Remote assessment and management of patients with dizziness: development, validation, and feasibility of a gamified vestibular rehabilitation therapy platform.

Introduction: Dizziness is a growing public health concern with as many as 95 million adults in Europe and the United States experiencing vestibular hypofunction, which is associated with reduced quality of life, poorer health, and falls. Vestibular rehabilitation therapy (VRT) is effective in reducing symptoms and improving balance; however, limited access to qualified clinicians and poor patient adherence impedes optimal delivery. The goal of this study was to develop and evaluate the feasibility of a remote therapeutic monitoring VRT Platform application (APP) for the assessment and treatment of vestibular dysfunction. Methods: User-centered iterative design process was used to gather and integrate the needs of users (clinicians and patients) into the design at each stage of development. Commonly used vestibular patient-reported outcome measures (PROs) were integrated into the APP and adults with chronic dizziness were enrolled to evaluate validity and reliability of the APP compared to standard clinical measures (CLIN). Gaze stabilization exercises were gamified to provide an engaging experience and an off-the-shelf sensor captured eye and head movement to provide feedback on accuracy of performance. A prospective, pilot study design with pre-and post-treatment assessment assessed feasibility of the APP compared to standard VRT (CLIN). Results: Participants with dizziness wanted a summary rehabilitation report shared with their clinicians, felt that an app could help with accountability, and believed that a gaming format might help with exercise adherence. Clinicians felt that the app should include features to record and track eye and head movement, monitor symptoms, score accuracy of task performance, and measure adherence. Validity and reliability of the digital PROs (APP) were compared to scores from CLIN across two sessions and found to have good validity, good to excellent test-retest reliability, and excellent usability (≥88%ile). The pilot study demonstrated feasibility for use of the APP compared to CLIN for treatment of vestibular hypofunction. The mean standard system usability score of the APP was 82.5 indicating excellent usability. Discussion: Both adult patients with chronic dizziness and VRT clinicians were receptive to the use of technology for VRT. The HiM-V APP is a feasible alternative to clinical management of adults with chronic peripheral vestibular hypofunction.

Pioneer factor ETV2 safeguards endothelial cell specification by recruiting the repressor REST to restrict alternative lineage commitment.

Mechanisms of cell fate specification remain a central question for developmental biology and regenerative medicine. The pioneer factor ETV2 is a master regulator for the endothelial cell (EC) lineage specification. Here, we studied mechanisms of ETV2-driven fate specification using a highly efficient system in which ETV2 directs human induced pluripotent stem cell-derived mesodermal progenitors to form ECs over two days. By applying CUT&RUN, single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses, we characterized the transcriptomic profiles, chromatin landscapes, dynamic cis-regulatory elements (CREs), and molecular features of EC cell differentiation mediated by ETV2. This defined the scope of ETV2 pioneering activity and identified its direct downstream target genes. Induced ETV2 expression both directed specification of endothelial progenitors and suppressed acquisition of alternative fates. Functional screening and candidate validation revealed cofactors essential for efficient EC specification, including the transcriptional activator GABPA. Surprisingly, the transcriptional repressor REST was also necessary for efficient EC specification. ETV2 recruited REST to occupy and repress non-EC lineage genes. Collectively, our study provides an unparalleled molecular analysis of EC specification at single-cell resolution and identifies the important role of pioneer factors to recruit repressors that suppress commitment to alternative lineages.

Therapeutic Inhibition of LincRNA-p21 Protects Against Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy is an adaptive response to pressure overload aimed at maintaining cardiac function. However, prolonged hypertrophy significantly increases the risk of maladaptive cardiac remodeling and heart failure. Recent studies have implicated long noncoding RNAs in cardiac hypertrophy and cardiomyopathy, but their significance and mechanism(s) of action are not well understood. METHODS: We measured lincRNA-p21 RNA and H3K27ac levels in the hearts of dilated cardiomyopathy patients. We assessed the functional role of lincRNA-p21 in basal and surgical pressure-overload conditions using loss-of-function mice. Genome-wide transcriptome analysis revealed dysregulated genes and pathways. We labeled proteins in proximity to full-length lincRNA-p21 using a novel BioID2-based system. We immunoprecipitated lincRNA-p21-interacting proteins and performed cell fractionation, ChIP-seq (chromatin immunoprecipitation followed by sequencing), and co-immunoprecipitation to investigate molecular interactions and underlying mechanisms. We used GapmeR antisense oligonucleotides to evaluate the therapeutic potential of lincRNA-p21 inhibition in cardiac hypertrophy and associated heart failure. RESULTS: lincRNA-p21 was induced in mice and humans with cardiomyopathy. Global and cardiac-specific lincRNA-p21 knockout significantly suppressed pressure overload-induced ventricular wall thickening, stress marker elevation, and deterioration of cardiac function. Genome-wide transcriptome analysis and transcriptional network analysis revealed that lincRNA-p21 acts in trans to stimulate the NFAT/MEF2 pathway. Mechanistically, lincRNA-p21 is bound to the scaffold protein KAP1. lincRNA-p21 cardiac-specific knockout suppressed stress-induced nuclear accumulation of KAP1, and KAP1 knockdown attenuated cardiac hypertrophy and NFAT activation. KAP1 positively regulates pathological hypertrophy by physically interacting with NFATC4 to promote the overactive status of NFAT/MEF2 signaling. GapmeR antisense oligonucleotide depletion of lincRNA-p21 similarly inhibited cardiac hypertrophy and adverse remodeling, highlighting the therapeutic potential of inhibiting lincRNA-p21. CONCLUSIONS: These findings advance our understanding of the functional significance of stress-induced long noncoding RNA in cardiac hypertrophy and demonstrate the potential of lincRNA-p21 as a novel therapeutic target for cardiac hypertrophy and subsequent heart failure.

The long noncoding RNA CARDINAL attenuates cardiac hypertrophy by modulating protein translation.

One of the features of pathological cardiac hypertrophy is enhanced translation and protein synthesis. Translational inhibition has been shown to be an effective means of treating cardiac hypertrophy, although system-wide side effects are common. Regulators of translation, such as cardiac-specific long noncoding RNAs (lncRNAs), could provide new, more targeted therapeutic approaches to inhibit cardiac hypertrophy. Therefore, we generated mice lacking a previously identified lncRNA named CARDINAL to examine its cardiac function. We demonstrate that CARDINAL is a cardiac-specific, ribosome-associated lncRNA and show that its expression was induced in the heart upon pathological cardiac hypertrophy and that its deletion in mice exacerbated stress-induced cardiac hypertrophy and augmented protein translation. In contrast, overexpression of CARDINAL attenuated cardiac hypertrophy in vivo and in vitro and suppressed hypertrophy-induced protein translation. Mechanistically, CARDINAL interacted with developmentally regulated GTP-binding protein 1 (DRG1) and blocked its interaction with DRG family regulatory protein 1 (DFRP1); as a result, DRG1 was downregulated, thereby modulating the rate of protein translation in the heart in response to stress. This study provides evidence for the therapeutic potential of targeting cardiac-specific lncRNAs to suppress disease-induced translational changes and to treat cardiac hypertrophy and heart failure.

Allele-Specific Suppression of Variant MHC With High-Precision RNA Nuclease CRISPR-Cas13d Prevents Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as MYH7 are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored. METHODS: We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the MYH7 variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct MYH7 SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human MYH7 allelic pathogenic SNVs. RESULTS: Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous MYH7 allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human MYH7 pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human MYH7 analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy. CONCLUSIONS: Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.

Efficient and reproducible generation of human iPSC-derived cardiomyocytes using a stirred bioreactor.

In the last decade human iPSC-derived cardiomyocytes (hiPSC-CMs) proved to be valuable for cardiac disease modeling and cardiac regeneration, yet challenges with scale, quality, inter-batch consistency, and cryopreservation remain, reducing experimental reproducibility and limiting clinical translation. Here, we report a robust cardiac differentiation protocol that uses Wnt modulation and a stirred suspension bioreactor to produce on average 124 million hiPSC-CMs with >90% purity using a variety of hiPSC lines (19 differentiations; 10 iPSC lines). After controlled freeze and thaw, bioreactor-derived CMs (bCMs) showed high viability (>90%), interbatch reproducibility in cellular morphology, function, drug response and ventricular identity, which was further supported by single cell transcriptomes. bCMs on microcontact printed substrates revealed a higher degree of sarcomere maturation and viability during long-term culture compared to monolayer-derived CMs (mCMs). Moreover, functional investigation of bCMs in 3D engineered heart tissues showed earlier and stronger force production during long-term culture, and robust pacing capture up to 4 Hz when compared to mCMs. bCMs derived from this differentiation protocol will expand the applications of hiPSC-CMs by providing a reproducible, scalable, and resource efficient method to generate cardiac cells with well-characterized structural and functional properties superior to standard mCMs.

Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease.

Rare coding mutations cause ∼45% of congenital heart disease (CHD). Noncoding mutations that perturb cis-regulatory elements (CREs) likely contribute to the remaining cases, but their identification has been problematic. Using a lentiviral massively parallel reporter assay (lentiMPRA) in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we functionally evaluated 6,590 noncoding de novo variants (ncDNVs) prioritized from the whole-genome sequencing of 750 CHD trios. A total of 403 ncDNVs substantially affected cardiac CRE activity. A majority increased enhancer activity, often at regions with undetectable reference sequence activity. Of ten DNVs tested by introduction into their native genomic context, four altered the expression of neighboring genes and iPSC-CM transcriptional state. To prioritize future DNVs for functional testing, we used the MPRA data to develop a regression model, EpiCard. Analysis of an independent CHD cohort by EpiCard found enrichment of DNVs. Together, we developed a scalable system to measure the effect of ncDNVs on CRE activity and deployed it to systematically assess the contribution of ncDNVs to CHD.

Molecular and Spatial Signatures of Mouse Embryonic Endothelial Cells at Single-Cell Resolution.

BACKGROUND: Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized. METHODS: We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs). RESULTS: From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including Etv2+Bnip3+ EC progenitors. Spatial transcriptomic analysis of 15 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (Eln, Dkk2, Vegfc, and Egfl8), venous (Fam174b and Clec14a), and capillary (Kcne3) marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages. CONCLUSIONS: Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.

A Genomic Link From Heart Failure to Atrial Fibrillation Risk: FOG2 Modulates a TBX5/GATA4-Dependent Atrial Gene Regulatory Network.

BACKGROUND: The relationship between heart failure (HF) and atrial fibrillation (AF) is clear, with up to half of patients with HF progressing to AF. The pathophysiological basis of AF in the context of HF is presumed to result from atrial remodeling. Upregulation of the transcription factor FOG2 (friend of GATA2; encoded by ZFPM2) is observed in human ventricles during HF and causes HF in mice. METHODS: FOG2 expression was assessed in human atria. The effect of adult-specific FOG2 overexpression in the mouse heart was evaluated by whole animal electrophysiology, in vivo organ electrophysiology, cellular electrophysiology, calcium flux, mouse genetic interactions, gene expression, and genomic function, including a novel approach for defining functional transcription factor interactions based on overlapping effects on enhancer noncoding transcription. RESULTS: FOG2 is significantly upregulated in the human atria during HF. Adult cardiomyocyte-specific FOG2 overexpression in mice caused primary spontaneous AF before the development of HF or atrial remodeling. FOG2 overexpression generated arrhythmia substrate and trigger in cardiomyocytes, including calcium cycling defects. We found that FOG2 repressed atrial gene expression promoted by TBX5. FOG2 bound a subset of GATA4 and TBX5 co-bound genomic locations, defining a shared atrial gene regulatory network. FOG2 repressed TBX5-dependent transcription from a subset of co-bound enhancers, including a conserved enhancer at the Atp2a2 locus. Atrial rhythm abnormalities in mice caused by Tbx5 haploinsufficiency were rescued by Zfpm2 haploinsufficiency. CONCLUSIONS: Transcriptional changes in the atria observed in human HF directly antagonize the atrial rhythm gene regulatory network, providing a genomic link between HF and AF risk independent of atrial remodeling.

In vivo proximity proteomics uncovers palmdelphin (PALMD) as a Z-line-associated mitigator of isoproterenol-induced cardiac injury.

Z-lines are core ultrastructural organizers of cardiomyocytes that modulate many facets of cardiac pathogenesis. Yet a comprehensive proteomic atlas of Z-line-associated components remain incomplete. Here, we established an adeno-associated virus (AAV)-delivered, cardiomyocyte-specific, proximity-labeling approach to characterize the Z-line proteome in vivo. We found palmdelphin (PALMD) as a novel Z-line-associated protein in both adult murine cardiomyocytes and human pluripotent stem cell-derived cardiomyocytes. Germline and cardiomyocyte-specific palmd knockout mice were grossly normal at baseline but exhibited compromised cardiac hypertrophy and aggravated cardiac injury upon long-term isoproterenol treatment. By contrast, cardiomyocyte-specific PALMD overexpression was sufficient to mitigate isoproterenol-induced cardiac injury. PALMD ablation perturbed transverse tubules (T-tubules) and their association with sarcoplasmic reticulum, which formed the Z-line-associated junctional membrane complex (JMC) essential for calcium handling and cardiac function. These phenotypes were associated with disrupted localization of T-tubule markers caveolin-3 (CAV3) and junctophilin-2 (JPH2) and the reduction of nexilin (NEXN) protein, a crucial Z-line-associated protein that is essential for both Z-line and JMC structures and functions. PALMD was found to interact with NEXN and enhance its protein stability while the Nexn mRNA level was not affected. Together, this study discovered PALMD as a potential target for myocardial protection and highlighted in vivo proximity proteomics as a powerful approach to nominate novel players regulating cardiac pathogenesis. Highlights: In vivo proximity proteomics uncover novel Z-line components that are undetected in in vitro proximity proteomics in cardiomyocytes.PALMD is a novel Z-line-associated protein that is dispensable for baseline cardiomyocyte function in vivo.PALMD mitigates cardiac dysfunction and myocardial injury after repeated isoproterenol insults.PALMD stabilizes NEXN, an essential Z-line-associated regulator of the junctional membrane complex and cardiac systolic function.

Reduced Mitochondrial Protein Translation Promotes Cardiomyocyte Proliferation and Heart Regeneration.

BACKGROUND: The importance of mitochondria in normal heart function are well recognized and recent studies have implicated changes in mitochondrial metabolism with some forms of heart disease. Previous studies demonstrated that knockdown of the mitochondrial ribosomal protein S5 (MRPS5) by small interfering RNA (siRNA) inhibits mitochondrial translation and thereby causes a mitonuclear protein imbalance. Therefore, we decided to examine the effects of MRPS5 loss and the role of these processes on cardiomyocyte proliferation. METHODS: We deleted a single allele of MRPS5 in mice and used left anterior descending coronary artery ligation surgery to induce myocardial damage in these animals. We examined cardiomyocyte proliferation and cardiac regeneration both in vivo and in vitro. Doxycycline treatment was used to inhibit protein translation. Heart function in mice was assessed by echocardiography. Quantitative real-time polymerase chain reaction and RNA sequencing were used to assess changes in transcription and chromatin immunoprecipitation (ChIP) and BioChIP were used to assess chromatin effects. Protein levels were assessed by Western blotting and cell proliferation or death by histology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Adeno-associated virus was used to overexpress genes. The luciferase reporter assay was used to assess promoter activity. Mitochondrial oxygen consumption rate, ATP levels, and reactive oxygen species were also analyzed. RESULTS: We determined that deletion of a single allele of MRPS5 in mice results in elevated cardiomyocyte proliferation and cardiac regeneration; this observation correlates with improved cardiac function after induction of myocardial infarction. We identified ATF4 (activating transcription factor 4) as a key regulator of the mitochondrial stress response in cardiomyocytes from Mrps5+/- mice; furthermore, ATF4 regulates Knl1 (kinetochore scaffold 1) leading to an increase in cytokinesis during cardiomyocyte proliferation. The increased cardiomyocyte proliferation observed in Mrps5+/- mice was attenuated when one allele of Atf4 was deleted genetically (Mrps5+/-/Atf4+/-), resulting in the loss in the capacity for cardiac regeneration. Either MRPS5 inhibition (or as we also demonstrate, doxycycline treatment) activate a conserved regulatory mechanism that increases the proliferation of human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: These data highlight a critical role for MRPS5/ATF4 in cardiomyocytes and an exciting new avenue of study for therapies to treat myocardial injury.

Spatiotemporal cell junction assembly in human iPSC-CM models of arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that causes life-threatening arrhythmias and myocardial dysfunction. Pathogenic variants in Plakophilin-2 (PKP2), a desmosome component within specialized cardiac cell junctions, cause the majority of ACM cases. However, the molecular mechanisms by which PKP2 variants induce disease phenotypes remain unclear. Here we built bioengineered platforms using genetically modified human induced pluripotent stem cell-derived cardiomyocytes to model the early spatiotemporal process of cardiomyocyte junction assembly in vitro. Heterozygosity for truncating variant PKP2R413X reduced Wnt/ß-catenin signaling, impaired myofibrillogenesis, delayed mechanical coupling, and reduced calcium wave velocity in engineered tissues. These abnormalities were ameliorated by SB216763, which activated Wnt/ß-catenin signaling, improved cytoskeletal organization, restored cell junction integrity in cell pairs, and improved calcium wave velocity in engineered tissues. Together, these findings highlight the therapeutic potential of modulating Wnt/ß-catenin signaling in a human model of ACM.

Fibre-infused gel scaffolds guide cardiomyocyte alignment in 3D-printed ventricles.

Hydrogels are attractive materials for tissue engineering, but efforts to date have shown limited ability to produce the microstructural features necessary to promote cellular self-organization into hierarchical three-dimensional (3D) organ models. Here we develop a hydrogel ink containing prefabricated gelatin fibres to print 3D organ-level scaffolds that recapitulate the intra- and intercellular organization of the heart. The addition of prefabricated gelatin fibres to hydrogels enables the tailoring of the ink rheology, allowing for a controlled sol-gel transition to achieve precise printing of free-standing 3D structures without additional supporting materials. Shear-induced alignment of fibres during ink extrusion provides microscale geometric cues that promote the self-organization of cultured human cardiomyocytes into anisotropic muscular tissues in vitro. The resulting 3D-printed ventricle in vitro model exhibited biomimetic anisotropic electrophysiological and contractile properties.

Tbx5 maintains atrial identity by regulating an atrial enhancer network.

Understanding how the atrial and ventricular chambers of the heart maintain their distinct identity is a prerequisite for treating chamber-specific diseases. Here, we selectively inactivated the transcription factor Tbx5 in the atrial working myocardium of the neonatal mouse heart to show that it is required to maintain atrial identity. Atrial Tbx5 inactivation downregulated highly chamber specific genes such as Myl7 and Nppa , and conversely, increased the expression of ventricular identity genes including Myl2 . Using combined single nucleus transcriptome and open chromatin profiling, we assessed genomic accessibility changes underlying the altered atrial identity expression program, identifying 1846 genomic loci with greater accessibility in control atrial cardiomyocytes compared to KO aCMs. 69% of the control-enriched ATAC regions were bound by TBX5, demonstrating a role for TBX5 in maintaining atrial genomic accessibility. These regions were associated with genes that had higher expression in control aCMs compared to KO aCMs, suggesting they act as TBX5-dependent enhancers. We tested this hypothesis by analyzing enhancer chromatin looping using HiChIP and found 510 chromatin loops that were sensitive to TBX5 dosage. Of the loops enriched in control aCMs, 73.7% contained anchors in control-enriched ATAC regions. Together, these data demonstrate a genomic role for TBX5 in maintaining the atrial gene expression program by binding to atrial enhancers and preserving tissue-specific chromatin architecture of atrial enhancers.