Date Published: August 13, 2019
Publisher: Public Library of Science
Author(s): Adam B. Schroer, Junaith S. Mohamed, Melinda D. Willard, Vincent Setola, Emily Oestreich, David P. Siderovski, Antonio Musaro.
Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair.
Regulators of G protein Signaling (RGS proteins) are intracellular GTPase-accelerating proteins (GAPs) that attenuate the G protein-dependent signals that cells receive from their external environment [1, 2]. The RGS protein family member RGS12 is unique in interacting with multiple signaling pathways, including those associated with growth and survival cues from receptor tyrosine kinases (RTKs) and mitogen-activated protein kinases (MAPKs), G protein-coupled receptors (GPCRs), and Ras GTPases [3–9]. It was previously reported that skeletal muscles of developing mouse embryos express RGS12 , suggesting a potential role for this unique RGS family member in the skeletal muscle developmental process; however, little has since been reported regarding potential function(s) of RGS12 in the signaling pathways underlying the myogenesis program active during both development and muscle repair. With regards to the latter, adult skeletal muscle has a remarkable regenerative capacity, largely made possible by a specialized population of stem cells—satellite cells—found in a characteristic niche between the sarcolemma and basal lamina of myofibers [11–13]. Myogenesis requires strictly regulated signaling events involving the activation (and subsequent proliferation) of quiescent satellite cells, expression of muscle-specific genes, and differentiation into new muscle fibers during repair or fusion into existing fibers during growth . Muscle regeneration begins with satellite cell activation by alterations to their niche and by factors released during injury, resulting in MYF5 and MYOD induction and several cycles of proliferation. Although some activated satellite cells remain in their niche and return to quiescence as a reservoir, other daughter cells migrate along the sarcolemma then differentiate and fuse with either damaged fibers or with other myoblasts to form repaired or de novo myofibers, respectively. This process is characterized by PAX7 down-regulation and up-regulation of muscle-specific genes (e.g., MRF4, myogenin) .
The results presented herein, from both cell culture and mouse model studies, illuminate a previously underappreciated role of RGS12 in muscle precursor-cell function. Our original report , that RGS12 expression is temporospatially regulated in the developing mouse embryo, with strong expression in developing skeletal muscle, was highly suggestive evidence that fueled these present studies. We have since found Rgs12 expressed in mouse adult TA muscle, and its expression increased following injury and in dystrophic muscle, further suggesting a role for RGS12 in muscle regeneration. The dynamic regulation of Rgs12 mRNA expression seen in muscle precursor cells during myogenesis is consistent with a potential role for the encoded G protein- and MAPK-scaffold in coordinating myogenic signals: Rgs12 is expressed in satellite cells and proliferating myoblasts, and reduced as myoblasts undergo differentiation and fusion into myotubes. Moreover, RGS12 protein is seen to bind to endosomally enriched phosphoinositides in vitro and to be localized to early endosomes in cultured myoblasts, suggesting that its scaffolding properties  may localize specific molecular interactions (i.e., Ras/Raf/MEK/ERK signaling cascade components) to certain cytosolic sub-regions within the activated satellite cell itself, the trans-amplifying cell destined for differentiation, or both.