Role of the cytoskeleton in muscle transcriptional responses to altered use

Abstract

In this work, the interaction between the loss of a primary component of the skeletal muscle cytoskeleton, desmin, and two common physiological stressors, acute mechanical injury and aging, were investigated at the transcriptional, protein, and whole muscle levels. The transcriptional response of desmin knockout (des^−/−) plantarflexors to a bout of 50 eccentric contractions (ECCs) showed substantial overlap with the response in wild-type (wt) muscle. However, changes in the expression of genes involved in muscle response to injury were blunted in adult des^−/− muscle compared with wt (fold change with ECC in des^−/− and wt, respectively: Mybph, 1.4 and 2.9; Xirp1, 2.2 and 5.7; Csrp3, 1.8 and 4.3), similar to the observed blunted mechanical response (torque drop: des^−/− 30.3% and wt 55.5%). Interestingly, in the absence of stressors, des^−/− muscle exhibited elevated expression of many these genes compared with wt. The largest transcriptional changes were observed in the interaction between aging and the absence of desmin, including many genes related to slow fiber pathway (Myh7, Myl3, Atp2a2, and Casq2) and insulin sensitivity (Tlr4, Trib3, Pdk3, and Pdk4). Consistent with these transcriptional changes, adult des^−/− muscle exhibited a significant fiber type shift from fast to slow isoforms of myosin heavy chain (wt, 5.3% IIa and 71.7% IIb; des^−/−, 8.4% IIa and 61.4% IIb) and a decreased insulin-stimulated glucose uptake (wt, 0.188 μmol/g muscle/20 min; des^−/−, 0.085 μmol/g muscle/20 min). This work points to novel areas of influence of this cytoskeletal protein and directs future work to elucidate its function.

skeletal muscle is adept at adapting to physiological stressors. It is in this way that muscle is able to balance the mechanical and metabolic needs of the body for efficient movement. However, the mechanisms of muscular adaptation are more complex than a simple linear change in mass in response to a change in loading (17). Muscles experience a wide variety of stresses from mechanical loading to oxidative damage to metabolic dysregulation (5, 11). The discussion of which of these “stressors” is most relevant to muscle physiology depends heavily on the context of the question. Experimentally, physiologists stress tissues in different ways depending on the topic of interest: mechanical insult, biochemical disruption, electrical stimulation, etc. (3, 58, 60). The choice of experimental stress is important to the framework of the hypothesis and the interpretation of the study's results. Accordingly, it is possible that the choice of experimental stress could define the role of a given protein or pathway of interest if, for example, one focuses on mechanics or biology alone when both may play equally important or potentially interrelated roles.

From a physiological standpoint, muscle frequently experiences combinatorial stress. An intense bout of exercise can induce both mechanical and oxidative muscle damage and activate a host of inflammatory and metabolic pathways, and we know from exercise studies in humans that the response to stress can vary considerably depending on whether the muscle is young or old, healthy or diseased (22, 36). The interaction between different stress types is particularly relevant for muscle as it is a primary target in interventional physical therapies. A particular strength training program may be exceptionally effective for a muscle wasting disease but ineffective for a metabolic disease because the interaction between the stress of each disease and the applied mechanical stress are creating fundamentally different muscular responses.

The purpose of this study was to use two experimental paradigms at the extremes of the spectrum of physiological stress, namely, acute mechanical stress and chronic aging stress, to investigate the role of the cytoskeletal protein desmin in muscle physiology. On the one hand, a bout of eccentric contractions applies a brief and purely mechanical stress to the tissue, while on the other, aging involves a host of biological pathways to combat the cumulative effects of oxidative stress, inflammation and cellular senescence on a completely different time scale. These models were employed in the presence and absence of desmin, the predominant intermediate filament protein in muscle and a primary component of the muscular cytoskeleton. Mutations in the desmin gene cause a myopathy in humans characterized by muscle weakness and structural disorganization that progressively worsens with age (13). Studies in the desmin knockout (des^−/−) mouse have begun to investigate some of the hallmarks of desminopathy, including disorganized and misaligned sarcomeres (52), lowered stress production (28, 49), increased fatiguability (28) and a lowered response to eccentric contraction (ECC)-induced injury (49). The mechanism for this myopathy is not fully understood, and some previous studies have suggested it to be primarily mechanical in nature (27, 51), while others have proposed biochemical aspects (40). The altered mechanical response of isolated muscle from the des^−/− mouse to ECC (49) and the alterations to the desmin cytoskeleton following ECC in wild-type (wt) mice (30) suggest that desmin may play a role in mediating ECC-induced injury. This, combined with the progressive myopathy exhibited by muscles lacking desmin, makes the des^−/− mouse an interesting model to study the effect of combinatorial stress on skeletal muscle. Additionally, investigating the interaction of the disease state with the muscle's response to acute mechanical and chronic biological stress could provide some clues as to the roles of desmin in mechanical and biochemical aspects of muscle function over the short and long term.

Fold changes of individual genes involved in extracellular matrix (ECM) regulation and inflammation have been published previously for control muscles from this microarray dataset (39) and are not repeated here. Instead, gene expression data were investigated broadly by a top-down approach that focuses on interactions between desmin and the stressors of aging and ECC injury. Our hypothesis in this study was that the absence of desmin would significantly alter the transcriptional response of skeletal muscle to the acute mechanical stress of eccentric contraction and the chronic stress of aging. We further hypothesized that the combination of these two stressors would create a unique transcriptional state, different from either individual stress state, with alterations not only localized to the mechanical integrity of the cytoskeleton, but spread across the spectrum of muscle physiology.

MATERIALS AND METHODS

Experimental Design

Experiments were performed on muscles from wt 129/Sv (Taconic Farms, Germantown, NY) and des^−/− 129/Sv (41) mice at two ages: “young” (7–9 wk, 29.1 ± 1.2 g; wt n = 5, des^−/− n = 5) and “adult” (>12 mo, 24.9 ± 1.3 g; wt n = 5, des^−/− n = 4). The young time point was selected as the onset of sexual maturity and the adult time point as the fraction of the mouse lifetime corresponding to the average age of clinical symptom presentation in human desminopathies (13). One hind limb from each mouse was subjected to an exercise protocol of 50 ECC, while the contralateral limb served as a control. Twelve hours after the ECC protocol, tibialis anterior (TA) muscles were harvested, flash-frozen, and processed for microarray analysis, a time determined to maximize the expression of muscle-specific injury response genes (3). The TA muscle was chosen for analysis as it is the largest of the dorsiflexor muscle group and is known to exhibit signs of injury following ECC (3). All procedures were performed in accordance with the National Institutes of Health Guide for the Use and Care of Laboratory Animals and were approved by the University of California and Department of Veterans Affairs Committees on the Use of Animal Subjects in Research.

Eccentric Exercise

ECCs were applied to the mouse plantarflexors as previously described (3). In brief, animals were continually anesthetized with 2% isoflurane at 2 l/min and secured in a custom designed jig. One foot was secured to a plate attached to a rotational bearing that provided both precise rotational displacements and torque measurements (custom modified model 360B; Aurora Scientific, Ontario, Canada). The animal's knee was positioned and fixed such that the ankle angle (α) could be precisely controlled by the rotation of a footplate. Sterile subcutaneous 28-gauge needle electrodes (Grass Instruments, Braintree, MA) were placed in the vicinity of the right peroneal nerve, ∼0.5 mm under the skin, just lateral to the midline and distal to the knee joint for stimulation of the dorsiflexor muscle group. Proper placement of electrodes and optimum stimulation parameters were determined as previously described (3).

After stimulation parameters were defined and the hind limb secured, initial measurements of isometric torque were taken at α = 90°, which was determined to be the angle of peak isometric dorsiflexion torque (29). The foot was then dorsiflexed to α = 52°, and isometric torque was recorded again. Eccentric contractions were elicited by stimulating the peroneal nerve for 400 ms while rotating the foot through 76° of plantarflexion (α = 52–128°) once per minute for 50 min. The dorsiflexor muscle group thus initially contracted isometrically for 150 ms, following which the muscles were stretched at a rate of 520°/s while stimulation continued, resulting in a rapid torque rise (Fig. 1A). After the ECC bout, the ankle was returned to α = 90°, and isometric torque was again recorded. The contralateral leg was subjected to isometric torque measurements at α = 90° only and served as a control for ECC injury. Mice were then returned to their cages and allowed to recover for 12 h. After the recovery period, isometric torque was recorded in both the exercised and contralateral leg at α = 90° and the TA muscle from each leg was dissected, flash-frozen in liquid nitrogen, and stored at −80°C. Animals were then euthanized by cervical dislocation.

Fig. 1.

Desmin knockout (des^−/−) dorsiflexors exhibit a differential response to an eccentric contraction (ECC) bout compared with wild type (wt). A: a sample ECC torque record resulting from stimulation of the hind-limb dorsiflexors (gray). The hatched bar marks the time of stimulation and is superimposed on ankle angle (α). While α remains constant at 52°, the contraction is isometric (denoted by a horizontal bar above the torque trace), but as α increases, the contraction becomes eccentric as dorsiflexors are simultaneously stimulated and stretched. B: des^−/− dorsiflexors produce less isometric torque initially (PRE), exhibit a smaller force drop following the ECC bout (PREPOST), and show less recovery 12 h postbout (POSTRECOVERY). Bars indicate P < 0.05 compared with arrow. C: young des^−/− dorsiflexors experience less isometric torque drop than wt during the ECC bout. Isometric torque prior to every other ECC is plotted as a function of ECC number for clarity. The torque decrease is quantified by arrows to the right of the plot for each group, and the torque drop as a percentage of initial torque is noted. Similar results were seen for adult groups (not shown). *P < 0.05 as determined by 2-way ANOVA with repeated measures and Tukey post hoc correction.

Microarray Processing

RNA was extracted from whole TA muscles of wt and des^−/− mice with a combination of standard TRIzol (Invitrogen, Carlsbad, CA) and RNeasy (Qiagen, Valencia, CA) protocols. Muscles were homogenized on ice in a rotor-stator homogenizer in 0.5 ml TRIzol. We then added 0.1 ml of chloroform, and the sample was vigorously vortexed for 15 s followed by centrifugation. The supernatant was combined with an equal volume of 70% ethanol and filtered through the RNeasy spin column. The column was washed, incubated with RNase-free DNase (Qiagen), washed again, and eluted as described in the manufacturer's protocol. RNA concentration was determined by the absorbance at 260 nm, and RNA purity was defined by the 260 nm-to-280 nm absorbance ratio. Individual Affymetrix microarrays (“GeneChip” Mouse Genome 430A 2.0 Array; Affymetrix, Santa Clara, CA) were used for each muscle (young wt n = 10, young des^−/− n = 10, adult wt n = 10, adult des^−/− n = 8). RNA processing for the GeneChip, including stringent quality control measures, was performed by the Gene Chip Core at the Department of Veterans Affairs San Diego Health Care System (San Diego, CA).

Genespring software (SiliconGenetics, Redwood City, CA) was used to identify genes that were differentially expressed as a function of genotype, age, and treatment. Three independent probe set algorithms were used for background subtraction and normalization (MAS5, RMA, and GCRMA), and each feature was normalized per chip and per gene as previously described (54). Normalized expressions of identified genes, excluding putative genes and expressed sequence tags, were subjected to three-way ANOVA (main factors of desmin, ECC, and age) with a significance level set to 0.05 and a Benjamini and Hochberg false discovery rate multiple testing correction for present features. Hierarchical clustering was performed on conditions with a Pearson centered distance metric and a centroid linkage rule. In accordance with Minimum Information About a Microarray Experiment standards, microarray data and annotations have been deposited via Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/ accession number GSE41363).

Gene Classification

To investigate the biological context of transcriptional changes, we investigated the role of significant genes in various muscle pathways. Gene function was investigated with a combination of the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/pubmed/), gene ontology (GO) classification (http://www.ebi.ac.uk/GOA/), and a broad literature search for studies on the gene or protein of interest in skeletal muscle. Genes were then classified into one of six categories according to the function that most likely predominated in the framework of skeletal muscle physiology (53): cytoskeletal, ECM, excitation-contraction coupling (EX), metabolism, inflammation, and remodeling. The cytoskeletal category included genes involved in cell structure, such as intermediate filaments, and genes encoding contractile proteins, such as myosin isoforms. ECM genes included genes encoding ECM components, such as collagens and proteoglycans, and genes involved in signaling proliferation or breakdown, such as matrix metallopeptidases. The EX category included genes involved in the function of the neuromuscular junction and genes involved in calcium transport into and out of the sarcoplasmic reticulum. Metabolic genes included those involved in the glycolytic or the fatty acid metabolism pathways as well as genes involved in mitochondrial function and utilization of energy stores such as ATP. The inflammatory category included genes encoding known inflammatory cell markers, genes involved in response to stress, such as heat shock, and genes promoting or inhibiting portions of the inflammatory pathways. Finally, genes involved in remodeling included those identified as having anabolic or catabolic functions in muscle, such as growth factors and the ubiquitination pathway, genes involved in cell cycle control, genes specific to satellite cells or myoblasts and genes involved in angiogenesis. Genes with significant age × desmin interactions are listed in Supplementary Table S1 and genes with significant ECC × desmin interactions are listed in Supplementary Table S2 with their classification and a PubMed identification number (http://pubmed.gov PMID) to a paper or papers used in determining that classification.¹

The identified categories will inherently have different quantities of gene components and thus GO analyses were used to determine whether a pathway was over- or underrepresented. GO analyses were performed with a Web-based Gene Set Analysis Toolkit (WebGestalt; http://bioinfo.vanderbilt.edu/webgestalt/).

Quantitative Real-Time PCR

To validate GeneChip expression values and obtain expression levels for genes not included in the Mouse Genome 430A 2.0 Array, isolated RNA samples were subjected to quantitative real-time PCR (QPCR). Extracted RNA was diluted 1:5 with DNase/RNase-free water, and 1 μl of each sample was reverse transcribed by standard protocols (Superscript III, Invitrogen). Amplification of cDNA was performed with the Eppendorf MasterCycler GradientS (Hamburg, Germany) with primers specific to the genes of interest. All primers were tested for cross-reactivity with other transcripts using nBLAST and Oligo (version 6.6; Molecular Biology Insights, Cascade, CO). Samples were run at least in triplicate on a 96-well plate with each well containing 10 μl volume made up of the KAPA SYBR FAST Master Mix (2×) Universal (KAPA Biosystems) and forward and reverse primers.

Amplification conditions consisted of an initial hold at 95°C for 2 min was followed by 40 cycles of denaturing at 95°C for 15 s, followed by annealing/extension at 68°C for 40 s. A successful reaction was confirmed by the observation of a single reaction product on an agarose gel and a single peak on the DNA melting temperature curve determined at the end of the reaction. The standard curve method was used to express QPCR results with the “cycles to threshold” value representing the PCR cycle number at which the SYBRgreen signal was increased above the threshold. Expression of each gene was normalized to its mean value.

Myosin Heavy Chain Isoform Determination

Myosin heavy chain (MyHC) isoforms were identified by the gel electrophoresis technique previously described (55). Briefly, TA muscles (des^−/− n = 6, wt n = 6) were homogenized and centrifuged, and the myofibril-rich pellet was washed and resuspended in buffer supplemented with a protease cocktail (5 μl of 100 mmol/l PMSF, 10 μg/μl leupeptin, and 10 μg/μl pepstatin A). Protein was then diluted to a concentration of 0.125 mg/ml across all homogenates. Separation of MyHC isoforms was performed with SDS-PAGE on polyacrylamide gels (16 cm × 22 cm, 0.75 mm thickness) for 22 h at 275 V at 4°C. Stacking and resolving gels were 4 and 8% polyacrylamide, respectively. Following migration, gels were silver stained according to manufacturer's instructions (Bio-Rad, Hercules, CA). MyHC isoform (I, IIa, IIx, and IIb) bands were identified by their relative electrophoretic mobilities. Band intensity was quantified by densitometry (Quantity One, Bio-Rad).

Isolated Skeletal Muscle Insulin Stimulation

Insulin sensitivity was measured in isolated soleus muscles from young (des^−/− n = 8, wt n = 7) and adult (des^−/− n = 6, wt n = 5) mice by the 2-deoxyglucose uptake (2DOGU) technique. The soleus muscle was chosen for these measures because its small size allows oxygenation through the entire muscle belly, a requirement for accurate 2DOGU measurements. In brief, mice were fasted for 4 h, weighed, and then anaesthetized via intraperitoneal injection (Nembutal 150 mg/kg). Fasting glucose was measured by the glucose oxidase method with a blood glucose meter (Contour, Bayer), and blood sampled from the tail vein; the epidydimal fat pad was dissected and weighed. For insulin stimulation, paired soleus muscles were incubated at 35°C for 30 min in oxygenated (95% O₂, 5% CO₂) flasks of Krebs-Henseleit buffer (KHB) containing: 0.1% BSA, 2 mM Na-pyruvate, and 6 mM mannitol. One muscle per pair was incubated without insulin and the contralateral muscle with insulin [60 μU/ml (0.36 nM)]. After 30 min, muscles were transferred to a second flask and incubated at 35°C for 20 min in KHB plus 0.1% BSA, 9 mM [¹⁴C]mannitol (0.053 mCi/mmol, PerkinElmer), and 1 mM [³H]2DG (6 mCi/mmol, PerkinElmer) with the same insulin concentration as in the first incubation. After the second incubation phase, muscles were blotted on ice-cold filter paper, trimmed, freeze-clamped, and then were stored (−80°C). 2DOGU rate was calculated as previously described (8).

Paired soleus muscles were then homogenized. Equal amounts of protein (30 μg) were boiled for 5 min in 1× Laemmli sample buffer and separated on 7% gels by SDS-polyacrylamide gel electrophoresis (PAGE) as previously described (37). Primary antibodies used for immunoblotting were from Cell Signaling Technology (p-AktSer473 cat. #9271, Akt cat. #9272). Antibody binding was detected with an enhanced chemiluminescence horseradish peroxidase substrate detection kit (Bio-Rad). Imaging and band quantification were carried out with a ChemiDoc XRS-plus imaging system (Bio-Rad).

Data Processing

Torque data were acquired via customized LabView software and analyzed using Matlab. All data were subjected to a one-, two-, or three-way ANOVA with repeated measures where appropriate. Significance was set at α < 0.05, and all error bars represent means ± SE. Statistical tests used for specific datasets are as listed in the text.

RESULTS

ECC Induced Injury

Des^−/− dorsiflexors experienced a unique response to the ECC bout compared with wt (Fig. 1B). Initial isometric torque was significantly lower in des^−/− muscles compared with wt at both ages (P < 0.05). However, during the ECC bout, torque dropped significantly more in the wt groups (P < 0.001; young 32.7 ± 3.6%, adult 55.5 ± 6.1%) than in des^−/− (P > 0.05; young 24.7 ± 4.1%, adult 30.3 ± 10.9%). After 12 h of recovery, isometric torque increased significantly in the young wt group (P < 0.01) though it did not fully return to its pre-ECC value, consistent with our previously published data (3). However, torque did not increase significantly in des^−/− groups (P > 0.1). Thus, des^−/− dorsiflexors produced lower isometric torque, experienced less torque drop following ECC, and subsequently exhibited reduced recovery. This is also evident in torque measurements during the course of the ECC bout where a larger progressive decrease in torque is seen in wt muscles relative to des^−/− (Fig. 1C). Though des^−/− muscles initially produce significantly less isometric torque, there was no longer a significant difference between genotypes by ECC number 26 (asterisks, Fig. 1C).

Gene Expression as a Function of Desmin, Age, and ECC

Microarray samples were divided according to three grouping variables, desmin (des^−/−, wt), age (young, adult), and ECC (ECC, control), and subjected to three-way ANOVA (Fig. 2). Desmin and age were the most significant factors in differential gene expression of muscle evidenced by 1,880 genes with a main effect of desmin and 1,384 genes with a main effect of age identified by the ANOVA. Interestingly, there were 2,011 genes with a significant age × desmin interaction, indicating that not only is des^−/− muscle inherently different from wt, it also ages differently. In contrast to desmin and age, the effect of ECC was minor, with only 437 genes with a main effect, and there were 77 genes with a significant desmin × ECC interaction, suggesting that des^−/− muscle responds differently to ECC compared with wt at the transcriptional level, as well as at the mechanical level (see above). There were no genes with a significant age × ECC interaction, suggesting that the ECC response is similar at both ages, and there were no genes with a significant desmin × age × ECC interaction. Expression of eight of the differentially regulated genes was evaluated by qPCR, which recapitulated 29 out of 38 (76%) significant main effects and interactions determined by three-way ANOVA (data not shown).

Fig. 2.

Three-way ANOVA identifies differentially expressed genes as a function of desmin, age, and ECC. The number of genes with a main effect of desmin (des^−/−, wt), age (young, adult), and ECC (ECC, control) are noted in circles. The number of genes with significant interaction terms are noted in the overlap between appropriate circles. The main effect of desmin, the main effect of age, and the interaction between desmin and age accounted for the largest number of differentially expressed genes.

Effect of Desmin Deletion on Transcriptional Response to ECC

Of the 77 genes with a significant interaction between desmin and ECC, the majority (65) also had a significant main effect of treatment, suggesting that the same genes are involved in the des^−/− and wt response to ECC, but regulation of their expression is different between genotypes. Of the genes that could be classified, 62% were classified as being involved in muscle remodeling (Fig. 3A, Supplementary Table S1). Several genes known to be primary players in muscle remodeling in response to injury are included in this category, including myosin binding protein H (Mybph), xin actin-binding repeat containing 1 (Xirp1), BCL2-associated athanogene 3 (Bag3), regulator of calcineurin 1 (Rcan1), musculoskeletal, embryonic nuclear protein 1 (Mustn1), and myogenic transcription factors myogenic differentiation 1 (Myod1) and activating transcription factor 3 (Atf3) (3, 6, 25, 58). Genes classified in the inflammation category (18% of classified genes) are primarily involved in the cellular response to stress and include several heat shock proteins. Representation of the other categories was low compared with remodeling and inflammation (<10% of classified genes). Consistent with gene classification results, GO pathway analysis identified the genes with a significant ECC × desmin interaction as being primarily involved in MAP kinase signaling (GO:0033549, P < 0.05 and GO:0017017, P < 0.05), a cascade thought to be activated in skeletal muscle by exercise (15).

Fig. 3.

Differential gene expression in des^−/− muscle after ECC. A: the function of genes with a significant desmin × ECC interaction term (the 77 genes indicated in the overlap of the red and green circles in Fig. 2) as determined by 3-way ANOVA was investigated and categorized according to 6 divisions important to muscle physiology. Remodeling was the most represented category, containing 62% of the genes. EX, excitation-contraction coupling. B: a heat map of expression of genes classified in A illustrates patterns of gene expression in young des^−/− (control, n = 5 chips; ECC, n = 5 chips) and wt (control, n = 5 chips; ECC, n = 5 chips). A clear difference is apparent between control and ECC wt muscle, visualized by the green and red color schemes, respectively. Both control and ECC des^−/− samples have intermediate expressions for the majority of genes. Hierarchical clustering is indicated at the top of the heat map by horizontal connecting lines indicating that both des^−/− control and ECC samples are more similar to wt ECC than to wt control.

A hierarchical clustering algorithm based on the 77 genes with significant desmin-ECC interactions separated young des^−/− control and ECC samples into one cluster that was identified as having expression patterns that were more similar to wt ECC than to wt control. If the expression of each gene in each group is compared in a heat map (Fig. 3B), the wt ECC group clearly differentiates itself from wt control with its predominantly red color scheme, indicating that the majority of genes are upregulated with ECC. By comparison, the des^−/− ECC group shows a blunted increase in expression relative to control, indicating a dampened response to ECC. However, inspection of the heat map shows that the expression of these genes in des^−/− control samples is intermediate between wt control and wt ECC, suggesting higher expression of “injury-response” genes in control des^−/− muscle compared with control wt muscle.

Effect of Desmin Deletion on the Transcriptional Response to Aging

Genes with a significant age × desmin interaction were classified according to function (Supplementary Table S2). The majority of these genes fell into the categories of remodeling (38%) and metabolism (24%), though ECM (12%), inflammation (11%), cytoskeleton (9%), and EX (6%) categories were also well represented (Fig. 4A). This suggests that des^−/− muscle ages differently compared with wt across the spectrum of physiology from fuel consumption to structure. Consistent with gene classification results, GO pathway analysis identified the genes with a significant age × desmin interaction as being primarily involved in regulation of transcription and biosynthetic process (GO:0045449, P < 0.001 and GO:0010556, P < 0.001) and cellular metabolic process and regulation of metabolic process (GO:0044237, P < 0.001 and GO:0019222, P < 0.001).

Fig. 4.

Gene expression is differentially regulated in des^−/− muscle with age. A: the function of genes with a significant desmin × age interaction term (the 2,011 genes indicated in the overlap of the green and blue circles in Fig. 2) as determined by 3-way ANOVA was investigated and categorized according to 6 divisions important to muscle physiology. Substantial differences in gene expression in aging des^−/− muscle compared with wt are seen in every category, though remodeling and metabolism are the most represented. B: a heat map of expression of genes classified in A illustrates patterns of gene expression in control muscle from des^−/− (young, n = 5 chips; adult, n = 4 chips) and wt (young, n = 5 chips; adult, n = 5 chips). Expression values for genes are categorized according to the divisions defined in A. Surprisingly, hierarchical clustering, as indicated by horizontal lines at the top of the heat map, groups young des^−/− with adult wt and adult des^−/− with young wt. C: expression of the myostatin gene (Mstn) as determined by QPCR. Expression increases with age in the des^−/− samples but remains unchanged in wt resulting in a significant desmin-age interaction. Bars indicate P < 0.05 compared with arrow.

Interestingly, hierarchical clustering on these genes identified the young des^−/− and adult wt as having similar expression patterns, suggesting that des^−/− muscle may be experiencing signs of accelerated aging (Fig. 4B). However, when the heat map of gene expression is subdivided into physiological categories, clear differences in expression patterns are seen between groups in different categories (Fig. 4B, compare cytoskeleton, EX, and metabolism), indicating that more changes are occurring in des^−/− muscle than simply accelerated aging.

Remodeling, inflammation, and fibrosis in des^−/− muscle.

Many genes identified as major players in the muscle hypertrophy and atrophy pathways were differentially regulated with age in des^−/− muscle, including thymoma viral proto-oncogene 1 (Akt1), phosphatidylinositol 3-kinase, forkhead box O3 (Foxo3), myogenin (Myog), and myogenic transcription factor 5 (Myf5) (12, 53). Additionally, several genes were differentially regulated that have been implicated in mechanotransduction: Jun-B oncogene (Junb), FBJ osteosarcoma oncogene (Fos), mitogen-activated protein kinase kinase 1 (Map2k1), and mitogen-activated protein kinase 14 (Mapk14) (7, 26, 53). Myostatin, a protein involved in the negative regulation of skeletal muscle growth, was not included in the mouse GeneChip, and thus expression values of the myostatin gene (Mstn) were determined by QPCR (Fig. 4C). Mstn expression values increased significantly in both control and ECC des^−/− muscle with age but trended toward decreasing with age in wt groups, resulting in a significant desmin-age interaction as determined by three-way ANOVA.

Our previously published data indicated increased expression in des^−/− muscle of genes we identified as major players in the ECM or inflammatory pathways (39). A three-way ANOVA including ECC samples as well as controls indicated that many of these genes have a significant age × desmin interaction as suggested by fold change data. These include several fundamental components of the inflammatory pathway, including interleukin 6 (Il6) and its receptor (Il6ra), interferon gamma receptor 1 (Ifngr1) and suppressor of cytokine signaling 3 (Socs3), which is a known activator of nuclear factor-κβ (53, 56). Additionally, genes involved in the fibrotic pathway were differentially regulated with age, including transforming growth factor-β (Tgfb1) and its receptor (Tgfbr3) and CCAAT/enhancer binding protein (C/EBP), beta (Cebpb), which is involved in the regulation of fibrotic inflammatory signals (48, 50). Consistent with differential regulation of the fibrotic pathway, genes involved in the structure of the ECM are also differentially regulated with age including the genes for collagens I, III, IV, V, VI, and XV (Col1a1, Col3a1, Col4a1, Col4a2, Col5a1, Col5a2, Col5a3, Col6a1, Col6a2, Col15a1) and laminins β1 and γ1 (Lamb2, Lamc1). These results are consistent with recent studies that have identified signs of increased inflammation and regeneration in des^−/− muscle as well as a progressive accumulation of ECM with age (28, 39).

Fiber type switching in des^−/− muscle.

The majority of genes with a significant desmin-age interaction that were categorized as being part of the muscle cytoskeleton were components of the contractile apparatus associated with the slow (type I) fiber type (Fig. 5A). These included the slow muscle isoforms of myosin heavy chain (Myh7), myosin light chain (Myl3), the T, I, and C components of the troponin complex (Tnnt1, Tnni1, Tnnc1), tropomyosin (Tpm3), and the Z-disk associated protein myozenin (Myoz2) (9, 53). Additionally, several genes classified as being part of EX were also part of the slow fiber pathway including ATPase (Atp2a2), calsequestrin (Casq2), and calcium/calmodulin-dependent protein kinase II, alpha (Camk2a), which encodes a protein, αKap, involved in directing CamKII to its specific substrates in the sarcoplasmic reticulum (61). The expression of all of these genes was increased more than twofold in des^−/− muscle compared with wt in adult samples, but not in young (Fig. 5A). Interestingly, one of the primary genes involved in the control of fiber type switching from fast to slow, peroxisome proliferative-activated receptor, gamma, coactivator 1 alpha (Ppargc1a, a.k.a. PGC-1α) is twofold downregulated in adult des^−/− muscle compared with wt, which might suggest a preferentially fast fiber type in adult des^−/− muscle. However, endothelial PAS domain protein 1 (Epas1, a.k.a. HIF-2α), a mediator of the PGC-1α fiber type switch (47, 61), was elevated twofold in adult des^−/− muscle.

Fig. 5.

Des^−/− muscle experiences a fiber type switch from fast to slow with age. A: a schematic of genes involved in the slow fiber program including fold changes in expression in adult des^−/− muscle over adult wt. Slow isoforms of genes are shown below the dotted division, and fast isoforms are shown above. The majority of slow isoform genes have high fold changes, indicating increased expression of the slow fiber program in adult des^−/− muscle. Three genes not specific to fast or slow fibers but involved in the fiber type switch are depicted on the dotted division. B: myosin heavy chain gel quantification shows a significant increase in the slower type IIa myosin heavy chain and a significant decrease in the fastest type IIb in adult des^−/− compared with adult wt. *P < 0.05 between genotypes.

Consistent with an increase in genes associated with the slow fiber type transition, a change in the composition of MyHC isoforms was measured by gel electrophoresis. A significant increase in the slower isoform percentage, %IIa (wt 5.3 ± 0.3%, des^−/− 8.4 ± 1.0%), and a significant decrease in the fastest isoform percentage, %IIb (wt 71.7 ± 1.8%, des^−/− 61.4 ± 3.9%), was found in adult des^−/− muscle compared with adult wt (Fig. 5B).

Peripheral fat accumulation and insulin resistance in des^−/− mice.

Several genes with a significant desmin × age interaction that were categorized as being involved in metabolism regulate the insulin signaling pathway (Fig. 6A). Specifically, three negative regulators of insulin signaling were substantially increased in adult des^−/− muscle compared with adult wt, including fetuin beta (Fetub), a putative inhibitor of insulin receptor tyrosine kinase activity, and Toll-like receptor 4 (Tlr4), an inhibitor of kappaB kinase beta (Ikbkb), which when activated are known to lead to inhibition of IR substrate (IRS) signaling (35, 43, 57). Additionally, an inhibitor of thymoma viral proto-oncogene 1 (Akt1), tribbles homolog 3 (Trib3), was increased in adult des^−/− muscle (31). Consistent with a negative regulation of the insulin signaling pathway, serum/glucocorticoid-regulated kinase 1 (Sgk1), a downstream target of PI3K that is also involved in glucose transporter type 4 (Slc2a4, a.k.a. GLUT-4) translocation, was 2.5-fold lower in adult des^−/− muscle (20). This combined with a nearly fivefold increase in Trib3 expression, an inhibitor insulin-stimulated activation of Akt, suggests that insulin-stimulated glucose uptake by des^−/− muscle may be impaired. Additionally, three isoforms of pyruvate dehydrogenase kinase (Pdk2, Pdk3, Pdk4), which inhibit pyruvate dehydrogenase activity, were differentially regulated with age in des^−/− muscle, suggesting further potential alterations in glucose metabolism (59).

Fig. 6.

Des^−/− mice have increased fat mass and decreased muscular insulin sensitivity. A: a schematic of genes involved in insulin-stimulated glucose uptake in muscle including fold changes in expression in adult des^−/− muscle over adult wt. Three genes that block the insulin signaling pathway have high positive fold changes, while several genes involved in the pathway have negative fold changes resulting in a collective downregulation of the pathway. Genes involved in glucose use in the mitochondria also have high fold changes, indicating potential alterations in glycolysis downstream of glucose uptake at the sarcolemma. B: young and adult des^−/− mice have a significantly increased fat pad-to-body mass ratio over wt mice. At both ages the fractional fat pad mass is more than doubled in des^−/− mice. C: insulin-stimulated glucose uptake (ISGU) in the soleus muscle is significantly impaired in the young des^−/− group compared with wt. ISGU decreased significantly with age in both genotypes. Adult des^−/− muscle maintained a lower level than adult wt though not significantly so. D: young and adult des^−/− mice have significantly decreased blood glucose levels following 4 h of fasting. E: insulin-stimulated Akt phosphorylation is significantly reduced in isolated adult des^−/− soleus muscle compared with wt, though basal levels are unchanged. Insulin stimulation did not result in significantly increased Akt phosphorylation in adult muscle, and there was no difference between genotypes. Images of phosphorylated Akt and total Akt western blots are shown below the graph. Bars indicate P < 0.05 compared with arrow.

In addition to genes involved in muscle insulin signaling, two genes involved in signaling related to leptin, which controls appetite and feeding behavior, were upregulated in adult des^−/− muscle compared with wt. The leptin gene was not included on the GeneChip, but CCAAT/enhancer binding protein (C/EBP), alpha (Cebpa), an inhibitor of leptin was threefold increased and agouti-related protein (Agrp), a downstream target of leptin was >30-fold increased. Expression increases in both of these genes have been shown to promote appetite and weight gain (19, 33). Young and adult des^−/− mice have increased accumulation of peripheral fat compared with wt (P < 0.05) as determined by the epidydimal fat pad mass as a percentage of body mass (Fig. 6B). This increase was due only to an increase in fat mass, as body masses were not significantly different between genotypes.

Consistent with gene changes that suggest lower insulin sensitivity in des^−/− muscle, insulin-stimulated glucose uptake in isolated soleus muscles was significantly reduced in young des^−/− muscle compared with wt (P < 0.01) and was reduced in adult des^−/− muscle compared with adult wt, although this did not reach significance (P > 0.05) (Fig. 6C). Despite the aforementioned changes in gene expression and skeletal muscle insulin action, fasting blood glucose levels were significantly lower in young and adult des^−/− mice compared with wt (P < 0.05) (Fig. 6D). Insulin-stimulated Akt phosphorylation levels in the young des^−/− soleus were significantly reduced compared with young wt (P < 0.05) despite no significant difference in basal values (P > 0.1) (Fig. 6E). In adult muscles, however, there was not a significant increase in Akt phosphorylation after insulin stimulation over basal values, and no difference in the response between genotypes (P > 0.05).

DISCUSSION

The purpose of this study was to further define the function(s) of the muscle intermediate filament protein desmin, by subjecting skeletal muscles to physiological stressors. ECC and aging were chosen as they represent a range of chronicity and intensity of the types of stresses that muscles experience. The bout of 50 ECCs, though minimally invasive, still represents a substantial mechanical insult to muscle. In wt muscle, tetanic isometric torque drops by ∼50% after the bout and requires a full 7 days to recover to its pre-ECC value (3). Additionally, eccentrically exercised muscle shows significant histological signs of injury, including localized myofibrillar disruption, loss of membrane integrity, and infiltration of inflammatory cells (2, 44). Thus, we were surprised in the current study that desmin, or lack thereof, independent of injury or age, had the largest number of differentially regulated genes (Fig. 2). Though a smaller number of genes with larger transcriptional changes could have equal or greater effects physiologically, these data point to desmin loss affecting muscle function across the spectrum of muscle physiology. These data also indicate that not only are des^−/− muscles transcriptionally different from wt, the interaction between the desminopathy and the physiological stressors of injury and aging results in unique cellular responses and muscular adaptations. Genetic modifications to other cytoskeletal or extracellular matrix proteins in muscle, such as dystrophin, tenascin-C, and α7β1 integrin have demonstrated differential aging and response to injury as well (10, 32, 46), but the des^−/− phenotype appears to be unique in its effects on fiber type and metabolic regulation.

Desmin has long been hypothesized to play a role in muscular response to ECC-induced injury, but the nature of that role remains unclear (4, 30, 49). Desmin filaments distribute externally applied strain among sarcomeres, which may prevent excessive sarcomere lengthening by coupling sarcomeres to a stiffer “anchor” or promote excessive lengthening by coupling sarcomeres to sites of high strain. Either explanation fits with theoretical data (38). Expression data in des^−/− muscle indicate a muted response to ECC injury. ECC response genes identified in injured wt muscle were similarly regulated in des^−/− muscle with ECC but with smaller changes (Fig. 3B). This effect paralleled the muted mechanical response to ECC in des^−/− dorsiflexors (Fig. 1). However, expression of ECC response genes was elevated in the des^−/− control muscle compared with wt, suggesting that des^−/− muscle may already be undergoing some type of injury response prior to the imposed ECC bout, consistent with previously published data indicating increased inflammation, remodeling, and fibrosis in des^−/− muscle (28, 39). This result is consistent with published data from the mdx mouse, where a mutation to another cytoskeletal protein, dystrophin, results in increases in expression of genes involved in inflammation, proteolysis, and fibrosis with age (14, 46). However, mdx muscle has been reported to be more susceptible to a bout of ECC and not less, as reported for des^−/− muscle, so mechanisms of injury may differ (45). If sites of prior damage in des^−/− muscle are more susceptible to overextension induced by ECC, then without desmin to integrate the sarcomere lattice, further cytoskeletal disruption may not be incurred during repeated overextension of the same sarcomeres. It is also possible that the reduction in isometric force in wt muscle is due in part to dissociation of the desmin network at the injury site, since loss of desmin immunostaining in injured fibers is seen as soon as 5 min after ECC injury (30), and the ECC bout lasted for 50 min in this study. Further studies are required to test these hypotheses.

Many genes classified as being involved in the cytoskeleton, EX, and metabolism were also differentially regulated with age in des^−/− muscle. Based on the expression pattern of these genes, we identified two additional pathways altered in des^−/− muscle: the slow fiber pathway and the insulin signaling pathway. A transition to a slower fiber type was noted previously in the des^−/− soleus, but no change was seen in fast-twitch muscle such as the gastrocnemius (28). In this study, we were able to detect a significant increase in the slower MyHC isoform percentage (IIa) and a significant decrease in the fastest isoform percentage (IIb) in the predominately fast-twitch tibialis anterior muscle of adult des^−/− muscle compared with adult wt. The mechanism behind this transition to a slower fiber type is unclear. Fiber type transitions from fast to slow are sometimes seen in endurance-trained muscles (16) and in muscles with alterations in calcium handling or calcineurin (21). It is possible that, without desmin to keep sarcomeres laterally aligned, the sarcoplasmic reticulum is being damaged by contraction or stretch, resulting in chronically elevated calcium levels and a transition to slower fiber types (42). Additionally, in the absence of desmin, mitochondria accumulate in the subsarcolemmal space and may have altered function that could also influence fiber type (40). Though progressive inflammation, regeneration, and fibrosis are characteristic of dystrophic muscle, the switch to a slower fiber type is unique to des^−/− muscle as dystrophic mdx muscle has been shown to exhibit a switch to a faster fiber type (34).

Many of the metabolic genes differentially regulated with age in des^−/− muscle are involved in insulin signaling and glucose metabolism. The net effect of these transcriptional changes in the tibialis anterior muscle suggests an inhibition of the insulin signaling pathway in des^−/− muscle (Fig. 6A), which was supported by our findings of reduced insulin-stimulated glucose uptake (Fig. 6C) and Akt phosphorylation in isolated des^−/− soleus muscles (Fig. 6E). Though the predominately slow-twitch soleus muscle has different patterns of use than the fast-twitch TA muscle, both exhibit signs of reduced insulin sensitivity. The link between insulin resistance and cytoskeletal disruption due to desmin loss is unclear. Chronic inflammation has been linked to insulin resistance especially as mediated through Ikbkb signaling (1). Tlr4 is a receptor that is activated by the binding of saturated fatty acids, which recruits myeloid differentiation factor 88 (Myd88), which then leads to the activation of Ikbkb and serine phosphorylation of IRS and subsequent impairment of glucose uptake (23). Expression of both Tlr4 and Myd88 are increased over twofold in young des^−/− muscle compared with young wt, suggesting that changes in insulin signaling measured here may be related to the inflammation previously characterized in young des^−/− muscle. There is also evidence that muscle insulin sensitivity is transiently reduced by eccentric exercise induced muscle damage (24), supporting the hypothesis that repetitive muscle damage to des^−/− mice may be the initiating factor in the metabolic dysfunction measured here. However, whether or not inflammation is the driving factor in this effect is unclear. Interestingly, inflammation and muscle injury are also characteristic of dystrophic mdx muscle, which exhibits no alterations in insulin-stimulated glucose uptake, suggesting there may be another mediating factor.

Increased inflammation is not the only explanation for the observed increase in insulin resistance in des^−/− muscle. Muscle insulin sensitivity is closely tied to obesity, and des^−/− mice have significantly increased fat mass, so it is possible that increased fat was a causative mechanism for the differential gene regulation in insulin signaling and/or glucose uptake findings. However, insulin-mediated glucose uptake values are not correlated with percentage fat or fat pad mass (r² < 0.3, P > 0.05). Additionally, des^−/− mice have been shown to participate less in voluntary exercise than wt mice, so it is possible that their reduced insulin action and increased adiposity is due to reduced activity (18). Interestingly, Agrp, a protein that is primarily involved in appetite regulation, was one of the most highly upregulated genes in adult des^−/− muscle (>30-fold), It is tempting to speculate that increases in Agrp are leading to increased feeding, adiposity, and subsequent insulin resistance in des^−/− mice, but the mechanism for alterations in a cytoskeletal protein resulting in changes in a neuropeptide are completely unknown.

It is important to note when interpreting gene expression data that differences in mRNA levels do not always correlate to differences in protein accumulation. Expression of a few transcripts involved in fiber type switching and metabolic dysregulation was evaluated here at the protein level, but future studies will be needed to investigate whether other transcriptional changes correlate with changes in protein quantities or function. Additionally, though genes were assigned only one functional category in this work, they may have multiple functions physiologically, depending on the state of the muscle, which could span more than one category (53). References are provided for assigned genes in Supplementary Tables S1 and S2, which discuss the role of the gene or protein in muscle in more detail.

In conclusion, this study identified transcriptional alterations to pathways involved in muscle physiology due to the interaction between the stressors of desmin loss, injury, and aging. The differential response to injury and aging in muscle lacking desmin suggests that this protein plays a role in physiological adaptation to a spectrum of muscle stress. It is important to note that the observed transcriptional changes may indirectly result from the loss of desmin. However, through careful evaluation of these effects and comparisons to transcriptional changes observed in other models, we hope to identify intermediate steps in the development of desminopathy and, in future studies, target these hypothesized intermediates to directly test their role in linking desmin to the observed physiological changes. Though more studies are required to define this role, this study has pointed to new and promising areas of research into the function of desmin in skeletal muscle.

GRANTS

This work was supported by National Institute of Health Grants AR-40050, HD-050837, and P30 AR-058878-02 and the Department of Veterans Affairs.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: G.A.M., S.S., and R.L.L. conception and design of research; G.A.M. and S.S. performed experiments; G.A.M. and S.S. analyzed data; G.A.M., S.S., and R.L.L. interpreted results of experiments; G.A.M. prepared figures; G.A.M. drafted manuscript; G.A.M., S.S., and R.L.L. edited and revised manuscript; G.A.M., S.S., and R.L.L. approved final version of manuscript.