MAB1900 Sigma-AldrichAnti-Integrin β1D Antibody, CT cytoplasmic domain, clone 2B1

Recent studies indicate that matrix metalloproteinases (MMPs) and critical linkage proteins in the extracellular matrix (ECM) regulate skeletal muscle mass, although the effects of resistance training (RT) on protein expression and activity are unclear. Thus, the purpose of the present study was to investigate the effects of RT on MMP activity and expression of ECM-related proteins. Ten male Sprague-Dawley rats were randomly assigned to 1 bout (1B) or 18 bouts (18B) of electrical stimulation. The right gastrocnemius muscle was isometrically contracted via percutaneous electrical stimulation (five sets of 5 sec stimulation × five contractions/set with 5 sec interval between contractions and 3 min rest between sets) once (1B) or every other day for 5 weeks (18B). The left leg served as a control. Activity of MMP-2 and MMP-9, determined via gelatin zymography, was increased (P less than 0.05) immediately after 1B. However, MMP activation was not evident following 18B. No changes in collagen IV, laminin α2, α7-integrin, or ILK protein expression were detected immediately following 1B or 18B. However, β1-integrin protein expression was significantly increased (P less than 0.05) with 18B. Our results suggest that resistance exercise activates MMPs during the initial phase of RT but this response is attenuated with continuation of RT.

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a severe and fatal muscle-wasting disease with no cure. MDC1A patients and the dy(W-/-) mouse model exhibit severe muscle weakness, demyelinating neuropathy, failed muscle regeneration and premature death. We have recently shown that laminin-111, a form of laminin found in embryonic skeletal muscle, can substitute for the loss of laminin-211/221 and prevent muscle disease progression in the dy(W-/-) mouse model. What is unclear from these studies is whether laminin-111 can restore failed regeneration to laminin-α2-deficient muscle. To investigate the potential of laminin-111 protein therapy to improve muscle regeneration, laminin-111 or phosphate-buffered saline-treated laminin-α2-deficient muscle was damaged with cardiotoxin and muscle regeneration quantified. Our results show laminin-111 treatment promoted an increase in myofiber size and number, and an increased expression of α7β1 integrin, Pax7, myogenin and embryonic myosin heavy chain, indicating a restoration of the muscle regenerative program. Together, our results show laminin-111 restores muscle regeneration to laminin-α2-deficient muscle and further supports laminin-111 protein as a therapy for the treatment of MDC1A.

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.

Sarcospan (SSPN) is a core component of the major adhesion complexes in skeletal muscle, the dystrophin- and utrophin (Utr)-glycoprotein complexes (DGC and UGC). We performed a rigorous analysis of SSPN-null mice and discovered that loss of SSPN decreased DGC and UGC abundance, leading to impaired laminin-binding activity and susceptibility to eccentric contraction-induced injury in skeletal muscle. We show that loss of SSPN increased levels of α7β1 integrin. To genetically test whether integrin compensates for the loss of DGC and UGC function in SSPN-nulls, we generated mice lacking both SSPN and α7 integrin (DKO, double knockout). Muscle regeneration, sarcolemma integrity and fibrosis were exacerbated in DKO mice and were remarkably similar to muscle from Duchenne muscular dystrophy (DMD) patients, suggesting that secondary loss of integrin contributes significantly to pathogenesis. Expression of the DGC and UGC, laminin binding and Akt signaling were negatively impacted in DKO muscle, resulting in severely diminished specific force properties. We demonstrate that SSPN is a necessary component of dystrophin and Utr function and that SSPN modulation of integrin signaling is required for extracellular matrix attachment and muscle force development.

Filamin C is an actin-crosslinking protein that is specifically expressed in cardiac and skeletal muscles. Although mutations in the filamin C gene cause human myopathy with cardiac involvement, the function of filamin C in vivo is not yet fully understood. Here we report a medaka mutant, zacro (zac), that displayed an enlarged heart, caused by rupture of the myocardiac wall, and progressive skeletal muscle degeneration in late embryonic stages. We identified zac to be a homozygous nonsense mutation in the filamin C (flnc) gene. The medaka filamin C protein was found to be localized at myotendinous junctions, sarcolemma, and Z-disks in skeletal muscle, and at intercalated disks in the heart. zac embryos showed prominent myofibrillar degeneration at myotendinous junctions, detachment of myofibrils from sarcolemma and intercalated disks, and focal Z-disk destruction. Importantly, the expression of γ-actin, which we observed to have a strong subcellular localization at myotendinous junctions, was specifically reduced in zac mutant myotomes. Inhibition of muscle contraction by anesthesia alleviated muscle degeneration in the zac mutant. These results suggest that filamin C plays an indispensable role in the maintenance of the structural integrity of cardiac and skeletal muscles for support against mechanical stress.

Utrophin is normally confined to the neuromuscular junction (NMJ) in adult muscle and partially compensates for the loss of dystrophin in mdx mice. We show that Akt signaling and utrophin levels were diminished in sarcospan (SSPN)-deficient muscle. By creating several transgenic and knockout mice, we demonstrate that SSPN regulates Akt signaling to control utrophin expression. SSPN determined α-dystroglycan (α-DG) glycosylation by affecting levels of the NMJ-specific glycosyltransferase Galgt2. After cardiotoxin (CTX) injury, regenerating myofibers express utrophin and Galgt2-modified α-DG around the sarcolemma. SSPN-null mice displayed delayed differentiation after CTX injury caused by loss of utrophin and Akt signaling. Treatment of SSPN-null mice with viral Akt increased utrophin and restored muscle repair after injury, revealing an important role for the SSPN-Akt-utrophin signaling axis in regeneration. SSPN improved cell surface expression of utrophin by increasing transportation of utrophin and DG from endoplasmic reticulum/Golgi membranes. Our experiments reveal functions of utrophin in regeneration and new pathways that regulate utrophin expression at the cell surface.

Glioblastomas are characterized by an intense local invasiveness that limits surgical resection. One mechanism by which glioma cells enforce their migration into brain tissue is reorganization of tumour associated extracellular matrix (ECM). Collagen XVI is a minor component of connective tissues. However, in glioblastoma tissue it is dramatically upregulated compared to the ECM of normal cortex. The aim of this study is to delineate tumour cell invasion and underlying mechanisms involving collagen XVI by using a siRNA mediated collagen XVI knockdown model in U87MG human glioblastoma cells. Knockdown of collagen XVI resulted in decreased invasiveness in Boyden chamber assays, and in a reduction of focal adhesion contact numbers per cell. Gene expression was upregulated for protocadherin 18 and downregulated for kindlin-1 and -2. Proliferation was not affected while flow cytometric analysis demonstrated reduced β1-integrin activation in collagen XVI knockdown cells. We suggest that in glioblastoma tissue collagen XVI may impair the cell-cell interaction in favour of enhancement of invasion. The modification of the β1-integrin activation pattern through collagen XVI might be a molecular mechanism to further augment the invasive phenotype of glioma cells. Elucidating the underlying mechanisms of glioma cell invasion promoted by collagen XVI may provide novel cancer therapeutic approaches in neurooncology.

Duchenne muscular dystrophy, the most common form of childhood muscular dystrophy, is caused by X-linked inherited mutations in the dystrophin gene. Dystrophin deficiencies result in the loss of the dystrophin-glycoprotein complex at the plasma membrane, which leads to structural instability and muscle degeneration. Previously, we induced muscle-specific overexpression of Akt, a regulator of cellular metabolism and survival, in mdx mice at pre-necrotic (less than 3.5 weeks) ages and demonstrated upregulation of the utrophin-glycoprotein complex and protection against contractile-induced stress. Here, we found that delaying exogenous Akt treatment of mdx mice after the onset of peak pathology (greater than 6 weeks) similarly increased the abundance of compensatory adhesion complexes at the extrasynaptic sarcolemma. Akt introduction after onset of pathology reverses the mdx histopathological measures, including decreases in blood serum albumin infiltration. Akt also improves muscle function in mdx mice as demonstrated through in vivo grip strength tests and in vitro contraction measurements of the extensor digitorum longus muscle. To further explore the significance of Akt in myofiber regeneration, we injured wild-type muscle with cardiotoxin and found that Akt induced a faster regenerative response relative to controls at equivalent time points. We demonstrate that Akt signaling pathways counteract mdx pathogenesis by enhancing endogenous compensatory mechanisms. These findings provide a rationale for investigating the therapeutic activation of the Akt pathway to counteract muscle wasting.

Duchenne muscular dystrophy is caused by dystrophin mutations that lead to structural instability of the sarcolemma membrane, myofiber degeneration/regeneration and progressive muscle wasting. Here we show that myogenic Akt signaling in mouse models of dystrophy promotes increased expression of utrophin, which replaces the function of dystrophin thereby preventing sarcolemma damage and muscle wasting. In contrast to previous suggestions that increased Akt in dystrophy was a secondary consequence of pathology, our findings demonstrate a pivotal role for this signaling pathway such that modulation of Akt can significantly affect disease outcome by amplification of existing, physiological compensatory mechanisms.

In order to investigate a possible relationship between integrin degradation and water-holding capacity (WHC), integrin was quantified using western blot, and water mobility and distribution was measured by proton NMR T(2) relaxometry at 24h postmortem in pork (n=30) with a large variation in WHC (drip loss varying from 2.8% to 11.3%). Regression analyses revealed correlation coefficients of r=-0.32 (P=0.08) and r=0.40 (P=0.03) for the correlations between the content of integrin determined by western blot analysis and WHC determined as either drip loss or by NMR, respectively. Water mobility and distribution was also measured in 18 meat samples upon 7 days of aging, which revealed a correlation (r=0.54) between integrin content determined by western blot analysis 24h postmortem and the mobility of the myofibrillar water (T(21) relaxation time) at day 7. In contrast, no correlation could be established between integrin content 24h postmortem and WHC at day 7 determined by NMR as the amount of extramyofibrillar water (T(22) population) (r=-0.01). In conclusion, both visualisation by CLSM and quantification of integrin by western blot analyses of suggested that a strong link between integrin degradation and WHC in pork is questionable, whereas integrin degradation seems to have impact on the succeeding development in the mobility of the myofibrillar water.