Adjunctive β₂-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease

Abstract

Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation-independent mannose-6-phosphate receptor (CI-MPR)-mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA-knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA-KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI-MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β₂-agonist treatment enhanced CI-MPR-mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.—Farah, B. L., Madden, L., Li, S., Nance, S., Bird, A., Bursac, N., Yen, P. M., Young, S. P., Koeberl, D. D. Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease.

Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) has prolonged ventilator-free survival and muscle strength in patients with Pompe disease; however, the limitations of ERT have become increasingly evident, and many patients eventually become ventilator dependent. The enzyme dosages required for ERT in Pompe disease range up to 100-fold greater than those for other lysosomal disorders, which can be attributed, at least in part, to the large muscle mass and to the formation of anti-GAA antibodies. ERT depends on the uptake of recombinant lysosomal enzymes by the cation-independent mannose-6-phosphate receptor (CI-MPR) at the plasma membrane and trafficking to the lysosomes, and CI-MPR is present at low levels in the skeletal muscle postnatally (1).

Clenbuterol treatment has been previously shown to enhance the biochemical correction of muscle from ERT in GAA-knockout (KO) mice with Pompe disease (2); furthermore, clenbuterol at a lower dose and a second β₂-agonist (albuterol) increased rotarod latency and biochemical correction following β₂-agonist treatment, in comparison with ERT alone (3). Glycogen content was reduced significantly in all striated muscles evaluated with the exception of tibialis anterior by clenbuterol treatment, in comparison with ERT alone (3). Both of the β₂-agonists evaluated increased uptake of rhGAA in the cerebral and cerebellar hemispheres of the brain during ERT, and glycogen storage was reduced accordingly in the brain (3). Furthermore, Western blot analysis revealed that clenbuterol treatment increased the signal for CI-MPR in the extensor digitalis longus (EDL) muscle, cerebellum, and cerebrum (3).

The effect of CI-MPR on gene therapy has been evaluated by administering an adeno-associated virus (AAV) vector encoding human GAA driven by a liver-specific regulatory cassette (AAV-LSPhGAA) to establish a liver depot for GAA secretion. A low dose of AAV-LSPhGAA, 2 × 10¹⁰ vector particles (vp), was injected intravenously to GAA-KO mice with or without clenbuterol treatment (4). The effect of clenbuterol was evident when rotarod testing increased by 75% following vector administration and clenbuterol treatment, in comparison with vector administration alone. The effect of clenbuterol was further demonstrated by increased biochemical correction in striated muscles and the brain from this combination therapy, in comparison with the vector alone (4). These data demonstrated a synergistic effect on efficacy from AAV vector administration and clenbuterol treatment in mice with Pompe disease.

The direct transduction of nondividing myofibers has a distinct advantage for the treatment of a myopathy such as Pompe disease, especially for AAV vectors that remain almost exclusively episomal and are lost from dividing hepatocytes in the liver (5, 6). Muscle-restricted transgene expression with an AAV vector containing a muscle-specific regulatory cassette significantly reduced glycogen accumulations in the heart and skeletal muscle, while evading the transgene-directed T-cell responses otherwise directed against GAA expressed with a constitutive regulatory cassette in adult GAA-KO mice (7, 8). However, the dosage requirements to achieve biochemical correction of skeletal muscle were ∼10-fold higher with the vector containing the muscle-specific regulatory cassette, in comparison with the vector containing the liver-specific regulatory cassette (8, 9). Therefore, we have evaluated the effect of adjunctive β₂-agonist therapy in combination with the muscle-specific vector, thereby potentially reducing vector dosage requirements for the AAV vector by augmenting the trafficking of GAA to lysosomes.

MATERIALS AND METHODS

Preparation of AAV vectors

The vector AAV-MHCK7hGAApA contains the MHCK7 regulatory cassette, the human GAA cDNA, and a human growth hormone polyadenylation sequence flanked by the AAV2 terminal repeats (8). Briefly, HEK293 cells were transfected with an AAV vector plasmid, the AAV packaging plasmid (courtesy of Dr. James M. Wilson, University of Pennsylvania, Philadelphia, PA, USA; ref. 10), and pAdHelper (Stratagene, La Jolla, CA, USA). Cell lysate was harvested 48 h following infection, subjected to 3 freeze-thaw cycles, and isolated by sucrose cushion pelleting followed by 2 cesium chloride gradient centrifugation steps. AAV stocks were dialyzed against 3 changes of Hanks buffer, and aliquots were stored at −80°C. The number of vector DNA-containing particles was determined by DNase I digestion, DNA extraction, and Southern blot analysis. All viral vector stocks were handled according to U.S. National Institutes of Health (NIH) Biohazard Safety Level 2 guidelines.

Generation of muscle-specific CI-MPR-KO and double-KO (DKO) mouse models:

CI-MPR-KO mice were generated using a muscle-specific promoter [muscle creatine kinase (MCK)] and the cre/loxP conditional-KO system, as described previously (11). The muscle-specific CI-MPR-KO mice were crossed with GAA-KO mice to generate muscle-specific DKO mice. This mouse colony was subsequently screened to be GAA^−/−, M6PR^flox/flox, and MCK-Cre⁺. DKO mice were genotyped and bred as described previously (2). At the indicated time points postinjection, tissue samples were obtained and processed as described below. All animal procedures were done in accordance with Duke University Institutional Animal Care and Use Committee guidelines.

In vivo evaluation of AAV vector-mediated efficacy

GAA-KO mice (3 mo old) and DKO mice (6 mo old) were administered the AAV vector with or without clenbuterol. Clenbuterol (30 μg/ml) was provided continuously from d 1 in the drinking water, and vector was administered on d 1 by intravenous injection. Rotarod testing was performed as described previously (12). Wirehang testing was performed with 0.5-cm-mesh hardware cloth fixed to an 8- × 10-inch frame. Mice were placed on the wire mesh, which was slowly inverted 6 inches over a cage containing paper bedding. The latency, or time until the mouse fell off of the wire mesh, was recorded. GAA activity and glycogen content were analyzed as described previously (12). Histological processing and staining of brain was performed using a modified paraffin processing and staining protocol, as described previously (13). Quantification of vector DNA was performed as follows, using primers for human GAA and mouse β-actin (14). Plasmid DNA corresponding to 0.01–10 copies of human GAA gene (in 500 ng genomic DNA) was used in a standard curve.

LAMP2 detection by immunohistochemistry

Paraffin-embedded sections were deparaffinized, rehydrated, then blocked in PBS containing 5% chick serum and 0.1% Triton-X 100. Primary antibody for LAMP-2 (H4B4; 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied overnight at 4 C. Chicken anti-mouse conjugated 488 (1:200; Invitrogen, St. Louis, MO, USA) was used for detection and DAPI for counterstaining of nuclei. Images (×40, 5 images/slide) were acquired for blinded counting of LAMP-2⁺ muscle fibers. One-way ANOVA was performed using JMP Pro 10 software (SAS Insitute, Inc., Cary, NC, USA) to determine statistical significance of LAMP-2 quantification (P<0.05).

Western blotting

Protein was prepared by homogenization and sonication, as described for quantification of GAA and glycogen. Aliquots were taken from the protein samples, and protein concentration was determined by BCA Kit (Bio-Rad, Richmond, CA, USA). Laemmle sample buffer (250 mM Tris, pH 7.4; 2% w/v sodium dodecyl sulfate; 25% v/v glycerol; 10% v/v 2-mercaptoethanol; and 0.01% w/v bromphenol blue) was added to the remainder of the sample, followed by heating to 105°C for 5 min, and storage at −80°C. Equal amounts of each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immediately transferred to a polyvinylidene difluoride membrane (Bio-Rad) using Towbin transfer buffer (25 mM Tris, pH 8.8; 192 mM glycine; and 15% v/v methanol). Following transfer, membranes were blocked in 5% milk in PBST. Primary antibodies (AKT 2938, LC3B 2775, SQSTM1/p62 5114, and GAPDH D16H11; Cell Signaling Technologies, Danvers, MA, USA) were added to the membranes in 1% w/v bovine serum albumin in PBST and incubated overnight at 4°C. Membranes were washed 3 times in PBST, followed by incubation in anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology) in 1% w/v bovine serum albumin in PBST for 1 h at room temperature. Blots were again washed 3 times in PBST, and then once in PBS. Blots were developed with an enhanced chemiluminescence system (GE Healthcare, Pittsburgh, PA, USA), and images were acquired on a GelDoc XR+ imager (Bio-Rad).

Densitometry

Western blot bands were quantified using ImageJ software (NIH, Bethesda, MD, USA). LC3 turnover was analyzed by comparing the intensity of the LC3-II band to the internal control GAPDH, as is currently recommended practice (18). Results are shown as means ± sem. Significance between conditions (P<0.05) was ascertained by 2-tailed t test.

RESULTS

Treatment with clenbuterol has increased CI-MPR expression in muscle accompanied by increased CI-MPR mediated uptake of GAA and trafficking to the lysosome, where GAA was activated to reduce lysosomal glycogen (Fig. 1A). To study intracellular trafficking of GAA, a muscle-specific AAV vector, pseudotyped as AAV8 (AAV2/8-MHCK7hGAApA), in a 5-fold lower quantity than was previously required to clear glycogen storage from the heart (8), was administered intravenously to adult GAA-KO mice (2×10¹¹ vp). Biochemical efficacy was demonstrated by increased GAA activity in skeletal muscle at 12 wk following vector administration, in comparison with untreated GAA-KO mice (Fig. 1B). Clenbuterol further elevated GAA activity in the heart and diaphragm, in comparison with the vector alone. The combination therapy increased GAA activity to greater than the level observed in wild-type (WT) mice; however, GAA activity remained lower than WT levels in skeletal muscle (Fig. 1B). Vector administration reduced the glycogen content of the diaphragm, soleus, and EDL (Fig. 1C). The addition of clenbuterol significantly reduced glycogen content in the heart and all skeletal muscles evaluated, in comparison with the vector alone. However, all groups of GAA-KO mice had higher glycogen content than WT mice (<0.1 μm glucose/mg protein).

Figure 1.

Enhanced efficacy from clenbuterol treatment following AAV2/8-MHCK7hGAA administration. Vector-injected mice (2×10¹¹ vp/mouse) were untreated (n=8), treated with vector alone (n=10), or treated with clenbuterol (2) following vector administration (n=12), and analyzed at 12 wk. Statistically significant differences were demonstrated for clenbuterol-treated mice, in comparison with mice treated with vector alone (1-way ANOVA, Tukey posttest). A) Introduced GAA is bound to CI-MPR to allow trafficking to the lysosomes, where it is activated and subsequently reduces lysosomal glycogen. B) GAA activity. Normal GAA activity in WT mice is shown for comparison. C) Glycogen content. D) Urinary biomarker (Glc₄), analyzed as described previously (37). E) Correlation of glycogen content in different tissues with urinary Glc₄. *P < 0.05; **P < 0.01; ***P < 0.001.

The mean urinary glucose tetrasaccharide (Glc₄) biomarker of glycogen storage and turnover was significantly lower in GAA-KO mice following combination treatment compared with mice treated with vector only (Fig. 1D). Urinary Glc₄ correlated with heart glycogen content (R²=0.72) and not with muscle glycogen (Fig. 1E), indicating a major effect on the heart. These data suggested that the β₂-agonist treatment enhanced trafficking of GAA to lysosomes and activation within striated muscle, without increasing receptor-mediated uptake per se, in the context of muscle-specific GAA expression.

The dependence of efficacy from clenbuterol on CI-MPR expression was evaluated in CI-MPR-KO/GAA-KO (DKO) mice that lacked CI-MPR expression in muscle. GAA activity was only slightly increased by the addition of clenbuterol in diaphragm, while GAA activity remained unchanged in the heart and limb muscles (Fig. 2A). Glycogen content was only slightly reduced in the quadriceps, and unchanged in the heart and diaphragm, by the addition of clenbuterol (Fig. 2B). Thus, the additional biochemical efficacy from clenbuterol administration seemed to depend on CI-MPR expression, because GAA activity remained the same or increased only slightly following clenbuterol administration. The addition of clenbuterol had no effect on glycogen storage in the heart and diaphragm of DKO mice; however, glycogen content was slightly reduced in all of the limb muscles by clenbuterol, which raised the possibility of an additional mechanism for its activity (Fig. 2B). Vector DNA quantification was performed in heart to determine whether variability in transduction might underlie the differences between groups of mice; however, no significant differences were observed between any of the treatment groups, indicating equivalent transgene delivery in all groups (Fig. 2C).

Figure 2.

Effect of clenbuterol in vector-treated DKO mice. Vector-injected mice (2×10¹⁰ vp/mouse) were treated with vector alone (n=5) or with clenbuterol (2) following vector administration (n=6) and analyzed at 12 wk. Statistically significant differences were demonstrated for clenbuterol-treated mice, in comparison with mice treated with vector alone (paired t test). A) GAA activity. B) Glycogen content. C) Real-time PCR quantification of vector DNA in the liver of DKO mice (n=4/group). *P < 0.05; **P < 0.01; ***P < 0.001.

The effect of adjunctive clenbuterol on muscle function was investigated by performing rotarod and wirehang testing 12 wk following vector administration in GAA-KO and DKO mice (Fig. 3). Improved function on the wirehang test in GAA-KO mice only, not DKO mice, indicated dependence on CI-MPR for an increase in muscle strength. However, the rotarod latency increased for both GAA-KO and DKO mice following clenbuterol treatment, revealing beneficial roles of clenbuterol on this type of neuromuscular function even in absence of CI-MPR expression. Previously, clenbuterol increased rotarod performance in absence of ERT in GAA-KO, consistent with an effect of muscle hypertrophy (2). The wirehang latency of vector-injected DKO mice was markedly lower, in comparison with vector-injected GAA-KO mice (Fig. 3), consistent with the demonstrated lack of efficacy from AAV vector administration in DKO mice (4).

Figure 3.

Enhanced muscle function following clenbuterol treatment with AAV2/8-MHCK7hGAA (2×10¹⁰ vp) administration. Vector-injected GAA-KO mice were treated with vector alone (n=10) or with clenbuterol (2) following vector administration (n=12); vector-injected DKO mice were treated with vector alone (n=5) or with clenbuterol following vector administration (n=6); and all mice were analyzed at 12 wk. Statistically significant differences were demonstrated for clenbuterol treated mice, in comparison with mice treated with vector alone (paired t test). A) Rotarod latency. B) Wirehang latency. *P < 0.05; **P < 0.01.

Given the partial biochemical response of the quadriceps and other limb muscles in absence of CI-MPR expression, LAMP2 vacuolization was analyzed as a marker for glycogen accumulation (Fig. 4A). As expected, greater LAMP-2 expression was observed in GAA-KO mice compared to WT mice (15). Although vector treatment alone did not significantly decrease LAMP-2 expression, the addition of clenbuterol significantly reduced LAMP-2 expression (Fig. 4B), which resembled that observed in WT animals (Fig. 4A).

Figure 4.

Clenbuterol treatment decreases the presence of LAMP-2⁺ muscle fibers. A) Immunofluorescent detection of LAMP-2 in quadriceps shows decrease in expression with AAV + clenbuterol treatment, high expression in untreated GAA-KO control mice, and low expression in WT controls. Scale bars = 50 μm. B) Combination treatment of AAV + clenbuterol significantly decreased the abundance of LAMP-2 in both GAA-KO and DKO mice compared to mice treated with AAV only and untreated controls. *P < 0.05, **P < 0.01 vs. untreated controls; ^#P < 0.05 vs. WT.

A dysregulation of macroautophagy, a process by which cytosolic components are sequestered and delivered to lysosomes, has long been implicated in the pathology of Pompe's disease (16). Pompe disease is associated with an increase in the number of autophagosomes and a decrease in autophagic flux, as the lysosomal degradation of autophagosomal targets is impaired (17). We thus investigated whether vector administration affected this abnormal autophagy (Fig. 5). Treatment with the AAV vector significantly decreased the protein levels of LC3-II, a marker for autophagosomal formation (Fig. 5A, C and ref. 18). Moreover, this decrease was associated with an increase in autophagic flux, as the treatment also significantly decreased the abnormal accumulation of SQSTM1/p62 (Fig. 5A, B), implying an increase in autophagic flux (19). Adjunctive treatment with clenbuterol caused a nonstatistically significant increase in the levels of both LC3-II and SQSTM1/p62 compared with vector alone; however, when compared to the dramatic improvement seen in the clenbuterol-treated mice, it likely was not biologically significant. Furthermore, we investigated the AKT/mTOR pathway, which is known to inhibit autophagy (20). GAA-KO mice showed increased phosphorylation of AKT (Fig. 6A–C), and the mTOR substrate p70s6k (Fig. 6D–F), compared to the WT mice. Vector administration significantly reduced both of these phosphorylations, which were further reduced with adjunctive clenbuterol treatment (Table 1). Of note, the reduction of AKT/mTOR signaling by clenbuterol did not seem to correlate with decreased p62 in the clenbuterol-treated mice (Table 1), implying that any clenbuterol effect was mediated by a different pathway.

Figure 5.

GAA transduction increases autophagic flux in murine quadriceps. A) Immunoblot of SQSTM1/p62 (bottom band; ref. 38) and autophagosomal marker LC3. B) Densitometric analysis of p62 in the same mice. C) Densitometric analysis of LC3-II. Results displayed as means ± sd (n=4/group). *P < 0.05; **P < 0.01.

Figure 6.

GAA transduction reduces AKT and mTOR signaling in murine quadriceps. A) Immunoblot of phosphorylated and total AKT. B) Densitometric analysis of AKT phosphorylation in the same mice. Results displayed as means ± sem. C) Ratio of phosphorylated to total AKT. D) Immunoblot of phosphorylated and total p70s6k. E) Densitometric analysis of p70s6k phosphorylation in the same mice. F) Ratio of phosphorylated to total p70s6k. Results displayed as means ± sem. (n=4/group, except for WT: n=2). *P < 0.05; **P < 0.01; ***P < 0.001.

Table 1.

Effects of clenbuterol on autophagy in muscle

Treatment	Autophagosome number (LC3-II)	Autophagic flux (p62)	mTOR activity (p-p70s6k)	AKT activity (pAKT)
No treatment	↑↑↑↑	↓↓↓↓	↑↑↑↑	↑↑↑↑
Vector	↑↑	↓↓	↑↑↑	↑↑↑
Vector + clenbuterol	↑↑	↓↓	↑↑	↑↑

DISCUSSION

The current study was designed to address the question of whether the increased CI-MPR expression associated with clenbuterol administration might improve the trafficking of GAA to lysosomes in mice with Pompe disease, given the low expression of CI-MPR in striated muscle postnatally (1). The addition of clenbuterol following AAV vector-mediated GAA expression in muscle also reduced glycogen content in the heart and skeletal muscle. Muscle strength also was enhanced by adjunctive therapy with clenbuterol, similar to the biochemical correction that occurred in muscle. The effect of clenbuterol was mostly dependent on CI-MPR expression, consistent with the role of increased CI-MPR in mediating efficacy during GAA replacement in Pompe disease (2). Unexpectedly, glycogen content in skeletal muscle was slightly reduced in concert with clenbuterol-increased rotarod performance in DKO mice that lacked CI-MPR in muscle. The reduction in LAMP-2, observed in both the GAA-KO and DKO mouse strains, correlated with the reduction in glycogen content, similar to that observed with ERT treatment (1). Previously, clenbuterol slightly reduced the glycogen content of skeletal muscle by itself (3), which presumably did not depend on CI-MPR expression in absence of GAA replacement. Hence, clenbuterol proved to have both CI-MPR-dependent and independent benefits during muscle-targeted gene therapy in mice with Pompe disease.

One of the hallmarks of Pompe disease is an abnormal increase in autophagosomes. It was originally suspected that an increase in autophagy was responsible for the pathology of Pompe disease, but further research showed that there was actually a functional deficit in autophagy, despite the increased number of autophagosomes (17). Although the exact link between the block in autophagic flux and the pathology of Pompe's disease remains unclear, recent work has shown that restoration of autophagic flux following ERT correlated with improved muscle function in patients with Pompe disease (21). The improvement in function and autophagic flux following AAV vector administration agreed with this earlier finding. Clenbuterol, although further improving function, does not seem to further improve the autophagic flux, implying that its action is through a separate pathway.

The decrease in AKT and p70s6k in the GAA-KO mice that occurred after recovery from AAV treatment (Fig. 6) was an unexpected finding. Although we initially predicted that AKT levels should be higher in the treated mice, secondary to increased muscle mass and IGF-1 signaling (22), the opposite trend was observed. Only one previous study has investigated the role of AKT/mTOR in Pompe's disease, when Nishiyama et al. (23) found AKT signaling to be reduced in the fibroblasts of affected patients, with improvement in lysosomal delivery of GAA following activation of this pathway by insulin. Although the results from our study seem to contradict these previous findings, it is important to note that fibroblasts and skeletal muscle may respond differently to the loss of GAA, and AKT may play different roles in different tissues. Another interesting finding is that the LC3-II levels were highly increased (Fig. 5C) in the GAA-KO mice, which also showed increased mTOR signaling (Fig. 6). While mTOR has classically been known to inhibit the first step of autophagosomal formation, leading to a decrease in LC3-II protein levels (20), a recent study has shown that it may also inhibit lysosomal activity (24), and that effect may be predominating in this system. In our study, AAV vector transduction reduced the increased AKT signaling in GAA-KO mice, and the addition of clenbuterol further reduced pAKT (Table 1). Thus, the effect of adding clenbuterol was to reduce the abnormalities of autophagy further, in comparison with partial correction from vector administration alone. Moreover, clenbuterol and AAV transduction further enhanced clearance of glycogen in limb muscles above the amount observed from AAV transduction in both GAA-KO and DKO mice. The observation that clenbuterol treatment reduced glycogen storage in DKO mice confirms that clenbuterol works independently of GAA to significantly reduce glycogen content in the skeletal muscles of mice with Pompe disease (3), because AAV transduction with GAA was ineffective in DKO mice (Fig. 2), as described previously (4). Furthermore, clenbuterol's observed effects on reducing mTor signaling and glycogen content were similar to the reported effect of rapamycin, which inhibited mTor and reduced glycogen storage in the skeletal muscle of GAA-KO mice (25).

Adjunctive therapy with clenbuterol was beneficial in combination with ERT or gene therapy (2–4); however, clinical translation of this strategy will depend on the response of humans to clenbuterol. One critical factor will be the effective serum concentration of β₂-agonists. Limited data are available, but the effective concentration (EC₅₀) for clenbuterol was lower for humans and other higher mammals than for rodents. For example, the EC₅₀ for clenbuterol for the relaxation of rat smooth muscle was ∼10-fold higher than the EC₅₀ for equine or human smooth muscle (26–28). Moreover, the standard dosages of clenbuterol used clinically have increased muscle strength or increased muscle mass in several studies. Patients with chronic heart failure developed increased lean muscle mass after taking clenbuterol at 80 μg/d for 12 wk (29). Patients with amyotrophic lateral sclerosis (ALS) were stronger as determined by the myometer score for upper and lower extremities after taking clenbuterol, 60 μg/d, for 3 mo (30). A small group of patients with Duchenne muscular dystrophy developed increased power and volume of well-preserved muscle following 18 mo of taking clenbuterol, 30–40 μg/d (31). Therefore, standard dosages of clenbuterol might be beneficial in Pompe disease by increasing muscle strength, possibly through biochemical correction and improvement in autophagy (Table 1).

Our data also raise the intriguing possibility of further improving the treatment by using an mTOR inhibitor, such as rapamycin, in conjunction with clenbuterol, as well as gene therapy. Previous work has shown a beneficial effect by gene therapy in combination with rapamycin (25), as mTORC1 inhibition reduces glycogen synthesis. In addition, mTORC1 inhibition increases the activity of TFEB (32), a transcription factor known as the master regulator of autophagosomal and lysosomal biosynthesis (33). More intriguing, Spampanato et al. (34) have recently shown that TFEB overexpression leads to direct benefit in Pompe's disease models, not only by improving lysosomal function, but also through a new form of exocytosis in which autolysosomal glycogen is excreted from the cell. Although long-term rapamycin therapy can cause immunosuppression (35) and insulin resistance (36), these potential side effects may be acceptable, given the severe phenotype of Pompe's disease. The development of new mTOR inhibitors also may reduce some of these adverse effects.

Overall, our results suggest that the therapeutic role of clenbuterol can be expanded from the concept of enhanced receptor-mediated uptake of GAA to include enhanced trafficking of muscle-expressed GAA during gene therapy. Furthermore, its effects on AKT signaling indicate the potential benefit from a second mechanism independent of CI-MPR in limb muscle. Ultimately, clenbuterol could be beneficial in patients with late-onset Pompe disease for whom ERT is not an option, because it could reduce glycogen storage in limb muscles and result in positive effects on ambulation.