A novel GFP reporter mouse reveals Mustn1 expression in adult regenerating skeletal muscle, activated satellite cells and differentiating myoblasts.

AIM: Mustn1 has been implicated in myofusion as well as skeletal muscle growth and repair; however, the exact role and spatio-temporal expression of Mustn1 have yet to be fully defined. METHODS: Transgenic mice were generated with a 1512-bp sequence of the Mustn1 promoter directing the expression of GFP (Mustn1(PRO) -GFP). These mice were used to investigate the spatio-temporal expression of Mustn1(PRO) -GFP during skeletal muscle development and adult skeletal muscle repair, as well as various phases of the satellite cell lifespan (i.e. quiescence, activation, proliferation, differentiation). RESULTS: Mustn1(PRO) -GFP expression was observed within somites at embryonic day 12 and developing skeletal muscles at embryonic day 15 and 18. While uninjured adult tibialis anterior muscle displayed no detectable Mustn1(PRO) -GFP expression, cardiotoxin injury robustly elevated Mustn1(PRO) -GFP expression at 3 days post-injury with decreasing levels observed at 5 days and minimal, focal expression seen at 10 days. The expression of Mustn1(PRO) -GFP at 3 days post-injury consistently overlaid with MyoD although the strongest expression of Mustn1(PRO) -GFP was noted in newly formed myotubes that were expressing minimal levels of MyoD. By 5 days post-injury, Mustn1(PRO) -GFP overlaid in all myotubes expressing myogenin although cells were present expressing Mustn1(PRO) -GFP alone. The expression patterns of Mustn1(PRO) -GFP in regenerating muscle preceded the expression of desmin throughout the regenerative time course consistent with Mustn1 being upstream of this myogenic protein. Further, quiescent satellite cells located on freshly isolated, single myofibers rarely expressed Mustn1(PRO) -GFP, but within 24 h of isolation, all activated satellite cells expressed Mustn1(PRO) -GFP. Expression of Mustn1(PRO) -GFP in primary myoblasts diminished with prolonged time in proliferation media. However, in response to serum withdrawal, the expression of Mustn1(PRO) -GFP increased during myofusion (day 2) followed by declining expression thereafter. CONCLUSION: Mustn1(PRO) -GFP is expressed in activated satellite cells and myoblasts but continued time in proliferation media diminished Mustn1(PRO) -GFP expression. However, myoblasts exposed to serum withdrawal increased Mustn1(PRO) -GFP expression consistent with its demonstrated role in myofusion. The in vivo expression pattern of Mustn1 observed in regenerating and developing skeletal muscle is consistent with its presence in satellite cells and its critical role in myofusion.

Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil.

The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC. Taken together, these data demonstrate that the human neutrophil is able to catabolize 1-acyl-2-acetyl-GPC in a manner both quantitatively and qualitatively different from that of platelet-activating factor. The differential catabolism may regulate the relative proportion of these two bioactive phospholipids in the neutrophil.