The Protective Role Of MGPDH In Skeletal Muscle Regeneration Dysfunction Caused By Obesity And Diabetes Mellitus

Background:Mitochondrial glycerol-3-phosphate dehydrogenase(mGPDH)is a flavin-linked respiratory chain dehydrogenase that oxidizes glycerol 3-phosphate(G3P)to dihydroxyacetone phosphate(DHAP)with concurrent transfering of cytosolic reduction equivalents into the mitochondrial for oxidaton.mGPDH is an integral component of the mammalian respiratory chain and glycerophosphate(GP)-shuttle which connects mitochondrial and cytosolic processes and plays an important role in cell bioenergetics,both under physiological and pathological conditions.Skeletal muscle mass account for 40% of body weight,play an important role in body movement and metabolic homeostasis.Sufficient muscle mass is the foundation of its proper function,while myogenesis is central to the origins and maintenance of skeletal muscle fibers throughout lifetime.During its process,myogenic regeneration is considered as one of the most important phase,and under certain disease conditions,such as modern pandemic obesity and diabetes,the skeletal muscle regeneration is impaired,leading to the loss of muscle function and contributing to the global burden of these diseases.However,the mechanisms underlying the regeneration impairment are poorly understood and lack of efficient treatment.The goal of this study was to investigate the role of mGPDH in skeletal muscle regeneration and probe the positive effect of m GPDH in obesity and diabetes associated impairment of muscles healing after injury.Methods:1.Effects of mGPDH on skeletal muscle cell differentiation:1.1 mGPDH expression pattern during C2C12 cell differentiation were detected by Western blot and qRT-PCR.1.2 si RNA for knockdown of mGPDH gene and plasmid carrying mGPDH gene were introduced to test the effects of mGPDH during differentiation.m GPDH,myogenic regulation factors(MRFs)were detected by Western blot and qRT-PCR.The phenotypes of differentiation were detected by immunofluorescence for MyHC and the number of multinucleated fiber.2.Effects of mGPDH on skeletal muscle regeneration:2.1 m GPDH expression pattern during skeletal muscle regeneration were detected by Western blot and qRT-PCR.2.2 mGPDH knockout mice were introduced to investigate the skeletal muscle regeneration after injury:1)Skeletal muscle histological sections of m GPDH-KO mice and wildtype mice were stained with H&E,immunofluorescence and Masson to observe the regeneration process of skeletal muscle after CTX injury.2)Western blot and qRT-PCR were done to test the level of m GPDH and myogenic markers of myogenin and myh3.3.Mechanism of m GPDH regulated skeletal muscle regeneration:3.1 ATP level and the oxygen consumption rate were detected to observe the energy change upon mGPDH interruption or overexpression during cell differentiation.3.2 Mitochondrial content and respiratory enzyme were detected by qRT-PCR to observe the changes of mitochondrial amount and function regulated by mGPDH.3.3 AMPK inhibitor compound C was used and Western-blot,q RT-PCR and immunofluorescence were done to test the influence of m GPDH on AMPK pathway activation during differentiation.4.Effects of mGPDH addition on the regeneration during obesity and diabetes:4.1 Western blot and IHC staining were used to detect the m GPDH expression level in skeletal muscles from obese patients and normal subjects.4.2 Western blot and q RT-PCR were used to detect the mGPDH expression level in skeletal muscles from obese and diabetic mice at base level or after CTX injury.4.3 q RT-PCR and H&E staining were used to test the effects of m GPDH activation on myogenic markers expression and regeneration precess after CTX injury.Results:1.Effects of mGPDH on skeletal muscle cell differentiation:(1)Western blot and q RT-PCR showed that mGPDH expression was increased during C2C12 differentiation.(2)Immunofluorescence stain showed that MyHC+ multinucleated myotube were less and smaller than control after m GPDH been knockdown by si RNA,on the other hand,more and bigger fibers were presented after m GPDH overexpression.(3)Western blot and qRT-PCR proved that m GPDH knockdown or overexpression affected the expression of myogenic regulators myogenin and MyHC.2.Effects of mGPDH on skeletal muscle regeneration:(1)Western blot and q RT-PCR showed that mGPDH expression was increased during skeletal muscle regeneration.(2)H&E,immunofluorescence and masson stains showed that mice lack of m GPDH have difficulties in skeletal muscle repairement.Western-blot and q RT-PCR proved that mGPDH-KO mice have difficulties in activation of myogenic regulators.3.Mechanism of how m GPDH regulates skeletal muscle regeneration:(1)ATP level and the oxygen consumption rate were decreased upon m GPDH interruption,and increased upon m GPDH overexpression after 2 days of differentiation.(2)Mitochondria content and respiratory enzyme were decreased upon mGPDH interruption,and increased upon mGPDH overexpression after 2 days of differentiation.(3)Western-blot showed that mGPDH affected the activation of AMPK pathway.Western-blot and immunofluorescence exhibited that AMPK inhibitor compound C interrupt the positive effect of m GPDH on differentiation.4.Activation of mGPDH rescued the regeneration dysfunction in obesity and diabetic mice: HE stain exhibited that the increasing of m GPDH accelerated the regeneration process in ob/ob and STZ mice upon skeletal muscle injury.qRT-PCR showed that AAV9 treatment increased the expression of mGPDH and myogenic regulators in ob/ob and STZ mice.Conclusion:1.mGPDH plays an important role in regulation of myoblast differentiation process.2.mGPDH knockout mice have impaired capability in skeletal muscle regeneration after injury.3.mGPDH regulates skeletal muscle regeneration through activating the AMPK pathway.4.Activation of mGPDH rescued the regeneration dysfunction in obesity and diabetic mice.