Time-of-day effects on ex vivo muscle contractility following short-term satellite cell ablation

Abstract

Muscle isometric torque fluctuates according to time-of-day with such variation owed to the influence of circadian molecular clock genes. Satellite cells (SCs), the muscle stem cell population, also express molecular clock genes with several contractile-related genes oscillating in a diurnal pattern. Currently, limited evidence exists regarding the relationship between SCs and contractility, although long-term SC ablation alters muscle contractile function. Whether there are acute alterations in contractility following SC ablation and with respect to the time-of-day is unknown. We investigated whether short-term SC ablation affected contractile function at two times of day and whether any such alterations led to different extents of eccentric contraction-induced injury. Using an established mouse model to deplete SCs, we characterized muscle clock gene expression and ex vivo contractility at two times-of-day (morning: 0700 and afternoon: 1500). Morning-SC⁺ animals demonstrated ∼25%–30% reductions in tetanic/eccentric specific forces and, after eccentric injury, exhibited ∼30% less force-loss and ∼50% less dystrophin^negative fibers versus SC⁻ counterparts; no differences were noted between Afternoon groups (Morning-SC⁺: −5.63 ± 0.61, Morning-SC⁻: −7.93 ± 0.61; N/cm²; P < 0.05) (Morning-SC⁺: 32 ± 2.1, Morning-SC⁻: 64 ± 10.2; dystrophin^negative fibers; P < 0.05). As Ca⁺⁺ kinetics underpin force generation, we also evaluated caffeine-induced contracture force as an indirect marker of Ca⁺⁺ availability and found similar force reductions in Morning-SC⁺ vs. SC⁻ mice. We conclude that force production is reduced in the presence of SCs in the morning but not in the afternoon, suggesting that SCs may have a time-of-day influence over contractile function.

NEW & NOTEWORTHY Muscle isometric torque fluctuates according to time-of-day with such variation owed to molecular clock regulation. Satellite cells (SCs) have recently demonstrated diurnal characteristics related to muscle physiology. In our work, force production was reduced in the presence versus absence of SCs in the morning but, not in the afternoon. Morning-SC⁺ animals, producing lower force, sustained lesser degrees of injury versus SC⁻ counterparts. One potential mechanism underpinning lower forces produced appears to be lower calcium availability.

INTRODUCTION

Skeletal muscle comprises 40%–50% of body mass in mammals and plays vital roles in contractile function and locomotion (1–3). Recent evidence demonstrates that muscles have circadian molecular clocks residing within cell nuclei, whose gene expression oscillates daily (4). The rhythmicity of molecular clocks regulates numerous processes in muscle such that numerous physiological and metabolic events are time-of-day dependent (5–7). In this regard, muscle isometric torque differs with respect to time-of-day (8) with the mechanism underpinning this variation linked to the molecular clock. Alterations to force production in several clock-gene knockout mouse models provide further support for time-of-day regulations (9–11). Physiologically, different levels of maximal force induce varying degrees of injury (12, 13), and consequently, diurnal changes in force production may also lead to varying degrees of injury and repair according to time-of-day. Recent evidence in support of this contention is that the extent of muscle injury and repair following major injury (cardiotoxin) varies according to time-of-day (14).

Satellite cells (SCs), resident muscle stem cells responsible for muscle repair, also house molecular clocks that diurnally express clock genes over 24 h (15). Further characterization of the quiescent SC transcriptome demonstrates that several contractile-related genes are diurnally expressed (15). However, it is unclear whether diurnally expressed SC-specific clock genes and contractile-related genes impact muscle contractile function (16, 17). Alterations in ex vivo whole muscle contractile function have been reported after long-term SC ablation in an overload model of hypertrophy (17), although such alterations may be secondary to changes in muscle morphology (i.e., increased fibrosis) rather than being directly attributable to the absence of SCs. Whether SCs directly affect contractile function in the short term remains unknown.

Using an established mouse model to deplete SCs, we investigated whether short-term SC ablation affects muscle force production at two different times of day (0700 Morning-SC⁺, -SC⁻ and 1500 Afternoon-SC⁺, -SC⁻), and, if such differences exist, whether this alters contractile injury-induced force loss and loss of cytoskeletal protein (dystrophin).

METHODS

Animals

Pax7^CreERT2/+; Rosa26^DTA/+ mice (n = 24), ages 4–6 mo of mixed sex (Jackson Laboratories, Bar Harbor, ME, Stock Nos. 017763 and 010527, respectively) were used for these experiments (16, 18–20). All animal experiments were performed with the approval of the Northwestern University Institutional Animal Care and Use Committee. Mice had ad libitum access to food/water and were housed on a 14:10 light-dark cycle (lights on at 0600). Morning experiments were undertaken at 0700 (1 h into the light phase) and afternoon experiments were carried out at 1500 (9 h into the light phase). Inducible depletion of SCs was accomplished through Cre-Lox mediated Pax7⁺ cell ablation via five consecutive days of oral gavage of either tamoxifen (2 mg/mL) or vehicle (peanut oil) followed by a 10-day washout period, similar to our prior studies (19). Experiments were performed immediately after euthanasia at each time point.

Muscle Isolation and Experimental Apparatus

Immediately after euthanasia, 5.0 silk sutures were tied to proximal and distal EDL (extensor digitorum longus) tendons, dissected, and mounted between a force transducer (Aurora 300C, Aurora Scientific, ON, Canada) and length motor in a custom bath of Ringer’s solution (137 mM NaCl, 5 mM KCl, 1 mM NaH₂PO₄, 24 mM NaHCO₃, 2 mM CaCl₂, 1 mM MgSO₄, and 11 mM glucose containing 10 mg/L curare, pH 7.5) at 37°C with platinum electrodes straddling the muscle as previously described (21–23). Force, length, and time records were recorded on both an oscilloscope and a custom LabVIEW program.

Isometric Tetanic and Caffeine-Specific Forces

All contractile forces were normalized to physiological cross-sectional area (PCSA). A total of three maximal isometric tetanic contractions were administered at 100 Hz, 400 ms duration, 9–12 V, and the maximal value of the three was used similar to previous protocols (21–23). Each contraction was separated by 3 min rest. Caffeine contracture force was induced to estimate maximal unstimulated Ca⁺⁺-induced force via caffeine acting directly on the sarcoplasmic reticulum (SR) RyR1 receptor (24–26). Muscle tetanic tension was evaluated first in Ringer’s and after 3 min, replaced with Ringer’s containing 50 mM caffeine. For caffeine contracture, the muscle was not stimulated but tension was allowed to rise to a plateau over a 30-min time period until it peaked. Peak caffeine contracture force was recorded and subsequently expressed in units of specific force (N/cm²).

Eccentric Contraction

To evaluate maximal eccentric specific force, muscle was stimulated isometrically for 200 ms, then lengthened by 15% of L_f at a velocity of 2 L_f/s. Muscle was stimulated for a total of 400 ms. A single bout of 10 injurious eccentric contractions was performed with 3-min rest between contractions to avoid the confounding effects of fatigue (22, 23). A maximum isometric tetanic contraction was administered 3 min before and 5 min following the 10 eccentric contractions to quantify the extent of isometric force-loss, widely used as an indirect marker of injury (27–29). Linear regression was used to assess the relationship between maximum eccentric force and isometric tetanic force loss following injury.

Gene Expression

Genes of interest were analyzed using the QX200 AutoDG Droplet Digital PCR system (Bio-Rad). Muscle RNA extraction was performed as previously described (30, 31) and was analyzed for quality using Nanodrop-2000 and quantified using Qubit. An absorbance ratio of <1.8 was used to qualify the RNA and the quantification was used to equilibrate the samples to 1 ng/μL before ddPCR (droplet digital PCR).

A total of 5 μL of the equilibrated sample was aliquoted into the 96-well ddPCR plate along with 17 μL of a master mix (One-Step RT-ddPCR advanced kit for probes). The master mix also consisted of florescence-labeled ddPCR primers (Bio-Rad) for the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and the gene of interest (Bmal1, CLOCK, Cry1, Per2). The plate was then placed into the AutoDG to generate up to 20,000 droplets per well followed by reverse transcription. The plate was read using the QX200 droplet reader to visualize exact gene copy numbers per microliter of relative samples.

Immunohistochemistry

Flash-frozen EDL muscles, stored at −80°C, were transferred to a −25°C cryostat to section for immunohistochemical staining of SCs and myofiber perimeter (described in detail in Supplemental Methods).

Statistical Analyses

Individual groups were analyzed using unpaired t tests. A simple linear regression was used to analyze the relationship between tetanic and caffeine-specific forces, as well as eccentric specific force and isometric tetanic force loss. Gene expressions of muscle molecular clock genes were analyzed by two-way ANOVA for the main effects of time-of-day and treatment. Specific statistical tests are indicated in figure legends. All statistical analyses were performed using Prism 9.0 (GraphPad, San Diego, CA). All data in the results are reported as means ± standard error of mean (SE). Significance level (α) was set to 0.05 for all parametric tests.

RESULTS

Gene Expression

The pattern of muscle clock gene expression from morning to afternoon was similar in SC⁺ animals compared with SC⁻. A main effect of time-of-day was observed in Cry1 and Per2 genes (P < 0.05) (Fig. 1, C and D). Muscle clock gene expression was unaltered by SC ablation at either time point compared with SC⁺ animals (Fig. 1, A–D).

Figure 1.

Muscle molecular clock gene expression. A–D: gene expression of muscle molecular clock components Bmal1, CLOCK, Cry1, Per2 (respectively) in morning and afternoon animals represented in units of change in fold expression. All data are shown as means ± SE. Clock gene expression was compared through two-way-ANOVA for the main effects of time-of-day and treatment (**P < 0.01) (n = 4–5/group).

Eccentric-Specific Force and Contractile Injury Following 10 Eccentric Contractions

Although eccentric force in Morning-SC⁺ was reduced compared with Afternoon-SC⁺ animals, we note that such differences did not reach statistical significance (Morning-SC⁺: 19.88 ± 1.42, Afternoon-SC⁺: 25.56 ± 2.80 all values in N/cm²) (P = 0.09). Eccentric-specific force of Morning-SC⁺ animals was reduced compared with SC⁻ counterparts, whereas no differences were observed between afternoon groups (Morning-SC⁺: 19.88 ± 1.42, Morning-SC⁻: 27.81 ± 1.89, Afternoon-SC⁺: 25.56 ± 2.80, Afternoon-SC⁻: 25.26 ± 2.88 all values in N/cm²) (P < 0.05) (Fig. 2, A–F). Tetanic force loss and dystrophin^negative fibers (32, 33) were used to determine the extent of injury across groups (Fig. 2, C and D and G and H, Supplemental Fig. S2). Morning-SC⁺ animals exhibited reduced force loss and lesser amounts of dystrophin^negative fibers following 10 eccentric contractions compared with Morning-SC⁻ counterparts (Tetanic force loss: Morning-SC⁺: −5.63 ± 0.61, Morning-SC⁻: -7.93 ± 0.61; N/cm²) (Dystrophin^negative fibers: Morning-SC⁺: 32 ± 2.1, Morning-SC⁻: 64 ± 10.2) (P < 0.05) (Fig. 2, C and D). No differences in force loss or dystrophin^negative fibers were observed between afternoon groups (Tetanic force loss: Afternoon-SC⁺: −6.81 ± 0.39, Afternoon-SC⁻: −6.01 ± 0.4; N/cm²) (Dystrophin^negative fibers: Afternoon-SC⁺: 32 ± 5.0, Afternoon-SC⁻: 32.7 ± 10.7) (P > 0.05) (Fig. 2, G and H). An overall negative association was found between force loss and maximal eccentric force (Overall r² = 0.39, P < 0.01) (Fig. 3A).

Figure 2.

Eccentric-specific force and contractile injury in Morning and Afternoon groups. A and E: eccentric-specific forces of morning and afternoon groups (N/cm²). B and F: representative figure of experimental contractile-injury protocol consisting of a pre-eccentric tetanic contraction, 10 eccentric contractions, and post-eccentric tetanic contraction. C and G: extents of tetanic force-loss in morning and afternoon groups (N/cm²). D and H: Dystrophin^negative fibers in morning and afternoon groups. All data are shown as means ± SE. All groups were compared through unpaired t tests (*P < 0.05) (**P < 0.01) (n = 3–5/group).

Figure 3.

The extent of contractile force-loss is a function of maximum eccentric specific force. A linear regression between max eccentric force and tetanic force loss. All data shown are raw data points (n = 18).

Isometric Tetanic and Caffeine-Specific Forces

Tetanic specific force of Morning-SC⁺ animals was reduced compared with SC⁻ animals (Morning-SC⁺: 18.66 ± 2.12, Morning-SC⁻: 24.3 ± 1.07; N/cm²) (P < 0.05) (Fig. 4A), whereas no differences were observed between afternoon groups (Afternoon-SC⁺: 23.22 ± 1.53, Afternoon-SC^-: 21.45 ± 1.40; N/cm²). Caffeine-induced contracture force, reflective of maximal Ca⁺⁺ release and/or availability to contractile units, was reduced in Morning-SC⁺ mice compared with SC⁻ counterparts (Morning-SC⁺: 4.23 ± 0.24, Morning-SC⁻: 5.059 ± 0.16, N/cm², n = 6, P < 0.05) (Fig. 4B). Caffeine-contracture force was significantly correlated with maximal tetanic force (r² = 0.95, P = 0.001) (Fig. 4C). Maximum tetanic force measured immediately before measuring caffeine forces confirmed initial findings of reduced forces in Morning-SC⁺ compared with SC⁻ animals (Morning-SC⁺: 24.57 ± 1.10, Morning-SC⁻: 29.41 ± 0.56, N/cm², n = 6, P < 0.05).

Figure 4.

Tetanic and caffeine contracture-specific forces in Morning-SC⁺ vs. Morning-SC⁻ animals. A: tetanicspecific forces of morning groups (N/cm²). B: caffeine contracture-specific forces of Morning-SC⁻ vs. Morning-SC⁺. C: linear regression between maximal tetanic force and caffeine contracture force. All data are shown as means ± SE. All groups were compared through unpaired t tests (*P < 0.05) (n = 4-5/group) (caffeine data: n = 3/group).

Satellite Cell Ablation and Myofiber Characteristics

Pax7 abundance was significantly reduced (75%) in the tamoxifen-treated group (Total SCs: SC⁺ 36.3 ± 4.66 vs. SC⁻ 12.66 ± 3.71) (P < 0.05) (Supplemental Fig. S1A). Supplemental Fig. S1, C and D are a representative image of our SC ablation, respectively. Myofiber area was not different between SC⁺ and SC⁻ animals (SC⁺ 1,528.57 ± 148.63 vs. SC⁻ 1,501.54 ± 128.79; units in µm²) (Supplemental Fig. S1B).

DISCUSSION

We used an established mouse model to deplete SCs to characterize muscle clock gene expression and ex vivo skeletal muscle force production in the morning or afternoon in the presence and absence of SCs (Morning-SC⁺, -SC⁻ Afternoon-SC⁺, -SC⁻). Morning-SC⁺ animals demonstrated reduced tetanic and eccentric specific force compared with SC⁻ animals, although no differences were observed between Afternoon SC⁺/SC⁻ groups. In addition, Morning-SC⁺ animals, who produced lower forces, exhibited lower levels of contractile injury-induced force loss and cytoskeletal protein loss (less dystrophin^negative fibers) versus SC⁻ counterparts, but no such differences were noted between afternoon groups for these outcomes. To identify possible mechanisms for the lower forces observed in Morning-SC⁺ animals, we assessed caffeine-induced contracture force (a surrogate for maximal Ca⁺⁺ availability to contractile units) and found that Morning-SC⁺ harbored lower forces versus SC⁻ counterparts. Collectively, these data suggest that contractile function was influenced by SCs according to time-of-day.

Mounting evidence suggests that force production varies with respect to time-of-day (8), which may, in part, be explained by clock-gene regulation of contractile function. In this regard, Per, Bmal1, and CLOCK-mutant animals have previously exhibited alterations (reductions/increases) in ex vivo force production (9–11). In addition, SC molecular clock genes and contractile-related genes demonstrate a 24-h diurnal expression pattern (4, 15), although how such oscillations affect muscle contractility is unclear. Currently, there is limited evidence to suggest SCs play a role in contractile function as few studies have assessed baseline contractile function in whole muscle preparations following SC ablation (16, 17, 34). In a recent report, Bachman et al. (34) reported reduced force in prepubertal SC⁻ mice, although in mature mice, such force differences were not observed. Fry et al. (17) examined the long-term effects of SC depletion following an overload model of hypertrophy and found that whole muscle contractile function was reduced in SC⁻ mice, which they attributed to excess extracellular matrix (ECM) accumulation. In the present study, we assessed whole muscle force in the presence or absence of SCs and with respect to time-of-day after short-term (10–14 days) SC ablation. We found that SCs differentially regulated contractile properties according to time-of-day following short-term ablation, with Morning-SC⁺ animals displaying lower forces compared with SC⁻ animals.

Eccentric contractions produce the greatest contractile force and consequently lead to greater contractile-induced muscle damage/injury compared with concentric or isometric contractions (28, 35–37). As different eccentric forces can induce varying levels of injury (12, 13), we evaluated if time and SC-dependent differences in force observed in morning groups would lead to differences in two markers of contractile injury: force loss and loss of cytoskeletal protein dystrophin. Post-injury force loss and loss of various cytoskeletal proteins such as dystrophin have been widely used as markers for muscle damage across a variety of contractile injury models (12, 28, 32, 33, 35, 38–42). After administration of an injury protocol (22, 23), we found that Morning-SC⁺ animals exhibited reduced force loss and lower amounts of dystrophin^negative fibers post-injury compared with Morning-SC⁻ counterparts, with no differences in these markers between Afternoon SC⁺/SC⁻ animals. Our results support previous reports (12, 13) that higher forces lead to higher force losses reflective of greater magnitudes of injury with our data suggesting that time-of-day SC influence may be a secondary influence underlying this observation. Further support for this hypothesis comes from recent evidence in mice demonstrating that the magnitude of injury and repair following major muscle injury (cardiotoxin) differs according to the time-of-day injury is sustained (14). Although higher sample sizes are probably required to detect statistical significance, we observed lower eccentric force in Morning-SC⁺ versus Afternoon-SC⁺ animals (P = 0.09). This may be relevant for future studies that determine if human muscle repair following eccentric contractions differs by time-of-day similar to toxin-induced injury-repair models reported in animals (14).

As the maximal force production and extent of contractile injury was reduced in Morning SC⁺ versus Morning-SC⁻ animals, we sought to determine a potential mechanism to explain such reductions in baseline force. In this regard, evidence has shown molecular clocks harbor a connection to Ca⁺⁺-related contractile proteins and signaling pathways (4, 10, 43, 44). Interactions between Ca⁺⁺ and contractile proteins can induce post-translational modifications (i.e., phosphorylation) capable of altering force production (4) and previous evidence has demonstrated post-translational modifications differ according to exercise timing (45). Although we did not directly measure any contractile protein phosphorylation kinetics, given the critical role Ca⁺⁺ plays in force generation, we evaluated caffeine-induced contracture forces, as an indirect measure of maximal Ca⁺⁺ availability, between morning groups. Similar to our findings in tetanic/eccentric force, we observed a reduction in caffeine-contracture force in Morning-SC⁺ compared with SC⁻ counterparts, suggesting lower tetanic/eccentric forces in Morning-SC⁺ animals may be due to a reduced volume of Ca⁺⁺ available to contractile units. Although our measure of Ca⁺⁺ was indirect, we note that a similar approach has previously been reported as a valid surrogate measure for the evaluation of Ca⁺⁺ volume (24–26). In addition, linear regression analyses revealed a strong association (r² = 0.95, P < 0.01) between caffeine-contracture force and maximal tetanic force.

In conclusion, we report reduced ex vivo force production in the presence of SCs in the morning but not in the afternoon. Furthermore, Morning-SC⁺ animals demonstrating reduced force production also experienced lesser extents of injury-induced force loss and dystrophin^negative fibers. One potential mechanism underpinning lower maximal forces observed in Morning-SC⁺ animals may be lower Ca⁺⁺ availability for force generation. Although we attribute time-of-day findings to be regulated, in part, by time-sensitive molecular clocks within SCs, our model of inducible depletion ablates the entire SC and is not confined to molecular clocks. Accordingly, we cannot rule out the possibility that another cellular mechanism in SCs outside of the molecular clock may be contributing to differences in force production. Taken collectively, the results from the current study suggest that SC time-of-day characteristics may be an important consideration in future experiments that investigate the mechanisms underpinning the diurnal nature of contractile function.

DATA AVAILABILITY

All data used within the results and to create figures are included in the material of this manuscript. Additional analysis and files can be provided upon request.

SUPPLEMENTAL DATA

Supplemental Figs. S1 and S2 and Supplemental Methods: https://doi.org/10.6084/m9.figshare.25514272.

GRANTS

This work was supported, in part, by a Novo Nordisk Foundation Challenge Grant NNF14OC0011493 (to J.A.H.), and the National Institutes of Health Grant HD094602 (to S.D.). This work was supported in part by the Research Career Scientist Award No. IK6 RX003351 from the United States (U.S.) Department of Veterans Affairs Rehabilitation R&D (Rehab RD) Service (to R.L.L).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.E.K., S.D., and J.A.H. conceived and designed research; R.E.K., G.M., F.D., O.L.-K., and S.D. performed experiments; R.E.K. and S.D. analyzed data; R.E.K., R.L.L., S.D., and J.A.H. interpreted results of experiments; R.E.K. and S.D. prepared figures; R.E.K. drafted manuscript; R.E.K., R.L.L., S.D., and J.A.H. edited and revised manuscript; R.E.K., R.L.L., S.D., and J.A.H. approved final version of manuscript.