Attenuation of the unfolded protein response and endoplasmic reticulum stress after mechanical unloading in dilated cardiomyopathy.

Abnormal intracellular calcium (Ca(2+)) handling can trigger endoplasmic reticulum (ER) stress, leading to activation of the unfolded protein response (UPR) in an attempt to prevent cell death. Mechanical unloading with a left ventricular assist device (LVAD) relieves pressure-volume overload and promotes reverse remodeling of the failing myocardium. We hypothesized that mechanical unloading would alter the UPR in patients with advanced heart failure (HF). UPR was analyzed in paired myocardial tissue from 10 patients with dilated cardiomyopathy obtained during LVAD implantation and explantation. Samples from healthy hearts served as controls. Markers of UPR [binding immunoglobulin protein (BiP), phosphorylated (P-) eukaryotic initiation factor (eIF2α), and X-box binding protein (XBP1)] were significantly increased in HF, whereas LVAD support significantly decreased BiP, P-eIF2α, and XBP1s levels. Apoptosis as reflected by C/EBP homologous protein and DNA damage were also significantly reduced after LVAD support. Improvement in left ventricular dimensions positively correlated with P-eIF2α/eIF2α and apoptosis level recovery. Furthermore, significant dysregulation of calcium-handling proteins [P-ryanodine receptor, Ca(2+) storing protein calsequestrin, Na(+)-Ca(2+) exchanger, sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA2a), ER chaperone protein calreticulin] was normalized after LVAD support. Reduced ER Ca(2+) content as a causative mechanism for UPR was confirmed using AC16 cells treated with a calcium ionophore (A23187) and SERCA2a inhibitor (thapsigargin). UPR activation and apoptosis are reduced after mechanical unloading, which may be mediated by the improvement of Ca(2+) handling in patients with advanced HF. These changes may impact the potential for myocardial recovery.

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.

Leupeptin-based inhibitors do not improve the mdx phenotype.

Calpain activation has been implicated in the disease pathology of Duchenne muscular dystrophy. Inhibition of calpain has been proposed as a promising therapeutic target, which could lessen the protein degradation and prevent progressive fibrosis. At the same time, there are conflicting reports as to whether elevation of calpastatin, an endogenous calpain inhibitor, alters pathology. We compared the effects of pharmacological calpain inhibition in the mdx mouse using leupeptin and a proprietary compound (C101) that linked the inhibitory portion of leupeptin to carnitine (to increase uptake into muscle). Administration of C101 for 4 wk did not improve muscle histology, function, or serum creatine kinase levels in mdx mice. Mdx mice injected daily with leupeptin (36 mg/kg) for 6 mo also failed to show improved muscle function, histology, or creatine kinase levels. Biochemical analysis revealed that leupeptin administration caused an increase in m-calpain autolysis and proteasome activity, yet calpastatin levels were similar between treated and untreated mdx mice. These data demonstrate that pharmacological inhibition of calpain is not a promising intervention for the treatment of Duchenne muscular dystrophy due to the ability of skeletal muscle to counter calpain inhibitors by increasing multiple degradative pathways.

Activin IIB receptor blockade attenuates dystrophic pathology in a mouse model of Duchenne muscular dystrophy.

Modulation of transforming growth factor-ß (TGF-ß) signaling to promote muscle growth holds tremendous promise for the muscular dystrophies and other disorders involving the loss of functional muscle mass. Previous studies have focused on the TGF-ß family member myostatin and demonstrated that inhibition of myostatin leads to muscle growth in normal and dystrophic mice. We describe a unique method of systemic inhibition of activin IIB receptor signaling via adeno-associated virus (AAV)-mediated gene transfer of a soluble form of the extracellular domain of the activin IIB receptor to the liver. Treatment of mdx mice with activin IIB receptor blockade led to increased skeletal muscle mass, increased force production in the extensor digitorum longus (EDL), and reduced serum creatine kinase. No effect on heart mass or function was observed. Our results indicate that activin IIB receptor blockade represents a novel and effective therapeutic strategy for the muscular dystrophies.

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 adeno-associated 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 dystrophin-deficient 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 on striated muscle.

Anterior segment growth and peripheral neural pathways in chick.

PURPOSE: To learn if peripheral nerve pathways are necessary for corneal expansion and anterior segment growth under a 12-hr light:dark cycle or for the inhibition of corneal expansion under constant light rearing. METHODS: Recently hatched White Leghorn chicks under anesthesia received unilateral ciliary ganglionectomy (CGx), cranial cervical ganglionectomy (Sx), or section of the ophthalmic nerve (TGx), along with sham-operated and/or never-operated control cohorts. Chicks were reared postoperatively under either a 12-hr light:dark cycle or under constant light. After 2 weeks and with the chicks under anesthesia, corneal radii of curvature and diameters were obtained with a photokeratoscope, refractometry and A-scan ultrasonography were performed, and the axial and equatorial dimensions of enucleated eyes were measured with digital calipers. Corneal areas were calculated from corneal curvatures and diameters. RESULTS: Despite the rich peripheral innervation to the eye, the selective denervations performed here exerted remarkably limited effects on corneal expansion and anterior segment development in chicks reared under either lighting condition. Ophthalmic nerve section did reverse in large part the inhibition of equatorial expansion of the vitreous chamber occurring under constant light rearing. CONCLUSIONS: The ciliary, sympathetic, or ophthalmic peripheral nerve pathways to the eye are not required either for corneal expansion and anterior segment development under a 12-hr light:dark cycle or for the inhibition of corneal expansion under constant light rearing. The ocular sensory innervation may be a means for regulating vitreous cavity shape.

Local patterns of image degradation differentially affect refraction and eye shape in chick.

PURPOSE: To evaluate visual blur as a mechanism for modulating eye shape. METHODS: Chicks wore a unilateral full goggle or one of several goggles modified with apertures. After 2 weeks, eyes were measured with refractometry, ultrasound, and calipers, and three retinal regions were assayed for dopamine and DOPAC (3,4-dihydroxyphenylacetic acid). RESULTS: Goggled eyes were diffusely enlarged or enlarged predominantly along the axial dimension, depending on the goggle. Myopia developed under goggle types inducing primarily axial growth and under some of the goggles inducing diffuse eye expansion. Enlarged eyes remained emmetropic beneath other goggles that caused diffuse eye expansion. Reductions in retinal dopamine and DOPAC were proportional to the eye growth and refraction effects. CONCLUSIONS: Localized image degradation can cause myopia with predominantly axial expansion, myopia with more diffuse vitreous chamber expansion, or eye expansion without myopia. Robust expansion of the equatorial diameter alone was not observed. The associated alterations in retinal dopamine metabolism are consistent with a hypothesized role of dopaminergic amacrine cells in the visual regulation of eye growth. Besides refraction and overall size, visual blur can affect eye shape; but the goggle responses do not correspond to a simple summation of blur signals across the retina. Therefore, other mechanisms seemingly are needed to account for the full range of refractions and ocular shapes seen in chicks and, by analogy, in humans.

Stimulation of an alpha1-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 alphabetamADP 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 alphabetamADP, 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 alpha1 adrenergic receptor. The block of enzyme activity by norepinephrine was rapid, occurring within 1 min, and was similar at both 4 and 37 degrees 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 alpha-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.

Degradation of extracellular ATP by the retinal pigment epithelium.

Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and betagamma-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca(2+). Both responses were inhibited by thapsigargin and P2Y(1) receptor inhibitor MRS 2179. Message for both P2Y(1) and P2Y(12) receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca(2+) signaling in the RPE.

Emmetropisation under continuous but non-constant light in chicks.

It has been suggested that ambient lighting at night influences eye growth and might play a causal role in human myopia. To test this hypothesis, we reared newly hatched chicks under 12 hr light-dark or light-dim cycles with a light phase intensity of 1500 microW/cm(2) and variable dim phase intensities between 0.01 and 500 microW/cm(2). Other chicks were reared under constant light conditions with intensities between 1 and 1500 microW/cm(2). After three weeks, the chicks were examined by refractometry, ultrasound and caliper measurements of enucleated eyes. To relate ocular parameters with a retinal neurotransmitter likely involved in eye growth control, retinal and vitreal levels of dopamine and its principal metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were measured by high performance liquid chromatography with electrochemical detection in the light, dark and dim phases. Diurnal fluctuations in axial length and choroidal thickness also were measured twice daily by partial coherence interferometry (PCI) in chicks under light-dark and the two brightest light-dim conditions. The eyes of chicks reared under most light-dim conditions had refractions and ocular dimensions comparable to those reared under light-dark conditions. At dim phase light intensities of 10 microW/cm(2) and above, the day-night changes in retinal dopamine metabolism were not observed. The daily fluctuations of axial length and choroidal thickness were altered with rearing under the two brightest dim light intensities, compared to the light-dark condition. Rearing under constant light with intensities ranging between 1 and 1500 microW/cm(2) produced a shallow anterior chamber and other eye alterations previously described for constant light rearing even though rearing under continuous light that fluctuated between these same intensities generally permitted normal eye growth. Thus, continuous but fluctuating light exerts different developmental effects on the eye than constant non-fluctuating light. Light-dim rearing may be more relevant to daily human light exposures than other laboratory lighting conditions and may provide an opportunity to study developmental interactions of visual quality (e.g. blur, defocus, etc.) and features of the light-dark cycle under conditions that perturb daily rhythms in dopamine metabolism and ocular dimensions. Such studies also could provide mechanistic insights into whether and how daily rhythms in retinal dopamine metabolism, axial length or choroidal thickness modulate refractive development.

GABA, experimental myopia, and ocular growth in chick.

PURPOSE: To learn whether gamma-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.