Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis.

BACKGROUND: Recent interest in understanding cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow the abscission of daughter cardiomyocytes. METHODS: Here, we use a proteomics screen to identify adducin, an actin capping protein previously not studied in cardiomyocytes, as a regulator of sarcomere disassembly. We generated many adeno-associated viruses and cardiomyocyte-specific genetic gain-of-function models to examine the role of adducin in neonatal and adult cardiomyocytes in vitro and in vivo. RESULTS: We identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phospho-isoforms of α-adducin in identified Thr445/Thr480 phosphorylation of a short isoform of α-adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin. CONCLUSIONS: These results highlight an important mechanism for coordinating cytoskeletal morphological changes during cardiomyocyte mitosis.

CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants.

BACKGROUND: TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity. METHODS: We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy-associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation. RESULTS: CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts. CONCLUSIONS: TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.

TTN truncation variants produce sarcomere-integrating proteins of uncertain functional significance.

Titin (TTN) is one of the largest and most complex proteins expressed in humans, and truncation variants are the most prevalent genetic lesion identified in individuals with dilated cardiomyopathy (DCM) or other disorders of impaired cardiac contractility. Two reports in this issue of the JCI shed light on a potential mechanism involving truncated TTN sarcomere integration and the potential for disruption of sarcomere structural integrity. Kellermayer, Tordai, and colleagues confirmed the presence of truncated TTN protein in human DCM samples. McAfee and authors developed a patient-specific TTN antibody to study truncated TTN subcellular localization and to explore its functional consequences. A "poison peptide" mechanism emerges that inspires alternative therapeutic approaches while opening new lines for inquiry, such as the role of haploinsufficiency of full-length TTN protein, mechanisms explaining sarcomere dysfunction, and explanations for variable penetrance.

The Cardiac Sarcomere and Cell Cycle.

PURPOSE OF REVIEW: The lack of adult human cardiomyocyte proliferative capacity impairs cardiac regeneration such as after myocardial injury. The sarcomere, a specialized actin cytoskeletal structure that is essential for twitch contraction in cardiomyocytes, has been considered a critical factor limiting adult human cardiomyocyte proliferation through incompletely understood mechanisms. RECENT FINDINGS: This review summarizes known and emerging regulatory mechanisms connecting the human cardiomyocyte sarcomere to cell cycle regulation including structural and signaling mechanisms. Cardiac regeneration could be augmented through targeting the inhibitory effects of the sarcomere on cardiomyocyte proliferation.

Derivation of extra-embryonic and intra-embryonic macrophage lineages from human pluripotent stem cells.

Tissue-resident macrophages are increasingly recognized as important determinants of organ homeostasis, tissue repair, remodeling and regeneration. Although the ontogeny and function of tissue-resident macrophages has been identified as distinct from postnatal hematopoiesis, the inability to specify, in vitro, similar populations that recapitulate these developmental waves has limited our ability to study their function and potential for regenerative applications. We took advantage of the concept that tissue-resident macrophages and monocyte-derived macrophages originate from distinct extra-embryonic and definitive hematopoietic lineages to devise a system to generate pure cultures of macrophages that resemble tissue-resident or monocyte-derived subsets. We demonstrate that human pluripotent stem cell-derived extra-embryonic-like and intra-embryonic-like hematopoietic progenitors differentiate into morphologically, transcriptionally and functionally distinct macrophage populations. Single-cell RNA sequencing of developing and mature cultures uncovered distinct developmental trajectories and gene expression programs of macrophages derived from extra-embryonic-like and intra-embryonic-like hematopoietic progenitors. These findings establish a resource for the generation of human tissue resident-like macrophages to study their specification and function under defined conditions and to explore their potential use in tissue engineering and regenerative medicine applications.

Detecting Cardiovascular Protein-Protein Interactions by Proximity Proteomics.

Rapidly changing and transient protein-protein interactions regulate dynamic cellular processes in the cardiovascular system. Traditional methods, including affinity purification and mass spectrometry, have revealed many macromolecular complexes in cardiomyocytes and the vasculature. Yet these methods often fail to identify in vivo or transient protein-protein interactions. To capture these interactions in living cells and animals with subsequent mass spectrometry identification, enzyme-catalyzed proximity labeling techniques have been developed in the past decade. Although the application of this methodology to cardiovascular research is still in its infancy, the field is developing rapidly, and the promise is substantial. In this review, we outline important concepts and discuss how proximity proteomics has been applied to study physiological and pathophysiological processes relevant to the cardiovascular system.

Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions.

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, titin (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. Last, we engineered a full-length TTN protein reporter assay and used 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 the 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. Although 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.

Actinin BioID reveals sarcomere crosstalk with oxidative metabolism through interactions with IGF2BP2.

Actinins are strain-sensing actin cross-linkers that are ubiquitously expressed and harbor mutations in human diseases. We utilize CRISPR, pluripotent stem cells, and BioID to study actinin interactomes in human cardiomyocytes. We identify 324 actinin proximity partners, including those that are dependent on sarcomere assembly. We confirm 19 known interactors and identify a network of RNA-binding proteins, including those with RNA localization functions. In vivo and biochemical interaction studies support that IGF2BP2 localizes electron transport chain transcripts to actinin neighborhoods through interactions between its K homology (KH) domain and actinin's rod domain. We combine alanine scanning mutagenesis and metabolic assays to disrupt and functionally interrogate actinin-IGF2BP2 interactions, which reveal an essential role in metabolic responses to pathological sarcomere activation using a hypertrophic cardiomyopathy model. This study expands our functional knowledge of actinin, uncovers sarcomere interaction partners, and reveals sarcomere crosstalk with IGF2BP2 for metabolic adaptation relevant to human disease.

Sarcomere function activates a p53-dependent DNA damage response that promotes polyploidization and limits in vivo cell engraftment.

Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse imaging, in vitro cardiomyocyte replication patterns recapitulate the progressive mononuclear polyploidization and replicative arrest observed in vivo. Single-cell transcriptomics and chromatin state analyses reveal that polyploidization is preceded by sarcomere assembly, enhanced oxidative metabolism, a DNA damage response, and p53 activation. CRISPR knockout screening reveals p53 as a driver of cell-cycle arrest and polyploidization. Inhibiting sarcomere function, or scavenging ROS, inhibits cell-cycle arrest and polyploidization. Finally, we show that cardiomyocyte engraftment in infarcted rat hearts is enhanced 4-fold by the increased proliferation of troponin-knockout cardiomyocytes. Thus, the sarcomere inhibits cell division through a DNA damage response that can be targeted to improve cardiomyocyte replacement strategies.

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis.

There is ongoing debate as to whether cardiac complications of coronavirus disease-2019 (COVID-19) result from myocardial viral infection or are secondary to systemic inflammation and/or thrombosis. We provide evidence that cardiomyocytes are infected in patients with COVID-19 myocarditis and are susceptible to severe acute respiratory syndrome coronavirus 2. We establish an engineered heart tissue model of COVID-19 myocardial pathology, define mechanisms of viral pathogenesis, and demonstrate that cardiomyocyte severe acute respiratory syndrome coronavirus 2 infection results in contractile deficits, cytokine production, sarcomere disassembly, and cell death. These findings implicate direct infection of cardiomyocytes in the pathogenesis of COVID-19 myocardial pathology and provides a model system to study this emerging disease.

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis.

Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.

Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human TNNT2 Variants.

BACKGROUND: Pathogenic TNNT2 variants are a cause of hypertrophic and dilated cardiomyopathies, which promote heart failure by incompletely understood mechanisms. The precise functional significance for 87% of TNNT2 variants remains undetermined, in part, because of a lack of functional genomics studies. The knowledge of which and how TNNT2 variants cause hypertrophic and dilated cardiomyopathies could improve heart failure risk determination, treatment efficacy, and therapeutic discovery, and provide new insights into cardiomyopathy pathogenesis, as well. METHODS: We created a toolkit of human induced pluripotent stem cell models and functional assays using CRISPR/Cas9 to study TNNT2 variant pathogenicity and pathophysiology. Using human induced pluripotent stem cell-derived cardiomyocytes in cardiac microtissue and single-cell assays, we functionally interrogated 51 TNNT2 variants, including 30 pathogenic/likely pathogenic variants and 21 variants of uncertain significance. We used 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: Hypertrophic cardiomyopathy-associated TNNT2 variants caused increased cardiac microtissue contraction, whereas dilated cardiomyopathy-associated variants decreased contraction. TNNT2 variant-dependent changes in sarcomere contractile function induced graded regulation of 101 gene transcripts, including MAPK (mitogen-activated protein kinase) 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. On the basis of 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 variants of uncertain significance. CONCLUSIONS: Our study found that hypertrophic cardiomyopathy-associated TNNT2 variants increased cardiac microtissue contraction, whereas dilated cardiomyopathy-associated variants decreased contraction, both of which paralleled changes in myofilament calcium affinity. Transcriptomic changes, including NPPB levels, directly correlated with sarcomere function and can be used to predict TNNT2 variant pathogenicity.

A Contraction Stress Model of Hypertrophic Cardiomyopathy due to Sarcomere Mutations.

Thick-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 ß-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.

Force Generation via ß-Cardiac Myosin, Titin, and α-Actinin Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions.

Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that ß-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from ß-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis.

Integrative Analysis of PRKAG2 Cardiomyopathy iPS and Microtissue Models Identifies AMPK as a Regulator of Metabolism, Survival, and Fibrosis.

AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFß isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFß signaling.