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(Circulation Research. 2005;97:418.)
© 2005 American Heart Association, Inc.

Molecular Medicine

Transgenic Overexpression of Locally Acting Insulin-Like Growth Factor-1 Inhibits Ubiquitin-Mediated Muscle Atrophy in Chronic Left-Ventricular Dysfunction

P. Christian Schulze, Jennifer Fang, Kimberly A. Kassik, Joe Gannon, Mihaela Cupesi, Cathy MacGillivray, Richard T. Lee, Nadia Rosenthal

From the Cardiovascular Division (P.C.S., J.F., K.A.K., J.G., M.C., C.M., R.T.L.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; and Mouse Biology Programme (N.R.), European Molecular Biology Laboratory, Monterotondo/Rome, Italy.

Correspondence to P. Christian Schulze, MD, PhD, Department of Medicine, Boston University Medical Center, 80 E Concord St, Evans 124, Boston, MA 02118-2526. E-mail christian.schulze{at}bmc.org

	Abstract

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Metabolic abnormalities develop in various chronic diseases and lead to progressive catabolism with decrements in the skeletal musculature that result in muscle atrophy. We investigated pathways of skeletal muscle proteolysis using an experimental model of chronic left-ventricular dysfunction. Skeletal muscle atrophy developed in wild-type mice 12 weeks following myocardial infarction accompanied by an increase in total protein ubiquitination and enhanced proteasome activity, activation of Foxo transcription factors, and robust induction of the ubiquitin-protein ligase atrogin-1/MAFbx. Further studies identified skeletal muscle myosin as a specific target of ubiquitin-mediated degradation in muscle atrophy. In contrast, transgenic overexpression of a local isoform of insulin-like growth factor-1 prevented muscle atrophy and increased proteasome activity, inhibited skeletal muscle activation primarily of Foxo4, and blocked the expression of atrogin-1/MAFbx. These results suggest that skeletal muscle atrophy occurs through increased activity of the ubiquitin–proteasome pathway. The inhibition of muscle atrophy by local insulin-like growth factor-1 provides a promising therapeutic avenue for the prevention of skeletal muscle wasting in chronic heart failure and potentially other chronic diseases associated with skeletal muscle atrophy.

Key Words: insulin-like growth-1 • chronic heart failure • skeletal muscle • atrophy • gene expression

	Introduction

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Progressive metabolic abnormalities leading to a catabolic state have been observed in various chronic diseases, including heart and renal failure, AIDS, and cancer.^1–3 A common characteristic is the development of specific myopathic changes and a loss of lean muscle mass.⁴ In chronic heart failure, alterations in multiple anabolic and catabolic systems result in progressive catabolism leading to cardiac cachexia in advanced stages of the disease.^1,5 Early stages of chronic heart failure–related muscle atrophy correlate with reduced expression of insulin-like growth factor (IGF)-1,⁶ activation of systemic,⁵ and local⁶ markers of inflammation, as well as increased oxidative stress.⁷ However, the specific mechanisms underlying skeletal muscle dysfunction and progressive muscle atrophy in chronic heart failure are still incompletely understood.

Catabolic loss of muscle protein occurs primarily through enhanced protein breakdown mediated by the ubiquitin–proteasome pathway.⁸ The critical regulators of protein ubiquitination are ubiquitin-protein ligases E3 that catalyze the transfer of activated ubiquitin from an ubiquitin-conjugating enzyme (E2). Individual ubiquitin ligases selectively target specific proteins for ubiquitination and exhibit tissue-specific and inducible expression patterns.^9,10 Proteins marked by chains of ubiquitin are rapidly degraded through the 26S proteasome in a ATP-dependent process that yields peptides and intact ubiquitin.⁸ The crucial role of the ubiquitin–proteasome pathway has been demonstrated in several models of muscle atrophy that include starvation, uremia, denervation, sepsis, and diabetes mellitus.^9–12

The present study was undertaken to investigate whether activation of the ubiquitin–proteasome pathway contributes to muscle atrophy in the syndrome of chronic heart failure. Because progressive muscle atrophy in advanced stages of chronic heart failure correlates with low serum levels and reduced local expression of IGF-1,^6,13 we hypothesized that expression an MLC/mIgf-1 transgene encoding a locally acting isoform of IGF-1 normally induced in response to muscle damage,¹⁴ could prevent the development of heart failure–associated muscle atrophy and concomitant activation of the proteasome.

Here we show that muscle activation of the ubiquitin–proteasome pathway in the setting of chronic left-ventricular dysfunction (CLVD) is accompanied by selective induction of the muscle-specific ubiquitin ligase atrogin-1/MAFbx (Muscle Atrophy F-box), presumably through the activation of Foxo transcription factors in the skeletal muscle. Transgenic supplementation of the mIgf-1 isoform prevents muscle atrophy and activation of the proteasome. Further, overexpression of mIgf-1 specifically inhibits activation of Foxo4, the most abundant of these factors in skeletal muscle, and blocks expression of atrogin-1/MAFbx. These studies establish a role for ubiquitin-mediated proteolytic degradation in muscle atrophy accompanying CLVD and highlight the potential therapeutic value of supplementing the local mIgf-1 isoform in the treatment of progressive muscle wasting accompanying heart failure.

	Materials and Methods

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Animal Model
Myocardial infarction was induced under anesthesia in 8- to 12-week-old male FVB mice (wild-type [WT]) and in an FVB transgenic mouse line (MLC/mIgf-1) with skeletal muscle-restricted expression of the Exon1-Ea isoform of the rat Igf-1 gene.¹⁴ Progressive cardiac dysfunction was induced by ligation of the left coronary artery, whereas sham-operated animals underwent the same procedure without ligation of the coronary artery. All mice underwent echocardiography after 2 and 12 weeks and were euthanized after 12 weeks by injection of pentobarbital. The investigation conformed to the NIH Guide for the Care and Use of Laboratory Animals (Publication No. 85-23, revised 1996) and was approved by the Harvard Medical School Standing Committee on Animals.

Histology
Organs were removed, fixed in 4% paraformaldehyde, and embedded in paraffin for further histological analysis. Tissue samples were cut in 5-µm thick sections and stained using hematoxylin and eosin, as well as sirius red. Morphological analysis of muscle fiber cross-sectional area was performed on tissue scans using ImagePro software (ImagePro Plus 4.5, Media Cybernetics).

Detection of Apoptosis
The rate of apoptosis was assessed by terminal deoxynucleotidyltransferase–mediated dUTP-biotin nick-end labeling. Further, immunohistochemistry was performed using antibodies against cleaved caspase-3 (Cell Signaling). Using light microscopy, the percentage of dark brown nuclei was analyzed and expressed as percentage of total nuclei as described previously.¹⁵

Vascular Density
Capillaries were stained immunohistochemically using antibodies directed against lectin (Molecular Probes). Capillary density was measured by counting random fields on skeletal muscle sections using light microscopy as described previously.¹⁵ Total capillary number was corrected for muscle fibers and expressed as capillaries per muscle fiber ratio.

Cell Culture
SOL8 cells (American Type Culture Collection) were cultured in DMEM containing 20% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were incubated with tumor necrosis factor- (20 ng/mL), interleukin-1ß (50 ng/mL), IGF-1 (10 ng/mL), and dexamethasone (Dexa; 10 ng/mL). Further, the pharmacological inhibitors Wortmannin (100 nmol/L; Sigma), U126 (10 µmol/L; Calbiochem), PD169316 (100 nmol/L; Calbiochem), and rapamycin (20 ng/mL; Cell Signaling) were used in the present study.

Real-Time Polymerase Chain Reaction
Total RNA (100 ng) was assessed by real-time polymerase chain reaction (PCR) (LightCycler; Roche) using primers for atrogin-1/MAFbx (sense: 5'-GAC TGG ACT TCT CGA CTG CC-3'; antisense: 5'-TCA GCC TCT GCA TGA TGT TC-3') and ß-tubulin (sense: 5'-CTG GGC TAA AGG CCA C-3'; antisense: 5'-AGA CAC TTT GGG CGA G-3'). The expression was normalized to expression levels of ß-tubulin.

Western Blot Analysis
Protein levels were analyzed by Western blot analysis using specific monoclonal antibodies for the detection of myosin heavy chain (MF20; Developmental Studies Hybridoma Bank) or ubiquitin (P4D1; Santa Cruz Biotechnology). Polyclonal antibodies against phosphorylated Akt, total Akt, phosphorylated Foxo1, -3, and -4, total Foxo, phosphorylated mammalian target of rapamycin (mTOR), p70^s6k, total mTOR, phosphorylated p38 mitogen-activated protein kinase (MAPK), total p38 MAPK, p42/44 MAPK, and extracellular signal regulated kinase 1/2 were from Cell Signaling. After incubation with horseradish peroxidase–conjugated secondary antibody, specific bands were visualized by enzymatic chemiluminescence (Perkin Elmer).

Immunoprecipitation
Thirty microliters of protein A Sepharose beads were incubated with 1 µg of anti-ubiquitin antibody. Total protein (300 µg) from the soluble fraction of the muscle lysates were incubated with antibody bead complexes for 2 hours rotating at 4°C. Beads were centrifuged and washed 3 times with 0.5 mL of lysis buffer and 1 time with 0.5 mL of ice-cold PBS. The beads were resuspended in SDS sample buffer, incubated at 95°C for five minutes, centrifuged, and the supernatant electrophoresed through a SDS-PAGE system. After transfer to a polyvinylidene difluoride membrane, further immunoblotting was performed.

Proteasome Activity
Protein lysates were incubated at 37°C with proteasome assay buffer containing 10 µmol/mL SLLVT–7-amino-4-methylocoumarin (Calbiochem) as substrate for the chymotrypsin-like activity of the proteasome. Specificity was tested by using MG132 (10 µmol/L; Sigma) and lactacystin (20 µmol/L; Sigma) to inhibit 20S proteasome activity. Fluorescence of free 7-amino-4-methylocoumarin as a measure of proteasome activity was assessed in intervals over 60 minutes on a temperature-controlled fluorescence reader (Perkin Elmer).

Statistical Analysis
All experiments were performed at least 3 times, and data are expressed as mean±SEM. Data were analyzed by Student t test for comparisons of 2 data groups. One-way ANOVA with Tukey post hoc analysis was used for the analysis of data sets of more than 2 groups. A probability value of P<0.05 was considered statistically significant. The statistical analysis was performed using the GraphPad InStat software package (Version 3.05 for Windows 95/NT).

	Results

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Assessment of an Animal Model of CLVD
Thirty-nine WT and 34 MLC/mIgf-1 transgenic mice were included in the study (online Table available at http://circres.ahajournals.org). The animals were randomly assigned to a group undergoing ligation of the left coronary artery or sham surgery. Perioperative mortality and total mortality over the study period was comparable between WT and MLC/mIgf-1 (P=not significant [NS]). Echocardiographic assessment demonstrated the expected development of progressive left-ventricular enlargement and systolic dysfunction following left coronary artery ligation (online Table). The echocardiographic analysis revealed comparable infarct sizes in both groups (percentage infarcted myocardium of total left-ventricular circumference: 33.2±9.3% in WT animals versus 33.2±6.4% in MLC/mIgf-1 transgenic animals; P=NS). Functional parameters of the left-ventricular myocardium were assessed at different levels of the left ventricle to analyze changes in ventricular structure during the remodeling process. Myocardial infarction induced comparable increases in wall thickness of the noninfarcted ventricular wall and ventricular volume in WT and MLC/mIgf-1 mice. Myocardial infarction induced pronounced dilation of the infarcted ventricles without differences between WT and transgenic animals (left-ventricular end-diastolic diameter [LVEDD] at the level of the papillary muscle: 3.19±0.59 mm in WT animals versus 3.19±0.46 mm in MLC/mIgf-1 transgenic animals; LVEDD apex: 3.56±1.59 mm in WT animals versus 3.47±0.77 mm in MLC/mIgf-1 transgenic animals; both P=NS). Myocardial infarction significantly reduced myocardial fractional shortening without differences between the 2 groups (online Table). Heart weight corrected for body weight was comparable between the 2 groups both at baseline and following myocardial infarction, indicating no specific cardiac effects in animals with skeletal muscle-restricted overexpression of mIgf-1 compared with WT animals.

Skeletal Muscle Atrophy and Activation of the Ubiquitin–Proteasome Pathway in CLVD
Assessment of skeletal muscle revealed a reduction in muscle fiber cross-sectional area in WT animals with CLVD (Figure 1A and 1B). This reduction was paralleled by a decrease in isolated muscle weight (online Table). Anti-ubiquitin immunoblotting revealed an increase in ubiquitinated substrates in soluble fractions of atrophying muscles (Figure 1C). The activity of the 20S proteasome was measured by assessment of the chymotrypsin-like proteolytic activity. WT animals with CLVD and muscle atrophy displayed an increase in 20S proteasome activity in skeletal muscle by 40% (P<0.05 versus controls) (Figure 1D). No increase in the rate of apoptosis in skeletal muscle myocytes was detected in animals with left-ventricular dysfunction. However, an increased rate of vascular apoptosis (199±77% of sham-operated animals; P<0.05) was found in muscle specimens of WT animals following myocardial infarction. Notably, signs of vascular rarefaction were found in skeletal muscle of animals with CLVD following myocardial infarction. Vascular density (capillary to myocyte ratio) decreased in skeletal muscle of animals with CLVD by 24% (P<0.05 versus sham surgery).

View larger version (59K): [in this window] [in a new window]   Figure 1. Muscle atrophy and increased activity of the proteasome in mice with CLVD is prevented by transgenic overexpression of mIgf-1. A, Skeletal muscle atrophy develops in mice with CLVD in several muscle groups. Overexpression of mIgf-1 results in increased muscle fiber cross-sectional area and blocks muscle atrophy in the setting of CLVD. B, Histological analysis of skeletal muscle at baseline and after development of CLVD (Sirius red staining). C, Increased protein ubiquitination in skeletal muscle of mice with CLVD is inhibited in muscle of transgenic animals with CLVD (top, anti-ubiquitin immunoblot of total muscle lysates from the quadriceps muscle; bottom, loading control, Comassie stain). D, Activity of the 20S proteasome increases in several atrophying muscles in CLVD. Overexpression of mIgf-1 blocks enhanced 20S proteasome activity in CLVD. EDL indicates M extensor digitorum longus; SOL, M soleus; QUA, M quadriceps. (One-way ANOVA followed by Tukey post-hoc analysis: *P<0.05 vs WT sham, #P<0.05 vs myocardial infarction WT animals; n=6 animals per data point.) Incubation of muscle lysates with the proteasome inhibitors lactacystin and MG132 significantly reduced 20S proteasome activity compared with control. P<0.001 vs control (n=3 per data point).

Transgenic Overexpression of mIgf-1 Prevents Activation of the Ubiquitin–Proteasome Pathway and Muscle Atrophy in CLVD
Next, we assessed skeletal muscle activation of the proteolytic ubiquitin–proteasome pathway in MLC/mIgf-1 transgenic animals. Muscle fiber cross-sectional area of MLC/mIgf-1 mice was increased at baseline because of the anabolic function of the locally overexpressed mIgf-1 isoform.¹⁶ In contrast to WT animals, MLC/mIgf-1 mice with CLVD experienced no reduction of muscle fiber cross-sectional area (Figure 1A and 1B). Further, transgenic overexpression of mIgf-1 prevented the increase in total muscle protein ubiquitination following myocardial infarction (Figure 1B). Finally, the increase in proteasome activity was prevented in skeletal muscle of MLC/mIgf-1 transgenic mice with CLVD (Figure 1C). In contrast to WT animals with CLVD, MLC/mIgf-1 mice with CLVD did not show changes in the rate of vascular apoptosis, indicating that local IGF-1 might regulate vascular apoptosis in skeletal muscle (131±31% of sham-operated animals; P=NS). Overexpression of mIgf-1 tended to increase capillary density; however, this increase did not reach statistical significance (+13% versus WT; P=NS). Overexpression of mIgf-1 reduced but did not completely block the loss in capillary density in animals with CLVD (–24±8% in WT versus –15±6% in mIgf-1 animals; P=0.08).

IGF-1 Blocks Expression of atrogin-1/MAFbx–Mediated Ubiquitination in Muscle Cell Cultures
To identify potential structural targets of the ubiquitin–proteasome pathway in muscle atrophy, we challenged SOL8 myogenic cell cultures with dexamethasone and cytokines as well as starvation and analyzed protein ubiquitination status with immunoprecipitation, which effectively isolates ubiquitinated protein complexes as detected by anti-ubiquitin immunoblotting (Figure 2A). Whereas Western blot analysis showed no differences in overall total myosin heavy-chain (MyHC) protein, 24-hour dexamethasone treatment induced a robust increase in MyHC ubiquitination (Figure 2B), which was also seen after serum withdrawal and tumor necrosis factor- and interleukin-1ß (data not shown). Thus, myosin ubiquitination may contribute to muscle cell atrophy.

View larger version (27K): [in this window] [in a new window]   Figure 2. Ubiquitin-mediated proteolysis of MyHC and effects of IGF-1 on expression of atrogin-1/MAFbx in vitro. A, Immunoprecipitation of ubiquitin-protein conjugates from total quadriceps muscle lysates using anti-ubiquitin antibodies. B, Analysis of MyHC ubiquitination in vitro. Whereas Western blot analysis showed no differences in overall protein levels between dexamethasone-treated (DEX) (10 ng/mL for 24 hours) cells and controls, ubiquitination of MyHC is robustly enhanced under dexamethasone compared with controls. C, In SOL8 cells, serum starvation for 72 hours increases atrogin-1/MAFbx expression, which is reduced by incubation with 20% FCS and stimulation with IGF-1 (10 ng/mL) for 3 hours. Gene expression of atrogin-1/MAFbx was assessed by quantitative real-time PCR (*P<0.001 vs control; #P<0.01 vs starvation; n=5 per data point; one-way ANOVA followed by Tukey post-hoc analysis). D, IGF-1 stimulation (10 ng/mL) for 30 minutes leads to phosphorylation of Akt, mTOR, and p70sk6. E, Pharmacological inhibition of p42/44 MAPK and p38 MAPK has no effects on IGF-1–induced suppression of atrogin-1/MAFbx expression. Inhibition of PI3 kinase completely and inhibition of mTOR partially inhibits IGF-1 effects (*P<0.001 vs control, #P<0.05 vs starvation; n=5 per data point; one-way ANOVA followed by Tukey post-hoc analysis).

E3-ligases are ubiquitin-protein–conjugating enzymes with a crucial role in the molecular cascade of protein ubiquitination, which marks them for rapid proteolytic degradation.^9,10 Induction of the E3-ligase atrogin-1/MAFbx in the setting of muscle atrophy was first confirmed in SOL8 cells starved by serum withdrawal for 72 hours. This effect was reduced by stimulation with 20% serum for 3 hours and completely normalized by addition of recombinant IGF-1 to the media for 3 hours (Figure 2C).

Analysis of the underlying pathways mediating the suppressive effects of IGF-1 on atrogin-1/MAFbx expression in SOL8 cells revealed robust activation of Akt, mTOR, and p70^s6k (Figure 2D), which all have been demonstrated to contribute to the prohypertrophic and antiatrophic effects of IGF-1 in skeletal muscle.^14,17–21 Pharmacological inhibition of p42/44 MAPK and p38 MAPK did not affect suppression of atrogin-1/MAFbx expression by IGF-1. In contrast, the effects of IGF-1 on the expression of atrogin-1/MAFbx in SOL8 cultures were completely blocked by phosphatidylinositol 3 (PI3) kinase inhibition and reduced by mTOR inhibition (Figure 2E).

Transgenic Expression of mIGF-1 Blocks Polyubiquitination of MyHC in CLVD-Induced Muscle Atrophy
To determine whether MyHC was a target of ubiquitin-mediated proteolytic degradation in vivo, we performed similar analyses on mouse skeletal muscle and found an increased fraction of ubiquitinated MyHC in WT animals with CLVD (Figure 3A). Expression of the MLC/mIgf-1 transgene blocked the increase in MyHC ubiquitination in skeletal muscle of animals with chronic CLVD. We also analyzed expression of atrogin-1/MAFbx in several muscle groups of mice with CLVD (Figure 3B). Quantitative real-time PCR revealed that transcripts of atrogin-1/MAFbx were strongly induced in atrophying muscles of animals with CLVD (3.48±1.38 versus 1.21±0.32 arbitrary units; P<0.01 versus WT sham). In MLC/mIgf-1 transgenic muscle, expression of atrogin-1/MAFbx remained at basal levels following the development of CLVD (Figure 3C). These results support a model wherein mIgf-1 blocks the degradation of myosin in CLVD by repressing the induction of atrogin-1/MAFbx expression.

View larger version (17K): [in this window] [in a new window]   Figure 3. MyHC ubiquitination and skeletal muscle expression of atrogin-1/MAFbx in vivo. A, Increased ubiquitination of MyHC in atrophying quadriceps skeletal muscle 12 weeks after coronary artery ligation and development of CLVD. Expression of the MLC/mIgf-1 transgene prevents the increase in ubiquitination of MyHC in skeletal muscles of animals with CLVD. B, Expression of atrogin-1/MAFbx increases in skeletal muscles of animals with CLVD. C, Quantitative real-time PCR revealed increased atrogin-1/MAFbx expression in the atrophying quadriceps muscle of animals with CLVD, which is prevented by transgenic overexpression of mIgf-1 (white bars, sham surgery; black bars, myocardial infarction; *P<0.05 vs WT sham; n=6 animals per data point; one-way ANOVA followed by Tukey post-hoc analysis).

Transgenic Overexpression of mIgf-1 Selectively Inhibits Activation of Specific Foxo Transcription Factors in Muscles of Mice With CLVD
To delineate the signals responsible for the prevention of CLVD-induced muscle atrophy and atrogin-1/MAFbx activation in MLC/mIgf-1 transgenic muscle, we monitored the activation status of the Akt/mTOR/p70^s6k pathway in the setting of chronic heart failure (Figure 4A and B). Phosphorylated Akt (which represents the active form of the kinase) was reduced in atrophied skeletal muscle of WT mice with CLVD (>50% reduction versus sham surgery; P<0.05). CLVD did not change the activity of mTOR or p70^s6k, suggesting that the mTOR/p70^s6k pathway is not implicated in heart failure–induced muscle atrophy.

View larger version (43K): [in this window] [in a new window]   Figure 4. Reduced of Akt phosphorylation and activation of Foxo factors in atrophying skeletal muscle in CLVD is prevented in MLC/mIgf-1 mice. A, Mice with CLVD show reduced Akt activity and increased activation of Foxo4 in skeletal muscle compared with controls. No changes on the mTOR/p70sk6 pathway are detectable in skeletal muscle of mice with CLVD. Transgenic overexpression of MLC/mIgf-1 in CLVD muscle activates mTOR, blocks repression of Akt, and inhibits Foxo transcription factors by enhancing Foxo phosphorylation. B, Quantitative analysis of data in A (white bars, WT; gray bars, MLC/mIgf-1; empty bars, sham surgery; striped bars, myocardial infarction). *P<0.05 vs WT sham (n=3 per data point); one-way ANOVA followed by Tukey post-hoc analysis.

In contrast, no reduction of Akt activity was observed in the muscles of MLC/mIgf-1 transgenic mice with CLVD. Increased levels of phosphorylated mTOR and p70^s6k in MLC/mIgf-1 transgenic muscle were also maintained after induction of CLVD, implicating this pathway in the maintenance of muscle mass and integrity in response to MLC/mIgf-1 overexpression.

The Akt/mTOR/ p70^s6k pathway has been implicated in the restriction of Foxo transcription factor activity that, in turn, controls the atrogin-1/MAFbx gene.²² Foxo transcription factors are activated by dephosphorylation, and repression of Akt activity in the muscles of WT mice with CLVD was accompanied by selective activation of Foxo4 by dephosphorylation (>70% reduction versus sham surgery; P<0.05) without significant changes in the other 2 Foxo transcription factors 1 and 3 (Figure 4A and 4B). Notably, activation of Foxo4 was blocked by mIgf-1 overexpression, as documented by increased Foxo4 phosphorylation (Figure 4A and 4B).

Taken together, these results implicate that skeletal muscle atrophy in the setting of CLVD is accompanied by reduced Akt activation, leading to activation of Foxo transcription factors without significantly affecting the mTOR and p70^s6k pathway. Overexpression of mIgf-1 activates both the PI3K/Akt/Foxo pathway as well as the mTOR/p70^s6k pathway, indicating that supplemental mIgf-1 expression might inhibit muscle atrophy through several intracellular signaling events.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we used an animal model of CLVD to implicate the ubiquitin–proteasome pathway in skeletal muscle atrophy accompanying heart disease and to explore potential therapeutic avenues for its prevention. Induction of the ubiquitin–proteasome pathway in atrophying skeletal muscle was accompanied by Foxo activation and expression of the atrophy-related ubiquitin-protein ligase atrogin-1/MAFbx. These changes are prevented by transgenic overexpression of mIgf-1.

CLVD results in profound metabolic changes that affect various endocrine systems and leads to an imbalance of catabolic over anabolic function.⁵ Patients with chronic heart failure exhibit elevated levels of proinflammatory cytokines,^1,5 an imbalance between catabolic and anabolic steroids,⁵ insulin resistance,²³ and oxidative stress.²⁴ chronic heart failure induces structural skeletal muscle abnormalities that include mitochondria,²⁵ changes in enzyme distribution from slow toward fast glycolytic isoforms,²⁶ a shift from slow twitch to fast twitch fibers,²⁵ and local cytokine expression,⁶ as well as a catabolic loss of muscle bulk in advanced stages of the disease.^1,5,13 Intriguingly, a reduced skeletal muscle expression of IGF-1 has been demonstrated in patients with chronic heart failure,¹³ as well as in animal models of CLVD.^6,27,28 These findings formed the rationale for the present experimental study.

Enhanced protein breakdown, a central event in catabolic loss of muscle proteins, is primarily mediated by the ubiquitin–proteasome pathway.⁸ In animal models of starvation,⁹ diabetes,¹¹ and sepsis,¹² this pathway has been identified as the central mediator of protein degradation. Indeed, ubiquitinated conjugates and genes that encode components of the ubiquitin–proteasome pathway increase during atrophy,^9,29 whereas inhibition of the 26S proteasome prevents progression of muscle atrophy.³⁰ Several structural muscle proteins (eg, myosin and troponin) are subject to ATP-dependent degradation through the 26S proteasome.^31,32

The present study identifies increased activity of the ubiquitin–proteasome pathway as a mechanism contributing to skeletal muscle atrophy in CLVD caused by enhanced MyHC polyubiquitination. This finding is consistent with previous reports demonstrating degradation of myosin through the ubiquitin–proteasome pathway in other settings,³¹ as well as increased polyubiquitination of MyHC in dilated cardiomyopathy³³ and in cancer cachexia.³² However, the molecular and structural characteristics underlying this mechanism are still unclear. The biochemical measurements performed in this study used total muscle tissue lysates. Whereas myocytes are the highly predominant portion of muscle tissue, other cells and tissues, such as extracellular matrix and vasculature, constitute normal skeletal muscle. Therefore, pathological abnormalities in those compartments might also have contributed to the observed functional derangements of skeletal muscle.

Ubiquitin-protein ligases play a crucial role in mediating ubiquitination of specific substrates. Elevated levels of the ubiquitin-protein ligase atrogin-1/MAFbx in skeletal muscle of mice with CLVD correlates well with other studies documenting increased atrogin-1/MAFbx expression in muscle atrophy incurred by immobilization, denervation, hindlimb suspension, and starvation.^9,10 The induction of atrogin-1/MAFbx expression before muscle weight loss suggests its involvement in development and progression of muscle protein loss.⁹ In support of this possibility, overexpression of atrogin-1/MAFbx induces atrophy in vitro, whereas muscle atrophy was prevented in animals with targeted deletion of atrogin-1/MAFbx.¹⁰

Maintenance of Akt/mTOR/p70^s6k signaling is 1 potential mechanism whereby expression of the MLC/mIgf-1 transgene abrogates muscle atrophy and breakdown of muscle structural proteins. Indeed, intramuscular administration of IGF-1 protein induces Akt phosphorylation, inhibits denervation-induced muscle loss, and blocks the increased expression of MAFbx and MuRF1.²⁰ Although no direct inhibitors of Akt signaling have been identified in atrophying skeletal muscle to date, its repression in various stages of muscle atrophy,²² as well as anticatabolic effects of constitutively active Akt,²⁰ implicate this pathway in maintenance of muscle mass. Intriguingly, exogenous administration of growth hormone to rats with CLVD counters the decline in skeletal muscle function, structure, and morphology.³⁴ Activation of the Akt pathway also occurs downstream of the ß₂-adrenergic receptor through the second messenger cAMP. In chronic heart failure, the expression of ß-adrenergic receptors is suppressed through enhanced noradrenergic stimulation. Thus, the anticatabolic effects of transgenic overexpression of mIgf-1 in vivo compensate for a reduced intrinsic stimulation of Akt signaling in skeletal in the setting of CLVD.

Foxo factor–mediated transcriptional activation has been associated with enhanced proteolysis and muscle atrophy.^20,22 In mammals, the forkhead transcription factors include Foxo1 (FKHR), Foxo3 (FKHRL1), and Foxo4 (AFX).²² Increased expression of Foxo1 occurs during muscle atrophy,²⁹ and phosphorylation of Foxo1, Foxo3, and Foxo4 leads to nuclear exclusion inhibiting their transcriptional activity.^20,22 Overexpression of constitutively active Foxo enhances activity of the atrogin-1/MAFbx promoter and increases levels of atrogin-1/MAFbx mRNA, whereas gene silencing of Foxo1-Foxo3 through RNA interference blocks atrogin/MAFbx promoter activity.²² In muscle cell cultures, expression of a dominant negative Akt molecule leads to robust induction of Foxo transcription factors and activation of a Foxo-dependent gene program, which is characteristic for muscle atrophy.²⁰ Overexpression of Akt in skeletal muscle²² and heart³⁵ induces phosphorylation of Foxo transcription factors, suppresses the expression of Foxo-dependent atrogenes, and induces myocyte hypertrophy. In the present study, MLC/mIgf-1 expression elicited a robust phosphorylation of Foxo4 (AFX) which was induced in muscle of both sham and CLVD animals. Notably, the mIgf-1 transgene did not alter absolute Foxo protein levels and did not significantly affect the phosphorylation of Foxo1 or Foxo3. AFX is the most abundant forkhead transcription factor expressed in skeletal muscle,³⁶ and, therefore, its inactivation may afford particularly dramatic prevention of muscle wasting.

It has been demonstrated by several groups that the expression of atrogin-1/MAFbx in muscle atrophy is controlled by Foxo transcription factors,^20,22 and activation of mTOR signaling has been implicated in myocyte hypertrophy in response to IGF-1 stimulation.¹⁷ In agreement with these observations, stimulation of SOL8 myoblasts with IGF-1 protein led to an activation of the mTOR pathway with enhanced phosphorylation of mTOR and p70^s6k. Inhibition of this pathway by rapamycin reduced the effects of IGF-1 on levels of atrogin-1/MAFbx, indicating that this pathway participates in the regulation of atrogene expression. Notably, this has recently been confirmed by another group.³⁷

Interestingly, transgenic overexpression of mIgf-1 had only modest effects on activation of the mTOR pathway in vivo. Because phosphorylation of mTOR and p70^s6k are not altered in CLVD, we conclude that this pathway is not implicated in muscle atrophy in the setting of chronic heart failure. In contrast, CLVD induced significant changes in the Akt/Foxo pathway, suggesting its pivotal role in mechanisms of muscle atrophy. One might speculate that Akt/Foxo signaling regulates muscle atrophy, whereas mTOR activation is required for muscle hypertrophy.³⁸ The exact role of these pathways in mechanisms of muscle metabolism, however, requires further investigation.

Consideration of IGF-1 isoforms is critical in the interpretation of current studies on the effects of supplementary growth factors. Exogenously administered IGF-1 induces muscle hypertrophy through autocrine and paracrine mechanisms,³⁹ and muscle-specific overexpression of a circulating IGF-1 isoform results in profound muscle growth mediated through increased protein synthesis and DNA accretion.⁴⁰ Overexpression of the locally acting mIgf-1 isoform counters the decline in muscle mass in mdx mice,⁴¹ in a mouse model of amyotrophic lateral sclerosis,⁴² in senescence,⁴³ and in response to angiotensin II infusion.²⁸ Gene transfer of mIgf-1 under the control of muscle-specific regulatory elements prevents age-related loss of skeletal muscle mass and function, even when administered at senescence.⁴⁴ Additionally, mIGF-1 may act as a potent regenerative agent, as increased stem cell recruitment to sites of muscle injury was observed in mice expressing the MLC/mIgf-1 transgene. When isolated from MLC/mIgf-1 muscles, these progenitor cells exhibit accelerated myogenic differentiation and induce muscle-specific markers in cocultured bone marrow cells.⁴⁴ From these and other analyses (N. Winn and N. Rosenthal, manuscript in preparation), it is likely that locally produced mIgf-1 counteracts atrophy through signal-transduction pathways that are distinct from those activated by circulating IGF-1.

	Acknowledgments

 
This work was supported, in part, by grants from the NIH (PO1 HL64858 to RTL), the Deutsche Akademie der Naturforscher Leopoldina (BMBF-LPD) (9901/8-41 to P.C.S.), and the Muscular Dystrophy Association (to N.R.).

	Footnotes

 
Original received January 28, 2005; revision received June 22, 2005; accepted July 19, 2005.

	References

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