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.

No abstract

Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes span the nuclear envelope and transduce force from dynamic cytoskeletal networks to the nuclear lamina. Here we show that LINC complexes also signal from the nuclear envelope to critical regulators of the actin cytoskeleton. Specifically, we find that LINC complexes that contain the inner nuclear membrane protein Sun2 promote focal adhesion assembly by activating the small GTPase RhoA. A key effector in this process is the transcription factor/coactivator complex composed of SRF/Mkl1. A constitutively active form of SRF/Mkl1 was not sufficient to induce focal adhesion assembly in cells lacking Sun2, however, suggesting that LINC complexes support RhoA activity through a transcription-independent mechanism. Strikingly, we also find that the inner nuclear membrane protein Sun1 antagonizes Sun2 LINC complexes and inhibits RhoA activation and focal adhesion assembly. Thus different LINC complexes have opposing roles in the transcription-independent control of the actin cytoskeleton through the small GTPase RhoA.

In vertebrates, the nuclear pore complex (NPC), the gate for transport of macromolecules between the nucleus and the cytoplasm, consists of approximately 30 different nucleoporins (Nups). The Nup and SUMO E3-ligase Nup358/RanBP2 are the major components of the cytoplasmic filaments of the NPC. In this study, we perform a structure-function analysis of Nup358 and describe its role in nuclear import of specific proteins. In a screen for nuclear proteins that accumulate in the cytoplasm upon Nup358 depletion, we identified proteins that were able to interact with Nup358 in a receptor-independent manner. These included the importin α/β-cargo DBC-1 (deleted in breast cancer 1) and DMAP-1 (DNA methyltransferase 1 associated protein 1). Strikingly, a short N-terminal fragment of Nup358 was sufficient to promote import of DBC-1, whereas DMAP-1 required a larger portion of Nup358 for stimulated import. Neither the interaction of RanGAP with Nup358 nor its SUMO-E3 ligase activity was required for nuclear import of all tested cargos. Together, Nup358 functions as a cargo-and receptor-specific assembly platform, increasing the efficiency of nuclear import of proteins through various mechanisms.

Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1) is the major transport receptor for the export of proteins from the nucleus. It binds to nuclear export signals (NESs) that are rich in leucines and other hydrophobic amino acids. The prediction of NESs is difficult because of the extreme recognition flexibility of CRM1. Furthermore, proteins can be exported upon binding to an NES-containing adaptor protein. Here we present an approach for identifying targets of the CRM1-export pathway via quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture. With this approach, we identified >100 proteins from HeLa cells that were depleted from cytosolic fractions and/or enriched in nuclear fractions in the presence of the selective CRM1-inhibitor leptomycin B. Novel and validated substrates are the polyubiquitin-binding protein sequestosome 1, the cancerous inhibitor of protein phosphatase 2A (PP2A), the guanine nucleotide-binding protein-like 3-like protein, the programmed cell death protein 2-like protein, and the cytosolic carboxypeptidase 1 (CCP1). We identified a functional NES in CCP1 that mediates direct binding to the export receptor CRM1. The method will be applicable to other nucleocytoplasmic transport pathways, as well as to the analysis of nucleocytoplasmic shuttling proteins under different growth conditions. Molecular &

Background: Pathogenic TNNT2 variants are a cause of hypertrophic (HCM) and dilated (DCM) cardiomyopathies, which promote heart failure by incompletely understood mechanisms. Additionally, the precise functional significance for 87% of TNNT2 variants remains undetermined partially due to a lack of functional genomics studies. The knowledge of which and how TNNT2 variants cause HCM and DCM could improve heart failure risk determination, treatment efficacy, and therapeutic discovery, as well as provide new insights into cardiomyopathy pathogenesis. Methods: We created a toolkit of human induced pluripotent stem cell (hiPSC) models and functional assays using CRISPR/Cas9 to study TNNT2 variant pathogenicity and pathophysiology. Using hiPSC-derived cardiomyocytes (hiPSC-CMs) in cardiac microtissue and single cell assays, we functionally interrogated 51 TNNT2 variants, including 30 pathogenic/likely pathogenic variants and 21 variants of unknown significance (VUS). We utilized RNA-sequencing to determine the transcriptomic consequences of pathogenic TNNT2 variants, and adapted CRISPR/Cas9 to engineer a transcriptional reporter assay to assist prediction of TNNT2 variant pathogenicity. We also studied variant-specific pathophysiology using a thin filament-directed calcium reporter to monitor changes in myofilament calcium affinity. Results: HCM-associated TNNT2 variants caused increased cardiac microtissue contraction, while DCM-associated variants decreased contraction. TNNT2 variant-dependent changes in sarcomere contractile function induced graded regulation of 101 gene transcripts, including MAPK signaling targets, HOPX , and NPPB . We distinguished pathogenic TNNT2 variants from wildtype controls using a sarcomere functional reporter engineered by inserting tdTomato into the endogenous NPPB locus. Based on a combination of NPPB reporter activity and cardiac microtissue contraction, our study provides experimental support for the reclassification of 2 pathogenic/likely pathogenic variants and 2 VUSs. Conclusions: Our study found that HCM-associated TNNT2 variants increased cardiac microtissue contraction, while DCM-associated variants cause decreased contraction, both of which paralleled changes in myofilament calcium affinity. Transcriptomic changes, including NPPB levels, directly correlated with sarcomere function and can be utilized to predict TNNT2 variant pathogenicity.

Highlights d BioID identifies 324 actinin proximity partners through sarcomere assembly d IGF2BP2 directly binds actinin's rod domain through its KH domain d IGF2BP2-actinin interactions determine ETC transcript localization d Actinin-IGF2BP2 interactions regulate metabolic adaptation to sarcomere function

Background: Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, TTN functions and therapeutic targets. Methods: We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Finally, we engineered a full-length TTN protein reporter assay and utilized next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. Results: An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR-ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome-editing using SpCas9 and a TTNtv-specific guide RNA restored TTN protein reading frame, which increased full length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. Conclusions: An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. While both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a "one-and-done" genome editing strategy to target ∼30% of DCM-associated TTNtvs.