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Heterotopic ossification (HO), or the pathologic formation of bone within soft tissues, is a significant complication following severe injuries as it impairs joint motion and function leading to loss of the ability to perform activities of daily living and pain. While soft tissue injury is a prerequisite of developing HO, the exact molecular pathology leading to trauma-induced HO remains unknown. Through prior investigations aimed at identifying the causative factors of HO, it has been suggested that additional predisposing factors that favor ossification within the injured soft tissues environment are required. Considering that chondrocytes and osteoblasts initiate physiologic bone formation by depositing nanohydroxyapatite crystal into their extracellular environment, we investigated the hypothesis that deposition of nanohydroxyapatite within damaged skeletal muscle is likewise sufficient to predispose skeletal muscle to HO. Using a murine model genetically predisposed to nanohydroxyapatite deposition (ABCC6-deficient mice), we observed that following a focal muscle injury, nanohydroxyapatite was robustly deposited in a gene-dependent manner, yet resolved via macrophage-mediated regression over 28 days post injury. However, if macrophage-mediated regression was inhibited, we observed persistent nanohydroxyapatite that was sufficient to drive the formation of HO in 4/5 mice examined. Together, these results revealed a new paradigm by suggesting the persistent nanohydroxyapatite, referred to clinically as dystrophic calcification, and HO may be stages of a pathologic continuum, and not discrete events. As such, if confirmed clinically, these findings support the use of early therapeutic interventions aimed at preventing nanohydroxyapatite as a strategy to evade HO formation. Electronic supplementary material The online version of this article (10.1007/s00223-018-0502-5) contains supplementary material, which is available to authorized users.

A genome-wide analysis is given of DNA methylation in mouse embryonic stem cells in which both Gsk-3α and Gsk-3β have been genetically deleted. DNA methylation patterns are compared to those of wild-type cells. More than 75% of known imprinted loci have reduced DNA methylation in the Gsk-3–knockout cells., Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α−/−;Gsk-3β−/− ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α−/−;Gsk-3β−/− ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3–dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3–mediated signal transduction in the epigenome.

Peripheral vasoconstriction and plasma catecholamine concentrations were studied in 37 dogs after cervical disc fenestration and salivary gland excision, laparotomy for intestinal anastomoses and cystotomy, or laparotomy for repair of diaphragmatic rupture, gastrotomy, and pyloromyotomy. Meperidine (4.4 mg/kg) was administered before extubation of 12 dogs undergoing laparotomy. Heart rate, respiratory frequency, indirect blood pressure, rectal temperature, toe web temperature, and plasma concentrations of epinephrine and norepinephrine were determined before induction of anesthesia, after intubation, after extubation, at sternal recumbency, and at standing. All dogs were hypothermic during surgery. After surgery, peripheral hypothermia (large rectal-toe web temperature gradients) increased from a mean of 4.6°C after intubation to a mean of 10.4°C when the dogs initially stood. Heart and respiratory rates and blood pressures during recovery were similar to those before anesthesia. Mean plasma catecholamine concentrations were neither significantly higher during recovery than before surgery nor were they increased in any surgical group, including the dogs not treated with meperidine. After anesthesia, 15% of the epinephrine and 12% of the norepinephrine samples were more than two standard deviations above the mean of the preanesthetic concentrations of all dogs. The ratio of all dogs with an epinephrine concentration more than two standard deviations above the mean of baseline epinephrine concentrations was greater at sternal recumbency than before anesthesia and the ratio of dogs with an increased epinephrine concentration at sternal recumbency was greater in the laparotomy dogs (9 of 24) than in the cervical surgery dogs (0 of 12). Decreased peripheral blood flow during recovery from anesthesia appears to be a homeostatic attempt of conserving body heat to correct the hypothermia produced by anesthesia and peripheral vasodilation.

Studies have shown that alterations of the magnesium (Mg2+) concentration can alter the response of certain vasoactive compounds. Responses of rabbit thoracic aorta to verapamil, diltiazem, nitroglycerin and isoproterenol were examined in zero Mg2+ (0.0 M), N Mg2+ (1.2 mM), 2 N Mg2+ (2.4 mM) and 4 N Mg2+ (4.8 mM). Preconstriction was induced with norepinephrine and cumulative dose-response curves were obtained for the vasodilators. The dose-response curve for isoproterenol was shifted to the right in zero Mg2+ while there was no effect on the other vasodilators. Elevation of the Mg2+ concentration to 4.8 mM produced a shift to the left in the dose-response curves for diltiazem, nitroglycerin and isoproterenol with no effect on verapamil. Therefore, Mg2+ deficiency does not appear to affect the vasodilatory actions of the agents tested except for the beta-receptor agonist, isoproterenol. Elevated Mg2+, however, potentiated the actions of isoproterenol, nitroglycerin and diltiazem, but not verapamil.