Towards understanding EC coupling protein expression with age

Abstract

Sarcopenia is characterised by a decrease in muscle specific force that can only partially be attributed to muscle atrophy. Changes in fiber type composition of muscle have also been observed in humans with age. Additionally, it is suggested that excitation contraction coupling (EC coupling) is impaired in aging by uncoupling of the two calcium channels facilitating EC coupling, the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1), perhaps due to a decrease in expression of the DHPR alpha1s subunit which has been demonstrated in rodents. Changes in DHPR beta1a subunit expression have also been reported in aging mice but not investigated in humans. The 12kDa FK506 binding protein (FKBP12) stabilizes RyR1 in the closed state. Its dissociation from RyR1 causes a ""leaky"" channel and decreased EC coupling. Furthermore, RyR1 has two variably spliced regions, ASI and ASII, thought to be developmentally regulated. The ASI region, which lies close to a DHPR beta1a binding site, affects EC coupling. The juvenile isoform (ASI(-)RyR1) shows enhanced calcium release during EC coupling and its overexpression is linked to myopathy in myotonic dystrophy. Regeneration in aging muscle due to increased denervation and reinnervation can give rise to immature muscle fibers with higher levels of the juvenile isoforms of some proteins. Therefore the aim of this study was to investigate the fiber type distribution as well as levels of RyR1, DHPR alpha1s, beta1a, and FKBP12 in aged human muscle from 42 donors (aged 40-90yr) undergoing knee (vastus medialis) and hip replacements (gluteus minimus and gluteus medius). The levels of ASI splice variant transcripts in the muscle were investigated using RT-PCR. Furthermore, the effect of the addition of beta1a to recombinant ASI splice variant channels in lipid bilayers was investigated. Vastus medialis was predominantly fast twitch, whereas gluteus minimus and gluteus medius were predominantly slow twitch fibers. Contrary to expectation, age affected fiber type composition differently in the three human muscles. Also, contrary to rodent studies, no significant difference in the human expression levels of the RyR1, DHPR alpha1s and beta1a or FKBP12 with age was found. The ASI(+)RyR1:ASI(-)RyR1 ratio showed no significant change with age, however, an unexpected strong correlation between the fiber type and splice variant was found. In muscle with a high fraction of slow-twitch fibers, ASI(-)RyR1 predominates and vice versa. This novel finding suggests the ASI splice variants are fiber type specific rather than developmentally regulated. Finally, novel lipid bilayer results showed that ASI(+)RyR1 was activated more by 10nM beta1a than ASI(-)RyR1, but was similarly activated by 50nM beta1a, indicating a higher affinity of ASI(+)RyR1 for beta1a. Although no significant difference in expression levels of EC coupling proteins with age in humans was found, the discovery of fiber type specificity in RyR1 splice variants is important. This has not been shown before and provides a paradigm shift in understanding skeletal muscle changes in myotonic dystrophy. The discovery of different isoform affinities for beta1a may explain differences between EC coupling in fast and slow twitch fibers and changes in EC coupling in myotonic dystrophy.