Mass spectrometry-based proteomic analysis of middle-aged vs. aged vastus lateralis reveals increased levels of carbonic anhydrase isoform 3 in senescent human skeletal muscle

Abstract

The age-related loss of skeletal muscle mass and associated progressive decline in contractile strength is a serious pathophysiological issue in the elderly. In order to investigate global changes in the skeletal muscle proteome after the fifth decade of life, this study analysed total extracts from human vastus lateralis muscle by fluorescence difference in-gel electrophoresis. Tissue specimens were derived from middle-aged (47-62 years) vs. aged (76-82 years) individuals and potential changes in the protein expression profiles were compared between these two age groups by a comprehensive gel electrophoresis-based survey. Age-dependent alterations in the concentration of 19 protein spots were revealed and mass spectrometry identified these components as being involved in the excitation-contraction-relaxation cycle, muscle metabolism, ion handling and the cellular stress response. This indicates a generally perturbed protein expression pattern in senescent human muscle. Increased levels of mitochondrial enzymes and isoform switching of the key contractile protein, actin, support the idea of glycolytic-to-oxidative and fast-to-slow transition processes during muscle aging. Importantly, the carbonic anhydrase (CA)3 isoform displayed an increased abundance during muscle aging, which was independently verified by immunoblotting of differently aged human skeletal muscle samples. Since the CA3 isoform is relatively muscle-specific and exhibits a fibre type-specific expression pattern, this enzyme may represent an interesting new biomarker of sarcopenia. Increased levels of CA are indicative of an increased demand of CO₂-removal in senescent muscle, and also suggest age-related fibre type shifting to slower-contracting muscles during human aging.

Figure 1

Two-dimensional (2D) fluorescent difference in-gel electrophoretic analysis of proteins during human skeletal muscle aging. Shown are fluorescent 2D gels of middle-aged (A–D) vs. aged (I–L) vastus lateralis muscle preparations, as well as pooled standards (E–H and M–P). Specimens MA1 to MA4 were derived from 47-, 55-, 59- and 62-year-old muscle tissue, and specimens OA1–OA4 were derived from 76-, 77-, 81- and 82-year-old muscle tissue, respectively. MA, middle-aged; OA, old-aged.

Figure 2

Proteomic identification of changed proteins during human skeletal muscle aging. Shown is a difference in-gel electrophoresis master gel of total protein extracts from vastus lateralis muscle. Skeletal muscle-associated proteins with a significantly different expression level are marked by circles and are numbered 1–19. Table I displays detailed listing of muscle proteins with a changed abundance in aged contractile tissue. The pH values of the first dimension gel system and molecular mass standards of the second dimension are indicated on the top and on the left of the panel, respectively.

Figure 3

Immunoblot analysis of carbonic anhydrase CA3 in aged human skeletal muscle. Shown are representative immunoblots labelled with antibodies to the CA3 isoform. Lanes 1 and 2 represent middle-aged (MA) vs. old-aged (OA) vastus lateralis (VL) muscle specimens, respectively. Immunoblot panels show individual middle-aged samples vs. individual aged samples as follows: (A) 47 vs. 76 years, (B) 55 vs. 77 years, (C) 59 vs. 81 years, and (D) 62 vs. 82 years. (E) The comparative immunoblot analysis was statistically evaluated using an unpaired Student’s t-test (n=4; ^*P<0.05).

Figure 4

Diagrammatic overview of molecular and cellular changes during skeletal muscle aging. The flowchart summarises major age-related changes in contractile patterns and muscle metabolism as revealed by proteomic analyses of young adult vs. middle-aged vs. aged human vastus lateralis muscle.