A new study on mouse models has revealed transcripts encoding a longevity-determining protein that traverse through the systemic circulation in miniscule shuttles called extracellular vesicles (EVs) are a key mediator of youthfulness in muscle tissue.

It’s common knowledge that muscles weaken with age. They become visibly smaller and less resilient to injury. Aged muscles don’t heal as well after damage because instead of restoring original muscle tissue, scar tissue is deposited instead.

It is also widely known that infusing blood from a young animal into an aged animal, through a protocol called ‘heterochronic parabiosis’, restores youthful features in aging muscles. Heterochronic parabiosis connects the circulatory systems of two animals such that the older animal is exposed to the circulating factors from the younger animal and vice versa. What has not been known until now, is the identity of the circulating factors in young blood that mediate this rejuvenating effect.

Scientists from the University of Pittsburgh have now pinpointed that transcripts of the longevity-protein (Klotho) in circulating EVs contribute to the systemic regulation of regeneration in aged skeletal muscles.

The authors show, the beneficial effect of young blood on aged muscle regeneration is decreased when the blood is depleted of EVs. This indicates aging of muscle tissue accompanied by the loss of muscle function and impaired repair, may be driven by aged EVs that carry fewer copies of these longevity-promoting instructions than EVs in young animals.

These new findings reported in an article in the journal Nature Aging titled, “Regulation of aged skeletal muscle regeneration by circulating extracellular vesicles,” that offers new insights into the dwindling capacity for muscle regeneration with age.

Extracellular vesicles (EVs) in the blood of young animals have more Klotho transcripts (left), indicated by green strands, compared to circulating EVs of aged animals (right). Age-related declines in circulating EV- derived Klotho transcripts result in a blunted skeletal muscle functional regeneration of injured aged animals, which can be reversed, at least in part, by treating injured aged skeletal muscle with young EVs. Graphic created with BioRender.com. [Fabrisia Ambrosio & Amrita Sahu]
“We’re really excited about this research for a couple of reasons,” says Fabrisia Ambrosio, PhD, director of rehabilitation for UPMC International, associate professor of physical medicine and rehabilitation at Pitt and senior author of the paper. “It helps us understand the basic biology of how muscle regeneration works and how it fails to work as we age. Taking that information to the next step, we can think about using extracellular vesicles as therapeutics to counteract these age-related defects.”

 

“We wondered if extracellular vesicles might contribute to muscle regeneration because these couriers travel between cells via the blood and other bodily fluids. Like a message in a bottle, EVs deliver information to target cells,” says Amrita Sahu, PhD, postdoctoral fellow in the Department of Physical Medicine and Rehabilitation at Pitt and first author of the paper.

Amrita Sahu, Ph.D., postdoctoral fellow in the Department of Physical Medicine and Rehabilitation at the University of Pittsburgh is the lead author of the paper [UPMC]
The research team isolated and characterized EVs from young and aged mouse serum–the fraction of blood remaining once blood cells and clotting factors have been removed from it. Upon injecting EVs from young mice into old mice with injured muscles, they observed, mice that received young serum showed enhanced muscle regeneration and functional recovery compared to those that received a placebo treatment. But the young serum’s restorative properties were lost when EVs were removed from it, indicating that EVs are pivotal in transmitting the rejuvenating effects from the young to aged mice.

 

Fabrisia Ambrosio, PhD, director of rehabilitation for UPMC International and associate professor of physical medicine and rehabilitation at the University of Pittsburgh, is senior author of the paper [Source: UPMC]
“In this work, we used imaging flow cytometry to compare structural and compositional features of young and aged EVs at the resolution of single nanoparticles. This approach allowed us to evaluate shifts in circulating EV subpopulation dynamics with increasing age.  We employed a machine learning approach on the imaging flow cytometry data to classify young versus aged EVs,” says Ambrosio. “Ultimately, we anticipate that this information can provide important cues into which sub-populations of EVs exert the greatest contribution to skeletal muscle healing and how these subpopulations change over time.”

Using machine learning classifiers, the authors found, aging shifts the nucleic acid, but not protein, fingerprint of circulating EVs. The authors show EVs deliver anti-aging Klotho mRNA to muscle progenitor cells. They also show EVs collected from old mice carried fewer copies of Klotho mRNA than those from young mice, resulting in less Koltho protein in muscle progenitor cells.

Earlier work from Ambrosio’s lab had identified Klotho as an important regulator of regenerative capacity in muscle progenitor cells and that this protein declines with age. The new study shows that age-related shifts in EV cargo contribute to decreased Klotho in aged muscle stem cells.

“EVs may be beneficial for boosting regenerative capacity of muscle in older individuals and improving functional recovery after an injury,” says Ambrosio.

The research reported in the current paper was supported by the National Institutes of Health and University of Pittsburgh Medical Center (UPMC) Enterprises.

“As a next step”, says Ambrosio, “we are primarily focusing on the development of novel EV-based technologies to enhance muscle healing in older individuals. Specifically, we are investigating methods to engineer EVs with molecular cargoes, including Klotho transcripts, with the goal of optimizing the downstream benefits of EVs on target muscle stem function. We are also investigating non-invasive approaches, such as exercise, to increase Klotho transcript levels in aged EVs.”

In future studies, Ambosio and co-author Radosveta Koldamova, MD, PhD, professor of environmental and occupational health at Pitt’s Graduate School of Public Health, will also explore the role of EVs in reversing age-related decline in cognition.