Abstract Body: Dilated cardiomyopathy (DCM), characterized by systolic dysfunction, is a significant cause of heart failure. Myosin heavy chain 7 (MYH7) pathogenic variants are common causes of DCM; however, no specific disease-modifying therapy for MYH7 DCM exists. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing the pathogenic MYH7 E848G DCM variant, we confirmed hypocontractility at the single cell level as assessed with traction force microscopy. hiPSC-CMs were cultured on polyacrylamide hydrogels with physiological stiffness in 15x100 µm patterned Matrigel-coated rectangles, each containing one cell. Upon 1 Hz electrical stimulation, contracting cells displaced fluorescent beads embedded within the gel, which were measured to estimate twitch force. MYH7^E848G/+ hiPSC-CMs had reduced maximum twitch force (97 ± 11 nN, n=46 cells) compared to MYH7^+/+ hiPSC-CMs (179 ± 18 nN, n=49 cells), recapitulating the disease phenotype. RNAseq analysis of MYH7^E848G/+ hiPSC-CMs compared to isogenic control MYH7^+/+ hiPSC-CMs found significant downregulation of desmin (DES) (76.4 ± 8.2% reduction, n=4 bio reps), which encodes a muscle-specific type III intermediate filament. Desmin protein expression in MYH7^E848G/+ hiPSC-CMs was significantly attenuated relative to MYH7^+/+ hiPSC-CMs (90.9 ± 1.8% reduction, n=4 bio reps), matching gene expression data. Gene expression of the transcription factor MEF2C, a known transactivator of DES, was also reduced (40.4 ± 7.0% reduction, n=4 bio reps), suggesting MYH7^E848G/+ suppresses the MEF2C-DES signaling axis. We hypothesized restoring desmin protein levels may improve contractility in MYH7^E848G/+ hiPSC-CMs. To test this, we created an adeno-associated virus (AAV9) packaged with full-length desmin cDNA fused with a T2A self-cleaving peptide and mApple fluorescent reporter. hiPSC-CMs were transfected with this mApple-T2A-DES AAV at 1 MOI and contractility was assessed via traction force microscopy. mApple⁺ MYH7^E848G/+ hiPSC-CMs had increased maximum twitch force (141 ± 11 nN, n=75 cells) relative to mApple^- MYH7^E848G/+ hiPSC-CMs (65 ± 20 nN, n=21 cells), approaching the maximum twitch force of mApple⁺ MYH7^+/+ hiPSC-CMs (165 ± 10 nN, n=87 cells). In conclusion, suppression of the MEF2C-DES signaling axis is a potential novel mechanism by which pathogenic MYH7 variants cause DCM. By leveraging this mechanistic insight, we found that rescuing desmin insufficiency can partially restore contractile function.