Proteomic and high-resolution microscopy analysis of the role of Dysferlin in membrane remodelling after myocardial infarction

Introduction:

Dysferlin, a large 230 kDa transmembrane protein with multiple Ca2+-binding C2-domains, is thought to aid in membrane repair by sealing sarcolemmal lesions upon Ca2+ entry. Mutations in Dysferlin are linked to dilated cardiomyopathy. However, its role in protecting membrane nanodomains in ventricular myocytes after myocardial infarction remains unclear.

Methods:

Myocardial infarction (MI) in adult wild-type (WT) and Dysferlin knockout (KO) mice was induced by ligating the left anterior descending artery. Sham-operated animals served as controls. Following heart extraction one week post-MI in n=5 mice per group, left ventricles were dissected into infarction zone, infarction border zone and remote zone under microscopic control. Left ventricular (LV) samples were lysed by pressure cycling technology, digested using 2% SDS and amine magnetic beads, and analysed by data-independent acquisition mass spectrometry (DIA-MS) on the Bruker timsTOF Pro. Additionally, we investigated the cardiac Dysferlin interactome in absence and presence Ca2+ via co-immunoprecipitation and DIA-MS of short run SDS-PAGE purified and in gel trypsin digested LV samples. Ultimately, super-resolution stimulated emission depletion (STED) microscopy was used to investigate the subcellular localization, function and interaction partners of Dysferlin in the MI border zone.

Results:

One week post-MI, echocardiography revealed larger infarct sizes and decreased LV ejection fraction in KO compared to WT mice (mean±SEM: 21±2% vs. 28±2%, n=20/30 mice, P<0.05). We reproducibly quantified 4.360 proteins including large transmembrane proteins across all LV samples, thereby highlighting genotype- and MI zone-specific proteomic changes. In the MI border zone, 725 differentially abundant proteins were identified in WT vs. KO samples, underscoring Dysferlin's crucial role in LV remodelling post-MI. Co-immunoprecipitation experiments from WT vs. KO LV tissues revealed previously unknown cardiac protein interactions with the Ca2+ release channel Ryanodine receptor type 2 and the gap junctional hemichannel Connexin-43. Consequently, STED microscopy was applied to study the subcellular localization and function of Dysferlin at Ca2+ release units and the intercalated disc membrane folds, revealing Dysferlin to protect specific membrane nanodomains in the MI border zone.

Conclusions:

In a spatial proteomics approach, we uncovered the proteomic basis of specific LV zones in MI by DIA-MS. Dysferlin deficiency further worsened LV remodelling post-MI as shown in deep proteomic profiling. Co-immunoprecipitation experiments and DIA-MS analysis characterized the cardiac Dysferlin interactome, including protein interactions with crucial Ca2+ release and membrane proteins in cardiomyocytes, which were confirmed by super-resolution microscopy. Therefore, Dysferlin emerges as a novel therapeutic target to maintain membrane integrity in cardiac tissue and prevent from cell death post-MI.