by David Conesa, Blas Echebarria, Leif Hove-Madsen, Yohannes Shiferaw, Enrique Alvarez-LacalleIntracellular calcium waves in cardiac myocytes propagate through a fire-diffuse-fire mechanism in which calcium released from one RyR2 cluster diffuses to neighboring clusters and triggers their activation. Yet propagation faces a fundamental physical difficulty: the calcium signal must cross distances of 1–2 μm between Z-planes while being attenuated by cytosolic buffering and diffusion, and at the same time the release site depletes its local sarcoplasmic reticulum calcium store. How waves propagate efficiently despite these constraints has remained unclear. We developed a three-dimensional computational model of mouse ventricular myocytes at submicrometer resolution to address this question. Our central finding is that co-localization of calsequestrin2 (CASQ2) with RyR2 clusters is required for robust wave propagation. In a physiological model, where CASQ2 is concentrated at release sites as observed experimentally, calcium waves propagate reliably across the cell with velocities that match the experimental range. In contrast, when CASQ2 is distributed uniformly throughout the sarcoplasmic reticulum, keeping total CASQ2 unchanged, the wavefront stalls. These results identify CASQ2–RyR2 co-localization as a key structural requirement for effective calcium wave propagation in ventricular myocytes.