GPR81 mediated metabolic reprogramming alleviates skeletal muscle aging

Introduction: Skeletal muscle aging, or sarcopenia, is characterized by age-associated loss in skeletal muscle mass and strength. One of the hallmarks of skeletal muscle senescence is fat infiltration or myosteatosis, leading to enhanced lipid accumulation and impaired fatty acid metabolism. Understanding the mechanisms leading to impaired lipid metabolism and developing therapies to reverse or prevent myosteatosis might be crucial for healthy aging of the skeletal muscle. The novel lactate receptor GPR81 has been shown to govern adipocyte fatty acid metabolism, but its function in skeletal muscle is eluded.

Materials and

Methods: To study the role of GPR81 in skeletal muscle function and aging, we knocked down GPR81 in young healthy myoblasts using a lentiviral vector (cells transduced with empty vectors served as controls; Y_Empty) and assessed hallmarks of muscle senescence. Cellular bioenergetics, especially lipid oxidation was evaluated using the seahorse analyzer and metabolic dyes such as Mitotracker, TMRM and BODIPY. We also assessed if senescence could be reversed using the GPR81 agonist 3-chloro-5-hydroxy BA (CHBA) both, in-vitro in a replicative senescence model of human myoblasts, and in-vivo using the Hutchinson-Gilford progeria model of accelerated aging (LAKI). Changes in markers of aging as well as cellular metabolism was assessed using immunohistochemistry analysis and seahorse analyzer. Mouse health was monitored by recording animal weight and assessing motor activity using the open field activity assay. Skeletal muscle regeneration was assessed after cardiotoxin (CTX) injury, and force was recorded by measuring isometric force production in live animals.

Results, Conclusions, and Discussions: GPR81 is expressed in skeletal muscle, and its expression is lost as the muscle ages (Fig. A). We found that knockdown of GPR81 in young myoblasts (Y_shGPR81) lead to an increase in DNA damage, accumulation of reactive oxygen species (ROS) and impaired differentiation to myotubes. Further, GPR81 knockdown also resulted in accumulation of lipid droplets in myoblasts (Fig. B) and impaired lipid oxidation leading to lipotoxicity. Conversely, treatment of senescent myoblasts (S) with CHBA (S_CHBA) enhanced lipid oxidation (Fig. C) leading to a decrease in lipid accumulation to the level of young controls (Y; Fig. D), ultimately resulting in decreased DNA damage, ROS accumulation and enhanced ability to form myotubes. In the progeria mouse model of accelerated aging, we found that administration of CHBA intraperitoneally (LAKI-CHBA) for a month improved their health and increased ambulation in the open field assay. Further, CHBA decreased accumulation of intramyocellular lipid droplets, as seen by decreased Sudan Black B staining (Fig. D, E) and enhanced the number of Pax7 positive satellite cells (Fig. F). Further, CHBA enhanced muscle function after injury, as seen by an increase in skeletal muscle force production after CTX injury (Fig. G). In conclusion, we demonstrate the role of the lactate receptor GPR81 in muscle function and aging. GPR81 plays a key role in skeletal muscle lipid metabolism, and senescence associated loss of GPR81 leads to myosteatosis. Further, we demonstrate that agonists of GPR81 such as CHBA can be used as a potential therapy to reverse age-associated loss muscle function.

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