The KSR2-calcineurin complex regulates STIM1-ORAI1 dynamics and store-operated calcium entry (SOCE).

Store-operated calcium entry (SOCE) is the predominant Ca(2+) entry mechanism in nonexcitable cells and controls a variety of physiological and pathological processes. Although significant progress has been made in identifying the components required for SOCE, the molecular mechanisms underlying it are elusive. The present study provides evidence for a direct involvement of kinase suppressor of Ras 2 (KSR2) in SOCE. Using lymphocytes and fibroblasts from ksr2(-/-) mice and shKSR2-depleted cells, we find that KSR2 is critical for the elevation of cytosolic Ca(2+) concentration. Specifically, our results show that although it is dispensable for Ca(2+)-store depletion, KSR2 is required for optimal calcium entry. We observe that KSR2 deficiency affects stromal interaction molecule 1 (STIM1)/ORAI1 puncta formation, which is correlated with cytoskeleton disorganization. Of interest, we find that KSR2-associated calcineurin is crucial for SOCE. Blocking calcineurin activity impairs STIM1/ORAI1 puncta-like formation and cytoskeleton organization. In addition, we observe that calcineurin activity and its role in SOCE are both KSR2 dependent.

Macrophage-secreted myogenic factors: a promising tool for greatly enhancing the proliferative capacity of myoblasts in vitro and in vivo.

In this work we set out to determine if the murine macrophage J774 cell line can be used to produce myogenic growth factors. Activated J774 macrophages were grown in serum-free conditions. The macrophage-conditioned medium (MCM) was then used to treat cultures of primary myoblasts and regenerating muscle tissue, in vitro and in vivo respectively. MCM activity in vitro was tested by analyzing the expression of muscle-specific transcription factors, in parallel with the proliferation and differentiation rates of the cells. The macrophage-secreted factors greatly enhanced the proliferative potential of both rat and human primary myoblasts and were found to be highly muscle-specific. In vivo, MCM administration markedly enhanced the regenerative processes in damaged muscles. The ability to produce large amounts of macrophage-secreted myogenic factor(s) in the absence of serum holds great promise for its biochemical characterization and successive application in therapeutic protocols, both for ex vivo gene therapy and for muscle repair.

Inhibition of fasL sustains phagocytic cells and delays myogenesis in regenerating muscle fibers.

Macrophage-muscle cell interactions are complex, and the majority is unknown. The persistence of inflammatory cells in skeletal muscle could be critical for myofiber viability. In the present paper, we show that FasL plays a role in the resolution of muscle inflammation. We analyzed inflamed muscles of normal mice treated from day 3 to day 8 with a FasL inhibitor (Fas-Ig) or with control Ig. Treated muscles were collected at 3, 5, and 10 days. The treatment with recombinant Fas-Ig protein induced a severe persistence of inflammatory cells at 5 days (115,000+/-27,838 vs. 41,661+/-6848, p<0.01) and 10 days from injury (145,500+/-40,850 vs. 5000+/-1000, p<0.001). Myofiber regeneration was highly impaired (37+/-14 vs. 252+/-28, p<0.01). Apoptosis of phagocytic cells was absent during Fas-Ig treatment (0.9+/-0.6 vs. 1300+/-150, p<0.0001), but apoptotic, mononucleated cells appeared at day 10, 2 days after the suspension of Fas-Ig administration. The time course of FasL expression during muscle inflammation, at mRNA and protein level, reveals a peak during myoblast proliferation. The peak of FasL expression coincides with the peak of apoptosis of phagocytic cells. In situ hybridization shows the co-expression of FasL and MyoD mRNA in mononucleated cells, i.e., myoblasts. Experiments on the myoblast cell culture confirmed the expression of FasL in myoblasts. The findings shown here indicate one of the pathways to control myoblast-macrophage interaction and might be relevant for the control of inflammatory cells in muscle tissue. Perhaps altering FasL expression with recombinant proteins could ameliorate inflammation in degenerative myopathies and up-regulate muscle regeneration.

Dystrophin deficient myotubes undergo apoptosis in mouse primary muscle cell culture after DNA damage.

Apoptosis has been demonstrated to occur in differentiated myocardial muscle, neonatal skeletal muscle and skeletal myoblasts in response to injury. In this report, we studied differentiated normal and dystrophin deficient murine skeletal muscle cell cultures that have been injured by a pulse of cis-platinum (2 h). Forty-eight hours after DNA damage, dystrophin positive myotubes appeared almost normal though some myoblasts showed DNA fragmentation. On the other hand, dystrophin deficient myotubes presented progressive degeneration via apoptosis detected either by TUNEL or by nuclear morphology. Degeneration of mdx muscle fibers was confirmed by counting both the number of myotubes observed by contrast phase microscopy and myonuclei viewed by immunoreaction for MyoD. A 6-fold decrease in the number of muscle cells was observed in the dystrophin-deficient cell culture compared to the parental culture (P < 0.001). Direct evidence of degenerating myotubes displaying MyoD- and TUNEL-positive nuclei was obtained. Like myoblasts, differentiated dystrophin deficient myotubes were able to degenerate via apoptosis, showing that mature dystrophin deficient cells are fragile and undergo apoptosis when subjected to a mild injury which would normally be repaired in parental cells.