Cardiac excitation-contraction (E-C) coupling requires dyads, the nanoscopic microdomains formed adjacent to Z-lines by apposition of transverse (T)-tubules and junctional sarcoplasmic reticulum (jSR). Disruption of dyad architecture and function are common features of diseased cardiomyocytes. However, little is known about the mechanisms that modulate dyad organization during cardiac homeostasis and disease. Here, we used proximity proteomics in intact, living hearts to identify proteins enriched near dyads.