A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics.

Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious, requiring 2 h of mass spectrometry time per single muscle fiber; 50 fibers would take approximately 4 days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 min total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 h. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Ninety-four proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, oxidative phosphorylation, and muscle structure and contractile function. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.

Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics.

Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious requiring two hours of mass spectrometry time per single muscle fiber; 50 fibers would take approximately four days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 minutes total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 hours. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Sixty-five proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, muscle structure and regulation. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.

Single muscle fibre contractile function with ageing.

Ageing is accompanied by decrements in the size and function of skeletal muscle that compromise independence and quality of life in older adults. Developing therapeutic strategies to ameliorate these changes is critical but requires an in-depth mechanistic understanding of the underlying physiology. Over the past 25 years, studies on the contractile mechanics of isolated human muscle fibres have been instrumental in facilitating our understanding of the cellular mechanisms contributing to age-related skeletal muscle dysfunction. The purpose of this review is to characterize the changes that occur in single muscle fibre size and contractile function with ageing and identify key areas for future research. Surprisingly, most studies observe that the size and contractile function of fibres expressing slow myosin heavy chain (MHC) I are well-preserved with ageing. In contrast, there are profound age-related decrements in the size and contractile function of the fibres expressing the MHC II isoforms. Notably, lifelong aerobic exercise training is unable to prevent most of the decrements in fast fibre contractile function, which have been implicated as a primary mechanism for the age-related loss in whole-muscle power output. These findings reveal a critical need to investigate the effectiveness of other nutritional, pharmaceutical or exercise strategies, such as lifelong resistance training, to preserve fast fibre size and function with ageing. Moreover, integrating single fibre contractile mechanics with the molecular profile and other parameters important to contractile function (e.g. phosphorylation of regulatory proteins, innervation status, mitochondrial function, fibre economy) is necessary to comprehensively understand the ageing skeletal muscle phenotype.