Programme Contents

Human aging and expression of proteins interacting with the ryanodine receptor in skeletal muscle

Sarcopenia is the progressive decline in muscle function with age, characterized by reduced force generating capacity and loss of muscle mass. Aging skeletal muscle shows a decrease in specific force that is only partially attributed to muscle atrophy. A change in fibre type distribution has also been observed, with fast twitch fibres decreasing and slow twitch fibres increasing. It has been suggested that excitation contraction coupling (ECC) may be impaired in aging by an uncoupling of the two Ca²⁺ channels facilitating ECC, the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1). This uncoupling is possibly due to a decrease in expression of the DHPR α_1s subunit (Delbono et al., 1995; Renganathan et al., 1997). In skeletal muscle, the C-terminal tail of the DHPR β_1a subunit activates the RyR1 and this interaction may contribute to EC coupling (Rebbeck et al., 2011). Apart from the DHPR, there are many other proteins that modulate RyR1 activity. One of these is the FK506 binding protein 12 (FKBP12) which stabilizes the RyR1 in the closed state and its dissociation from the RyR1 causes a leaky channel. A decreased EC coupling Ca²⁺ transient has also been ascribed to the dissociation of FKBP12 from the RyR1 due to increased nitrosilation occurring in aged muscle (Andersson et al., 2011).

Human muscle samples were obtained from 42 donors (age 40-90) undergoing knee and hip replacements. The samples were taken from the gluteus minimus, gluteus medius or vastus medialis depending on the operation. Details of the samples can be seen in the Table below. The muscle was immediately processed in the operating theatre in phosphate buffered saline containing 1mM EGTA and snap frozen in liquid nitrogen. The fibre type distribution in muscle homogenate was determined for each muscle sample by SDS PAGE using an 8% polyacrylamide (99:1) gel containing 35% glycerol and a runtime of 20h at 140V and 4°C. Densitometry was used to determine the slow and fast twitch percentages. Expression levels of the DHPR α_1s and β_1a subunits relative to that of actin were determined in muscle homogenates using western blot and densitometry. Muscle homogenates were used as we have found that most of the β_1a is lost during microsomal vesicle preparation. The levels of FKBP12 to RyR1 were determined in microsomal vesicles prepared from the same donors using western blot and densitometry.

Thus far we have analysed the fibre distribution of 6 vastus medialis (4 female, 2 male), 5 gluteus minimus (2 female, 3 male) and 2 gluteus medius (1 female, 1 male) samples. There is a higher fraction of fast twitch fibres in vastus medialis, whereas gluteus minimus has a higher fraction of slow twitch fibres. Clear trends are visible in the female vastus medialis and gluteus minimus samples where fast twitch fibre percentage decreases with age and the slow twitch percentage increases. More data points are needed for the male muscles. Preliminary western blot results show a decrease of FKBP12 relative to the RyR1 with age. Levels of α_1s and β_1a are being processed. It appears that the levels of several of the proteins that modulate the RyR1 and are associated with it may change with age and could affect the release of Ca²⁺ during ECC and muscle function.

Andersson DC, Betzenhauser MJ, Reiken S, Meli AC, Umanskaya A, Xie W, Shiomi T, Zalk R, Lacampagne A & Marks AR. (2011). Cell Metabolism 14, 196-207.

Delbono O, O'Rourke KS & Ettinger WH. (1995). Journal of Membrane Biology 148, 211-222.

Rebbeck RT, Karunasekara Y, Gallant EM, Board PG, Beard NA, Casarotto MG & Dulhunty AF. (2011). Biophysical Journal 100, 922-930.

Renganathan M, Messi ML & Delbono O. (1997). Journal of Membrane Biology 157, 247-253