This study investigates the role of REEP5 in maintaining organelle integrity and redox homeostasis in cardiac tissue (specifically cardiomyocytes). Using AAV9-mediated shRNA knockdown in postnatal mice, we acheived a cardiac-specific depletion of REEP5. Hearts were harvested 4 weeks post-injection and subjected to subcellular fractionation to isolate microsomal (SR/ER-enriched), mitochondrial, and cytosolic cellular compartmetns. Each fraction was analyzed using data-independent acquisition (DIA) LC-MS/MS on a Q Exactive Plus instrument and the resulting dataset caputres an organelle-specific proteomic profile following depletion of REEP5. Loss of REEP5 leads to changes in the proteomic profiles of targets involved in regulating SR/ER membrane dynamics, ER-mitochondrial communications, mitochondrial morphology and oxidative stress. This work highlights REEP5 as an important regulator of ER-mitochondrial communication and cardiac homeostasis.