Background and Purpose: Mavacamten (MAVA) is a novel small molecule inhibitor of cardiac myosin, mitigating cardiomyocyte hypercontractility in patients with hypertrophic obstructive cardiomyopathy (HOCM). Despite its recent approval for clinical use, the transcriptional and functional impacts of MAVA remain not well understood. In this study we investigate the effects of MAVA across diverse cardiac models, including healthy female porcine cardiomyocytes and myocardial slices, human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs), cardiac organoids and living myocardial slices (LMSs) derived from patients with HOCM. Experimental Approach: Long‐term LMS culture facilitated continuous force measurements, while SarcTrack and MUSCLEMOTION analyses were used to evaluate contractility in cardiomyocytes and cardiac organoids. Transcriptome profiling of MAVA‐treated HOCM hiPSC‐CMs and HOCM LMSs allowed in‐depth examination of gene expression signatures in response to MAVA treatment. Key Results: Across all models tested, MAVA demonstrated robust force inhibition. In primary disease models, MAVA showed little effect on time to peak or relaxation times and even reduced contraction and relaxation velocities. By contrast, in engineered human HOCM models, MAVA accelerated both contraction and relaxation, suggesting potential model‐specific effects. Transcriptome analyses revealed that MAVA treatment not only influenced contraction regulation but also significantly altered cytoskeleton organization, muscle stretch response, and metabolic pathways. Notably, in LMSs derived from three HOCM patients, MAVA treatment up‐regulated myosin binding protein H (MyBPH) expression, suggesting that MyBPH may also be involved in contraction regulation. Conclusion and Implications: These data suggest that MAVA not only inhibits force within the sarcomere but also influences transcriptional pathways in model‐specific manner
