microRNA-based interventions against loss of muscle mass and function resulting from in utero and early post-natal protein restriction.

The reduction in muscle mass and strength that occur during ageing has a major impact on the quality of life of older individuals. Older people demonstrate loss of confidence in walking and reduced mobility which in turn leads to loss of independence and social isolation. These changes occur partly because we lose a large proportion of the muscle cells (called muscle fibres), but also the muscle cells that we retain are weak. It is currently unknown how muscle fibres are lost during ageing. There is considerable evidence that poor maternal nutrition leads to a number of changes in muscle of the offspring that result in reduced function. Muscle strength is also compromised in older individuals who did not grow well in early life, and studies suggest that maternal, developmental and nutritional factors are important. microRNAs are small molecules that regulate gene expression resulting in different sets of proteins being present in the cells. Through this, microRNAs regulate cell functions. It is established that most biological processes, including muscle growth and wasting and ageing are, or are likely to be, regulated by microRNAs. The levels of microRNAs in muscle and other tissues change during ageing and upon changes in diet. As microRNAs can regulate the expression of many genes, and therefore physiological processes, they are likely candidates to regulate the effects of poor diet on muscle.
We hypothesise that a reduction in protein intake during foetal and early neonatal life results in modified microRNA-target interactions in muscles of the offspring and this leads to loss of muscle mass and function which has long term effects on the number of muscle fibres and this ultimately adversely influences whether older individuals can maintain good muscle function as they age. It is not possible to directly examine this possibility in humans and this project will therefore use mouse models. We will determine the effect of the reduced protein intake in utero, or in the early post-natal period, on muscle fibre number and muscle mass and function in adulthood and ageing and whether restoration of microRNA levels in muscle will prevent the loss of muscle mass and function associated with in utero and/or early post-natal protein restriction.
The research proposed is of high importance and we anticipate that the outcomes of this study will lead to a greater understanding of the role that diet plays on the processes underlying the loss of muscle mass and musculoskeletal function in older individuals and hence to the logical development of interventions to correct these processes.

Nutrition, particularly low protein intake during foetal and/or early neonatal life has a long-term effect on skeletal muscle and neuromuscular homeostasis of the offspring, but little is known about the mechanisms involved. microRNAs are potent gene regulators, their muscle expression changes during ageing and following dietary interventions and they are an attractive candidate mechanism for regulating muscle changes resulting from unbalanced early life nutrition. We hypothesise that (1) in utero and/or early post-natal life, protein restriction disrupts miRNA-target interactions in muscles of the offspring and adversely affects neuromuscular homeostasis leading to a reduction in the number and size of myofibres; this in turn leads to premature reduced muscle mass as the mice age and (2) restoration of miRNA levels in muscle will prevent the loss of muscle mass and function associated with maternal and/or early post-natal protein restriction.
The project will use an established model of reduced protein intake (8% dietary protein) in mice. Offspring of protein-deficient dams will be maintained either with protein deficient dams or cross-fostered to lactating females fed control diet (20% protein) and vice versa. Mice will be culled at weaning, or weaned onto the deficient or the control diet until 3, 21 and 24 months. At specific ages, skeletal muscle structure and function will be assessed. Changes in miRNA and their predicted targets expression will be examined using RNA sequencing. miRNA:target interactions will be modelled using systems biology approaches and validated using qPCR and western blotting, as well as miRNA gain- and loss-of-function approaches in vitro. Using in vivo tail vein injections of miRNA mimics and antagomiRs we will determine whether restoration of miRNA:target interactions corrects the deficits in neuromuscular homeostasis, muscle fibre number and strength induced by in utero and/or post-natal protein restriction.

Researchers in the area of nutrition, geriatrics, gerontology and frailty will be direct beneficiaries upon completion of this research.
In the longer term the impact of this research will be on the health and quality of life of the elderly. Thus, local and national charities and policy-makers may benefit from regular contact with the research group throughout the project to disseminate information to promote and guide healthy ageing. An example of the approach used to facilitate this will be through attendance at the Institute of Ageing and Chronic Disease annual 'lay' Open Day by interested parties such and local and national charities and local MPs. This provides a forum for briefing such groups in both general terms and with specific information related to this project. In addition, local school children and teachers will be invited to attend the Open Day where we will hold specific interactive discussions to inform both schoolchildren and teachers. This will demonstrate the specific need for this research, inform about outcomes and provide an educational forum for increasing awareness of musculoskeletal biology. This approach will also help identify areas which school outreach parties may usefully focus on when small groups of active research staff (at all career levels), including the applicants and postdoctoral scientist funded on this project will visit local school. Full details for these activities are provided in the Pathways to Impact section.
The timescale of development of appropriate interventions deriving from the current work may be several years. However, if successful, the prevention of age-related loss of skeletal muscle mass and function would lead to an improvement in quality of life for elderly individuals with a major economic impact on UK health and social care costs.