In vivo proximity labeling reveals altered protein networks in PLN-R14del cardiomyopathy

Background Phospholamban (PLN) is a pivotal regulator of calcium handling and excitation–contraction coupling in cardiomyocytes. The pathogenic variant PLN-R14del (p.Arg14del) causes cardiomyopathy, often leading to severe heart failure and arrhythmias. We recently demonstrated that abnormal sarcoplasmic reticulum (SR) clustering caused by the mutant protein is the primary disease-causing mechanism in a PLN-R14del knock-in mouse. However, the mechanisms driving this phenotype remain unknown. Purpose To uncover mechanisms underlying SR disorganization by elucidating the protein interaction network of PLN-R14del. Methods A BioID2 proximity-labeling approach was applied in wild-type (WT) and homozygous PLN-R14del (R14Δ/Δ) mice. Mice were injected with AAV9 vectors encoding PLN-R14del-BioID2, PLN-BioID2, or a Luciferase control, followed by one week of biotin supplementation. Cardiac function was assessed by echocardiography. PLN-BioID2 fusion protein expression and biotinylation efficiency were evaluated by Western blotting and immunofluorescent staining. Biotinylated peptides from trypsin digested cardiac lysates were enriched using streptavidin beads and analyzed by mass spectrometry. Results PLN-BioID2 fusion proteins were robustly expressed in cardiomyocytes and induced efficient protein biotinylation without affecting cardiac function. Mass spectrometry analysis revealed a highly enriched set of biotinylated proteins in PLN–BioID2 cardiac lysates that were absent in control lysates. Endogenous PLN was detected in both WT and R14Δ/Δ lysates, supporting incorporation into native PLN pentameric complexes. Notably, p62, a marker of abnormal PLN-SR clusters, was selectively enriched in R14Δ/Δ lysates. Gene Ontology analysis revealed enrichment of cardiac muscle contraction, electrical conduction, and muscle development in WT hearts. In contrast, R14Δ/Δ hearts showed enrichment of pathways associated with impaired protein folding, intracellular transport, protein localization, and endoplasmic reticulum stress. Conclusion PLN–BioID2 proximity labeling reliably captures the native PLN interactome in vivo. Moreover, the distinct enrichment of protein quality control and ER stress pathways in PLN-R14del hearts indicates mutation-specific remodeling of the PLN interactome associated with proteostatic dysfunction.