Researchers analyzed skeletal muscle biopsies of patients with persistent fatigue and malaise post-exertion to determine the underlying mechanisms of long COVID. In a recent study posted to the medRxiv* preprint server, a team of researchers from Germany analyzed skeletal muscle biopsies of patients with persistent fatigue and malaise post-exertion to determine the underlying mechanisms of long coronavirus disease (long COVID).
Background
A commonly observed symptom after acute viral infections is persistent muscle fatigue, and a subset of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have experienced a continuation of their coronavirus disease 2019 (COVID-19) symptoms, as well as newly occurring symptoms well after recovery. These symptoms include muscle weakness, persistent fatigue, post-exertional malaise (PEM), neurocognitive impairments, and myalgia. Since the types of symptoms, as well as their severity and manifestations, vary across patients, determining the underlying mechanisms of long COVID or post-acute sequelae of COVID-19 (PASC) has proven difficult. Furthermore, even though inflammatory myopathy has been observed in severe COVID-19-associated fatality cases, the impact of the virus on skeletal muscle tissue has not been well-studied.
About the study
In the present study, eleven patients were included above 18 with SARS-CoV-2 infection diagnosed by a positive polymerase chain reaction (PCR) test and experiencing PEM and muscle fatigue for a minimum of six months after recovering from the SARS-CoV-2 infection. An approval for vastus lateralis muscle biopsy with no contraindications to the process was required. Detailed neurological and clinical examinations consisting of handgrip strength tests, sensory tests, tests for muscle strengths of various muscle groups, a six-minute walk test, and a test of reflexes were performed. Additionally, serum samples were collected, and the participants responded to online questionnaires on fatigue and underwent a magnetic resonance imaging (MRI) scan of the proximal lower extremities. Biopsy samples of the vastus lateralis muscle were also obtained. For the control cohort, cryopreserved skeletal muscle samples from individuals between the ages of 18 and 65 that were collected before the onset of the COVID-19 pandemic in December 2019 were used. It was ensured that the samples were obtained from individuals without a history of cancer, inflammatory disease, or mitochondriopathy, without high creatinine levels or pathological electromyogram, and not undergoing any immunosuppressive therapy or corticosteroid treatment.
The study included a healthy disease control cohort (HDC), for which it was ensured that the samples had no immunohistochemical or histopathological abnormalities, and a control cohort of samples with type-2b atrophy (2BA), in which the samples showed selective atrophy of type-2b muscle fibers. Virological analyses included quantitative reverse transcription–polymerase chain reaction to detect and quantify SARS-CoV-2 ribonucleic acid (RNA), enzyme-linked immunosorbent assays to determine anti-SARS-CoV-2 immunoglobulin G (IgG) levels, and electrochemiluminescence immunoassay to detect the spike and nucleocapsid antigens. The serum samples were also subjected to a range of myositis-specific and myositis-associated autoantibodies and antinuclear antibody assays. Furthermore, the routinely used enzymological and histological stains and immunohistochemical stains were used to visualize the biopsy samples. Populations of immune cells were also quantified, and semiquantitative scoring was done to determine the degree of major histocompatibility complex (MHC) class I and class II upregulation and the type-2b-fiber atrophy. Additionally, electron microscopy and morphometry analyses were conducted to determine the capillarity of the samples. The study also included in-depth proteomics and RNA sequence analyses of the samples.
Results
The results showed that the skeletal muscle samples from long COVID patients had fewer capillaries, and the basement membranes of their capillaries were thicker when compared to the two historical cohorts HDC and 2BA. The long COVID patients’ skeletal muscle tissue samples also had a higher number of CD169+ macrophages than the historical cohorts, even though there was no obvious evidence of myositis. Although there were no detectable levels of SARS-CoV-2 in the muscle tissues, analysis of the transcriptomic data revealed that compared to the historical cohorts, the skeletal muscle samples from long COVID patients presented distinct genetic signatures of immune dysregulation and changes in metabolic pathways. The transcriptome profiles also indicated an increase in the expression of extracellular matrix remodeling and angiogenesis genes and a decrease in the expression of genes involved in mitochondrial functioning and metabolic processes. The authors believe that acute SARS-CoV-2 infections could have caused long-lasting structural modifications to the skeletal muscle microvasculature, which could explain the fatigue post-exertion.
Conclusions
Overall, the results indicated that the skeletal muscles of patients with long COVID symptoms such as PEM and persistent fatigue exhibit changes in the number and structure of the capillaries, as well as genetic signatures of immune dysregulation, upregulation of angiogenesis-related genes and a decrease in the expression of genes involved in the mitochondrial activity. These findings could explain the underlying mechanisms of long COVID symptoms of muscle pain and fatigue following exercise.
Preprint of cited research available in medRxiv (Feb. 16, 2023):
https://www.medrxiv.org/content/10.1101/2023.02.15.23285584v1
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