Abstract Diastolic heart disease (DHD) is the leading cause of death and disability worldwide. With no FDA approved therapies to improve diastolic function, the NIH has placed special emphasis on collaborative initiatives bridging basic science and clinical data to understanding the molecular mechanism of DHD. The hallmarks of diastolic heart disease are a stiffening left ventricle and an inability for the heart muscle to relax, both of which occur in DHD by mechanisms that are poorly understood. Building on recent work that the cardiac microtubule network (MTN) can both stiffens and impairs the relaxation of failing cardiomyocytes, in Aim 1 we determine the relative contribution of the MTN to myocardial stiffness in intact preparations of HTN and DHD ventricular myocardium. We isolate the myocyte and fibrotic contribution to stiffening and use complimentary reductionist approaches to isolate the relative contributions of actomyosin activity, passive titin stiffness, and the MTN to myocyte mechanics. In Aim 2 we extend these findings into a clinically analogous ex-vivo working myocardial slice preparation to dissect the MTN impediment to the phases of diastole in HTN and DHD. The working preparation allows us to contextualize the findings in Aim 1 with the emergent properties arising from cyclically contracting loaded myocardium. Thus, by carefully designing a series of complementary assays we will resolve the molecular contributions to cardiac stiffening and impaired relaxation in DHD, and in doing so determine the potential for MTN-targeting therapies in DHD.