PROJECT SUMMARY/ABSTRACT Dilated cardiomyopathy (DCM) is associated with impaired systolic/pump function of the heart, which can lead to heart failure and death. It is estimated that 1 in 250 adults in the U.S. have DCM, with ~40% of these cases being attributed to genetic causes. A common characteristic of DCM is reduced stiffness of titin. Titin is a molecular spring that provides passive tension to the heart by functioning within contractile units known as myofibrils. It is believed that re-establishing titin spring function (i.e. stiffening titin) in DCM patients could provide an innovative, disease-modifying approach to treat cardiomyopathy. Unpublished findings from our collaborator, Dr. McKinsey, reveal a remarkable ability of the cytoplasmic protein, histone deacetylase 6 (HDAC6), to control the stiffness of titin. The long-term objective of the proposed work is to develop an HDAC6-selective small molecule inhibitor as a ‘titin stiffener’ to improve systolic function and treat DCM in humans. This is in-line with the mission of the NHLBI, which includes treatment of heart disease to enhance the health of individuals so they can live longer and more fulfilling lives. One specific aim is to rank-order novel HDAC6 inhibitors (discovered by Eikonizo Therapeutics) for their ability to increase titin stiffness in cultured adult rat cardiomyocytes, and for their ability to improve systolic cardiac function in a short-term mouse model of heart failure. In a second specific aim, compounds that advance through these initial filters will be tested for efficacy in a rat model of DCM characterized by severe titin softening. This rat model recapitulates many elements of RBM20 (RNA-binding motif protein 20, a splicing factor that targets titin) cardiomyopathy, an aggressive and early onset genetic DCM in humans. This drug discovery proposal has the potential to facilitate the development of transformative therapies to treat DCM in humans.