Heart failure is often marked by stiffening of cardiac tissue that impairs the heart’s ability to relax. The microtubule network – a part of the cardiomyocyte cytoskeleton – provides an internal stiffness that can impede cardiomyocyte contraction and relaxation. We have recently found that cardiomyocyte microtubule network stiffness is tightly regulated by post-translational detyrosination and that microtubules, detyrosination, and cytoskeletal cross-linkers are consistently elevated in heart failure, with concomitant increases in cardiomyocyte stiffness. Our findings that reducing detyrosination lessens microtubule network density and contractile defects in cardiomyocytes from patients with advanced heart failure supports detyrosination as a therapeutic target. At the same time, these findings raise important questions about the processes driving remodeling of the microtubule network in heart failure and the consequences of sustained increases in the microtubule network over time. Accordingly, the proposed research will test the hypothesis that remodeling of the cardiac microtubule network is a reversible adaptation to altered mechanical stress, which when sustained, contributes to pathological hypertrophy and contractile dysfunction. Studies under three aims will address the multiple components of this hypothesis. Aim 1 experiments will determine if mechanical stress is sufficient to drive cell-autonomous remodeling of the microtubule network using a mechanobiology toolkit to isolate the contribution of three key mechanical stressors (pre-load, after-load, and matrix stiffness) on microtubule network remodeling. Aim 2 experiments will extend our mechanical manipulations to the tissue and organ level to characterize microtubule network remodeling under relevant in vivo contexts. Aim 3 studies will employ in vitro and in vivo genetic manipulations to determine whether sustained increases in detyrosination contribute to pathologic cardiac hypertrophy in the presence and absence of chronic pressure overload. Our overall study design uses novel and complementary experimental approaches to both exploit strengths of model systems and mitigate their shortcomings. This includes primary cardiomyocytes from human myocardium to complement findings from animal models and engineered tissue constructs. This cross-species, multi-scale approach balances the dual goals of reductionist rigor and integrative relevance that furthers ultimate clinical translation. Together this work will provide insight into the causes of microtubule network changes in heart failure and help determine if preventing or reversing these changes is therapeutically beneficial.