Myogenesis in vitro involves myoblast cell cycle arrest, migration, and fusion to form multinucleated myotubes. Extracellular matrix (ECM) integrity during these processes is maintained by the opposing actions of matrix metalloproteinase (MMP) proteases and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs). Here, we report that TIMP-2, MMP-2, and MT1-MMP are differentially expressed during mouse myoblast differentiation in vitro. A specific role for TIMP-2 in myogenesis is demonstrated by altered TIMP-2(-/-) myotube formation. When differentiated in horse serum-containing medium, TIMP-2(-/-) myotubes are larger than wild-type myotubes. In contrast, when serum-free medium is used, TIMP-2(-/-) myotubes are smaller than wild-type myotubes. Regardless of culture condition, myotube size is directly correlated with MMP activity and inversely correlated with beta1 integrin expression. Treatment with recombinant TIMP-2 rescues reduced TIMP-2(-/-) myotube size and induces increased MMP-9 activation and decreased beta1 integrin expression. Treatment with either MMP-2 or MMP-9 similarly rescues reduced myotube size, but has no effect on beta1 integrin expression. These data suggest a specific regulatory relationship between TIMP-2 and beta1 integrin during myogenesis. Elucidating the role of TIMP-2 in myogenesis in vitro may lead to new therapeutic options for the use of TIMP-2 in myopathies and muscular dystrophies in vivo.
Extracellular matrix (ECM) molecules play critical roles in muscle function by participating in neuromuscular junction (NMJ) development and the establishment of stable, cytoskeleton-associated adhesions required for muscle contraction. Matrix metalloproteinases (MMPs) are neutral endopeptidases that degrade all ECM components. While the role of MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), has been investigated in many tissues, little is known about their role in muscle development and mature function. TIMP-2 -/- mice display signs of muscle weakness. Here, we report that TIMP-2 is expressed at the NMJ and its expression is greater in fast-twitch (extensor digitorum longus, EDL) than slow-twitch (soleus) muscle. EDL muscle mass is reduced in TIMP-2-/- mice without a concomitant change in fiber diameter or number. The TIMP-2-/- phenotype is not likely due to increased ECM proteolysis because net MMP activity is actually reduced in TIMP-2-/- muscle. Most strikingly, TIMP-2 colocalizes with beta1 integrin at costameres in the wild-type EDL and beta1 integrin expression is significantly reduced in TIMP-2-/- EDL. We propose that reduced beta1 integrin in fast-twitch muscle may be associated with destabilized ECM-cytoskeletal interactions required for muscle contraction in TIMP-2-/- muscle; thus, explaining the muscle weakness. Given that fast-twitch fibers are lost in muscular dystrophies and age-related sarcopenia, if TIMP-2 regulates mechanotransduction in an MMP-independent manner it opens new potential therapeutic avenues.
Understanding social relationships and organization in colonial bat species can provide valuable insight into species ecology and potentially aid in conservation efforts of rare bat species. We applied social network analysis to describe social relationships and organization in 3 colonies of Rafinesque's big-eared bats {Corynorhinus rafinesquU) roosting in bottomland hardwood forests in Kentucky. We radiotracked 48 adult big-eared bats to 64 day-roosts over 549 bat-days during the summers of 2009-2011. We measured homophily, network centralization, density, transitivity, and core-periphery structure of networks of bats sharing common roosts, and we measured degree centrality of nodes (bats or roosts) within networks. Pattems of ties within each colony were homophilous by sex (E-I index = -0.87). Males were consistently the least central nodes in bat networks. Bat network centralization ranged from 1.2% to 40% among colonies, and roost network centralization ranged from 17% to 40%. The colony exhibiting the least centralized and most dense bat network also occupied habitat with low roost availability. This roost network was highly centralized, with bats frequently aggregating at a single roost. The colony with the most centralized and least dense bat network occupied habitat with a greater availability of roosts, resulting in diffuse networks of bats and roosts. Transitivity decreased after young became volant in the colony with highest roost availability. Our findings suggest that social structure in colonies of Rafinesque's big-eared bats is affected by the sex of individuals in colonies, reproductive season, and tbe preponderance of available day-roosting habitat.
Circulating levels of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs), are altered in human obesity and may contribute to its pathology. TIMP-2 exerts MMP-dependent (MMP inhibition and pro-MMP-2 activation) and MMP-independent functions. To assess the role of TIMP-2 in a murine model of nutritionally induced obesity, weight gain in wild-type and TIMP-2 deficient [knockout (KO)] mice fed a chow or high-fat diet (HFD) was determined. The effects of diet on glucose tolerance and insulin sensitivity, as well as pancreatic β-cell and adipocyte physiology, were assessed. Chow-fed TIMP-2 KO mice of both sexes became obese but maintained relatively normal glucose tolerance and insulin sensitivity. Obesity was exacerbated on the HFD. However, HFD-fed male, but not female, TIMP-2 KO mice developed insulin resistance with reduced glucose transporter 2 and pancreatic and duodenal homeobox 1 levels, despite increased β-cell mass and hyperplasia. Thus, although β-cell mass was increased, HFD-fed male TIMP-2 KO mice develop diabetes likely due to β-cell exhaustion and failure. TIMP-2 mRNA, whose expression was greatest in sc adipose tissue, was down-regulated in HFD-fed wild-type males, but not females. Furthermore, HFD increased membrane type 1-MMP (MMP-14) expression and activity in male, but not female, sc adipose tissue. Strikingly, MMP-14 expression increased to a greater extent in TIMP-2 KO males and was associated with decreased adipocyte collagen. Taken together, these findings demonstrate a role for TIMP-2 in maintaining extracellular matrix integrity necessary for normal β-cell and adipocyte physiology and that loss of extracellular matrix integrity may underlie diabetic and obesogenic phenotypes.
Five millennia ago, the North American Beaver (Castor canadensis) was extirpated from the Southern High Plains by droughts. Beaver were found to be largely absent from the Llano Estacado, despite exhaustive search efforts. Here we report the first definitive evidence of an extant C. canadensis population recolonizing the Llano Estacado.
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