Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Normal and Dystrophic Mice

Background: Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies. Methodology/Principal Findings: Here we describe a unique method of myostatin inhibition utilizing recombinant adenoassociated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophindeficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose–dependent manner. Conclusions/Significance: Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition o

Stimulation of an α1-adrenergic receptor downregulates ecto-5′ nucleotidase activity on the apical membrane of RPE cells

The purines ATP and adenosine play an important role in the communication between the photoreceptors and the retinal pigment epithelium (RPE). While the RPE is known to release ATP into subretinal space, the source of extracellular adenosine is unclear. In other tissues, ecto-nucleotidases mediate the consecutive dephosphorylation of ATP to AMP, and AMP is converted to adenosine by ecto-5′ nucleotidase (CD73). This study identifies ecto-5′ nucleotidase on RPE cells and investigates modulation of enzyme activity. The RPE was the most active site of 5′AMP dephosphorylation in the posterior rat eye. The ecto-5′ nucleotidase inhibitor αβmADP prevented the production adenosine by the apical membrane of the bovine RPE. Cultured human ARPE-19 cells expressed mRNA and protein for ecto-5′ nucleotidase. The production of phosphate from 5′AMP by ARPE-19 cells was inhibited by αβmADP, but the ecto-alkaline phosphatase inhibitor levamisole had no effect. Degradation of 5′AMP was blocked by norepinephrine, epinephrine and phenylephrine, with inhibition by antagonists prazosin and corynanthine implicating the α1 adrenergic receptor. The block of enzyme activity by norepinephrine was rapid, occurring within 1 min, and was similar at both 4 and 37°C, consistent with cleavage of the enzyme from its GPI anchor. HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath. In the apical face of the bovine-RPE eyecup, norepinephrine reduced the production of phosphate from 5′AMP, suggesting that both receptor and enzyme face sub-retinal space. In conclusion, RPE cells express ecto-5′ nucleotidase, with activity on the apical membrane, and stimulation of α-1 adrenergic receptors downregulates activity. As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time

Rescue of Dystrophic Skeletal Muscle by PGC-1α Involves a Fast to Slow Fiber Type Shift in the mdx Mouse

Increased utrophin expression is known to reduce pathology in dystrophin-deficient skeletal muscles. Transgenic over-expression of PGC-1α has been shown to increase levels of utrophin mRNA and improve the histology of mdx muscles. Other reports have shown that PGC-1α signaling can lead to increased oxidative capacity and a fast to slow fiber type shift. Given that it has been shown that slow fibers produce and maintain more utrophin than fast skeletal muscle fibers, we hypothesized that over-expression of PGC-1α in post-natal mdx mice would increase utrophin levels via a fiber type shift, resulting in more slow, oxidative fibers that are also more resistant to contraction-induced damage. To test this hypothesis, neonatal mdx mice were injected with recombinant adeno-associated virus (AAV) driving expression of PGC-1α. PGC-1α over-expression resulted in increased utrophin and type I myosin heavy chain expression as well as elevated mitochondrial protein expression. Muscles were shown to be more resistant to contraction-induced damage and more fatigue resistant. Sirt-1 was increased while p38 activation and NRF-1 were reduced in PGC-1α over-expressing muscle when compared to control. We also evaluated if the use a pharmacological PGC-1α pathway activator, resveratrol, could drive the same physiological changes. Resveratrol administration (100 mg/kg/day) resulted in improved fatigue resistance, but did not achieve significant increases in utrophin expression. These data suggest that the PGC-1α pathway is a potential target for therapeutic intervention in dystrophic skeletal muscle

Rescue of Dystrophic Skeletal Muscle by PGC-1α Involves a Fast to Slow Fiber Type Shift in the mdx Mouse

Increased utrophin expression is known to reduce pathology in dystrophin-deficient skeletal muscles. Transgenic over-expression of PGC-1α has been shown to increase levels of utrophin mRNA and improve the histology of mdx muscles. Other reports have shown that PGC-1α signaling can lead to increased oxidative capacity and a fast to slow fiber type shift. Given that it has been shown that slow fibers produce and maintain more utrophin than fast skeletal muscle fibers, we hypothesized that over-expression of PGC-1α in post-natal mdx mice would increase utrophin levels via a fiber type shift, resulting in more slow, oxidative fibers that are also more resistant to contraction-induced damage. To test this hypothesis, neonatal mdx mice were injected with recombinant adeno-associated virus (AAV) driving expression of PGC-1α. PGC-1α over-expression resulted in increased utrophin and type I myosin heavy chain expression as well as elevated mitochondrial protein expression. Muscles were shown to be more resistant to contraction-induced damage and more fatigue resistant. Sirt-1 was increased while p38 activation and NRF-1 were reduced in PGC-1α over-expressing muscle when compared to control. We also evaluated if the use a pharmacological PGC-1α pathway activator, resveratrol, could drive the same physiological changes. Resveratrol administration (100 mg/kg/day) resulted in improved fatigue resistance, but did not achieve significant increases in utrophin expression. These data suggest that the PGC-1α pathway is a potential target for therapeutic intervention in dystrophic skeletal muscle.This article is published as Selsby JT, Morine KJ, Pendrak K, Barton ER, Sweeney HL (2012) Rescue of Dystrophic Skeletal Muscle by PGC-1α Involves a Fast to Slow Fiber Type Shift in the mdx Mouse. PLoS ONE 7(1): e30063. doi: 10.1371/journal.pone.0030063.</p

experimental myopia, and ocular growth in chick. Invest Ophthalmol Vis Sci.

PURPOSE. To learn whether ␥-aminobutyric acid (GABA) participates in retinal mechanisms that influence refractive development. METHODS. White leghorn chicks, some of which wore a unilateral goggle to induce myopia, received daily intravitreal injections of agonists or antagonists to the major GABA receptor subtypes. Eyes were studied with refractometry, ultrasound, and calipers. Retinas of other chicks wearing unilateral goggles were assayed for GABA content. RESULTS. Antagonists to GABA A or GABA A0r (formerly known as GABA C ) receptors inhibited form-deprivation myopia. GABA A antagonists showed greater inhibition of myopic growth in the equatorial than the axial dimension. A GABA A0r antagonist displayed parallel inhibition in the axial and equatorial dimensions. A GABA A0r agonist but not GABA A agonists altered the myopic refraction of goggled eyes. GABA B receptor antagonists, more so than an agonist, also slowed development of myopia, inhibiting axial growth more effectively than equatorial expansion of goggled eyes. When administered to nongoggled eyes, GABA A or GABA A0r agonists or antagonists also altered eye growth, chiefly stimulating it. Only a GABA A agonist induced a myopic refraction. Several of these agents stimulated eye growth in the axial, but not the equatorial, dimension. Retinal GABA content was slightly reduced in goggled eyes. CONCLUSIONS. GABA A , GABA A0r , and GABA B receptors modulate eye growth and refractive development. The anatomic effects of these drugs reinforce the notion that eye shape and not just eye size is regulated. A retinal site of action is consistent with the known ocular localizations of GABA and its receptors and with the altered retinal biochemistry in form-deprived eyes

PGC-1α induced changes in muscle function.

<p>Following four (n = 7) or six (sol n = 6; EDL n = 13) weeks of PGC-1α over-expression muscle function in the soleus and EDL was altered.</p><p>*indicates significantly different from corresponding control. Wk – week, sol – soleus, EDL – extensor digitorum longus, CSA – cross sectional area.</p

PGC-1α over-expression reduces disease-related muscle injury.

<p>10× micrographs of six week old soleus muscles following H and E staining (A and B). (C) The total areas of necrotic, H&E negative, or regenerating cells were quantified and is expressed as a percent of the total soleus area. In addition, laminin was detected with a fluorescently labeled antibody (not shown) in order to determine central nucleation. (D) PGC-1α caused a reduction in central nucleation. N = 5/group; * indicates p<0.05.</p

Protein expression following 6 weeks of PGC-1α over-expression.

<p>Slow genes are shown in black (utrophin – A, slow myosin heavy chain – B) oxidative genes are shown in gray (cytochrome C – C, uncoupling protein-1 – D, complex IV subunit IV – E, myoglobin – F, Hsp 60 – G), pathway genes are shown in white (Sirt-1 – H, nuclear respiratory factor -1 – I, p38 – J, phospho-p38 – K), and controls have diagonal lines (Actin – L, Troponin – M, Spectrin – N). Relative change compared to control limbs (n = 6/group) (O). * indicates p<0.05 compared to control limbs.</p