In cardiomyocytes, dyads are nanoscale structures formed by the juxtaposition of T-tubules, a network of tubular invaginations of the plasma membrane, and regions of the endoplasmic reticulum specialized for Ca2+ release, known as the junctional sarcoplasmic reticulum (jSR). Dyads are positioned adjacent to Z-lines, such that sarcomere Z-lines, jSR, and T-tubules co-localize in a regular, transverse, linear pattern. Dyads mediate excitation-contraction (E-C) coupling, which converts rapidly propagating plasma membrane electrical signals into coordinated Ca2+ transients throughout the cardiomyocyte, resulting in synchronized, forceful sarcomere contraction. A hallmark of failing cardiomyocytes is disorganization of dyads, which disrupts Ca2+ handling and results in decreased contraction and increased risk of arrhythmia. The molecular mechanisms underlying dyad architecture and positioning have remained a mystery, despite their importance to heart homeostasis and disease. Our preliminary data establish a hierarchy for dyad formation in which a little studied protein, CMYA5, tethers jSR to sarcomere Z-lines, and T-tubules associate with jSR to form dyads. We further show that CMYA5 is required for normal dyad architecture, fidelity of E-C coupling, and regulation of RYR2 Ca2+ release activity. Mice lacking CMYA5 had dilated cardiomyopathy and were sensitized to develop severe cardiac dysfunction in response to pressure overload. In failing human hearts, loss of T-tubule and jSR organization were coupled to perturbed CMYA5 localization. Our studies establish CMYA5 as a novel entry point to study mechanisms responsible for dyad architecture and positioning adjacent to Z-lines, and implicate abnormalities of CMYA5-dependent mechanisms in the disorganization of dyads in human heart failure3–5, which contributes to heart failure pathogenesis. Building on these novel observations, we will pursue the following Specific Aims to gain further insights into the function of CMYA5 in regulating CM Ca2+ release and E-C coupling: (1) Investigate CMYA5 regulation of RYR2 activity. We will test the hypothesis that CMYA5 interaction with RYR2 regulates RYR2 Ca2+ release by controlling RYR2 channel activity and RYR2 channel clustering. (2) Identify mechanisms by which CMYA5 tethers RYR2/jSR to Z-lines. We will test the hypothesis that CMYA5 anchors RYR2/jSR at Z-lines through interaction with currently unknown bridging proteins. (3) Evaluate contribution of CMYA5 mislocalization to dyad disruption in human and experimental heart disease. This proposal will reveal novel mechanisms responsible for the subcellular organization of dyads, hallmark nanostructures of CMs that are essential for normal E-C coupling. Elucidation of these mechanisms will provide insights into the mechanisms that perturb dyads in human heart failure, exacerbating contractile dysfunction and arrhythmia, and may lead to avenues to protect E-C coupling in inherited and acquired forms of heart disease.