Regeneration of Transgenic Skeletal Muscles with Altered Timing of Expression of the Basic Helix-Loop-Helix Muscle Regulatory Factor MRF4

Abstract

In regenerating muscle cells, muscle regulatory factor (MRF) 4 is normally the last of the four MRFs to be expressed. To analyze how the timing of MRF4 expression affects muscle regeneration, we compared regeneration after local freeze injury of muscles from wild-type mice with muscles from transgenic mice in which MRF4 expression was under control of an ϳ1.6-kb fragment of the myogenin promoter. Three days after injury, masseter and tibialis anterior (TA) muscles in wild-type mice expressed little or no MRF4 mRNA; whereas these muscles in transgenic mice expressed abundant MRF4 mRNA from both the transgene and the endogenous gene. Thus, MRF4 up-regulation was accelerated in transgenic compared to wild-type regenerating muscles, and expression of the transgene appeared to activate, perhaps indirectly, expression of the endogenous MRF4 gene. At 11 days after injury, regeneration, as measured by cross-sectional area and density of regenerated fibers, was significantly impaired in transgenic TA compared to wild-type TA, whereas at 19 days after injury both transgenic and TA muscle fibers had fully recovered to preinjury values. Regeneration of masseter muscles, which normally regenerate much less completely than TA muscles, was unaffected by the transgene. Thus, the timing of MRF4 up-regulation, as well as additional muscle-specific factors, can determine the progress of muscle regeneration. Regeneration of skeletal muscles after injury and formation of skeletal muscles during development are both regulated by a family of muscle-specific, basic helixloop-helix transcription factors, including muscle regulatory factor (MRF) 4, myogenin, MyoD, and Myf-5.

Platelet-Rich Plasma and Skeletal Muscle Healing: A Molecular Analysis of the Early Phases of the Regeneration Process in an Experimental Animal Model

“…We were not surprised to find no modulation of Mrf4 myogenic factor at the stages of regeneration selected in this study, since Mrf4 expression has been reported in the later stages of regeneration [31]. The same could also to be true for the myogenin protein level, where the lack of protein modulation may depend on the early observation window selected.…”

Regulatory interactions between muscle and the immune system during muscle regeneration

“…This negative effect of altered MRF-4 activation on muscle differentiation is also apparent in muscle regeneration in vivo. Overexpression of MRF4 in a transgenic mouse line caused defective muscle regeneration following injury (85). Presumably, any regulatory role played by p38 in muscle regeneration following injury can be attributable to the p38␣ isoform; genetic ablation of all other p38 isoforms had no effect on muscle fiber growth during regeneration following CTX injection (92).…”

“…IL-4 can also contribute to shifting macrophages from the cytotoxic M1 phenotype to the M2c phenotype that promotes tissue repair (30,31). However, IL-4 can also have direct effects on muscle regeneration, by promoting satellite cell activation and differentiation (42,85). Thus, M2 macrophages can play a central role in regulating regeneration in mdx dystrophy by attenuating the potentially cytolytic M1 phenotype and acting directly on satellite cells to drive their function in muscle repair.…”

Molecular and Cellular Mechanism of Muscle Regeneration

“…The elevated expression and activity of p38 late in muscle differentiation leads to increased MRF4 phosphorylation and, as a consequence, a decline in desmin and skeletal -actin expression [165]. In fact, overexpression of MRF4 in a transgenic mouse line caused defective muscle regeneration following injury [166]. Therefore, TNF--dependent signaling regulates various aspects of the muscle regenerating process (immune response, and proliferation and differentiation of satellite cells) through different downstream mediators (NF-κB, JNK, and p38) [ Figure 2].…”