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. 2013 Dec 17;22(2):390–396. doi: 10.1038/mt.2013.263

Morpholino Treatment Improves Muscle Function and Pathology of Pitx1 Transgenic Mice

Sachchida Nand Pandey 1, Yi-Chien Lee 1,2, Toshifumi Yokota 3, Yi-Wen Chen 1,4,*
PMCID: PMC3916049  PMID: 24232919

Abstract

Paired-like homeodomain transcription factor 1 (PITX1) was proposed to be part of the disease mechanisms of facioscapulohumeral muscular dystrophy (FSHD). We generated a tet-repressible muscle-specific Pitx1 transgenic mouse model which develops phenotypes of muscular dystrophy after the PITX1 expression is induced. In this study, we attempted to block the translation of PITX1 protein using morpholinos. Three groups of the transgenic mice received intravenous injections of phosphorodiamidate morpholino oligomers (PMO) (100 mg/kg), octaguanidinium dendrimer-conjugated morpholino (vivo-morpholino) (10 mg/kg), or phosphate-buffered saline (PBS) after the PITX1 expression was induced. Immunoblotting data showed that PITX1 expression in the triceps and quadriceps was significantly reduced 70% and 63% by the vivo-morpholino treatment, respectively. Muscle pathology of the mice treated with the vivo-morpholino was improved by showing 44% fewer angular-shaped atrophic myofibers. Muscle function determined by grip strength was significantly improved by the vivo-morpholino treatment. The study showed that systemic delivery of the vivo-morpholino reduced the PITX1 expression and improved the muscle phenotypes. Aberrant expression of DUX4 from the last unit of the D4Z4 array has been proposed to be the cause of FSHD. The findings of this study suggest that the same principle may be applied to suppress the aberrantly expressed DUX4 in FSHD.

Introduction

Facioscapulohumeral muscular dystrophy (FSHD) is a dominant muscular dystrophy with a prevalence of 1:20,000, which is the third common muscular dystrophy.1 The majority of patients with FSHD carry a deletion of the D4Z4 repeats in the 4q35 subtelomeric region. Unaffected individuals have 11–150 copies of the D4Z4 repeats while patients have only 1 to 10 copies.2,3,4,5 Each D4Z4 repeat contains a double homeobox protein 4 (DUX4), which is cytotoxic when ectopically expressed in cells based on in vivo and ex vivo studies.6,7,8,9 Recent studies showed that a combination of two genomic features is required to cause FSHD. First, the contraction of the D4Z4 repeats, which leads to DNA hypomethylation of the D4Z4 region and allows DUX4 mRNA to be transcribed.10 Second, an intact polyadenylation signal in the region distal to the last repeat of D4Z4, which allows DUX4 transcripts from the last D4Z4 repeat to be polyadenylated thus stable for protein translation. The combination leads to the aberrant expression of DUX4 and downstream molecular changes involved in FSHD.11,12

Our previous study showed that paired-like homeodomain transcription factor 1 (PITX1) was specifically upregulated in the muscle of patients with FSHD and may mediate DUX4-induced myopathy in FSHD via the regulatory pathway involving DUX4, PITX1, and p53.11,13,14 To study the effect of PITX1 overexpression in skeletal muscles, we generated a tet-repressible muscle-specific Pitx1 transgenic mouse model in which expression of PITX1 in skeletal muscles can be controlled by oral administration of doxycycline.13 We showed that overexpression of PITX1 in skeletal muscles lead to phenotypes of muscular dystrophy, which share similarity to FSHD, including muscle atrophy, muscle weakness, and angular-shaped atrophic myofibers.13 In this study, we used the Pitx1 transgenic model to investigate the feasibility of suppressing a pathogenic protein in vivo using morpholinos.

Phosphorodiamidate morpholino oligomers (PMOs) are short chains of ~25 morpholino subunits. Each subunit is comprised of a nucleic acid base, a morpholino ring, and a nonionic phosphorodiamidate intersubunit linkage.15 PMOs have been used to block translation initiation by targeting the 5′ UTR of a gene; to modify pre-mRNA splicing in the nuclei by targeting splice junctions or splice regulatory sites; and to inhibit miRNA processing and activity by targeting mature miRNA or pri-miRNA.16 Several advantages of using morpholinos to suppress gene expression include:

  1. Morpholinos have higher binding affinity than equivalent DNA-based antisense oligos, which allows them to invade RNA secondary structure therefore increases the probability of designing effective oligos.

  2. With their requirement for greater complementarity with their target RNAs, morpholinos cause less off-target expression modulation.

  3. The morpholinos are stable in cells and do not induce immune responses.15,17,18

In this study, we used two types of morpholinos, PMO and octaguanidinium dendrimer-conjugated morpholino (vivo-morpholino). The octaguanidium dendrimer conjugation improves delivery of the morpholino by increasing its ability of penetrating the cell membrane.19 The findings will help us to evaluate the strategy as a potential therapeutic mean for FSHD.

Results

In this study, we evaluated the PMO and vivo-morpholino targeting the same 25 bases DNA sequence located at the translation start site of the Pitx1 mRNA transcript. To improve the delivery efficiency, the vivo-morpholino was conjugated with a triazine core scaffold featuring eight guanidinium head groups to facilitate cell penetration.19 Considering the higher delivery efficiency and potential toxicity of the vivo-morpholino, a lower dosage (10 mg/kg) was used, in comparison to the dosage of the PMO (100 mg/kg). Both morpholinos were delivered by intravenous injections weekly for 6 weeks. In this study, we would like to determine whether the morpholino treatments can suppress PITX1 protein expression and prevent or slow down the disease progression. Therefore, we chose to start the morpholinos injections at the same time when the Pitx1 overexpression was induced in the Pitx1 transgenic mice. Expression of the PITX1 protein, muscle function of the Pitx1 transgenic mice and muscle pathology were evaluated after the treatments.

The vivo-morpholino against Pitx1 suppressed PITX1 protein expression in the Pitx1 transgenic mice

To determine whether the overexpressed PITX1 protein was knocked down by the morpholinos, immunoblotting was conducted using proteins from the triceps and quadriceps of the mice treated with either the PMO or vivo-morpholino. The Pitx1 transgenic mice treated with phosphate-buffered saline (PBS) were used to determine the PITX1 expression level without treatment, while the baseline expression of PITX1 in normal muscles was determined using muscles from littermates that carry only one of the two transgenes. Our results showed that 70% of the PITX1 expression was knocked down by the vivo-morpholino treatment (P < 0.05) in the triceps compared to the PBS treatment (Figure 1a), while the PMO did not significantly affect the PITX1 expression. Similarly, the vivo-morpholino treatment reduced 63% of the PITX1 expression in the quadriceps (P < 0.05) (Figure 1b), while the PMO treatment had no effect on PITX1 expression. Immunohistochemistry was performed to visualize localization of the PITX1 proteins in myonuclei (Figure 2). The results showed no visible PITX1 staining in control muscles while a large number of PITX1-positive nuclei were observed in muscle sections of the Pitx1 transgenic mice treated with PBS. The vivo-morpholino treatment reduced the number of PITX1-positive nuclei, while PMO treatment did not show obvious effect. The results of immunoblotting and immunohistochemistry showed that the vivo-morpholino treatment, but not the PMO treatment, reduced the expression of PITX1 protein significantly.

Figure 1.

Figure 1

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Suppression of paired-like homeodomain transcription factor 1 (PITX1) protein expression in skeletal muscle by vivo-morpholino treatment. Expression levels of PITX1 protein in (a) triceps and (b) quadriceps of the Pitx1 transgenic mice and control mice. Data are presented as mean ± SEM, control mice received PBS (n = 5); Pitx1 transgenic mice received PBS (n = 4); Pitx1 transgenic mice received PMO (n = 3); Pitx1 transgenic mice received VM (n = 5). “*” indicates P < 0.05 and “**” indicates P < 0.01. C, control mice; DT, Pitx1 transgenic; PBS, phosphate-buffered saline; PMO, phosphorodiamidate morpholino oligomers; VM, vivo-morpholino.

Figure 2.

Figure 2

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The paired-like homeodomain transcription factor 1 (PITX1) protein is reduced by vivo-morpholino treatment in the quadricep muscle. (a) PITX1 expression is not detected in control mice. Nuclear localization of the PITX1 in muscles of the Pitx1 transgenic mice (TRE-Pitx1/mCK-tTA) treated with (b) PBS or (c) PMO. (d) Vivo-morpholino treatment reduced the number of Pitx1 positive nuclei. H&E staining of control mice showed (e) healthy muscle fibers. Pitx1 transgenic (TRE-Pitx1/mCK-tTA) mice treated with (f) PBS and (g) PMO showed large number of angular shape fibers. (h) Pitx1 transgenic (TRE-Pitx1/mCK-tTA) mouse treated with the vivo-morpholino showed fewer angular fibers in the muscles. Arrows indicate myofibers with central nuclei and asterisks indicate angular atrophic myofibers. Scale bar: 100 µm.

The PMO and vivo-morpholino used in this study were designed to block protein translation instead of inducing RNA degradation. To determine whether the vivo-morpholino treatment also affect the expression of Pitx1 at the mRNA level, we performed real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and showed that the mRNA level of the Pitx1 in mice treated with vivo-morpholino was not affected by the treatment (Figure 3).

Figure 3.

Figure 3

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The vivo-morpholino treatments did not affect Pitx1 mRNA level in the skeletal muscles of the Pitx1 transgenic mice. Data are presented as mean ± SEM, Pitx1 transgenic mice received PBS (n = 4); Pitx1 transgenic mice received VM (n = 5). PBS, phosphate-buffered saline; VM, vivo-morpholino.

The vivo-morpholino treatment reduced atrophic myofibers in the Pitx1 transgenic mice

To examine the pathological improvement of the Pitx1 transgenic (TRE-Pitx1/mCK-tTA) mice treated with the PMO and vivo-morpholino, histology of the triceps and quadriceps of the mice was examined and percentages of angular-shaped myofibers were determined (Figure 2). The control samples showed no angular-shaped atrophic fibers, while a large number of angular-shaped atrophic fibers (16%) and some fibers with central nuclei were observed in muscle sections of the Pitx1 transgenic mice treated with PBS. Mice treated with PMO did not show improvement of pathology (21%). However, the mice treated with the vivo-morpholino showed a 44% (P < 0.05) reduction of angular-shaped atrophic fibers (9%) (Figure 4). In addition, the PITX1 expression was highly correlated (r2 = 0.84) with the percentage of angular fibers in each sample. The results showed that vivo-morpholino treatment reduced PITX1 expression and the number of atrophic myofibers.

Figure 4.

Figure 4

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The Pitx1 transgenic mice (TRE-Pitx1/mCK-tTA) treated with the vivo-morpholino showed significantly lower percentage of angular fibers than the control. Data are presented as mean ± SEM, control mice received PBS (n = 5); Pitx1 transgenic mice received PBS (n = 4); Pitx1 transgenic mice received PMO (n = 3); Pitx1 transgenic mice received VM (n = 5). “*” indicates P < 0.05 and “**” indicates P < 0.01. PBS, phosphate-buffered saline; PMO, phosphorodiamidate morpholino oligomers; VM, vivo-morpholino.

The vivo-morpholino treatment improved grip strength of the Pitx1 transgenic mice without overt toxicity

To determine whether the muscle strength was improved by the PMO and vivo-morpholino treatments, grip strength of both the forelimb and hindlimb of the Pitx1 transgenic mice were performed. The Pitx1 transgenic mice treated with PBS were significantly weaker than the control mice as previously reported.13 The muscle strength of the Pitx1 transgenic mice was significantly improved by the vivo-morpholino treatment (P < 0.05). We did not observe significant improvement of grip strength in the mice treated with PMO (Figure 5a,b).

Figure 5.

Figure 5

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Grip strength of the Pitx1 transgenic mice (TRE-Pitx1/mCK-tTA) treated with the vivo-morpholino was significantly improved. Grip strength test showed increase of (a) forelimb and (b) hindlimb grip strength in the Pitx1 transgenic mice treated with the vivo-morpholino. Data are presented as mean ± SEM, control mice received PBS (n = 5); Pitx1 transgenic mice received PBS (n = 4); Pitx1 transgenic mice received PMO (n = 3); Pitx1 transgenic mice received vivo-morpholino (n = 5). “*” indicates P < 0.05. PBS, phosphate-buffered saline; PMO, phosphorodiamidate morpholino oligomers; VM, vivo-morpholino.

To determine whether the vivo-morpholino treatment caused hepatic or renal toxicity in the mice, blood serum enzymes were examined (Supplementary Figure S1). Alanine transaminase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), and bilirubin were tested for liver toxicity. Blood urea nitrogen and creatine were tested for kidney toxicity. Creatine kinase was tested for skeletal and cardiac muscle damage. These enzymes did not show significant changes in response to the PMO nor the vivo-morpholino treatments. We concluded that there was no obvious toxicity detected in mice treated with either type of morpholinos.

Discussion

Several approaches have been developed to suppress pathogenic proteins by sequestering or degrading the mRNA, including shRNA, miRNA, siRNA, and antisense oligonucleotides.20,21,22,23 Before DUX4 was considered as the causative gene of FSHD, several candidate genes in the D4Z4 region were investigated for their involvement in FSHD, including FSHD region gene 1, FSHD region gene 2, and adenine nucleotide translocator 1.24 shRNA and miRNA against FSHD region gene 1 were designed and delivered using adeno-associated viral vector and were shown to successfully reduce the FSHD region gene 1 expression in vivo.21,22 Later, miRNA against DUX4 was delivered by adeno-associated viral vector into mice ectopically expressing DUX4 and was able to rescue the pathology induced by DUX4 expression.23 In this study, we demonstrated that a modified antisense oligonucleotide, morpholino, can be used to successfully suppress the pathogenic PITX1 protein and rescue the disease phenotypes, which offers an additional strategy for treating FSHD. Using this approach, we can take advantages of using morpholino molecules, including higher affinity, more specificity, less off-target effects, and low immunogenicity.

Morpholinos are a short chain of morpholino rings. PMO have longer half-life comparing to oligonucleotides due to a substitution of the five-membered-ring sugar of DNA with six-membered-morpholino rings. In addition, negatively charged phosphate linkages are replaced by nonionic phosphorodiamidate linkages. The structural changes make PMO more stable.25 In addition, unlike other antisense approaches, morpholinos have high binding affinity with less off-target effects.15,17,18 Using morpholino to block the translation of specific mRNA is widely used to study functions of genes.26,27,28,29 For example, vivo-morpholinos were used to suppress dopamine receptor 1 (DRD1), vesicular monoamine transporter 2 (VMAT2), and glucose transporter 4 (GLUT4) in skeletal muscle to study their functions in a mouse model of voluntary physical activity.30 However, less work has been done by using this approach for treating diseases. Few cases were reported, such as using modified morpholinos to suppress expression of viral proteins in mouse models.31,32,33 Our approach showed that vivo-morpholinos can be delivered intravenously and block protein translation in vivo, which provides an additional strategy for treating diseases caused by aberrant expression of pathogenic proteins.

In addition to blocking translation, the steric blocking mechanism of morpholinos has also been used to alter splicing of pre-mRNAs. The application of morpholinos in blocking splicing has been extensively studied in animal models of Duchenne muscular dystrophy (DMD). PMO against exon 23 donor site was reported to restore dystrophin expression in muscles of the mdx mice.34,35,36 PMO has also been used to skip exon 6 and 8 in a dog model of DMD (CXMD) and exon 51 in human DMD to restore the dystrophin reading frame.37,38 The leakiness of the dystrophin-deficient myofibers was believed to contribute to the success of the treatment. Since PMO is not charged and has been shown difficult to be delivered into intact skeletal muscles, the leakiness of the myofibers allows the PMO to be delivered into the cells.39 In this study, the failure of delivering sufficient amount of PMO into the muscles of the Pitx1 mice supports the view that effective delivery of PMO relies on leaky membrane of the myofibers. In this study, we believed that the PMO could not be delivered into the muscle fibers efficiently to suppress PITX1 expression. Normal level of creatine kinase, an enzyme leaking out of muscles when damaged, in serum of all mice tested, further supported that the sarcolemmal membrane of the Pitx1 transgenic mice was intact. On the contrary, vivo-morpholino treatment showed efficacy in suppressing the PITX1 expression and rescued disease phenotypes in the Pitx1 transgenic mice although the vivo-morpholino was delivered at a lower dose. The result demonstrated that vivo-morpholino against a pathogenic protein effectively entered intact myofibers and suppressed the target gene.

The dosages of PMO and vivo-morpholino used in this study were determined based on previous studies in animal models.35,38,40,41 Previously, PMO at 100 mg/kg administered to mdx mice showed efficacy without toxicity.40 The modification of the vivo-morpholino significantly increases cellular uptake therefore much lower concentration is needed for delivery. The modification also increases toxicity of the vivo-morpholino. The toxicity of vivo-morpholino and PMO has been extensively studied in mdx mice.35,36,41,42 Vivo-morpholino and PMO showed no toxicity in mdx after treatment for as long as 5 and 12 weeks, respectively, determined by histological examination and serum enzyme levels of liver and kidney.35,42 mdx mice treated with up to 1.5 g/kg PMO for 6 months did not show signs of liver and kidney toxicity.36 Systemic delivery of vivo-morpholino in mdx mice (6 mg/kg) biweekly for 5 weeks did not show liver or kidney toxicity.35 A recent study showed that 5 weekly injections of vivo-morpholino (12 mg/kg) did not cause liver or kidney toxicity.41 While studies suggest that vivo-morpholino is generally well tolerated, PPMO, a PMO conjugated with arginine-riched peptides, targeting human exon 50 (AVI-5038) was reported to cause mild tubular degeneration in the kidney of cynomolgus monkeys which received weekly injection (9 mg/kg) for 4 weeks.39 Although the cause of toxicity was not known, one of the hypotheses was that the cationic property of PPMO caused off-target effects which lead to renal toxicity. We delivered the vivo-morpholino at a dose of 10 mg/kg weekly for 6 weeks and showed no detectable liver and kidney toxicity by blood serum enzyme readings, which agreed with the findings of previous studies.

Efficacious downregulation of a physiologically expressed target gene to lower than its normal expression level can potentially be toxic to the cells, therefore careful evaluation of the dosages and knock-down efficacy is necessary when the approach is used. In addition, genes that are not normally expressed in vital organs may be more suitable targets for this approach. DUX4 which expresses in germline cells but not other healthy tissues seems to be such a target.43 Generating a DUX4 mouse model has been challenging due to its cytotoxic nature when ectopically expressed. Previously, we reported that PITX1 was a direct transcriptional target of DUX4 and specifically upregulated in muscles of patients with FSHD. In our conditional muscle-specific Pitx1 transgenic model, overexpressing PITX1 lead to muscular dystrophy phenotypes including muscle atrophy, muscle weakness, and angular shaped atrophic myofibers, which was also reported in FSHD.13,44,45,46 In this study, we showed that protein expression level of PITX1 was positively correlated with the percentage of angular-shaped atrophic fibers in the muscle. The finding suggested that the upregulation of PITX1 in FSHD may contribute to this specific pathology. In addition, we demonstrated that the pathogenic protein, PITX1, can be suppressed by the vivo-morpholino without affecting the mRNA level. The findings provide a proof of principle of using vivo-morpholino to suppress a pathogenic protein in vivo. Considering DUX4 transcripts from the last D4Z4 repeat in the patients were stabilized by polyadenylation for translation11,12 and the aberrant expression of the DUX4 protein is believed to be the cause of FSHD, morpholinos that can block protein translation of the DUX4 may potentially be used to treat FSHD.

Materials and Methods

The tet-repressible muscle-specific Pitx1 transgenic mouse. All animal procedures were approved by the institutional animal care and use committee (IACUC) at the Children's National Medical Center in Washington, DC. Two transgenic mouse lines (TRE-Pitx1 and mCK-tTA) were crossbred in order to generate the tet-repressible muscle-specific Pitx1 transgenic mice (TRE-Pitx1/mCK-tTA) as previously reported.13 The TRE-Pitx1 mouse line carries a construct containing the Pitx1 gene, driven by a tetracycline response element (TRE). The mCK-tTA mouse line carries a construct containing the tTA gene, driven by a muscle specific creatine kinase promoter (mCK). The female mice received drinking water with doxycycline (200 μg/ml with 5.0% sucrose) in order to suppress the Pitx1 transgene expression in the pups in utero. Under these conditions, the TRE-Pitx1/mCK-tTA double transgenic mice were born at the expected Mendelian ratio. The pups continued to receive doxycycline through the mother's milk. After weaning, all mice were maintained on water treated with doxycycline until they were entered into an experimental regiment. For all experiments, littermates carrying a single transgene (Pitx1 or tTA) were used as controls. Male patients with FSHD generally are more affected than the female patients, including age of onset and muscle weakness.47,48 Therefore, we studied only male mice to reduce experimental variations in this study.

To identify mice carrying one or two of the transgenes (TRE-Pitx1 and mCK-tTA), we collected a piece of tail tip and isolated DNA for genotyping. The following primers were used for PCR amplification. For Pitx1, forward 5′-TGGAGGCCACGTTCC AAAG-3′ and reverse 5′-GTTCTTGAACCAGACCCGCAC-3′ were used. For tTA, forward 5′-acagcgcattagagctgctt-3′ and reverse 5′-Ccccttctaaagggcaaaag-3′ were used.

Real-time qRT-PCR. Real-time qRT-PCR was performed as previously described.49 Briefly, total RNA samples were isolated from triceps of mice treated with the vivo-morpholino and PBS. Total RNA (1 µg) was reverse transcribed using oligo (dT) primer (0.5 µg/µl). Primer sequences used for mouse Pitx1 gene were (forward) 5′-TGGAGGCCACGTTCCAAAG-3′ and (reverse) 5′-GTTCTTGAACCAGACCCGCAC-3′ for ribosomal RNA 18S: 5′-TAGCCTTCGCCATCACTGCCATTA-3′ and (reverse) 5′-AACCTGGCTGTACTTCCCATCCTT-3′. ABI 7900HT (Applied Biosystems, Foster City, CA) was used for mRNA quantification. Briefly, cDNA was added to SYBR Green PCR Master Mix (Applied Biosystems). Samples were amplified in triplicate by using the following thermal cycling conditions: 95 °C for 10 minutes, followed by 40 cycles of amplification at 95 °C for 15 seconds, followed by 60 °C for 1 minute to allow for denaturing and annealing extension. All primers were tested for nonspecific amplicons and primer dimers by visualizing PCR products on 2% agarose gels before performing qRT-PCR. The ΔΔCT method was used to determine expression values of Pitx1 relative to ribosomal RNA 18S as described previously.49 T-test was used to determine statistical significance (P < 0.05).

Morpholino injections. Morpholinos have been shown to effectively block translation when sequences near the translation start site are targeted (−80 to +20 where +1 is the A of the AUG translation start site).50 The morpholino oligos (5′-TCATGCCTCCCTTGAAGGCGTCCAT-3′) used in this study were designed and synthesize by Gene Tools, LLC (Philomath, OR), which targets the first 25 bases from the start codon (+1 to +25 where +1 is the A of the AUG) of the Pitx1 (NM_011097.2). In general, Gene Tools designs morpholino molecules by analyzing the first 25 bases of coding sequence then slide the 25-base window upstream until a 25-base target is found that satisfies the general requirements for a optimal morpholino oligo (i.e., 40–60% GC content without significant self-complementarity or stretches of 4 or more Gs) (http://www.gene-tools.com/node/18). In PMO, negatively charged phosphate linkages are replaced by noncharged backbone, which causes difficulty for delivering the molecule into the cells. To overcome the problem, vivo-morpholino was conjugated with a triazine core scaffold featuring eight guanidinium head groups, which increases delivery efficiency.19 In this study, both the PMO and vivo-morpholino were dissolved in PBS (100 µl) and delivered directly by intravenous injections. The doses of the PMO and vivo-morpholino used were 100 and 10 mg per kg of body mass, respectively. They were delivered by intravenous injections weekly for 6 weeks.

The oral doxycycline was discontinued to induce Pitx1 transgene expression when the mice were 8 weeks old. At the same time, total eight male TRE-Pitx1/mCK-tTA mice received either PMO (n = 3) or vivo-morpholino (n = 5) treatment weekly for 6 weeks. A group of TRE-Pitx1/mCK-tTA male littermates (n = 4) received weekly PBS injections as the control of the experiment. Male littermates carrying only one transgene (TRE-Pitx1 or mCK-tTA) received PBS injections (n = 5) for 6 weeks to provide a baseline value (normal expression level) of the PITX1. One week after the last injection, the mice were euthanized, and skeletal muscles including quadriceps and triceps were collected for protein and RNA assays.

Muscle functional test. The grip strength test (Grip Strength Meter; Columbus Instruments, Columbus, OH) was performed at week 6 after the Pitx1 transgene induction to determine the effects of treatment on muscle function. The test was performed according to previously published procedure.13 Briefly, the grip strength is tested by holding the mouse over the grid of the instrument to allow the mouse grip the steel bars. Then the mouse was pulled away from the force meter until it released the grid. The meter recorded the maximum force that was applied. The mouse was then tested once a day for five consecutive days; five measurements were recorded for each test. The largest measurements from each of the five tests were averaged and normalized to body weight (kg).

Histological and immunohistochemical assays. Immediately after muscle dissection, the whole muscle was snap-frozen in isopentane cooled with liquid nitrogen, then stored at −80 °C until sectioning. A Leica CM 1900 cryostat (Walldorf, Baden-Wurttenberg, Germany) was used to prepare cryosections for all the following histological analyses. Hematoxylin and eosin staining was conducted using 8 µm sections. Five random nonoverlapping fields of the tissue sections were imaged using Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan), RT slider camera (Diagnostic Instrument, Sterling Height, MI), and SPOT advanced software. Angular fibers and total fibers of each image were counted using Image J software (http://rsb.info.nih.gov/ij) in a blind manner to minimize bias. The counts of five field of each sample were averaged for statistical analysis using Student's t-test.

Immunohistochemistry was performed as previously described.13 Briefly, 4 µm cryosections were prepared. The sections were fixed using cold acetone, blocked using 10% horse serum and then incubated with a primary antibody at 4 °C overnight. The primary antibody used for PITX1 is a rabbit polyclonal antibody. After primary antibody incubation, the sections were washed three times with PBS. The ABC kit (Vectors Lab, Burlingame, CA) was then used to detect the PITX1. The sections were stained with nuclear dye Hoechst 33258 (1:2,500 dilution) to visualize nuclei. The muscle sections were imaged using Nikon Eclipse E800 microscope, RT slider camera, and SPOT advanced software.

Immunoblotting. Muscle sections were disrupted in lysis buffer (50 mmol/l Tris-HCl, pH 7.5, 150 mmol/l NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% sodium dodecyl sulfate, and protease inhibitor) with a hand sonicator for 10 seconds at room temperature. Protein concentration was determined by DC protein assay (Bio-Rad, Hercules, CA), and 50 µg of protein was loaded to 4–12% Bis-Tris NuPage Mini Gels (Invitrogen, Carlsbad, CA) then transferred to Hybond nitrocellulose membranes (Amersham Biosciences, Little Chalfont, UK). After blocking, the membrane was incubated with rabbit polyclonal anti-PITX1 antibody (1:2,500)11 followed by secondary antibodies conjugated with horseradish peroxidase (Amersham Biosciences). Chemiluminescent substrate (Pierce, Rockford, IL) was used to visualize the target protein on blue light autorad film (BioExpress, Kaysville, UT). Detection of loading control, vinculin, was similarly performed using a mouse monoclonal primary antibody against vinculin (1:20,000 dilution; Abcam, Cambridge, MA). Band density was measured using a GS 800 Calibrated Densitometer (Bio-Rad).

Measurement of serum enzymes and other component. Blood samples were collected immediately after the mice were euthanized. Serum was separated and stored at −80 °C. The levels of alkaline phosphatase (U/dl), alanine transaminase (U/dl), blood urea nitrogen (mg/dl), creatine kinase (KU/l), creatine (mg/dl), γ-glutamyltransferase (U/l), and total bilirubin (mg/l) were determined by Comparative Clinical Pathology Services, LLC (Columbia, MO).

SUPPLEMENTARY MATERIAL Figure S1. The morpholino treatments did not affect the levels of blood serum enzymes of the Pitx1 transgenic mice (TRE-Pitx1/mCK-tTA)

Acknowledgments

This study is supported by NIH/NIAMS1R01AR052027 and R01AR052027-03S1. Y-W.C. is partially supported by NIH/NICHD1R24HD050846 and DOD W81XWH-10-1-0659.

Supplementary Material

Supplementary Information
Click here for additional data file. (27.9KB, pdf)

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