Structure, Function and Drug Treatment of the Native Cardiac Sarcomere in Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disorder. Over 35% of the ~400 mutations identified in HCM patients occur in the motor protein β-myosin. In vitro studies of the R403Q mutant β-myosin revealed hypercontractility of myosin cross-bridge dynamics, including increased ATP turnover and more myosin molecules in the disorganized relaxed sate (DRX) that enables excessive β-myosin contributions to ventricular contractions. When treated with the only FDA-approved HCM-drug mavacamten, the biochemical and structural properties of the isolated mutant β-myosin resembled the WT control. However, direct observation of β-myosin’s conformational alterations due to HCM inducing mutations or mavacamten treatment have yet to be observed in situ. Cryo-electron tomography (cryo-ET) is a powerful imaging technique that enables the visualization of several native cellular macromolecular structures inside intact cells, such as intact myofibrils from cardiomyocytes. However, many of the cryo-ET studies of muscle have been limited to isolated subcellular structures in vitro rather than intact, unperturbed (cardio)myocytes. Additionally, studies often immersed the isolated contractile machinery in conformation inducing buffers before plunge freezing and cryo-ET, possibly further altering the native ratio of myosin active and inactive states. There is a direct need to identify if the isolation procedures utilized to obtain myofibrils result in similar β-myosin structural characteristics found in situ. Further, accurate depiction of native wild type cardiac β-myosin structural features are required to accurately understand how pathological mutations and treatments influence and affect cardiac function and health. To view these contractile proteins within their cellular context, technically difficult high pressure freezing (HPF) and cryo- focused ion beam (cryo-FIB) milling is needed to ensure pristine vitrification of the cells and their organelles. Few labs worldwide have successfully implemented an HFP/cryo-FIB milling/cryo-ET workflow for intact eukaryotic cells and tissues in their native state, including the lab of Dr. Daniela Nicastro, the sponsor of this application. Therefore, I aim to determine the native in situ structure of myofibrils within intact inducible pluripotent stem cell derived human cardiomyocytes using high pressure freezing, followed by cryo-FIB milling to generate thin lamellae that can be imaged by cryo-ET. I will apply this cryo-ET workflow also to characterize structural alterations that occur within sarcomeres due to β-myosin mutations with and without mavacamten treatment. Determining the structural alterations that occur due to mutant β-myosin and mavacamten treatment will provide important insight for future improvement and development of therapeutics able to improve the health of myopathy patients. Project Number: 1F32HL178175-01 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Dylan Wilburn | Institution: UT SOUTHWESTERN MEDICAL CENTER, DALLAS, TX | Award Amount: $75,520 | Activity Code: F32 | Study Section: Special Emphasis Panel[ZRG1 F10A-R (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F32HL17817501