Our results demonstrate the improvement of islet graft morphology and function by co-transplantation with MSCs. This improvement is attributable, at least in part, to the promotion of graft revascularization mediated by MSCs.

SUMMARY MyoD is a key regulator of skeletal myogenesis that directs contractile protein synthesis, but whether this transcription factor also regulates skeletal muscle metabolism has not been explored. In a genome-wide ChIP-seq analysis of skeletal muscle cells, we unexpectedly observed that MyoD directly binds to numerous metabolic genes, including those associated with mitochondrial biogenesis, fatty acid oxidation, and the electron transport chain. Results in cultured cells and adult skeletal muscle confirmed that MyoD regulates oxidative metabolism through multiple transcriptional targets including PGC-1β, a master regulator of mitochondrial biogenesis. We find that PGC-1β expression is cooperatively regulated by MyoD and the alternative NF-κB signaling pathway. Bioinformatics evidence suggests that this cooperativity between MyoD and NF-κB extends to other metabolic genes as well. Together, these data identify MyoD as a regulator of the metabolic capacity of mature skeletal muscle to ensure that sufficient energy is available to support muscle contraction.

Background The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury of donor organs. The islet isolation process, from pancreas procurement through islet collection, may activate p38MAPK leading to cytokine release and islet damage. This damage may be prevented by treating pancreata with a p38MAPK inhibitor (p38IH) prior to cold preservation. Methods Pancreata removed from Beagle dogs were infused with UW solution containing either the p38IH, SB203580 and Pefabloc (n=6) or vehicle (DMSO and Pefabloc) alone (n=7), through the pancreatic duct and preserved using the two-layer method. After 20–22 hours, islets were isolated and 3000 IEQ/kg were autotransplanted into the corresponding dog to monitor glucose metabolism. Results P38IH-treated pancreata yielded significantly more islets than control pancreata (IEQ/g: 2,134±297 vs. 1,477±145 IEQ/g or 65,012±9,385 vs. 45,700±5,103 IEQ/Pancreas; p<0.05). Apoptotic β-cell percentages assessed by LSC were lower in p38IH-treated than the controls (44±9.4% vs. 61.6±4.8%, p<0.05). TNF-α expression assessed by RT-PCR was significantly lower in the p38IH-treated group than controls. All dogs (3000 IEQ/kg) transplanted with p38IH-treated islets (n=5) became euglycemic vs. 4 of 5 dogs that received untreated islets. Plasma C-peptide levels following glucagon challenge were higher in animals receiving p38IH-treated islets (n=5) vs. untreated islets (n=4) (0.40±0.78 vs. 0.21±0.05 ng/mL, p<0.05). Conclusions Infusion of pancreata with UW solution containing p38IH through the duct prior to peservation suppresses cytokine release, prevents β cell apoptosis, and improves islet yield significantly with no adverse effect on islet function following transplantation. P38IH treatment of human pancreata may improve islet yield for use in clinical transplantation.

SUMMARY Skeletal muscle growth immediately following birth is a critical for proper body posture and locomotion. However, compared to embryogenesis and adulthood, the processes regulating the maturation of neonatal muscles is considerably less clear. Studies in the 1960s predicted that neonatal muscle growth results from nuclear accretion of myoblasts preferentially at the tips of myofibers. Remarkably, little information has been added since then to resolve how myoblasts migrate to the ends of fibers. Here, we provide insight to this process by revealing a unique NF-κB-dependent communication between NG2+ interstitial cells and myoblasts. NF-κB in NG2+ cells promotes myoblast migration to the tips of myofibers through cell-cell contact. This occurs through expression of ephrinA5 from NG2+ cells, which we further deduce is an NF-κB target gene. Together, results suggest that NF-κB plays an important role in the development of newborn muscles to ensure proper myoblast migration for fiber growth.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function.

Objectives We investigated whether the recovery of cultured human islets is improved through the addition of a p38α-selective mitogen activated protein kinase inhibitor, SD-282, to clinically used serum-free culture medium. Methods Immediately after isolation, islets were cultured for 24 hours in medium alone (control) or medium containing DMSO, 0.1 μM or 0.3 μM SD-282. Cytokine expression, apoptotic β cell percentage, and islet function were assessed post-culture. Results Expression of p38 and phosphorylated p38 in islets increased during culture. IL-6 mRNA expression in cultured islets, as well as IL-6, IL-8 and GM-CSF released into the medium were significantly reduced by adding SD-282. The apoptotic β cell percentage was significantly lower in islets cultured with 0.1 μM SD-282, but not 0.3 μM as compared to the control. Stimulation indices measured in vitro were higher, but without significance (p=0.06), and function of transplanted islets in diabetic NODscid mice was also better in 0.1 μM SD-282 group as compared to control. Conclusions Better islet function was obtained by adding 0.1 μM SD-282 to the serum-free culture medium. This improvement was associated with suppression of cytokine production and prevention of β cell apoptosis. However, this beneficial effect was diminished at a higher concentration.

Mitochondrial function is a key component of skeletal muscle health, and its dysfunction has been associated with a wide variety of diseases. Microplate-based respirometry measures aerobic respiration of live cells through extracellular changes in oxygen concentration. Here, we describe a methodology to measure aerobic respiration of intact murine skeletal muscle tissue. The tissues are not cultured, permeabilized, or enzymatically dissociated to single fibers, so there is minimal experimental manipulation affecting the samples prior to acquiring measurements.

Objectives Allogeneic mesenchymal stem cells (MSCs) and bone marrow cells (BMCs) were co-transplanted in NOD mice following none myeloablative preconditioning and the development of chimerism, insulitis, diabetes, and graft versus host disease (GVHD) were monitored. Methods Eight-weeks-old female NOD mice were injected intravenously with 2×107 BMCs and 5×105 MSCs from C57BL/6 mice following treatment with 2 intraperitoneal injections of anti-CD3 antibody (days −7 and −4), and 3Gy total body irradiation (day −1). Thereafter, blood glucose and chimerism were monitored on peripheral blood samples. Results Stable mixed chimerism (3->90% of donor phenotype) was induced in 63.2% of BMCs-MSCs-(n=19) and 45.0% of BMCs alone recipients (n=20, p=0.256). Insulitis was prevented and euglycemia persisted for >18 weeks in 89.5% of BMCs-MSCs recipients including those with <3% chimerism and 55% of BM alone recipients (p<0.05). In controls, 9.1% of mice receiving preconditioning treatment alone (n=11) and 16.7% of preconditioned mice receiving only MSCs (n=12) were non-diabetic. GVHD was not detected in all mice. Conclusion Co-injection of MSCs and BMCs increased the success rate in inducing chimerism and preventing insulitis and overt diabetes with no incidence of GVHD. Results also indicated that even micro-chimerism with <3% donor cells is sufficient for blocking autoimmunity.