Krüppel-like factor 10 regulates the contractile properties of skeletal muscle fibers in mice

Abstract

Introduction/Aims:

Klf10 is a member of the Krüppel-like family of transcription factors, which is implicated in mediating muscle structure (fiber size, organization of the sarcomere), muscle metabolic activity (respiratory chain), and passive force. The aim of this study was to further characterize the roles of Klf10 in the contractile properties of skeletal muscle fibers.

Methods:

Fifty-two single fibers were extracted from female wild-type (WT) and Klf10 knockout (KO) oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skinned muscles. Each fiber was immersed successively in relaxing (R), washing (W), and activating (A) solutions. Calcium was included in the activating solution to induce a maximum contraction of the fiber. The maximum force (F_max) was measured and normalized to the cross-sectional area to obtain the maximum stress (Stress_max). After a steady state in contraction was reached, a quick stretch-release was performed; the force at the maximum stretch (F_stretch) was measured and the stiffness was assessed.

Results:

Deletion of the Klf10 gene induced changes in the contractile parameters (F_max, Stress_max, Stiffness), which were lower and higher for soleus and EDL fibers compared with littermates, respectively. These measurements also revealed changes in the proportion and resistance of attached cross-bridges.

Discussion:

Klf10 plays a major role in the homeostasis of the contractile behavior of skeletal muscle fibers in a muscle fiber type–specific manner. These findings further implicate important roles for Klf10 in skeletal muscle function and shed new light on understanding the molecular processes regulating the contractility of skeletal muscle fibers.

1 |. INTRODUCTION

Krüppel-like transcription factor 10 (Klf10), also known as transforming growth factor-β (TGF-β)–inducible early gene-1 (TIEG1) is a member of the Krüppel-like family of transcription factors that regulates gene expression in multiple cell and tissue types.^1,2 Klf10 is implicated in multiple biological processes and diseases (i.e, osteoporosis,³ cancer,^4,5 and hypertrophic cardiomyopathy).⁶ Recently, Klf10 has been implicated in Duchenne muscular dystrophy, suggesting a role for Klf10 in the regulation of fibrosis.⁷

Loss of Klf10 expression results in multiple muscle phenotypes,^8,9 such as hyperplasia, hypertrophy, a decrease in succinate dehydrogenase, cytochrome c oxidase and menadione activities, ultrastructural muscle disorganization (smaller sarcomeres and absence of I bands), and changes in mitochondrial shape and respiration. All these phenotypes were only observed in female animals, with no significant defects or differences detected between male Klf10 knockout (KO) and wild-type (WT) mice. Similar sex-specific differences have also been observed in the skeleton on Klf10 KO mice where only female animals exhibited an osteopenic phenotype.¹⁰ The basis for sex-specific differences in bone are attributed to defects in estrogen signaling in Klf10 KO mice.³

Skeletal muscle must be characterized in passive¹¹ and active¹² conditions to achieve a comprehensive understanding of this tissue. Previous studies have only implemented passive stretch¹³ and compressive⁹ tests to reveal changes in elasticity of Klf10 KO muscle fibers. Herein we assess the role of Klf10 in the contractile properties of muscle fibers from slow-twitch (soleus) and fast-twitch (extensor digitorum longus [EDL]) muscles.

2 |. METHODS

2.1 |. Animals

Klf10 KO mice were generated using a neomycin targeting vector to delete approximately 5.5 kb of the Klf10 locus, including the entirety of exons 1 and 2.¹⁴ Klf10 KO animals utilized in this study were on a congenic C57BL/6 background. WT and Klf10 KO animals were derived from heterozygous breeding. Female animals, 3 months of age, were utilized in these experiments. The protocol was approved by the French Ministry of Higher Education, Research and Innovation (DUO-4776) and the ethics animal care and use committee of the Picardie region.

2.2 |. Transmission electron microscopy

Eight soleus and EDL muscles were harvested from 4 WT and 4 Klf10 KO mice using a published protocol.¹⁵ Transverse ultrathin (90 nm) sections were cut using a Leica UC7 ultramicrotome and were placed on copper grids. Five micrographs of each specimen were randomly captured across the muscle using a transmission electron microscope (TEM) (JEOL 1400Plus; JEOL, Ltd, Tokyo, Japan) operating at 80 kV with a magnification of 42 000×. ImageJ 1.46/Java 8 software (National Institutes of Health, Bethesda, Maryland) was used with the plugin “Analyze Particles” to determine the myosin (myo) cross-sectional area (myoCSA).¹⁶

2.3 |. Skinned muscle preparation

For this analysis, a second group of mice was utilized for TEM experiments. Soleus muscles were dissected from the right hindlimbs of 8 WT mice and 8 Klf10 KO mice, and then permeabilized at 4°C using a relaxing solution with different percentages of glycerol (12.5%, 25%, and 50%) added.¹³ Fiber bundles were stored in 50% glycerol relaxing solution at -20°C for 2 weeks.¹⁷

2.4 |. Active mechanical tests

Single muscle fibers were isolated from each Klf10 KO (N_soleus = 11, N_EDL = 16) and WT (N_soleus = 11, N_EDL = 14) skinned muscles. First, the fibers were placed in a small bath filled with the relaxing solution at 20°C. The extremities were connected to a force transducer (5 mN) and to a motor (Model 1400A; Aurora Scientific, Aurora, Ontario, Canada). The fiber length, diameter (D), and the distance between the successive sarcomeres were measured using a camera (UI-1220LE). The fibers were placed with an initial¹³ sarcomere length of 2.5 μm and immersed in a bath containing a washing solution (W) free of calcium for 8 seconds. To activate the actin-myosin cross-bridges, fibers were then immersed in another bath containing an active solution of calcium (pCa = 4.5), resulting in muscle fiber contraction and the measurement of the maximum force (F_max).^18,19 This value was measured as an average of data during 1 second when the force had reached a steady-state plateau. The maximum stress (Stress_max) was measured by dividing the maximum force F_max by the cross-sectional area (CSA) of the fiber. After 31 seconds of immersion in the calcium solution, a quick stretch-release cycle was performed with an amplitude (ε_stretch) of 0.2% of the fiber length during 1 second, and an edge at a speed of 1 fiber length per second.²⁰ The force (F_stretch) at the peak of the stretch was determined and the stiffness was calculated as follows:

$$\mathrm{Stiffness}=\frac{{\mathrm{F}}_{\mathrm{stretch}}-{\mathrm{F}}_{\max }}{{\epsilon }_{\mathrm{stretch}}\mathrm{.}\mathrm{CSA}}$$

2.5 |. Statistical analysis

SYSTAT version 11 (SYSTAT Software, Inc, San Jose, California) software was used and two-sample Mann-Whitney U tests were used to compare all parameters (myoCSA, D, stiffness, stress) as a function of genotype. P < .05 was considered significant.

3 |. RESULTS

The muscle fiber active force (Figure 1) revealed lower and higher forces for Klf10 KO soleus and Klf10 KO EDL muscle, compared with WT littermates, respectively. TEM acquisitions revealed a reduction in the mean number of myosin filaments per region of interest (Figure 2A,B) within the Klf10 KO muscles (N_Soleus = 90, N_EDL = 103) compared with the WT muscles (N_Soleus = 117, N_EDL = 112). TEM analysis of the myosin area (Figure 2C,D) also revealed a significant increase (P < .001) in cross-sectional area (myoCSA) in Klf10 KO soleus and EDL compared with WT littermates. No significant differences in mean ± SEM muscle fiber diameter (D) for the EDL (D_WT = 44 ± 6 μm, D_{Klf10_KO} = 44 ± 1 μm) or soleus (D_WT = 46 ± 3 μm, D_{Klf10_KO} = 39 ± 1 μm ; P = .065) a s afunctio no genotype were detected.

FIGURE 1.

Force behavior of the WT and Klf10 KO muscle fiber from soleus (A) and EDL (B) in the relaxing (R), washing (W), and activating (A) solutions. The dotted circle indicates the stretch-release test, which is enlarged in the figure below for soleus and EDL, respectively. The stretch-release test was performed at 30 seconds for both WT and Klf10 KO fiber. Abbreviations: EDL, extensor digitorum longus; F_max, maximum force; F_stretch, stretch force; WT, wild-type

FIGURE 2.

Region of interest (1500 × 1500 pixels, pixel size = 0.22 nm) from transmission electron microscopy of transversal section from soleus (A) and EDL (B) muscles for both genotypes (WT, Klf10 KO). The scatterplots depict that the individual data points between WT and Klf10 KO mice segregate, more obviously for EDL than soleus. Cross-sectional area of myosin (myoCSA) was measured in the region of interest in four soleus (C) and four EDL (D) muscles for both genotypes (WT, Klf10 KO). Myosin area is significantly different between WT and Klf10 KO fibers for both soleus and EDL muscles (P < .001): in soleus muscle (myoCSA_WT = 136.8 ± 1.4 nm², n = 466; myoCSA_{Klf10_KO} = 155.3 ± 1.3 nm², n = 360) and in EDL muscles (myoCSA_WT = 141.4 ± 0.8 nm², n = 448; myoCSA_{Klf10_KO} = 174.0 ± 1.4 nm², n = 412). All data is presented as mean ± standard error of mean and “N” is the number of myosin filaments used in the analysis. Abbreviations: KO, knockout; myoCSA, myosin cross-sectional area

The maximum normalized active force (Stress_max) was significantly (P = .001) lower for the Klf10 KO soleus fibers compared with the WT littermates (Figure 3A). Opposing results were observed for the EDL with Klf10 KO soleus fibers, showing significantly higher (P = .008) values compared with the WT littermates (Figure 3A). For the WT genotype, no significant difference was noted for maximum stress between soleus and EDL fibers.

FIGURE 3.

Boxplots of Stress_max values (A) and Stiffness (B) for soleus and extensor digitorum longus muscles as a function of genotype. **P < .01, ***P < .001

Results of stiffness measurements were similar to those for maximum stress, with significantly lower (P = .008) and higher (P = .004) values for the Klf10 KO soleus fibers and Klf10 KO EDL fibers, respectively, compared with the WT littermates (Figure 3B). No significant differences in stiffness were observed between WT soleus and WT EDL fibers.

4 |. DISCUSSION

In this study we have elucidated the impact of Klf10 expression on parameters related to muscle fiber contraction for oxidative and glycolytic muscles. In previous work we demonstrated that sarcomeric length is shorter in skeletal muscles of Klf10 KO mice.¹³ In spite of this phenotype, we conclude that the Klf10 KO sarcomere is functioning normally based on the data reported here.

Thus, the decrease in the maximum stress for Klf10 KO oxidative fibers compared with WT fibers may be indicative of a smaller proportion of attached cross-bridges or a weaker adhesion between the myosin head and the actin molecule during the isometric contraction.²¹ This possibility is supported by the TEM results showing a decreased density of myosin in soleus Klf10 KO fibers.

The weaker stiffness observed for the soleus Klf10 KO compared with WT fibers is consistent with reduced contractile properties. Indeed, the stretch applied after the active steady state reflects the resistance of the attached cross-bridges,¹² which are weaker in soleus Klf10 KO and associated with the reduced density of myosin.

In a previous study⁸ we demonstrated that the Klf10 KO soleus and EDL muscles exhibited hypertrophy, with an increase in the wet weight of Klf10 KO soleus (7.8 ± 0.6 mg) compared with WT soleus (6.3 ± 0.7 mg), and Klf10 KO EDL (8.3 ± 0.4 mg) compared with WT EDL (6.8 ± 0.4 mg). This may be one causative factor underlying the force measurement differences reported herein. Another potential explanation would be differences in myosin content or myosin isoforms, which need to be investigated in future studies. Although we have analyzed myofiber structures using various methods, it is difficult to compare the diameter of intrinsic structure (myosin) and the diameter of the entire muscle fiber. We previously demonstrated that the Klf10 KO sarcomeres are shorter than WT sarcomeres with a shorter H band where the myosin is located. Therefore, it could be assumed that the myosin may be compressed and this phenomenon may induce an increase of the CSA.

The increase in the contractile properties for Klf10 KO EDL fibers compared with littermate controls could be due to the homeostasis of the muscle in an effort to maintain the contractile properties.

In conclusion, we have demonstrated that Klf10 plays a crucial role in the homeostasis of the contractile behavior of skeletal muscle fibers and functions in a muscle type–specific manner.

In future studies, eccentric contraction force could be measured with repetitive stimulations to provide evidence for a greater role of Klf10 in muscle physiological forces. It is of interest to determine whether differences in calcium sensing, extracellular matrix composition, and excitation-contraction coupling exist in Klf10 KO muscles and whether such differences contribute to deficits in the force measurements described in this study.

Abbreviations:

A

activating solution

CSA

cross-sectional area

D

diameter

EDL

extensor digitorum longus

F_max

maximum force

F_stretch

stretch force

Klf

Krüppel-like factor

KO

knockout

Myo

myosin

pCa

-log(10) of the calcium concentration

R

relaxing solution

Stress_max

maximum stress

TEM

transmission electron microscopy

TIEG1

transforming max growth factor-β–inducible early gene-1

W

washing solution

WT

wild-type

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.