ABSTRACT: The structural and functional integrity of long-lived cells, like cardiomyocytes, depends heavily on robust degradation mechanisms, and disruption of these mechanisms can drive protein accumulation and cellular degeneration. Genetic variants in phospholamban (PLN), the main inhibitor of the sarco/endoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a), cause inherited cardiomyopathy characterized by the abnormal perinuclear accumulation of PLN protein resulting in a progressive loss of cardiomyocytes. However, the nature of these PLN structures, the mechanisms driving their formation, and their role in disease progression remain largely unresolved. Using a plna R14del zebrafish model, left ventricular tissue from end-stage PLN R14del cardiomyopathy patients, and pharmacological inhibition of SERCA2a in wild type (WT) cardiac slices, we demonstrate that abnormal perinuclear accumulation of PLN represents clusters of sarcoplasmic reticulum (SR)-derived PLN-containing vesicle-like structures that form due to impaired SERCA2a activity and increased cytosolic Ca²⁺ levels. Furthermore, we show that these SR-derived vesicle-like structures often localize adjacent to lysosomes. Interestingly, Ca²⁺dysregulation in plna R14del hearts leads to reduced lysosomal function, resulting in the accumulation of SR-derived vesicle-like structures at the microtubule organizing center (MTOC). Strikingly, the accumulation of vesicle-like structures at the MTOC is accompanied by reorganization of the linear microtubule network into asters, ion-channel mislocalization, sarcomere disorganization, and nuclear deformation. Restoration of lysosomal function through Ca²⁺ buffering or fasting reduces vesicle accumulation, restores microtubule organization, and rescues cellular architecture. Collectively, our study defines a Ca²⁺-sensitive pathway linking the formation of SR-derived vesicle-like structures, lysosomal dysfunction, and microtubule remodeling to pathological cellular rearrangements.
