Researchers at the Wellcome Sanger Institute and at Sun Yat-sen University have generated what they claim is the first comprehensive atlas of aging muscles in humans. The team applied single-cell technologies and advanced imaging to analyze human skeletal muscle samples from 17 individuals across the adult lifespan. Their resulting map provides new insights into the many complex processes underlying age-related muscle changes, and revealed new cell populations that may explain why some muscle fibers age faster than others. The study also identified compensatory mechanisms the muscles employ to combat aging and could point to strategies for therapies and interventions that improve muscle health and quality of life as we age.

This study was carried out as part of the international Human Cell Atlas initiative that aims to map every cell type in the human body.  Sarah Teichmann, PhD, at the Wellcome Sanger Institute, is co-founder of the Human Cell Atlas, and senior author of the team’s reported study. Commenting on the findings, Teichmann said, “Through the Human Cell Atlas, we are learning about the body in unprecedented detail, from the earliest stages of human development through to old age. With these new insights into healthy skeletal muscle aging, researchers all over the world can now explore ways to combat inflammation, boost muscle regeneration, preserve nerve connectivity, and more. Discoveries from research like this have huge potential for developing therapeutic strategies that promote healthier aging for future generations.”

Teichmann and colleagues described the study in a paper in Nature Aging titled “Human skeletal muscle aging atlas.”

Skeletal muscle makes up 40% of our body mass, is essential for movement and has pivotal roles in metabolism and immune regulation, the authors wrote. “The major components of skeletal muscle, the multinucleated myofibers (MFs), are classified into ‘slow-twitch’ (type I) and ‘fast-twitch’ (type IIA, type IIX and intermediate hybrid fibers) according to their contraction speed, structural protein composition and metabolic characteristics (oxidative versus glycolytic).” Slow-twitch fibers are designed more to enable endurance activities, while fast-twitch fibers enable more powerful, explosive movements. The myofibers are also surrounded by mononuclear muscle stem cells (MuSCs), which can generate new muscle after damage.

As we age, our muscles progressively weaken. This can affect our ability to perform everyday activities such as standing up and walking. For some people, muscle loss worsens, leading to falls, immobility, a loss of autonomy and a condition called sarcopenia. “Skeletal muscle aging is characterized by the loss of both muscle mass and strength, often leading to sarcopenia,” the team continued.

During aging, there is a selective decrease in both the number and size of fast-twitch MFs, they noted. “Furthermore, the number of MuSCs and their activation and proliferation in response to stimuli decrease with age.” However, the reasons why our muscles weaken over time have remained poorly understood, and most previous studies focused on one particular mechanism or cell type, “leaving a gap in our understanding of muscle aging as a whole.”

To generate their aging muscle atlas the researchers used both single-cell and single-nucleus sequencing techniques along with advanced imaging to analyse human muscle samples from 17 individuals aged 20 to 75 years. “… In the present study, we combined scRNA-seq and snRNA-seq to build a human skeletal muscle aging atlas that includes both MuSCs and MF nuclei as well as cells from the microenvironment,” they explained. Because a muscle fiber consists of just one cell, but many nuclei, it brings unique challenges for study. Both single-cell RNA sequencing and single-nucleus RNA sequencing methods were used to address these challenges. While single-cell RNA sequencing looks at individual cells, including less common types of muscle stem cells and other supporting cells, profiling muscle fibers is difficult. Single-nucleus RNA sequencing, on the other hand, can focus on the multiple cell nuclei scattered throughout the muscle cell, to better explore its genetics.

In total, the team carried out transcription profiling of 90,902 cells and 92,259 nuclei from from the samples. “This allowed us to investigate transcriptional changes of MuSCs, MFs and microenvironment cells during aging,” they stated.

The results showed that genes controlling ribosomes, responsible for producing proteins, were less active in muscle stem cells from aged samples. This impairs the cells’ ability to repair and regenerate muscle fibers as we age. Further, non-muscle cell populations within these skeletal muscle samples produced more of a pro-inflammatory molecule called CCL2, attracting immune cells to the muscle and exacerbating age-related muscle deterioration. “In the MuSC compartment, we found downregulation of ribosome assembly resulting in decreased MuSC activation as well as upregulation of pro-inflammatory pathways, such as NF-κB, and increased expression of cytokines, such as CCL2,” they stated. In the MF microenvironment, we found several cell types that expressed pro-inflammatory chemokines, such as CCL2, CCL3 and CCL4. These cytokines may mediate the recruitment of lymphoid cells into muscle and the pro-inflammatory environment of aged muscle.”

Age-related loss of a specific fast-twitch muscle fiber subtype, key for explosive muscle performance, was also observed. However, they discovered for the first time several compensatory mechanisms from the muscles appearing to make up for the loss. These included a shift in slow-twitch muscle fibers to express genes characteristic of the lost fast-twitch subtype, and increased regeneration of remaining fast-twitch fibre subtypes.

The team also identified specialized nuclei populations within the muscle fibers that help rebuild the connections between nerves and muscles that decline with age. Knockout experiments in lab-grown human muscle cells by the team confirmed the importance of these nuclei in maintaining muscle function. “Our atlas also highlights an expansion of nuclei associated with the neuromuscular junction, which may reflect re-innervation, and outlines how the loss of fast-twitch myofibers is mitigated through regeneration and upregulation of fast-type markers in slow-twitch myofibers with age,” the team stated.

Study co-senior author Hongbo Zhang, PhD, from Sun Yat-sen University, said, “In China, the U.K. and other countries, we have aging populations, but our understanding of the aging process itself is limited. We now have a detailed view into how muscles strive to maintain function for as long as possible, despite the effects of aging.” Study first author Veronika Kedlian, PhD, at the Wellcome Sanger Institute, added, “Our unbiased, multifaceted approach to studying muscle aging, combining different types of sequencing, imaging and investigation reveals previously unknown cellular mechanisms of aging and highlights areas for further study.”

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