January 8, 2025 | An international team of researchers hope to identify molecular and cellular level changes in muscle tissue that can be used to help diagnose and treat chronic fatigue syndrome (CFS) and long COVID. The quest is being pursued from both the physiological and electrical perspectives to better understand the muscle damage seen with the debilitating and still-mysterious conditions that share some striking similarities, according to Xuanhong Cheng, Ph.D., professor of bioengineering and materials science and engineering at Lehigh University.
This is “fundamental research,” she stresses, but growing scientific interest and grant funding for the study of these overlapping illnesses means much-needed hope is on the way for affected individuals. Recent estimates suggest that in the U.S. alone at least 1.3% of the population (roughly 3.3 million people) have CFS and, because many of the people with long COVID meet the criteria for CFS, its prevalence has further grown in the wake of the pandemic. In 2022, 6.9% of U.S. adults reported ever experiencing long COVID.
For her part in the multidisciplinary approach taken here, Cheng takes charge of measuring the electrical signature differences in healthy versus diseased muscle stem cells while her collaborators in Italy—respectively, Tiziana Pietrangelo, Ph.D., of Università degli Studi "G. d'Annunzio" Chieti – Pescara, and Stefano Cagnin, Ph.D., from the University of Padova—measure cell function differences and do molecular analysis of DNA and RNA to know what regulatory pathways and protein activities are altered.
The exploratory work is being funded by the National Institutes of Health, which launched a Researching COVID to Enhance Recovery (RECOVER) initiative in 2021 that was recently bolstered with a fresh infusion of $515 million in funding. Long COVID has been described as a “mass disabling event” (The Lancet, DOI: 10.1016/S0140-6736(24)01406-5), with no verifiable tests for diagnosing it or silver-bullet therapy to treat it, but the research attention it is now getting is expected to shine light on CFS—one of the most understudied and misunderstood conditions in modern medicine.
Even while the pandemic was still raging, it became apparent that CFS and long COVID had a lot in common including not only muscle fatigue but neuroinflammation and brain fog.
The project to study cellular changes in muscle tissue started with Pietrangelo, who had been working with muscle cells for a couple of decades, says Cheng. She was especially interested in oxidative stress in the muscle cells of the elderly and people with CFS.
Cheng, meanwhile, had spent most of the prior decade working on sensors for measuring the electrical impedance of single cells. After she and Pietrangelo met through common friends and began talking about their respective research interests, they decided to use the electric sensors to look at muscle cells from individuals with easy-to-fatigue muscles.
Initially, they used the electrical sensors on cultured muscle stem cells exposed to oxidative stress—an important environment in CFS muscles—to learn they could differentiate those from normal cultured cells, Cheng continues. They subsequently hypothesized that the sensors could also detect the oxidative stress in the stem cells of CFS patients.
In pondering their grant proposal, they realized the missing piece in further differentiating people who have CFS from healthy populations would require an understanding of how muscle fibers respond to stimulation by contracting. This is where Cagnin came in to focus on the gene expression in muscle stem cells and hopefully uncover disease-associated molecular changes via messenger RNA and microRNA analysis.
The pandemic was underway during the grant writing process and reports about long COVID were starting to emerge, extending their proposed study to the two conditions, says Cheng. “It could be,” they reasoned, “that individuals with long COVID also have [telltale] changes in their muscle tissue and muscle stem cell function... that could be used to identify individuals with damage [caused by the disease].”
Both CFS and long COVID oftentimes manifest in the muscles, she says. In chronic fatigue syndrome, as the name implies, muscle fatigue is the defining symptom. Individuals with CFS tire easily and can suffer pain from doing any sort of physical activity.
It is not easy to study individual muscle fibers, notes Cheng. “Most muscles are very long and not easy to isolate from patients, which is why we study muscle stem cells instead.” Muscle stem cells can be derived from a small biopsy sample and easily proliferate, she adds. It is believed that stem cells replenish muscle fibers in the tissue and, in individuals with CFS-associated muscle damage, “the muscle is probably not reproducing properly.”
Researchers are using changes detected in the muscle stem cells to “infer the damage to muscle tissue,” explains Cheng. That could potentially be used to define the changes happening in patients with CFS and long COVID and, longer term, serve as diagnostic markers of one disease or the other.
CFS is currently diagnosed based on patient symptoms and ruling out other diagnosable conditions, she says. “There is no single, clear biological marker that defines the disease.” And it’s the same story with long COVID.
The plan is to recruit 18 patients and 18 healthy controls into the study, which is still in the institutional review board process, says Cheng. The project is complicated by the fact that it involves foreign institutions. Patient enrollment will be happening mainly at Università degli Studi "G. d'Annunzio" Chieti – Pescara.
Participants will all be donating a small muscle biopsy sample, which involves a procedure no more painful than an injection of a drug or vaccine (she personally had one done, Cheng notes). The biopsy area feels a bit sore the next day, comparable to the delayed muscle soreness felt after exercising, and the scab falls off in a few days.
The study will employ a broadband electrical sensor that Cheng co-developed that can detect more signals than the Coulter counters that has been used for cell-counting purposes in labs for decades. Whenever cells pass through a tiny pinhole in the device, the current flowing through it gets blocked, enabling the counting.
With the broadband sensor, dipole elements in the molecules inhabiting biological tissue oscillate in response to the positive and negative side of electrodes in an alternating electric field, she explains. The dipoles respond differently to that external field and those differences in vibration can identify normal versus pathological cells based on the type and quantity of biomolecules. A very wide electrical frequency range is used to extract as many details as possible about that cellular response.
‘Starting Point’ of Disease
It is probably going to be a long time before the approach can be implemented clinically, says Cheng. But the idea, long-term, is to determine the electrical signal frequency ranges that can distinguish normal from diseased muscle tissue without the need for a biopsy for both diagnostic and treatment response purposes.
This will require extending the electrical signature obtained from the stem cells outside the body to intact muscle tissue. Pietrangelo is already using electrical impedance sensing to detect the function of muscles, so in the future it may be possible to combine that with the important electrical signal frequency ranges identified in the latest study to understand pathological changes in below-the-surface muscle tissue, she says.
For both CFS and long COVID there has been considerable debate about what should be the target tissue for diagnostic purposes, notes Cheng. “The general hypothesis is that the conditions are associated with a viral infection in the first place... but afterwards, why would people have problems in multiple different tissues and organs?”
Different theories are being pursued, including a potential problem with the brain and immune system (e.g., T cell exhaustion [PNAS, DOI: 10.1073/pnas.241511912]). “We’re not necessarily saying that the muscle is the starting point of disease, but it is the biggest organ [by total mass] and... is the more consistent tissue that’s affected,” she says.
The various lines of study are all important and ultimately complementary in trying to understand CFS and long COVID more comprehensively, Cheng adds. They are not “easy diseases” affecting a single organ or tissue that would be more easily rectified, but highly complex, multi-system conditions.