the quiescent (0.68 ± 0.26 vs. 0.91 ± 0.36 mN, P < 0.001) and fatigue-mimicking conditions (0.31 ± 0.13 mN vs. 0.42 ± 0.17 mN, P < 0.001). Similarly, MyHC I fibers generated ~29% and 32% lower size-specific P_o compared with IIa fibers in both the quiescent (110.3 ± 15.2 vs. 155.8 ± 19.2 kN·m⁻², P < 0.001) and fatigue-mimicking conditions (49.3 ± 10.0 kN·m⁻² vs. 72.7 ± 10.2 kN·m⁻², P < 0.001).

Force-pCa relationship and Ca²⁺sensitivity

Slow MyHC I Fibers.

There were no age or sex differences in pCa₅₀, activation threshold, or the slopes of the force-pCa relationship above (n₁) or below (n₂) pCa₅₀ for the MyHC I fibers in either the quiescent or severe fatigue mimicking conditions (Figure 3 and Table 3). The fatigue-mimicking condition markedly depressed the sensitivity of the myofilaments to Ca²⁺ as indicated by the rightward shift in the force-pCa relationship, however, the magnitude of the change in pCa₅₀ elicited by the fatigue-mimicking condition did not differ between young and old or men and women (Figure 3 and Table 3). Irrespective of age or sex, the force-pCa relationship parameters differed between the two conditions, where the quiescent condition had higher pCa₅₀ (5.94 ± 0.10 vs 5.30 ± 0.08, P < 0.001) and activation threshold (6.81 ± 0.09 vs 5.68 ± 0.12, P < 0.001), but a lower n₁ (2.61 ± 0.80 vs 2.84 ± 0.93, P = 0.013) and n₂ (2.73 ± 0.38 vs 6.82 ± 2.17, P < 0.001) compared with the fatigue condition.

Fig. 3.

Mean force-pCa relationship of myosin heavy chain (MyHC) I and IIa fibers. Single muscle fibers were activated in solutions containing free [Ca²⁺] ranging from pCa 6.5 to 4.5 in quiescent (pH 7 + 4 mM P_i, black) and pCa 5.8 to 4.5 in fatigue (pH 6.2 + 30 mM P_i, grey). A: pCa-force relationship where peak force (P_o) is expressed relative to the condition specific peak force (P_o) produced in pCa 4.5. B: pCa₅₀ calculated by fitting the data with Hill curves. C: Shift in pCa₅₀ with fatigue. There were no age or sex differences in pCa₅₀ in either condition. Significance level P < 0.05. The horizontal line in each box plot indicates the median, while the whiskers represent 1.5 times the upper- and lower-interquartile range. Each open circle is an individual fiber, with the number of fibers (n) displayed below the box plots, from 5–7 subjects. Data were analyzed using a mixed effects ANOVA.

Table 3.

Force-pCa parameters for the MyHC I fibers

		Men		Women		Combined			P-value
		Young	Older	Young	Older	Young	Old	Diff.	Age	Sex	Age*Sex
pH 7 + 4 mM Pi	n (N)	37 (5)	39 (6)	35 (5)	47 (7)	72 (10)	86 (13)
pCa50		5.93 ± 0.09	5.98 ± 0.10	5.92 ± 0.11	5.94 ± 0.10	5.92 ± 0.10	5.96 ± 0.10	↔	0.378	0.410	0.874
Activation Threshold		6.79 ± 0.11	6.81 ± 0.05	6.84 ± 0.10	6.82 ± 0.10	6.81 ± 0.11	6.82 ± 0.08	↔	0.953	0.180	0.458
n1		2.45 ± 0.63	2.57 ± 0.81	2.66 ± 0.95	2.73 ± 0.80	2.55 ± 0.80	2.66 ± 0.80	↔	0.291	0.207	0.764
n2		2.79 ± 0.36	2.85 ± 0.41	2.57 ± 0.32	2.70 ± 0.39	2.68 ± 0.36	2.77 ± 0.40	↔	0.482	0.154	0.608
pH 6.2 + 30 mM Pi
pCa50		5.29 ± 0.07	5.32 ± 0.09	5.29 ± 0.06	5.29 ± 0.07	5.29 ± 0.06	5.30 ± 0.08	↔	0.594	0.488	0.562
Activation Threshold		5.68 ± 0.11	5.68 ± 0.10	5.72 ± 0.14	5.66 ± 0.11	5.70 ± 0.13	5.67 ± 0.11	↔	0.517	0.946	0.203
n1		2.73 ± 0.83	2.96 ± 1.16	2.77 ± 1.04	2.87 ± 0.70	2.75 ± 0.93	2.91 ± 0.94	↔	0.196	0.937	0.744
n2		6.70 ± 2.21	7.32 ± 2.05	6.00 ± 2.09	7.12 ± 2.16	6.36 ± 2.17	7.21 ± 2.10	↔	0.073	0.329	0.508
ΔpCa50		−0.64 ± 0.07	−0.66 ± 0.08	−0.63 ± 0.10	−0.65 ± 0.07	−0.64 ± 0.09	−0.66 ± 0.08	↔	0.507	0.688	0.827

Data are presented as mean ± SD. n: number of fibers; N: number of subjects; pCa₅₀: [Ca²⁺] eliciting 50% P_o; Activation Threshold: the lowest [Ca²⁺] that elicits force; n₁: slope of the force-pCa relationship above pCa₅₀; n₂: slope of the force-pCa relationship below pCa₅₀. pCa_50, Activation Threshold, and ∆pCa₅₀ are shown in negative log units (pCa). Significance level P < 0.05.

Fast MyHC IIa Fibers.

Similar to the findings from the MyHC I fibers, there were minimal age or sex differences in pCa₅₀, activation threshold, or the slopes of the force-pCa relationship above (n₁) or below (n₂) pCa₅₀ for the MyHC IIa fibers in either the quiescent or severe fatigue mimicking conditions (Figure 3 and Table 4). However, in contrast to the MyHC I fibers, the activation threshold was ~1% greater in MyHC IIa fibers from older compared with young in the fatigue-mimicking condition (P = 0.042), with no age difference in the quiescent condition (P = 0.111). There was also an age by sex interaction (P = 0.005) in n₂ of the MyHC IIa fibers in the quiescent condition where young men had a higher n₂ (5.64 ± 1.60) compared with older men (4.50 ± 1.52, P = 0.033) but there were no differences between young and older women (P = 0.419). Similar to the findings from the MyHC I fibers, the fatigue-mimicking condition markedly depressed the sensitivity of the myofilaments to Ca²⁺ as indicated by the rightward shift in the force-pCa relationship, however, the magnitude of the change in pCa₅₀ elicited by the fatigue-mimicking condition did not differ between young and old or men and women (Figure 3 and Table 4). Irrespective of age or sex, the force-pCa relationship parameters differed between the two conditions, where the quiescent condition had higher pCa₅₀ (6.03 ± 0.10 vs 5.34 ± 0.06, P < 0.001) and activation threshold (6.52 ± 0.13 vs 5.75 ± 0.10, P < 0.001), but a lower n₁ (2.53 ± 0.74 vs 3.25 ± 1.07, P < 0.001) and n₂ (4.88 ± 1.66 vs 6.63 ± 2.04, P < 0.001) compared with the fatigue-mimicking condition.

Table 4.

Force-pCa parameters for the MyHC IIa fibers

		Men		Women		Combined			P-value
		Young	Older	Young	Older	Young	Old	Diff.	Age	Sex	Age*Sex
pH 7 + 4 mM Pi	n (N)	34 (5)	38 (6)	34 (5)	56 (7)	68 (10)	94 (13)
pCa50		6.01 ± 0.08	6.04 ± 0.10	5.99 ± 0.11	6.06 ± 0.09	6.00 ± 0.09	6.05 ± 0.09	↔	0.096	0.957	0.440
Activation Threshold		6.43 ± 0.11	6.57 ± 0.10	6.53 ± 0.15	6.54 ± 0.11	6.48 ± 0.14	6.55 ± 0.11	↔	0.111	0.303	0.216
n1		2.14 ± 0.39	2.58 ± 0.75	2.73 ± 0.63	2.62 ± 0.88	2.43 ± 0.60	2.60 ± 0.83	↔	0.825	0.361	0.103
n2		5.64 ± 1.60	4.50 ± 1.52†	4.33 ± 1.14	5.01 ± 1.89	4.98 ± 1.53	4.80 ± 1.76	↔	0.233	0.069	0.005
pH 6.2 + 30 mM Pi
pCa50		5.31 ± 0.04	5.35 ± 0.07	5.35 ± 0.05	5.35 ± 0.06	5.33 ± 0.05	5.35 ± 0.06	↔	0.468	0.348	0.604
Activation Threshold		5.70 ± 0.08	5.79 ± 0.11	5.74 ± 0.09	5.77 ± 0.11	5.72 ± 0.09	5.78 ± 0.11	↑ 1%	0.042	0.806	0.341
n1		2.95 ± 0.77	3.40 ± 0.96	3.24 ± 1.38	3.33 ± 1.07	3.09 ± 1.12	3.36 ± 1.02	↔	0.076	0.787	0.443
n2		6.92 ± 1.95	6.24 ± 2.08	6.88 ± 1.89	6.58 ± 2.16	6.90 ± 1.91	6.44 ± 2.12	↔	0.299	0.703	0.737
ΔpCa50		−0.69 ± 0.06	−0.69 ± 0.09	−0.64 ± 0.09	−0.71 ± 0.08	−0.67 ± 0.08	−0.70 ± 0.08	↔	0.115	0.406	0.152

Data are presented as mean ± SD. n: number of fibers; N: number of subjects; pCa₅₀: [Ca²⁺] eliciting 50% P_o; Activation Threshold: the lowest [Ca²⁺] that elicits force; n₁: slope of the force-pCa relationship above pCa₅₀; n₂: slope of the force-pCa relationship below pCa₅₀. pCa_50, Activation Threshold, and ∆pCa₅₀ are shown in negative log units (pCa).

^†Significantly different from young men. Boldfaced P-values highlight statistical significance at P < 0.05.

Fiber type differences.

Irrespective of age or sex, MyHC I fibers had a lower pCa₅₀ than IIa fibers in both the quiescent (MyHC I: 5.94 ± 0.10, MyHC IIa: 6.03 ± 0.10, P < 0.001) and the fatigue mimicking conditions (MyHC I: 5.30 ± 0.08, MyHC IIa: 5.34 ± 0.06, P < 0.001), and the change in pCa₅₀ with fatigue was less in MyHC I (0.65 ± 0.08) compared with IIa fibers (0.69 ± 0.08, P < 0.001). The activation threshold was higher in MyHC I (6.81 ± 0.09) compared with IIa fibers (6.52 ± 0.13, P < 0.001) in the quiescent condition but was lower in the fatigue mimicking condition (MyHC I: 5.68 ± 0.12, MyHC IIa: 5.75 ± 0.10, P < 0.001). n₁ was not different between MyHC I (2.61 ± 0.80) and IIa fibers (2.53 ± 0.74, P = 0.458) in the quiescent condition but was lower in MyHC I (2.84 ± 0.93) compared with IIa fibers (3.25 ± 1.07, P < 0.001) in the fatigue mimicking condition. In contrast, MyHC I fibers had a lower n₂ (2.73 ± 0.38) compared with IIa fibers (4.88 ± 1.66, P < 0.001) in the quiescent condition, with no differences (MyHC I: 6.82 ± 2.17, MyHC IIa: 6.63 ± 2.04, P = 0.296) in the fatigue mimicking condition.

Fiber morphology and MyHC distribution from immunohistochemistry (IHC)

To determine the fiber type-specific CSA and distribution with IHC, we analyzed 466 ± 162 fibers from the young (range 177–690 fibers) and 422 ± 197 fibers from the old adults (range 184–838 fibers). Consistent with the findings from the single fiber CSA data, MyHC I fiber CSA did not differ between young and old adults (P = 0.339) but was ~19% smaller in women (3413 ± 851 µm²) compared with men (4229 ± 948 µm², P = 0.048) (Table 5). Also consistent with the findings from the single fiber CSA data, MyHC IIa fiber CSA was ~29% smaller in fibers from old compared with young adults (P = 0.026) and ~28% smaller in women (2961 ± 1567 µm²) compared with men (4134 ± 920 µm², P = 0.044). Similarly, CSA of MyHC IIa/IIx hybrid fibers were ~39% smaller in old compared with young (P = 0.028) and ~44% smaller in women (1843 ± 857 µm²) compared with men (3265 ± 1335 µm², P = 0.013). When all fast fibers were combined (IIa, IIa/IIx, and IIx), the fibers from old were ~35% smaller compared with young (P = 0.027), but women (2525 ± 1669 µm²) and men did not differ (3651 ± 1119 µm², P = 0.081). Accordingly, the MyHC II/I ratio was markedly higher in the young compared with the old (P = < 0.001) indicating smaller fast compared with slow fibers in the old (Table 5), but there were no differences between men (0.87 ± 0.21) and women (0.75 ± 0.39, P = 0.376). There was also an age by sex interaction (P = 0.007) where the MyHC II/I ratio for young women (1.15 ± 0.19) was greater than old women (0.46 ± 0.18, P < 0.001), with no differences in the young (0.98 ± 0.22) and old men (0.77 ± 0.17, P = 0.295) (Table 5).

Table 5.

Fiber type distribution and cross-sectional area from immunohistochemistry

		Men		Women		Combined			P-value
		Young	Old	Young	Old	Young	Old	Diff.	Age	Sex	Age*Sex
MyHC I	N	5	6*	5*	7	10*	13*
CSA	µm2	3975 ± 1055	4441 ± 889	3241 ± 1144	3537 ± 642	3608 ± 1107	3954 ± 869	↔	0.339	0.048	0.829
Distribution	%	48.9 ± 19.7	51.2 ± 13.4	44.8 ± 12.6	54.5 ± 19.1	46.9 ± 15.7	53.0 ± 16.1	↔	0.404	0.959	0.606
Area	%	47.1 ± 19.9	55.4 ± 13.3	40.7 ± 9.7	64.0 ± 19.8	43.9 ± 15.1	60.0 ± 17.0	↑ 16%	0.035	0.879	0.292
MyHC IIa
CSA	µm2	4383 ± 905	3927 ± 961	4048 ± 1804	2185 ± 802	4215 ± 1357	2989 ± 1234	↓ 29%	0.026	0.044	0.160
Distribution	%	40.8 ± 18.6	39.5 ± 17.6	45.4 ± 9.5	39.7 ± 18.0	43.1 ± 14.1	39.6 ± 17.0	↔	0.618	0.736	0.759
Area	%	43.6 ± 18.3	37.3 ± 15.6	50.7 ± 9.1	32.1 ± 18.4	47.1 ± 14.2	34.5 ± 16.7	↔	0.083	0.887	0.378
MyHC IIa/IIx
CSA	µm2	3675 ± 857	2855 ± 1690	2691 ± 443	1359 ± 610	3238 ± 843	1982 ± 1355	↓ 39%	0.028	0.013	0.574
Distribution	%	7.8 ± 7.2	8.4 ± 11.7	8.2 ± 9.2	4.4 ± 4.9	8.0 ± 7.8	6.2 ± 8.6	↔	0.691	0.682	0.854
Area	%	7.1 ± 6.7	6.7 ± 9.8	6.9 ± 7.1	3.0 ± 4.8	7.0 ± 6.5	4.7 ± 7.4	↔	0.536	0.556	0.879
MyHC II
CSA	µm2	3798 ± 905	3529 ± 1346	3814 ± 1924	1604 ± 517	3806 ± 1417	2493 ± 1373	↓ 34%	0.027	0.081	0.077
Distribution	%	50.6 ± 20.2	48.0 ± 14.1	53.6 ± 12.1	45.3 ± 19.4	52.1 ± 15.8	46.5 ± 16.5	↔	0.453	0.981	0.691
Area	%	52.3 ± 20.3	44.1 ± 13.8	57.6 ± 9.4	35.9 ± 19.9	54.9 ± 15.2	39.7 ± 17.2	↓ 15%	0.048	0.838	0.353
MyHC II/I CSA Ratio		0.98 ± 0.22	0.77 ± 0.17	1.15 ± 0.19	0.46 ± 0.18	1.06 ± 0.21	0.60 ± 0.23	↓ 43%	<0.001	0.376	0.007

Data are presented as mean ± SD. N: number of subjects; CSA: cross-sectional area; Area: proportional cross-sectional area; Boldfaced P-values highlight statistical significance at P < 0.05.

^*1 old man and 1 young woman did not have IIa/IIx fibers.

The percent fiber type distribution did not differ between young and old or men and women (Table 5). In contrast, the proportional area of MyHC I fibers was ~16% greater in old compared with young (P = 0.035) but did not differ between women (54.3 ± 19.8%) and men (51.6 ± 16.3%, P = 0.879). The proportional area of MyHC IIa and MyHC IIa/IIx did not differ between young and old or men and women, however when all fast type fibers were combined, the proportional area was ~15% lower in old compared with young (P = 0.048) but did not differ between women (44.9 ± 19.3%) and men (47.8 ± 16.7%, P = 0.838) (Table 5).

DISCUSSION

The primary aim of the present study was to test the effects of age and sex on human single muscle fiber Ca²⁺ sensitivity in conditions mimicking quiescent (pH 7 + 4 mM P_i) and severely fatigued muscle (pH 6.2 + 30 mM P_i). Contrary to our hypotheses, we found no age differences in Ca²⁺ sensitivity of the slow or fast fibers in either the quiescent or fatigue-mimicking conditions. We confirmed previous findings from non-human studies (34–38) that elevated levels of H⁺ and P_i contribute to skeletal muscle fatigue by markedly depressing the sensitivity of the myofilaments to Ca²⁺. However, the metabolite induced depression in Ca²⁺ sensitivity did not differ in fibers from old compared with young men and women. Consistent with our previous studies (15, 26), we observed markedly lower absolute force (P_o) in the fast MyHC IIa fibers from the older adults, but no age differences in size-specific P_o, indicating that the age-related differences in P_o were explained by the smaller fiber size. These data suggest that fast fiber atrophy is a major factor contributing to the loss of force in older adults, but that the age-related increase in fatigability cannot be explained by an increased sensitivity of the muscle fibers to elevated H⁺ and P_i in maximal or submaximal Ca²⁺.

Calcium sensitivity in human limb skeletal muscle is preserved with aging and does not differ between men and women

Only six studies have investigated whether age-related alterations in Ca²⁺ sensitivity of limb skeletal muscle fibers are contributing to the decrements in contractile function in older adults, and the findings are equivocal (21–26). One important limitation of the previous studies is that they all used an activating condition of pH 7.0 + 0 mM P_i, whereas the physiological concentration of P_i in quiescent human skeletal muscle is ∼3–5 mM (27). This distinction is important because even small increases in [P_i] from 0 to 4 mM markedly depresses P_o in human muscle fibers, particularly in the slow MyHC I fibers (15, 29). Moreover, studies in non-human animals have demonstrated that elevated P_i alone depresses Ca²⁺ sensitivity (37, 49). In the present study, we used an activating condition that more closely mimics quiescent human skeletal muscle (pH 7 + 4 mM P_i) and found that the sensitivity of the myofilaments to Ca²⁺ (pCa₅₀ and activation threshold) is preserved with aging in both the slow MyHC I and fast MyHC IIa fibers in men and women. These data are in agreement with some studies reporting no age (24–26) or sex differences (21) in Ca²⁺ sensitivity, but in contrast to others that found decreased Ca²⁺ sensitivity in older compared with young adults, particularly in the fast fibers (21–23). Identifying the explanations for the discrepancies between studies is important because there is evidence of less stored Ca²⁺ in the sarcoplasmic reticulum (23) and a lower amplitude of the intracellular Ca²⁺ transient in fibers from old compared with young adults (50), which may cause even modest changes in Ca²⁺ sensitivity to become functionally relevant.

The potential explanations for the disparate findings between studies have been discussed in detail previously (21, 26, 47), and may include methodological differences, such as the age used to define the older adult cohort, using large increments in [Ca²⁺] to establish the force-pCa relationship (e.g., 0.2–0.25 pCa increments), not measuring and controlling sarcomere spacing and temperature, testing a low number of fibers, particularly the MyHC II fibers, and/or the MyHC II fibers were examined together by combining the IIa and IIx fibers with the hybrid IIa/IIx fibers. Another potential explanation is the selection bias that can commonly occur in single fiber studies, where the smaller more fragile fast fibers from the older adults are either not studied or fail the experimental procedures. To minimize the potential influence of each of these factors, in the present study we 1) measured and set the sarcomere spacing to 2.5 µm, 2) used small 0.1 pCa increments with 11–12 different [Ca²⁺] in each activating condition, 3) determined the fiber type based on the MyHC isoform(s) with SDS-PAGE, and 4) conducted experiments at a constant 20 °C with our custom single fiber microsystem, which minimized the amount of failed fiber experiments (i.e., only 16 of 404 fibers failed quality control criteria). In addition, we collected the largest human aging data set on the force-pCa relationship to date, testing 72 and 86 MyHC I and 68 and 94 MyHC IIa fibers from young and older adults, respectively. To test whether our results were influenced by selection bias, we verified that the age- and sex-related differences in fiber CSA were consistent between measurements obtained from the contractile mechanics experiments and those obtained via IHC. The data from this comprehensive approach provide compelling evidence that both aging and sex have little to no effect on the sensitivity of the myofilaments to Ca²⁺ in either slow or fast fibers.

Another potential explanation for the disparate findings across studies is the inherent limitation of using cross-sectional study designs to investigate human aging. For example, we did not observe the expected age difference in thigh lean mass in our cohort (Table 1), which may have contributed to the preserved Ca²⁺ sensitivity in the older adults. However, a lack of age-related reduction in thigh lean mass is unlikely to explain these findings, as Straight et al. reported reduced Ca²⁺ sensitivity with age in both MyHC I and IIa fibers, despite no observed age-related differences in leg fat-free mass (21). In addition, our cohort of older adults demonstrated several other hallmark features of aging skeletal muscle of the knee extensors, including markedly lower absolute and mass-specific mechanical force and power outputs (Table 1). It should also be noted that in contrast to our previous single fiber studies (15, 26), the older adults in the present study had lower physical activity levels compared with the young (Table 1). This is important because the amount of use and disuse of skeletal muscle has been shown to alter Ca²⁺ sensitivity in older adults. For example, 12-weeks of resistance exercise training elicited an increased Ca²⁺ sensitivity in older women (51) whereas short-term lower limb immobilization reduced Ca²⁺ sensitivity in older men (24, 25). However, the age differences in physical activity are unlikely to describe the findings in the present study, because despite lower physical activity levels in the older adults, we found no evidence for lower Ca²⁺ sensitivity in the fibers from the older adults.

The observation of a preserved Ca²⁺ sensitivity in both slow and fast fibers begs the question: what mechanisms underlie the accelerated loss of whole-muscle force and power relative to muscle mass with aging? Several mechanisms have been proposed, including reduced voluntary activation of the muscle by the nervous system (52, 53), infiltration of intermuscular adipose and fibrotic tissue (54–57), motor unit remodeling and neuromuscular junction instability (58), disrupted cross-bridge mechanics and Ca²⁺ handling (23, 59, 60), and/or the selective atrophy of the fast MyHC II fibers (15, 26, 29, 47). Although voluntary activation was not assessed in the present study, our prior work (9, 15, 61, 62) along with a meta-analysis (11) indicates that most community-dwelling, healthy older adults are able to activate their muscles to a similar extent as younger adults, and that the age-related losses in whole-muscle force and power are determined primarily by factors affecting the contractile properties within the muscle (8).

In the present study, the only contractile property reduced in fibers from older adults was the 33–34% lower absolute P_o of the fast MyHC IIa fibers. However, when absolute P_o was normalized to fiber size, there were no age-differences in size-specific P_o, suggesting a preserved intrinsic contractile function of the fast fibers with age. These findings align with a large body of evidence reporting similar or even higher size-specific force and power of the fast fibers in older compared with younger adults (15, 26, 29, 63–67), but is in contrast to others (23, 59, 68, 69). The explanation for the discrepancies between studies is unclear but, as discussed in detail previously (47), likely involves methodological challenges inherent to single fiber experiments, including small sample sizes and difficulty in accurately estimating fiber CSA from 2D images. Importantly, we also observed no age-related differences in Ca²⁺ sensitivity, which is a measure of contractile function that is independent of fiber CSA estimation, and further supports that intrinsic contractile function is preserved with age.

Given that fast fibers generate 41–47% greater size-specific force than slow fibers (Fig. 2C), a selective atrophy of the fast fibers would be expected to produce a disproportionately large reduction in whole-muscle force relative to the loss of muscle mass with age. This introduces a paradox, however, because the selective atrophy and/or loss of the more fatigable fast fibers should theoretically result in older adults being fatigue resistant. Indeed, older adults are typically less fatigable than young adults when performing both maximal (70–72) and submaximal isometric contractions (73–78). However, when older adults perform dynamic contractions, the age-related fatigue resistance is reversed in a velocity dependent manner, with minimal differences observed at slow-velocities but an increased fatigability in the older compared with young at moderate- to high-velocities (9, 13–15, 29, 40, 62, 72, 79, 80). Thus, although fast fiber atrophy may explain, at least in part, the disproportionate loss of whole-muscle force relative to muscle mass with aging, it cannot account for the increased fatigability observed during dynamic contractions. Future studies aimed at elucidating how aging alters both the contractile mechanics and bioenergetics in a fiber type specific manner will be essential for developing targeted strategies to preserve muscle power and fatigability in older men and women.

Elevated H⁺ and P_i are important mediators of skeletal muscle fatigue in humans by directly inhibiting cross-bridge function and decreasing the sensitivity of the myofilaments to Ca²⁺

Although we observed little to no effect of age or sex on Ca²⁺ sensitivity and the force-pCa relationship, elevating the concentrations of H⁺ and P_i markedly depressed force at all [Ca²⁺] and reduced the sensitivity of the myofilaments to Ca²⁺ in both young and older men and women. The reduced Ca²⁺ sensitivity manifested as a rightward shift in the force-pCa relationship, where the magnitude of the shift (∆pCa₅₀) did not differ between young and older men or women for either fiber type. These findings confirm previous non-human studies that elevated levels of H⁺ and P_i play an important role in the fatigue process in humans by directly impeding the cross-bridge and reducing the sensitivity of the myofilaments to Ca²⁺. Although our experimental approach did not permit mechanistic insight into how H⁺ and P_i alter Ca²⁺ sensitivity in human muscle, studies in non-human animals suggest that H⁺ decreases the affinity of the binding sites on troponin C (TnC) to Ca²⁺, whereas the P_i-induced reduction in Ca²⁺ sensitivity likely involves the effect of P_i on the cross-bridge rather than on TnC (16, 35, 36, 38). However, it is noteworthy that the magnitude of the shift in the force-pCa relationship elicited by the pH 6.2 + 30 mM P_i condition in humans at 20 °C (0.65–0.69 pCa units) was smaller than reported previously in rat fibers at both 15 °C (0.88–1.06 pCa units) and 30 °C (1.61–1.69 pCa units) in MyHC I and IIa fibers. The mechanisms for the discrepancies in the metabolite induced decrements in Ca²⁺ sensitivity between the mammalian species is unknown but may be due to differences in contractile kinetics between species, or perhaps because at pH 7.0 we used a more physiological [P_i] of 4 mM in the present study compared with the ~ 0 mM P_i condition used previously in rat fibers (34).

Another potential species difference is that in contrast to previous studies in rodents (34, 37), we observed a higher n₂ in response to elevated H⁺ and P_i. This finding is consistent with the increased Hill coefficient in human skeletal muscle fibers observed both after exercise (81–83) and when pH alone was reduced from 7.1 to 6.6 (39). The Hill coefficient, n, describes the steepness of the force-pCa relationship, and the slope of the forces below half-maximal (n₂) are thought to indicate the magnitude of the molecular cooperativity of the filaments caused by the myosin binding events (84). More specifically, the binding of Ca²⁺ to TnC is followed by a shift in the position of troponin I (TnI), allowing initial weak interactions between actin and myosin, and eventual strong binding of the cross-bridge (85, 86). The full activation of one troponin-tropomyosin complex by the myosin strong binding event increases the probability of strong-binding of neighboring cross-bridges which increases the force at low [Ca²⁺] (84, 86). The mechanism for the increased n₂ elicited by elevated levels of H⁺ and P_i is unclear. However, insight may be gleaned by recent studies on human skeletal muscle fibers conducted in saturating Ca²⁺ that have shown elevated levels of H⁺ and P_i to markedly reduce peak force and power, slow shortening velocity, and inhibit the low- to high-force transition step of the cross-bridge cycle (15, 29). The metabolite-induced slowing of the cross-bridge kinetics and prolonged attachment times (87) may increase the probability of neighboring cross-bridges to stay bound to actin, albeit with reduced force per bridge, that ultimately increases the molecular cooperativity of the filaments at submaximal [Ca²⁺]. Future studies that incorporate additional measures of cross-bridge binding kinetics are needed to determine the mechanisms for the increased n₂ with elevated levels of H⁺ and P_i in human skeletal muscle.

It is important to note that methodological factors could influence the interpretation of the Ca²⁺ sensitivity findings under the fatigue-mimicking condition. An effect of age or sex may emerge under fatigue-mimicking conditions at temperatures closer to physiological levels (e.g., 30–37 °C), as data from non-human animal models indicate the force–pCa relationship exhibits a greater metabolite induced shift at higher temperatures (34, 37). However, we have observed that human skeletal muscle fibers, particularly fast MyHC II fibers, deteriorate rapidly at 30 °C, making it difficult to obtain complete data sets from experiments involving multiple conditions and numerous contractions (15). Additionally, although older adults exhibit greater fatigability during moderate- to high-velocity dynamic contractions (e.g. (9)), they typically fatigue less than younger adults during isometric contractions (72, 78, 79), which may explain the preservation of Ca²⁺ sensitivity in our fatigue-mimicking experiments that can only be conducted using isometric contractions. Finally, we cannot rule out the possibility that the apparent preservation of Ca²⁺ sensitivity under the fatigue-mimicking condition reflects the lack of a statistically significant age-related increase in whole-muscle fatigability (P = 0.055). However, this explanation is unlikely, as the effect size in the present study (η²_p = 0.18) is nearly identical to that of our previous study using the same fatiguing protocol (η²_p = 0.19), in which a larger sample size produced a highly significant age effect (P < 0.001) (9).

Concluding remarks

The data in the present study provide novel evidence that, in addition to the direct effects elevated H⁺ and P_i have on depressing cross-bridge function in saturating Ca²⁺ (15, 29, 87), the metabolites are important mediators of fatigue in human skeletal muscle by decreasing the sensitivity of the myofilaments to Ca²⁺. Importantly, the Ca²⁺ sensitivity of slow and fast fibers were unaltered by age in conditions mimicking both quiescent and severe fatigue. In agreement with our previous findings (15, 26, 29), the lower absolute force of the fast fibers with age could be explained entirely by the differences in fiber size, rather than impaired intrinsic contractile function. We conclude that the age-related increase in fatigability cannot be attributed to a greater sensitivity of the cross-bridge to H⁺ and P_i in either maximal or submaximal Ca²⁺ and is likely explained by a greater accumulation of these metabolites within the working muscle of older adults (28).

Supplementary Material

Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.30661148

DATA AVAILABILITY

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