Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of calcium

The sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca²⁺ stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and dynamic exchange of PLB between pentamers and SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic variants of PLB: a cysteine substitution of Arg9 (R9C) and an in-frame deletion of Arg14 (R14del). Both variants are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized this mutation may also alter pentamer stability. Immunoblots revealed a significantly increased pentamer:monomer ratio for R14del-PLB compared to WT-PLB. We quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT. The data suggest that, like R9C, the R14del mutation stabilizes PLB in pentamers, decreasing its ability to regulate SERCA. The R14del mutation reduces the rate of PLB unbinding from pentamers after transient elevations of Ca²⁺, limiting the recovery of PLB-SERCA complexes. A computational model predicted that hyperstabilization of PLB pentamers by R14del impairs the ability of cardiac Ca²⁺ handling to respond to changing heart rates between rest and exercise. We postulate that impaired responsiveness to physiological stress contributes to arrhythmogenesis in human carriers of the R14del mutation.