Utilization Of Proximity-Labeling To Identify Sarcomere Assembly Dependent Interactions

The cardiac sarcomere is a multiprotein complex that generates pulsatile contractile force, enabling the heart to circulate vital nutrients, but its molecular components and dynamic interactions are incompletely understood. Cardiac alpha-actinin-2 (encoded by ACTN2 ; referred to as actinin) functions as the structural backbone of the Z-disc and is prone to mutations that result in human heart failure. We hypothesize that through identifying actinin PPIs at the Z-disc, we can identify new sarcomere functions and gain insights into heart failure pathogenesis. Until now, studying sarcomere PPIs has been challenging due to limitations in purifying intact sarcomere complexes and the inability to detect dynamic molecular interactions. To overcome this, we adapted BioID proximity proteomics to comprehensively study actinin PPIs in human induced pluripotent stem cells (hiPSCs) differentiated into cardiomyocytes (iCMs).We engineered an isogenic hiPSC line carrying an in-frame knock-in of BirA* , which encodes a promiscuous biotin ligase, at the ACTN2 locus. Upon differentiation into iCMs, the Actinin-BirA* fusion construct localizes to the Z-disc and, upon biotin supplementation, biotinylates vicinal proteins. We immunoprecipitated and analyzed Z-disc PPIs via quantitative tandem mass spectrometry. Among the >100 PPIs detected by this method, we identified known interactions including sarcomere and focal adhesion proteins, but also new interactions including proteins involved in translation and metabolism. Furthermore, we wanted to better understand which of these protein interactions are dependent on sarcomere assembly and function, potentially elucidating interactions involved in normal cardiomyocyte biology and disease states. Through disruption of sarcomere assembly in our Actinin-BirA* model, we were also able to identify sarcomere-dependent actinin interactions including specific proteins involved in translation, the unfolded protein response, and glycolysis. Further investigation will allow us to better understand the proteomic network around actinin and provide novel insights into human sarcomere biology.