SummaryThick-filament sarcomere mutations are a common cause of hypertrophic cardiomyopathy (HCM), a disorder of heart muscle thickening associated with sudden cardiac death and heart failure, with unclear mechanisms. We engineered four isogenic induced pluripotent stem cell (iPSC) models of β-myosin heavy chain and myosin-binding protein C3 mutations, and studied iPSC-derived cardiomyocytes in cardiac microtissue assays that resemble cardiac architecture and biomechanics. All HCM mutations resulted in hypercontractility with prolonged relaxation kinetics in proportion to mutation pathogenicity, but not changes in calcium handling. RNA sequencing and expression studies of HCM models identified p53 activation, oxidative stress, and cytotoxicity induced by metabolic stress that can be reversed by p53 genetic ablation. Our findings implicate hypercontractility as a direct consequence of thick-filament mutations, irrespective of mutation localization, and the p53 pathway as a molecular marker of contraction stress and candidate therapeutic target for HCM patients.

The nanoscale optoelectronic properties of materials can be especially important for polycrystalline photovoltaics including many sensor and solar cell designs. For thin film solar cells such as CdTe, the open-circuit voltage and short-circuit current are especially critical performance indicators, often varying between and even within individual grains. A new method for directly mapping the open-circuit voltage leverages photo-conducting AFM, along with an additional proportional-integral-derivative feedback loop configured to maintain open-circuit conditions while scanning. Alternating with short-circuit current mapping efficiently provides complementary insight into the highly microstructurally sensitive local and ensemble photovoltaic performance. Furthermore, direct open-circuit voltage mapping is compatible with tomographic AFM, which additionally leverages gradual nanoscale milling by the AFM probe essentially for serial sectioning. The two-dimensional and three-dimensional results for CdTe solar cells during in situ illumination reveal local to mesoscale contributions to PV performance based on the order of magnitude variations in photovoltaic properties with distinct grains, at grain boundaries, and for sub-granular planar defects.

18860-837-2048 (t) | travis.hinson@jax.org 19 20 Thick filament sarcomere mutations are the most common cause of hypertrophic cardiomyopathy (HCM), 21 a disorder of heart muscle thickening associated with sudden cardiac death and heart failure, with unclear 22 mechanisms. We engineered an isogenic panel of four human HCM induced pluripotent stem cell (iPSc) 23 models using CRISPR/Cas9, and studied iPSc-derived cardiomyocytes (iCMs) in 3-dimensional cardiac 24 microtissue (CMT) assays that resemble in vivo cardiac architecture and biomechanics. HCM mutations 25 result in hypercontractility in association with prolonged relaxation kinetics in proportion to mutation 26 pathogenicity but not calcium dysregulation. RNA sequencing and protein expression studies identified 27 that HCM mutations result in p53 activation secondary to increased oxidative stress, which results in 28 increased cytotoxicity that can be reversed by p53 genetic ablation. Our findings implicate 29 hypercontractility as an early consequence of thick filament mutations, and the p53 pathway as a 30 molecular marker and candidate therapeutic target for thick filament HCM. 31 32 35 hypertrophy (LVH) with preserved systolic contractile function 2 . In young athletes, HCM manifests as a 36 common cause of sudden cardiac death; while in adults, HCM is associated with heart failure that may 37 progress to require cardiac transplantation 3 . Over the last few decades, the genetic basis of HCM has been 38 demonstrated by inheritance of autosomal dominant mutations in components of the force-producing 39 sarcomere 4 . About two-thirds of HCM patients harbor heterozygous mutations in one of two sarcomere 40 genes: b-myosin heavy chain (MHC-b is encoded by MYH7) or myosin-binding protein C (MYBPC3) 4 . 41 Along with titin, MHC-b and MYBPC3 are located in the thick filament where ATP hydrolysis by MHC-42b is coupled to force generation through interactions with the actin-rich thin filament ( fig.1A). A 43 prevailing model suggests that HCM mutations alter cardiac force generation through dysregulation of 44 calcium handling 5, 6 . Whether MYBPC3 and MYH7 mutations result in HCM by shared or heterogeneous 45 mechanisms also remains unclear. 46 Recent functional studies of thick filament HCM mutations in reconstituted sarcomere and 47 motility assays 7, 8 . Equally puzzling, contractile studies of single cardiomyocytes from MYH6-R403Q +/-1 mouse models, which recapitulate LVH and fibrosis in vivo 9 , have produced similarly conflicting results 2 for the identical mouse model and strain 10, 11 . Human patient-specific induced pluripotent stem cell (iPSc) 3 HCM models of MYH7-R663H (arginine 663 substituted with histidine) have recapitulated some features 4 of HCM including cellular enlargement and altered calcium handling 6 , but mechanical phenotypes of 5 1 expertise in confocal microscopy. We also thank Bo Reese for RNA sequencing technical expertise. We 2 thank Samantha Harris for her generous contribution of MYBPC3 antibody.3 4 Sources of Funding:5

The environmental conditions associated with changing the hydration state of active pharmaceutical ingredients (API) are crucial to understanding their stability, bioperformance, and manufacturability. Identifying the dehydration event using < 1μg of material is an increasingly important challenge. Atomic Force Microscopy indentation mapping is implemented at controlled temperatures between 25-100°C, for nanoscale volumes of hydrated APIs exhibiting distinct dehydration behavior and anhydrous APIs as controls. For caffeine hydrate and azithromycin dihydrate, the relative mechanical modulus increases ~10-fold at dehydration temperatures. These are confirmed by conventional macroscopic measurements including Variable Temperature Powder X-ray Diffraction, Thermogravimetric Analysis, and Differential Scanning Calorimetry. Conversely, no such mechanical transition is observed for anhydrous ibuprofen or a proprietary anhydrous compound. AFM-based mechanical mapping is therefore demonstrated for smallvolume determination of temperature-induced solid-state dehydration events, which may enable spatially or temporally mapping for future studies of dehydration mechanisms and kinetics, as a function of commercially relevant nanoscale heterogeneities.