Towards understanding EC coupling protein expression with age

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

Unexpected dependence of RyR1 splice variant expression in human lower limb muscles on fiber-type composition

The skeletal muscle ryanodine receptor Ca2+ release channel (RyR1), essential for excitation-contraction (EC) coupling, demonstrates a known developmentally regulated alternative splicing in the ASI region. We now find unexpectedly that the expression of the splice variants is closely related to fiber type in adult human lower limb muscles. We examined the distribution of myosin heavy chain isoforms and ASI splice variants in gluteus minimus, gluteus medius and vastus medialis from patients aged 45 to 85 years. There was a strong positive correlation between ASI(+)RyR1 and the percentage of type 2 fibers in the muscles (r = 0.725), and a correspondingly strong negative correlation between the percentages of ASI(+)RyR1 and percentage of type 1 fibers. When the type 2 fiber data were separated into type 2X and type 2A, the correlation with ASI(+)RyR1 was stronger in type 2X fibers (r = 0.781) than in type 2A fibers (r = 0.461). There was no significant correlation between age and either fiber-type composition or ASI(+)RyR1/ASI(−)RyR1 ratio. The results suggest that the reduced expression of ASI(−)RyR1 during development may reflect a reduction in type 1 fibers during development. Preferential expression of ASI(−) RyR1, having a higher gain of in Ca2+ release during EC coupling than ASI(+)RyR1, may compensate for the reduced terminal cisternae volume, fewer junctional contacts and reduced charge movement in type 1 fibers

Regions of ryanodine receptors that influence activation by the dihydropyridine receptor ß1a subunit

BACKGROUD: Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca2+ release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR β1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of β1a activation of RyRs and the β1a binding site on RyR1. METHODS: we used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca2+ currents and Ca2+ transients in myotubes. RESULTS: We demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10–100 nM of the β1a subunit. Activation of RyR2 by 10 nM β1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for β1a. A reduction in activation was also observed when 10 nM β1a was added to the alternatively spliced (ASI(−)) isoform of RyR1, which lacks ASI residues (A3481-Q3485). It is notable that the equivalent region of RyR2 also lacks four of five ASI residues, suggesting that the absence of these residues may contribute to the reduced 10 nM β1a activation observed for both RyR2 and ASI(−)RyR1 compared to ASI(+)RyR1. We also investigated the influence of a polybasic motif (PBM) of RyR1 (K3495KKRRDGR3502) that is located immediately downstream from the ASI residues and has been implicated in EC coupling. We confirmed that neutralizing the basic residues in the PBM (RyR1 K-Q) results in an ~50 % reduction in Ca2+ transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for β1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of β1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca2+ release generated during skeletal EC coupling. CONCLUSIONS: We conclude that the β1a subunit likely binds to a region that is largely conserved in RyR1 and RyR2 and that this region is influenced by the presence of the ASI residues and the PBM in RyR1.The work was supported by grants from the Australian National Health and Medical Research Council, APP1020589 and APP APP1002589 to AFD, MGC, and PGB, Muscular Dystrophy Association (MDA275574) and National Institutes of Health (AR059646) to RTD, a Career development award (APP1003985) to NAB, an Australian Postgraduate Award to RTR, and an Australia National University postgraduate award to HW

The elusive role of the SPRY2 domain in RyR1

The second of three SPRY domains (SPRY2, S1085-V1208) located in the skeletal muscle ryanodine receptor (RyR1) is contained within regions of RyR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RyR1 channel gating. We examined the binding of the F loop (P1107-A1121) in SPRY2 to the ASI/basic region in RyR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling. A peptide with the F loop sequence and an antibody to the SPRY2 domain each enhanced RyR1 activity at low concentrations and inhibited at higher concentrations. A peptide containing the ASI/basic sequence bound to SPRY2 and binding decreased ∼10-fold following mutation or structural disruption of the basic residues. Binding was abolished by mutation of three critical acidic F loop residues. Together these results suggest that the ASI/basic and SPRY2 domains interact in an F loop regulatory module. Although a region that includes the SPRY2 domain influences EC coupling, as does the ASI/basic region, Ca2+ release during ligand- and depolarization-induced RyR1 activation were not altered by mutation of the three critical F loop residues following expression of mutant RyR1 in RyR1-null myotubes. Therefore the electrostatic regulatory interaction between the SPRY2 F loop residues (that bind to imperatoxin A) and the ASI/basic residues of RyR1 does not influence bi-directional DHPR-RyR1 signaling during skeletal EC coupling, possibly because the interaction is interrupted by the influence of factors present in intact muscle cells