Phosphoproteomic analysis of aged skeletal muscle

Abstract

Abstract. One of the most important post-translational modifications is represented by phosphorylation on tyrosine, threonine and serine residues. Since abnormal phosphorylation is associated with various pathologies, it was of interest to perform a phosphoproteomic profiling of age-related skeletal muscle degeneration. We used the fluorescent phosphospecific Pro-Q Diamond dye to determine whether changes in the overall phosphorylation of the soluble skeletal muscle proteome differs significantly between young adult and senescent fibres. As an established model system of sarcopenia, we employed 30-month-old rat gastrocnemius fibres. Following the mass spectrometric identification of 59 major 2-D phosphoprotein landmark spots, the fluorescent dye staining survey revealed that 22 muscle proteins showed a differential expression pattern between 3-month-and 30-month-old muscle. Increased phosphorylation levels were shown for myosin light chain 2, tropomyosin α, lactate dehydrogenase, desmin, actin, albumin and aconitase. In contrast, decreased phospho-specific dye binding was observed for cytochrome c oxidase, creatine kinase and enolase. Thus, aging-induced alterations in phosphoproteins appear to involve the contractile machinery and the cytoskeleton, as well as the cytosolic and mitochondrial metabolism. This confirms that sarcopenia of old age is a complex neuromuscular pathology that is associated with drastic changes in the abundance and structure of key muscle proteins.

Eccentric contraction-induced injury to type I, IIa, and IIa/IIx muscle fibers of elderly adults

“…Finally, an age-related increase in susceptibility to damage could be due to environmental factors. Elevations in reactive oxygen species (Fielding and Meydani 1997) and altered protein phosphorylation (Gannon et al 2008) are hallmarks of aging muscle. Acute oxidative stress exacerbates eccentric contraction-induced injury in old laboratory animals (Zerba et al 1990).…”

“…Acute oxidative stress exacerbates eccentric contraction-induced injury in old laboratory animals (Zerba et al 1990). Myosin light chain 2 shows an age-related increase in phosphorylation (Gannon et al 2008), an alteration that has been shown to increase susceptibility to contraction-induced damage in fast fibers from adult rodents (Childers and McDonald 2004). Desmin and tropomyosin, two other strain sensitive proteins, also show increased phosphorylation with aging (Gannon et al 2008).…”

DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue

“…Pyruvate isoform PK-M1 exists as 5 sub-species with differing isoelectric points in skeletal muscle tissues, whereby theses isozymes display varying degrees of phosphorylation and glycosylation [21,24]. Previous studies have clearly shown that the PK isoform exhibiting a pI of 6.6 and an apparent molecular mass of 57.8 kDa is several-fold decreased in senescent muscle fibres [21].…”

“…Recently, comparative subproteomics of the soluble myofibril-enriched fraction has shown distinct age-dependent effects on the contractile apparatus. The contractile protein exhibiting the most drastically changed expression was identified as the MLC2 isoform of the slowtype myosin light chain [23], which also exhibits enhanced phosphorylation levels in aged fibres [24]. Although fast and slow isoforms of myosin heavy chain switch in the majority of aged fibres, the dramatic increase in slow MLC2 expression was restricted to individual senescent cells [23].…”

“…Although fast and slow isoforms of myosin heavy chain switch in the majority of aged fibres, the dramatic increase in slow MLC2 expression was restricted to individual senescent cells [23]. Since posttranslational modifications play a central role in protein function and considerably increase protein diversity within the skeletal muscle proteome, it is important to stress that glycosylation, tyrosine nitration, and tyrosine/threonine phosphorylation are generally altered in senescent skeletal muscles [21,24,25], thereby complicating global protein expression analysis.…”