Age-related nitration/dysfunction of myogenic stem cell activator HGF.

Mechanical perturbation triggers activation of resident myogenic stem cells to enter the cell cycle through a cascade of events including hepatocyte growth factor (HGF) release from its extracellular tethering and the subsequent presentation to signaling-receptor c-met. Here, we show that with aging, extracellular HGF undergoes tyrosine-residue (Y) nitration and loses c-met binding, thereby disturbing muscle homeostasis. Biochemical studies demonstrated that nitration/dysfunction is specific to HGF among other major growth factors and is characterized by its locations at Y198 and Y250 in c-met-binding domains. Direct-immunofluorescence microscopy of lower hind limb muscles from three age groups of rat, provided direct in vivo evidence for age-related increases in nitration of ECM-bound HGF, preferentially stained for anti-nitrated Y198 and Y250-HGF mAbs (raised in-house) in fast IIa and IIx myofibers. Overall, findings highlight inhibitory impacts of HGF nitration on myogenic stem cell dynamics, pioneering a cogent discussion for better understanding age-related muscle atrophy and impaired regeneration with fibrosis (including sarcopenia and frailty).

A pilot study on nitration/dysfunction of NK1 segment of myogenic stem cell activator HGF.

Protein tyrosine residue (Y) nitration, a post-translational chemical-modification mode, has been associated with changes in protein activity and function; hence the accumulation of specific nitrated proteins in tissues may be used to monitor the onset and progression of pathological disorders. To verify the possible impact of nitration on postnatal muscle growth and regeneration, a pilot study was designed to examine the nitration/dysfunction of hepatocyte growth factor (HGF), a key ligand that is released from the extracellular tethering and activates myogenic stem satellite cells to enter the cell cycle upon muscle stretch and injury. Exposure of recombinant HGF (a hetero-dimer of α- and ß-chains) to peroxynitrite induces Y nitration in HGF α-chain under physiological conditions. Physiological significance of this finding was emphasized by Western blotting that showed the NK1 segment of HGF (including a K1 domain critical for signaling-receptor c-met binding) undergoes nitration with a primary target of Y198. Peroxynitrite treatment abolished HGF-agonistic activity of the NK1 segment, as revealed by in vitro c-met binding and bromodeoxyuridine-incorporation assays. Importantly, direct-immunofluorescence microscopy of rat lower hind-limb muscles from two aged-groups (2-month-old "young" and 12-month-old "retired/adult") provided in vivo evidence for age-related nitration of extracellular HGF (Y198). Overall, findings provide the insight that HGF/NK1 nitration/dysfunction perturbs myogenic stem cell dynamics and homeostasis; hence NK1 nitration may stimulate progression of muscular disorders and diseases including sarcopenia.

Epigenetic effects induced by the ectopic expression of Pax7 in 3T3-L1.

Although skeletal muscle cells and adipocytes are derived from the same mesoderm, they do not transdifferentiate in vivo and are strictly distinct at the level of gene expression. To elucidate some of the regulatory mechanisms underlying this strict distinction, Pax7, a myogenic factor, was ectopically expressed in 3T3-L1 adipose progenitor cells to perturb their adipocyte differentiation potential. Transcriptome analysis showed that ectopic expression of Pax7 repressed the expression of some adipocyte genes and induced expression of some skeletal muscle cell genes. We next profiled the epigenomic state altered by Pax7 expression using H3K27ac, an activating histone mark, and H3K27me3, a repressive histone mark, as indicators. Our results show that ectopic expression of Pax7 did not result in the formation of H3K27ac at loci of skeletal muscle-related genes, but instead resulted in the formation of H3K27me3 at adipocyte-related gene loci. These findings suggest that the primary function of ectopic Pax7 expression is the formation of H3K27me3, and muscle gene expression results from secondary regulation.