The Anthracycline Metabolite Doxorubicinol Abolishes RyR2 Sensitivity to Physiological Changes in Luminal Ca2+ through an Interaction with Calsequestrin.

The chemotherapeutic anthracycline metabolite doxorubicinol (doxOL) has been shown to interact with and disrupt the function of the cardiac ryanodine receptor Ca2+ release channel (RyR2) in the sarcoplasmic reticulum (SR) membrane and the SR Ca2+ binding protein calsequestrin 2 (CSQ2). Normal increases in RyR2 activity in response to increasing diastolic SR [Ca2+] are influenced by CSQ2 and are disrupted in arrhythmic conditions. Therefore, we explored the action of doxOL on RyR2's response to changes in luminal [Ca2+] seen during diastole. DoxOL abolished the increase in RyR2 activity when luminal Ca2+ was increased from 0.1 to 1.5 mM. This was not due to RyR2 oxidation, but depended entirely on the presence of CSQ2 in the RyR2 complex. DoxOL binding to CSQ2 reduced both the Ca2+ binding capacity of CSQ2 (by 48%-58%) and its aggregation, and lowered CSQ2 association with the RyR2 complex by 67%-77%. Each of these effects on CSQ2, and the lost RyR2 response to changes in luminal [Ca2+], was duplicated by exposing native RyR2 channels to subphysiologic (≤1.0 µM) luminal [Ca2+]. We suggest that doxOL and low luminal Ca2+ both disrupt the CSQ2 polymer, and that the association of the monomeric protein with the RyR2 complex shifts the increase in RyR2 activity with increasing luminal [Ca2+] away from the physiologic [Ca2+] range. Subsequently, these changes may render the channel insensitive to changes of luminal Ca2+ that occur through the cardiac cycle. The altered interactions between CSQ2, triadin, and/or junctin and RyR2 may produce an arrhythmogenic substrate in anthracycline-induced cardiotoxicity.

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

The skeletal muscle ryanodine receptor Ca(2+) 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 Ca(2+) 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.

BACKGROUND: Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca(2+) release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR ß1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of ß1a activation of RyRs and the ß1a binding site on RyR1. METHODS: We used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca(2+) currents and Ca(2+) 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 ß1a subunit. Activation of RyR2 by 10 nM ß1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for ß1a. A reduction in activation was also observed when 10 nM ß1a 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 ß1a 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 Ca(2+) transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for ß1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of ß1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca(2+) release generated during skeletal EC coupling. CONCLUSIONS: We conclude that the ß1a 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 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.