Long term molecular and epigenetic changes associated with muscle wasting

Abstract

Skeletal muscle plays an important role in wellbeing and health, with muscle mass and function influenced by factors including diet, physical activity, ageing and disease. Sarcopenia, the loss of muscle mass and function during ageing, is the major cause of functional decline and loss of independence in older adults, increasing the risk of falls, frailty, and physical disability. There is considerable variability in the rate individuals lose muscle mass and strength in old age and whether sarcopenia represents accelerated ageing or has specific mechanisms underlying its pathology is unknown. Furthermore, environmental exposures in prenatal and postnatal life may influence skeletal muscle development and function through epigenetic mechanisms, consistent with the developmental origins of health and disease (DOHaD) concept. In response to environmental factors, there is an adaptive response that results in changes in muscle mass and function, highlighting its plasticity. As a consequence, throughout the lifecourse, the environment can alter epigenetic mechanisms and affect the risk of developing non-communicable diseases. The loss of muscle mass during ageing has also been attributed in part to a reduced capacity of ageing muscle to repair itself. Muscle repair and regeneration depends on myogenic stem cells, referred to as satellite cells (SC). However, there is a decline in SC number and function as we age, impairing muscle regeneration and repair. Human primary myoblasts, committed stem cells of the muscle derived from vastus lateralis biopsies from 119 participants aged 73-83 years from the Hertfordshire Sarcopenia Study, were therefore used as a model in this study.
As epigenetic processes have also been implicated in the development of many ageing associated human diseases, an aim of this study was to use the Infinium Human MethylationEPIC BeadChip to examine changes in DNA methylation in human primary muscle myoblasts in relation to infant growth, postnatal infections, and sarcopenia and its definitional components. Sarcopenia and its definitional components, early life growth and frequency of childhood illnesses were found to be associated with differential patterns of DNA methylation. Twenty-one differentially methylated CpGs (dmCpGs) were identified to be correlated with sarcopenia, 13 with appendicular lean mass index (ALMi), 48 with grip strength and 3 with gait speed, with limited overlap between the dmCpGs associated with these measures. The top 100 dmCpG associated genes were enriched for pathways involved with cell TOR signalling, muscle structure development and Wnt signalling. Seven dmCpGs and 1 differentially methylated region (DMR) located in the BCAT1 gene and correlated with BCAT1 transcript levels in myoblasts, were identified to be associated with birthweight. BCAT1 encodes for the first enzyme in the branched chain amino acid catabolic pathway and has been previously linked to muscle growth and type 2 diabetes. Eight dmCpGs were found to be correlated with weight at 1 year, 6 correlated with conditional infant growth rate, 16 with frequency of childhood illnesses between birth and 1 year of age, and 53 with childhood illnesses between 1-5 years; the top pathways enriched were involved with glucose metabolism, myogenesis and stress responses.
To examine if differential methylation changes might be functionally important or influence the transcriptional activity of a gene, ATAC-seq was performed on the myoblasts to investigate the chromatin landscape. Changes in chromatin structure were found in relation to grip strength, with enriched pathways involved in muscle function and health. DNA methylation of 9 of the dmCpGs located in the BCAT1 DMR were correlated with chromatin structure, 2 of which were correlated with BCAT1 expression. Additionally, as the methylation levels of the BCAT1 dmCpGs were positively correlated to birthweight, this data suggests that a lower birthweight leads to a lower methylation level in the BCAT1 gene, which leads to a more open chromatin structure and ultimately leads to a lower expression, suggesting the recruitment of inhibiting transcription factors involved in this regulation. This could result in impaired amino acid metabolism, reduced muscle function and growth, and cause increased susceptibility to metabolic disorders in later life. Although changes in DNA methylation were found, the chromatin landscape was not majorly altered.
Furthermore, this study aimed to characterise myoblasts isolated from survivors of childhood severe acute malnutrition (SAM) in Jamaica. SAM occurs in children due to severe, sustained undernutrition, with major consequences being severe muscle wasting, low lean mass and weak grip strength. Therefore, this study aimed to phenotypically characterise myoblasts from SAM survivors using cell culture techniques. Myoblasts from adults who had survived the marasmus form of SAM in childhood were shown to have increased proliferation, early differentiation and senescence, while conversely having lower p53 expression, longer telomere length and lower ATP production despite a higher mtDNA copy number.
Taken together, the results demonstrate long term epigenetic alterations in human primary myoblasts arising from early life influences, furthering our understanding of the underlying molecular mechanisms of sarcopenia and SC function. Many potential directions to further develop and validate these findings can be undertaken to understand their functional significance.

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Identifiers

ORCID for Hanan Yasmin Sharkh: orcid.org/0000-0001-5599-3087

ORCID for Emma Garratt: orcid.org/0000-0001-5268-4203

ORCID for Keith Godfrey: orcid.org/0000-0002-4643-0618

ORCID for Karen A Lillycrop: orcid.org/0000-0001-7350-5489

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