Prostaglandin E2 (PGE2), a prostanoid synthesized from arachidonic acid via the cyclooxygenase pathway, is a modulator of physiological responses including inflammation, fever, and muscle regeneration. Several patents have been filed that are related to PGE2, one of them being directly related to skeletal muscles. In this report, we first summarize the key patents describing inventions for the utilization of PGE2 for either diagnostic or therapeutic purposes, including skeletal muscle. In the second part of our work we present new and exciting data that demonstrates that PGE2 accelerates skeletal muscle myogenic differentiation. Our discovery resulted from our recent and novel concept of bone-muscle crosstalk. Bone and muscle are anatomically intimate endocrine organs and we aimed to determine whether this anatomical intimacy also translates into a biochemical communication from bone cells to muscle cells at the in vitro level. The effects of MLO-Y4 osteocyte-like cell conditioned medium (CM) and three osteocyte-secreted factors, PGE2, sclerostin and monocyte chemotactic protein (MCP-3), on C2C12 myogenic differentiation were evaluated using morphological analyses, a customized 96-PCR gene array, and measurements of intracellular calcium levels. MLO-Y4 CM and PGE2, but not sclerostin and MCP-3, induced acceleration of myogenesis of C2C12 myoblasts that was linked with significant modifications in intracellular calcium homeostasis. This finding should further stimulate the pursuit of new patents to explore the use of PGE2 and the new concept of bone-muscle crosstalk for the development and application of inventions designed to treat muscle diseases characterized by enhanced muscle wasting, such as sarcopenia.

We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO-Y4 osteocyte-like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ~25%. MLO-Y4 and primary osteocyte CM (1–10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Since WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO-Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5ng/mL mirrored the effects of both primary osteocyte and MLO-Y4 CM by inducing nuclear translocation of β-catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO-Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100ng/mL) inhibited both the effects of MLO-Y4 CM and WNT3a on C2C12 cell differentiation. RT-PCR array results supported the activation of the Wnt/β-catenin pathway by MLO-Y4 CM and WNT3a. These results were confirmed by qPCR showing up-regulation of myogenic markers and two Wnt/β-catenin downstream genes, Numb and Flh1. We postulated that MLO-Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO-Y4 CM and WNT3a increased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca2+ signaling and the Wnt/β-Catenin pathway.

We have recently reported that a novel muscle-specific inositide phosphatase (MIP/MTMR14) plays a critical role in [Ca2+]i homeostasis through dephosphorylation of sn-1-stearoyl-2-arachidonoyl phosphatidylinositol (3,5) bisphosphate (PI(3,5)P2). Loss of function mutations in MIP have been identified in human centronuclear myopathy. We developed a MIP knockout (MIPKO) animal model and found that MIPKO mice were more susceptible to exercise-induced muscle damage, a trademark of muscle functional changes in older subjects. We used wild-type (Wt) mice and MIPKO mice to elucidate the roles of MIP in muscle function during aging. We found MIP mRNA expression, MIP protein levels, and MIP phosphatase activity significantly decreased in old Wt mice. The mature MIPKO mice displayed phenotypes that closely resembled those seen in old Wt mice: i) decreased walking speed, ii) decreased treadmill activity, iii) decreased contractile force, and iv) decreased power generation, classical features of sarcopenia in rodents and humans. Defective Ca2+ homeostasis is also present in mature MIPKO and old Wt mice, suggesting a putative role of MIP in the decline of muscle function during aging. Our studies offer a new avenue for the investigation of MIP roles in skeletal muscle function and as a potential therapeutic target to treat aging sarcopenia.

Walnut orchards suffer from a blight caused by the bacteria Xanthomonas arboricola pv. juglandis. These bacteria can be infected by viral bacteriophages and this study was carried out to isolate and characterize bacteriophages from walnut orchards located throughout the South Island of New Zealand. Twenty six X. arboricola phages were isolated from three hundred and twenty six samples of plant material representing phyllosphere and rhizosphere ecosystems. The phage isolates were characterized by host-range, plaque and particle morphology, restriction digest and phylogenetic analysis and stability under various storage conditions. From capsid and tail dimensions the bacteriophages were considered to belong to the double-stranded DNA families Podoviridae and Siphoviridae. Of the twenty six bacteriophages, sixteen belonged to Podoviridae and were found both in the phyllosphere and rhizosphere. In contrast, Siphoviridae were present only in the rhizosphere isolates. Phage genome sizes ranged from 38.0 to 52.0 kb from a Hind III restriction digestion and had in common a 400 kb fragment that was identical at the DNA level. Despite the similar restriction patterns, maximum parsimony bootstrap analysis showed that the phage were members of different groups. Finally, we hypothesise that these phage might have use in a biocontrol strategy and therefore storage stability and efficacy was tested. Titres declined more than 50% over a 12-months storage period. Deep-freezing temperatures (-34°C) increased while chloroform decreased the stability.