Creatine transporter content and localisation in human skeletal muscle: the effect of ageing and disuse

Luo, Dan (2020). Creatine transporter content and localisation in human skeletal muscle: the effect of ageing and disuse. University of Birmingham. Ph.D.

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Abstract

The maintenance of intramuscular creatine (Cr) is important for sustainability of skeletal muscle mass across the life-course. Creatine transporter (CreaT) is the only known specific Cr transporter which embedded in the cell membrane as a gate-keeper that transports Cr from blood into skeletal muscle. The impairments of structure and functions in CreaT will lead to insufficient intracellular Cr concentration, resulting in the disturbance of cellular energy homeostasis, metabolic disorders, muscle atrophy and neurological disorders. Impaired energy homeostasis could also have implications for dysregulated proteostasis, and muscle atrophy. Maintenance of proteostasis is essential to sustain skeletal muscle mass and therefore vital to health across the life-course. This is especially important to populations in the context of musculoskeletal ageing and scenarios of disease/injury. In this regard, detections of tissue CreaT content may aid the clinical diagnosis of diseases. As such, to understand the CreaT regulation mechanisms and its role in Cr metabolism can help the development of therapeutic interventions to improve intramuscular Cr levels and support muscle mass in health and disease. However, the role of CreaT in modulating Cr metabolism in skeletal muscle remains largely unexplored. The existing measurement techniques lack precision to fully explain the regulatory role of CreaT in different physiological scenarios of muscle deterioration. Accordingly, Chapter 2 of this thesis validated and optimized an immunofluorescence microscopy technique and provided a more precise approach to identify CreaT protein expression and distribution in human skeletal muscle. A new method of calculation was designed and developed to identify CreaT protein intensity in specific sub-cellular regions. By using these developed methods, Chapter 3 of this thesis investigated whether CreaT distribution and sub-cellular expression is altered in older age. A sub-set of quadriceps muscle biopsy samples from 6 young and 6 older healthy individuals were involved in study to identify CreaT content and distribution, Creatine kinase (CK) and high-energy phosphagens. No significantly difference of CreaT content was observed between young and older individuals but with a declined concentration of phosphocreatine (PCr) (P<0.05) in older muscles. CreaT protein localisation was determined to predominantly distribute at the cell peripheral in both young and old individuals (P<0.05) but with fibre type specific difference in old individuals. These results demonstrated that Cr metabolism did alter by ageing, however, CreaT content remains relatively unaffected. Chapter 4 of this thesis investigated the effect of immobilization on CreaT protein expression and distribution in human skeletal muscle. The 7-days leg immobilization trial was undertaken in which 15 healthy young individuals. The results elucidated that 7 days immobilization lead to a declined muscle isometric strength and fat free mass (P<0.01), in addition with a reduced type II muscle fibre cross-section area (CSA) (P<0.05). CreaT protein content was greater in immobilised leg compared control leg (P<0.01). CreaT was mainly distribute at the cell peripheral in both immobilised and control legs with fibre type specific difference (P<0.01). These outcomes determined that the greater muscle fibre CreaT expression with immobilization may have important implications for skeletal muscle energy production during disuse-induced atrophy and in the recovery of muscle mass upon re-loading.
In conclusion, this thesis provides a novel immunofluorescence approach to investigate CreaT activities in human skeletal muscle. This developed method provides valuable insight into the role of CreaT in alteration in intramuscular Cr metabolism across the spectrum of health and disease. In addition, this thesis enhances our understanding of CreaT protein expression and sub-cellular localisation in the role of Cr in skeletal muscle physiology.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):