Deficits in Ca2+ Release and in vivo Muscle Strength in Heterozygous I4895T RyR1 Knock-In Mice

The mutation from isoleucine to threonine of the skeletal isoform of the ryanodine receptor (RyR1) at residue 4898 results in severe Central Core Disease (CCD). Under homozygous expression (IT/IT), we reported a lack of Ca2+ release in response to electrical and pharmacological activation despite SR Ca2+ store content indistinguishable from control. Here we used heterozygous knock-in mice for the I4895T (IT/+; analogous to human I4898T) RyR1 mutation to determine the effects of the mutation on muscle strength and Ca2+ handling in flexor digitorum brevis (FDB) and interosseous muscle fibers.We compared in vivo muscle strength of wild-type (WT) and IT/+ mice. IT/+ mice exhibited significant weakness in both upper body and grip strength assays (4-paw peak grip force: 2400 ± 70 mN, n=8 and 2040 ± 80 mN, n=14 in WT and IT/+ mice, respectively). We also determined the magnitude of action potential- and ligand-evoked Ca2+ release in single intact FDB fibers using Ca2+ fluorometry. The magnitude of both electrically- and ligand-evoked Ca2+ release was significantly reduced in IT/+ fibers. Moreover, the maximum rate of change in mag-fluo-4 fluorescence during the rising phase of the electrically-evoked Ca2+ transient was significantly reduced in IT/+ fibers (WT 0.17 ± 0.01 ΔF/F/ms vs IT/+ 0.11 ± 0.01 ΔF/F/ms, n= 53, 56, respectively). Finally, the frequency (1.9 ± 0.5 and 0.8 ± 0.3 events/scan) and Ca2+ spark mass (5.9 ± 0.3 and 4.6 ± 0.2 μm3) of local Ca2+ release induced by osmotic shock (440 mOsm with sucrose, 750 lines/sec) were reduced in acutely dissociated IT/+ interosseous fibers compared to that of WT fibers. Together, these findings are consistent with the hypothesis that the IT mutation in the putative RyR1 selectivity filter significantly reduces Ca2+ flux through the channel.