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Impaired Expression of Notch Signaling Genes in Aged Human Skeletal Muscle

School of Exercise and Nutrition Sciences, Deakin University, Burwood, Victoria, Australia.

Address correspondence to David Cameron-Smith, PhD, School of Exercise and Nutrition Sciences, Deakin University, 221 Burwood Highway. Burwood, Victoria 3125, Australia. E-mail: david.cameron-smith{at}deakin.edu.au

	Abstract

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Notch signaling is essential for myogenesis and the regenerative potential of skeletal muscle; however, its regulation in human muscle is yet to be fully characterized. Increased expression of Notch3, Jagged1, Hes1, and Hes6 gene transcripts were observed during differentiation of cultured human skeletal muscle cells. Furthermore, significantly lower expressions of Notch1, Jagged1, Numb, and Delta-like 1 were evident in muscle biopsies from older men (60–75 years old) compared to muscle from younger men (18–25 years old). Importantly, with supervised resistance exercise training, expression of Notch1 and Hes6 genes were increased and Delta-like 1 and Numb expression were decreased. The differences in Notch expression between the age groups were no longer evident following training. These results provide further evidence to support the role of Notch in the impaired regulation of muscle mass with age and suggest that some of the benefits provided by resistance training may be mediated through the Notch signaling pathway.

The mammalian Notch receptors (Notch1, -2, -3, and -4) are transmembrane proteins composed of an extracellular region with multiple epidermal growth factor-like repeats necessary for ligand binding. The Notch signaling pathway is initiated when Notch receptor–bearing cells interact with Notch ligands expressed on adjacent cells. Humans have at least five Notch ligands (Jagged1 and -2, and Delta-like-1, -3, and -4), which are themselves transmembrane proteins with a number of epidermal growth factor-like repeats in their extracellular domain and a unique Delta/Serrate/Lag2 (DSL)-binding domain in the amino terminus necessary for receptor interaction. Notch–ligand interaction triggers two proteolytic cleavages that release the Notch intracellular domain (Notch^intra) from its plasma membrane tether, allowing it to translocate to the nucleus and bind to a transcriptional regulator known as CBF1/Su(H)/LAG-1. The activity of Notch^intra can be inhibited by Numb through ubiquination, which regulates the abundance and intracellular location of the signaling molecule. The Notch^intra–CBF1/Su(H)/LAG-1 complex recruits transcriptional coactivators that induce the gene expression of members of the Hairy-Enhancer of Split (HES) proteins. These proteins are basic helix-loop-helix (bHLH) DNA binding proteins that are thought to inhibit the expression and/or function of lineage-specifying genes such as MyoD (involved in myogenesis) (2). In skeletal muscle, Notch signaling contributes to muscle development, somitogenesis, as well as the proliferation and cell fate determination of muscle-specific satellite cells during postnatal myogenesis (3).

Mechanical loading has been shown to augment the proliferation and differentiation of satellite cells (4–7), which is thought to contribute to the repair and adaptation of the exercised muscle. Because the Notch signaling pathway has previously been implicated in the regenerative potential of rat muscle (1,8), we sought to examine whether components of this pathway were transcriptionally regulated in human skeletal muscle by exercise and during differentiation of human myoblasts in culture. We further examined whether the transcriptional regulation of Notch signaling was different between young and older human skeletal muscle both at rest and in response to a progressive 12-week heavy resistance exercise training program. It was hypothesized that: (i) the gene expression of members of the Notch signaling pathway would be increased during the differentiation of human primary cells in culture; (ii) reduced expression of these genes would be observed in the muscle of older compared to younger individuals; and (iii) increased messenger RNA (mRNA) expression would be observed following resistance training.

	METHODS

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Human Primary Skeletal Muscle Cell Culture
Human primary skeletal muscle cells were obtained from the vastus lateralis muscle of eight healthy volunteers (six men and two women, average age 25.1 ± 1.3 years). Informed written consent was obtained from each individual before participation in the study, after the nature, purpose, and risks of the study were explained. All experimental procedures involved in this study were formally approved by the Deakin University Ethics Committee. Human primary skeletal muscle cells were cultured according to the technique of Gaster and colleagues (9) with modifications. Briefly, the excised muscle was immersed and extensively washed in ice-cold minimum essential medium alpha modification (-MEM; Gibco, Invitrogen Corporation, Carlsbad, CA) before being minced and digested in 0.5% Trypsin–EDTA (Gibco). The supernatant containing the myoblasts was then collected, and the process was repeated another two times to break down any remaining tissue. Fetal bovine serum (Gibco) was subsequently added to the supernatant to a final concentration of 10%. The supernatant was filtered through a 100-µm filter to remove any connective tissue and then spun to collect the cells. The resulting cell pellet was resuspended in primary growth medium (-MEM, 10% fetal bovine serum, with penicillin at 50 IU/mL and streptomycin at 5 µg/mL) and was then seeded onto an uncoated flask and incubated at 37°C for 25 minutes to induce fibroblast attachment, leaving myoblasts suspended in the medium. The medium was aspirated and seeded onto an extracellular matrix–coated (Sigma, St. Louis, MO) flask. The resulting primary cell cultures were maintained in primary growth medium in humidified air at 37°C and 5% CO₂.

Study Design: Differentiation
Cells were initially plated in the primary growth medium until 80% confluence was attained. The plating medium was then removed, the cells were washed twice with phosphate-buffered saline, and the differentiation medium (-MEM containing 2% horse serum) was added. Medium was changed every 48 hours throughout the 72-hour differentiation time course. Following two washes with phosphate-buffered saline, cells were extracted for RNA and protein at 12, 24, 48, and 72 hours following the addition of the differentiation medium. The control consisted of cells actively growing in the primary growth medium extracted immediately before the addition of the differentiation medium (0 hours).

RNA extraction and reverse transcription.-- RNA from primary cell cultures was extracted using the RNA-Bee (Tel-Test, Friendswood, TX) reagent and protocols. RNA integrity and quantity were assessed on an Agilent 2100 Bioanalyzer with an RNA 6000 Nano LabChip Kit (Agilent Technologies, Palto Alto, CA). Reverse transcription was performed using the AMV reverse transcriptase kit (A3500; Promega, Madison, WI) protocols and reagents.

Study Design: Resistance Exercise Training
Participants.-- Sixteen healthy young men (18–25 years old) and 15 healthy older men (60–75 years old) who had not participated in regular strength exercise within a year prior to commencing the study were recruited (see Table 1 for participant details). A medical history questionnaire was used to identify and exclude participants with a diagnosed condition or illness that would endanger them during strenuous exercise. Older participants were required to undergo a complete medical screening including a 12-lead electrocardiogram exercise stress test to detect any underlying cardiopulmonary conditions. All participants were informed of the nature and risks of the study before their written informed consent was obtained.

Twelve-week exercise training.-- Following the initial acute exercise testing session, all participants completed 12 weeks of fully supervised progressive resistance exercise training 3 days each week, with a minimum of 48 hours of rest between exercise sessions. Initially, three training sessions were conducted using light resistance to familiarize the participants with the equipment, training protocol, and correct execution of the exercises. After the familiarization sessions, strength testing was performed to determine appropriate starting weights for all participants. Repetition maximum (RM) strength was estimated from their 5RM results for all exercises. Values of 5RM were retested at Week 6 and Week 12, and the training load was adjusted accordingly to ensure that the training was progressive.

Each training session was preceded by a 5-minute warm-up on a stationary cycle followed by a full set of exercises with light weights. The exercises consisted of leg press, bench press, seated row, leg extension, dumbbell shoulder press, and sit-up. Following the warm-up weights, participants completed 2 sets at the required intensity completing between 8 and 12 repetitions of each exercise. Specified rest periods were allowed between sets. Initially, the exercises were set to 50% of a participant' 1RM for 1 week followed by a progressive increase in the weights lifted each week until 80% of 1RM was attained at Week 6. The exercise intensity was set at 80% of 1RM for the remaining 6 weeks. An exercise specialist directly supervised the exercise sessions of every participant at every training session to verify compliance with the training protocol. At the end of the 12-week training program, participants again presented to the laboratory in the fasted state to complete the exercise trial consisting of a single bout of resistance exercise and collection of muscle biopsies. The exercise performed and the timing of the muscle biopsies were identical to those of the exercise trial completed by the participants before the commencement of the exercise training (see above).

Muscle Biopsy Procedure
The vastus lateralis muscle of the nondominant leg was sampled by the percutaneous needle biopsy technique (10) modified to include suction (11). Excised muscle tissue from each biopsy was immediately frozen and stored in liquid nitrogen for subsequent analysis. To minimize the potential for interference, serial biopsy samples were collected at least 2 cm from previous biopsy sites.

RNA Extraction and Reverse Transcription
RNA was extracted from 10 mg (wet weight) of muscle using the ToTALLY RNA Kit protocol and reagents (Ambion, Austin, TX). RNA integrity and quantity were assessed on an Agilent Bioanalyzer 2100 with an RNA 6000 Nano LabChip Kit (Agilent Technologies). Reverse transcription was performed using the AMV reverse transcriptase kit (A3500; Promega) protocols and reagents.

Primer Design
To perform polymerase chain reaction (PCR), specific primers were designed for all genes using Primer Express 3.0 software (Applied Biosystems, Foster City, CA) on sequences obtained from GenBank (see Table 1 for details). Where possible, primers were designed spanning intron–exon boundaries to prevent amplification of the target region from any contaminating DNA. Primer specificity was confirmed using Basic Local Alignment Search Tool (BLAST). Primers were purchased from GeneWorks (Adelaide, South Australia). Efficiency of PCR primers was confirmed by examining the dynamic range of responses for a series of dilutions of complementary DNA. Using the slopes of the lines, the efficiency (E) of target amplification was calculated using the equation E = (10^–1/slope) – 1.

Real-Time PCR Analysis
Real-time PCR was performed using the GeneAmp 7500 Sequence Detection System (Applied Biosystems). For the PCR step, reaction volumes of 20 µL contained SYBR Green 1 Buffer (Applied Biosystems), forward and reverse primers (see Table 2), and complementary DNA template. All samples were run in duplicate. Real-time PCR was run for 1 cycle (50°C for 2 minutes, 95°C for 10 minutes) followed by 40 cycles (95°C for 15 seconds, 60°C for 60 seconds), and fluorescence was measured after each of the repetitive cycles. A melting point dissociation curve generated by the instrument was used to confirm that only a single product was amplified. Data were analyzed using a comparative critical threshold (Ct) method in which the amount of target normalized to the amount of endogenous control relative to control value is given by 2^–Ct (Applied Biosystems). The efficacy of cyclophilin as endogenous control was examined using the equation 2^–Ct. No changes in the expression of this gene were observed (data not shown), so it was considered an appropriate endogenous control for this study.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). For the differentiation study, means were compared using a one-way analysis of variance and any significant differences were analyzed using Bonferroni's multiple comparison test. The differences in the gene expression between older and younger men were examined using an unpaired t test. The impact of age and training was compared using a two-way analysis of variance with repeated measures. Data are presented as mean ± standard error of the mean (SEM). Unless otherwise stated, a probability level of <.05 was adopted throughout to determine statistical significance.

	RESULTS

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Components of the Notch Signaling Pathway Are Regulated at the Transcriptional Level During Human Primary Skeletal Myoblast Differentiation
Human primary muscle cell cultures were grown to 80% confluence before being exposed to low-serum medium to induce differentiation. Differentiation was assessed by examining the expression of MHC protein by Western blot. A small degree of MHC expression was evident 12 hours after the induction of differentiation, with more marked expression detectable at 24, 48, and 72 hours postdifferentiation (Figure 1). Differentiation was accompanied by an increase in the mRNA expression of Notch3 (p <.001) by 24 hours (Figure 2), whereas no change was observed in the expression of Notch1 or Numb (data not shown). The expression of Jagged1 (2.5-fold), Hes1 (2.6-fold), and Hes6 (2-fold) transcripts all increased at 48 hours with the maximum expression of Hes6 (2.8-fold) evident at 72 hours postdifferentiation. Jagged2 and Delta-like 1 expression were not significantly altered at any time during differentiation (data not shown).

View larger version (8K): [in this window] [in a new window]   Figure 1. Western blot analysis of myosin heavy chain (MHC) protein expression over time in differentiating conditions. The cell extracts were subjected to electrophoresis through a 6% sodium dodecyl sulfate–polyacrylamide gel transferred to nitrocellulose membranes, and reacted to the myosin antibody (MY-35). Average MHC expression ± standard error of the mean of eight independent experiments was analyzed using densitometry, and is expressed as arbitrary density units. A representative blot is shown above the graph. Not significant (p =.09)

View larger version (12K): [in this window] [in a new window]   Figure 2. Messenger RNA expression analysis of components of Notch3 (A), Jagged1 (B), Hes1 (C), and Hes6 (D) following the induction of differentiation in human skeletal muscle primary cell cultures. The mean ± standard error of the mean of eight independent experiments is shown. *Significantly different from 0 hours (p <.05); **Significantly different from 0 hours (p <.01); ***Significantly different from 0 hours (p <.001)

View larger version (15K): [in this window] [in a new window]   Figure 3. The effects of heavy resistance exercise training on the expression of Notch1 (A), Notch3 (B), Jagged1 (C), Jagged2 (D), Delta-like 1 (E), Numb (F), and Hes6 (G) in young (white bars) and old (black bars) men. The men completed a single bout of extension exercise consisting of 3 sets of 12 repetitions of maximal leg extension exercise before and after 12 weeks of resistance exercise training. Vastus lateralis muscle samples were collected before the exercise (rest) as well as 2 hours postexercise. The mean ± standard error of the mean is presented. # Significantly different compared to young men (p <.05). mRNA = Messenger RNA

	DISCUSSION

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Effect of Age and Training Status on Notch Gene Expression
We next sought to determine whether human skeletal muscle from older individuals demonstrated reduced expression of Notch signaling genes. Interestingly, three of the genes measured (Notch1, Jagged1, and Delta-like 1) showed reduced expression in older skeletal muscle when compared to younger muscle at rest. To our knowledge, this is the first study to describe the effect of age on Notch expression in human skeletal muscle. Research in rats has described impaired injury-induced activation of the Notch ligand Delta with age resulting in inefficient regeneration of old muscle tissue (1). The results from our work extend these observations and indicate that reduced expression of Notch genes may contribute to the impaired regenerative capacity of older human skeletal muscle.

We next sought to determine if heavy resistance exercise training was capable of attenuating the decline in Notch gene expression evident with advancing age. Both younger and older men completed a single bout of resistance exercise both before and after a progressive 12-week heavy resistance exercise training program. No changes were observed in any of the genes examined 2 hours following an acute bout of exercise in the untrained state. Following training, however, the differences in expression patterns between the two age groups were no longer evident, suggesting that exercise training may have a beneficial effect on the expression of Notch genes in older individuals. Resistance exercise has been shown to have a positive effect on muscle mass, strength, and functional capacity in even very elderly individuals (18). Previous research has indicated that artificial activation of Notch signaling may "rescue" the impaired regenerative capacity of skeletal muscle in older rats (1). The data presented here seem to suggest that at least one mechanism by which exercise may exert these benefits is through the regulation of the expression of Notch signaling genes.

Resistance exercise training altered the expression of several Notch genes in the younger as well as the older individuals. Increased expression was observed in Notch1 and Hes6, a nonsignificant increase was observed in Jagged2, and Delta-like 1 and (to a lesser extent Notch inhibitor Numb) expression was reduced. Increased expression of Notch1 might enhance the capacity for Notch signaling and, subsequently, increased expression of its downstream targets, such as Hes6. The possible reasons for the marked reduction in Delta-like 1 are somewhat less clear. There are, however, numerous other Notch receptors (such as Delta-like 3 and 4, and Jagged 1 and 2). The specific role these receptors play in Notch signaling is currently not known. Each of these proteins may have unique cellular functions, and thus regulation (either up or down) of their expression may contribute to the development of specialized phenotypes. The similar changes in Notch gene expression observed between the two age groups with training indicate that the capacity of skeletal muscle to regulate these genes is not diminished with age. These findings may indicate that the regulation of Notch by exercise is not age-related and may in fact be a common signaling mechanism integral to exercise-induced skeletal muscle adaptation. The one notable exception to the increased expression of Notch genes with exercise training was observed with Numb. Numb expression was consistently lower in aged muscle, and this pattern was not altered at any time point following exercise training. Increased Numb expression has been found to attenuate Notch signaling and lead to the commitment of progenitor cells to the myoblast cell fate. Furthermore, asymmetric localization of Numb expression in actively proliferating myoblasts has suggested that Numb may be involved in cell fate determination and the maintenance of the satellite cell pool in mature muscle (8). As such, impaired expression of Numb in aged human skeletal muscle may be a potential mechanism, independent of resistance exercise, which contributes to the impaired regenerative capacity observed in this age group. The specific functions of Notch signaling in skeletal muscle postexercise remain undefined and provide an exciting avenue for further investigations.

Taken together, the results of the in vitro and in vivo experiments presented herein support a role for the Notch signaling pathway in the adaptive and/or regenerative capacity of human skeletal muscle. The increased expression of Notch genes during differentiation described here and elsewhere (12) as well as the increased expression following resistance exercise training, however, point toward an additional role in the regulation of later stages of myogenesis. However, much of the previous research presented to date suggests a role for Notch in the inhibition of myogenic differentiation (13–16). Although the proliferating myoblasts investigated in the present study demonstrated lower expression of Notch genes, we cannot discount a role for Notch in early proliferative events. Following injury, increased expression of Notch components are observed within satellite cells and promote the rapid expansion of the satellite cell progeny (8). The increased expression of Notch genes following exercise training may therefore reflect the residual activity of satellite cells within the muscle. The data presented here, however, do not distinguish the specific stage of myogenesis during which Notch genes are transcriptionally regulated, nor can we determine if the expression is predominantly associated with satellite cells or mature myofibers. It is, however, highly likely that Notch signaling expression and activation in skeletal muscle is essential at multiple stages of adult myogenesis, and its temporal and spatial regulation is tightly controlled (3).

The results of this study demonstrate for the first time a reduced expression of Notch signaling genes in aged human skeletal muscle. Furthermore, heavy resistance exercise training was able to alter the transcriptional profile of the genes investigated such that the differences observed between younger and older muscle pretraining were no longer evident. These results provide further evidence to support the role of Notch signaling in the impaired regulation of muscle mass with age, and suggest that some of the benefits provided by resistance exercise training may be mediated through the Notch signaling pathway. Further analysis of the impact of the Notch signaling pathway, including overexpression or knockout models, are required to further elucidate the contribution exerted by this pathway on muscle form and function.

	Acknowledgments

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We gratefully acknowledge and thank Dr. Andrew Garnham for his excellent medical skills and the willingness of the volunteers for making this research possible.

	Footnotes

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Decision Editor: Huber R. Warner, PhD

Received April 12, 2006

Accepted June 21, 2006

	References

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