King's College London

Sarcopenia, a condition characterised by progressive loss of skeletal muscle mass and strength, has been associated with an intrinsic deterioration of the force-generating capacity of individual myofibres. As the reduction in force-generation is greater than the loss of muscle fibre volume (specific force loss), a decrease in cellular quality may contribute to the aforementioned weakness. The work presented in this thesis aimed to further elucidate the mechanisms by which myofibre force is depressed during the ageing process, using a plethora of animal and human models, with specific reference to myonuclear proteins and organisation. Existent ex vivo methods to study myonuclear organisation in single muscle fibres did not fulfil the methodological necessities of this project. Thus, methods of varying degrees of automation were developed to study the (i) three-dimensional nuclear organisation, and (ii) nuclear pixel intensities of a range of antibodies, in confocal image stacks of isolated skinned single muscle fibres. Analyses were performed using custom-made MATLAB scripts and Fiji macros. Both methods were used in the present thesis and various related publications. Physiological and premature ageing are frequently associated with an accumulation of prelamin A, a precursor of lamin A, in the nuclear envelope of various cell types. The current work aimed to underpin the hitherto unknown mechanisms by which accumulation of prelamin A in the nucleus alters myonuclear organisation and muscle fibre function. By applying the methods developed in this work on single muscle fibres from various mouse models of lamin A misprocessing, results indicated that postnatal accumulation of farnesylated prelamin A and absence of Zmpste24 in myonuclei can lead to a low number of nuclei and enlarged myonuclear domains in single muscle fibres, concomitant with decreased transcriptional activity, reduction in contractile protein content and low specific force. ABSTRACT 3 Scientific studies on human ageing are limited by inclusion of sedentary cohorts. Hence, the role of myonuclear organisation is unknown in biological ageing, i.e. ageing that is not contaminated by inactivity-related pathologies. The final objective in this thesis was to verify whether levels of activity and age can lead to changes in nuclear organisation in single muscle fibres. Results showed an elevated number of nuclei in single muscle fibres from physically inactive patients and nuclear mispositioning in all the elderly cohorts. Transcriptional activity in the active cohort was >2x that of all other groups, revealing a possible association between level of physical activity, but not age. Consequently, biological ageing and muscle disuse do not share the same myonuclear alterations, demonstrating the importance of differentiating between the two conditions in ageing research. Future work should deepen the understanding of the mechanisms by which the persistence of farnesylated prelamin A and absence of Zmpste24 in myonuclei lead to a smaller nuclear population in single muscle fibres. Moreover, research should continue to characterise the specific contribution of muscle disuse to human ageing. Importantly, future studies should identify whether any correlation exists between the existence of prelamin A and progerin in single muscle fibres, and age and physical activity.