LAMP2 is a ubiquitously expressed protein critical for autophagy. Alternative splicing gives rise to three isoforms. However, the roles of major LAMP2 isoforms in the heart are not known. To address this knowledge gap, we generated lamp2a and lamp2b knockout (KO) mice to investigate the role of these isoforms in heart function and autophagy. Deletion of either Lamp2a or Lamp2b did not alter cardiac structure or function. Lack of all LAMP2 isoforms led to increased cardiac fibrosis and reduced survival during pressure overload, which were not observed in lamp2a or lamp2b KO mice. Also, LAMP2B loss did not affect levels of the autophagy markers LC3-II and SQSTM1/p62. Conversely, LAMP2A was upregulated in hearts lacking LAMP2B, potentially preserving autophagy and cardiac function. Reintroducing LAMP2A in lamp2 KO mice effectively reduced autophagosome accumulation and improved cardiac function. Overall, these data support LAMP2 isoform functional redundancy in the myocardium under pathological conditions.Abbreviations: AAV: adeno-associated virus; ACTA2: actin alpha 2, smooth muscle, aorta; CMA: chaperone-mediated autophagy; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LV: Left ventricle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NPPA: natriuretic peptide type A; NPPB: natriuretic peptide type B; SQSTM1/p62: sequestosome 1; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; TAC: transverse aortic constriction; WT: wild type.

Background: Danon disease is a fatal X-linked lysosomal storage disorder caused by loss of lysosome-associated membrane protein 2 (LAMP2), leading to severe cardiomyopathy and intellectual disability. Without advanced therapies, most patients progress to end-stage heart failure or early death. Prior studies suggest intercellular transfer of lysosomes from macrophages to cardiomyocytes, supporting the potential of hematopoietic stem cell transplantation as a therapeutic strategy. Aims: We evaluated whether ex vivo gene-modified hematopoietic stem and progenitor cells (HSPCs) could deliver LAMP2B to affected tissues and improve outcomes in a murine model of Danon disease. We hypothesized that transplantation of HSPCs transduced with a lentiviral vector encoding human LAMP2B (pALD-LAMP2B) would restore LAMP2B expression and ameliorate disease features. Methods: Sca-1 positive HSPCs were isolated from Lamp2 KO mice and transduced with pALD-LAMP2B (n=5) or left unmodified (n=6). Cells were transplanted into busulfan-conditioned 12–15-week-old Lamp2 KO recipients. Age-matched wild-type (WT, n=7) and untreated KO mice (n=3) served as controls. Six months post-transplant, invasive hemodynamics, immunofluorescence, and neurobehavioral testing were performed. Results: Immunofluorescence confirmed human LAMP2B expression in hearts of KO mice receiving gene-corrected HSPCs. These mice exhibited improved cardiac function compared to those receiving unmodified KO HSPCs (End diastolic pressure: p=0.005; Tau: p=0.06; Max dP/dt: p=0.28; Min dP/dt: p=0.30). In open field tests, KO mice had impaired locomotor activity versus WT, with reduced walking distance and speed. Mice treated with gene-corrected HSPCs showed improvement in these parameters toward WT levels, with favorable trends in immobility time, thigmotaxis, and exploratory behavior. Conclusions: Transplantation of gene-modified HSPCs restored LAMP2B expression in the heart and improved both cardiac and neurobehavioral outcomes in a Danon disease mouse model. This study provides the first evidence supporting cardiac-targeted HSPC-based gene therapy for a monogenic disease. Larger studies are warranted to confirm therapeutic efficacy and assess long-term outcomes.

Background: Cardiac troponin I (cTnI, encoded by TNNI3 ) inhibits contraction by preventing actin-myosin interaction. Pathogenic TNNI3 variants can cause cardiomyopathy, but no targeted therapies exist. We identified a family with restrictive cardiomyopathy carrying the cTnI A157V variant. Previously, we generated a homozygous mouse model (cTnI A158V, murine equivalent) that showed impaired cardiac relaxation on invasive hemodynamics but had normal lifespan and no cardiac hypertrophy or fibrosis. Aims: It is known that murine models of cardiomyopathies often exhibit milder phenotypes than humans, limiting their translational utility. Hence, to better model human disease, we generated a homozygous cTnI A158V pig model using CRISPR-Cas9. We aimed to characterize phenotypic and molecular differences between the mouse and pig models to gain insight into disease mechanisms. Methods: Heart tissue was collected from wild-type (WT) and homozygous A158V mice (9-10 months) and from WT and homozygous A158V pigs (2 months). Bulk RNA sequencing (RNA-seq) was performed on RNA extracted from A158V pig hearts (n=5) and compared to publicly available data from age-matched WT pigs (n=10) supplemented by one in-house WT control. Protein lysates were analyzed by Western blot. Isolated myofibrils were measured using the fast solution switching method. Results: A158V pigs had 100% mortality by 2 months (n=7), with gross hypertrophy and fibrosis. Myofibril studies showed significant prolongation of the linear relaxation phase in both A158V pigs (p<0.01) and mice (p<0.01) compared to respective WTs, with no significant differences in active tension in both models. Both models showed reduced phosphorylation of cTnI at serine 23/24, reaching significance in pigs (p=0.046) but not in mice (p=0.064). Unsupervised clustering of bulk-RNAseq data separated A158V from WT, with genotype explaining 44% and 53% of transcriptional variation in mice and pigs, respectively. Gene set enrichment analysis revealed significant upregulation of inflammatory pathways in A158V pigs only. Conclusions: Both A158V mouse and pig models recapitulate impaired cardiac relaxation seen in affected patients as shown by myofibril studies. However, the pig model more closely mirrors the human phenotype, including early mortality, hypertrophy, and fibrosis. Inflammatory pathway upregulation was observed only in A158V pigs, suggesting that inflammation may play a role in the severity or progression of disease.

Introduction: Cardiac troponin I (cTnI, TNNI3 gene) is a highly conserved subunit of the sarcomere that inhibits contraction by preventing actin-myosin interaction. Pathogenic TNNI3 variants can cause both hypertrophic and restrictive cardiomyopathies but lack targeted therapies. We identified a family with restrictive cardiomyopathy carrying a cTnI variant at amino acid 157 (A157V). Using CRISPR-Cas9, we generated a knock-in mouse model reflecting this mutation (A158V in mouse). Our previous studies showed that homozygous A158V mice had impaired cardiac relaxation on invasive hemodynamics but normal lifespan, no echocardiographic changes, and no fibrosis. Hypothesis: Myofibril mechanics and transcriptomic studies will better discriminate TNNI3 A158V mice from WT than previous phenotyping studies. Methods: Heart tissue was obtained from wild-type (WT) and homozygous A158V mice at 9-10 months. Myofibrils were isolated from heart tissues and mechanical measurements were completed using the fast solution switching method. RNA was extracted for bulk RNA sequencing. Results: Isolated myofibril studies showed significant prolongation of the linear relaxation phase in A158V mice versus controls (p=0.02) but no significant differences in active tension. Unsupervised clustering of bulk RNA samples separated A158V samples from controls, with 44% transcriptional variation derived from genotype. Cardiac changes in the A158V model were confirmed by significant depletion of pathways involved in structural components of the contractile apparatus, including contractile fiber, sarcomere, and I-Band. On an individual gene level, A158V mutated mice displayed higher expression of cardiac remodeling genes such as Timp1 , Postn , and Tnc . Conclusion: The TNNI3 A158V variant consistently produces impaired relaxation with myofibril studies showing prolonged linear relaxation phase. Transcriptomic studies highlight distinct clustering of TNNI3 A158V mice from WT, with prominent depletion in contractile apparatus pathways. Our findings suggest traditional methods to characterize mouse models may fail to capture disease phenotype in cardiomyopathies; however, myofibril studies and transcriptomic profiling may reveal earlier phenotypic changes.

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Background: Genetic sequencing is changing the way physicians practice medicine. Hypertrophic cardiomyopathy (HCM) is one of the most common inheritable cardiac diseases. Sarcoidosis is a multisystem disease involving granulomatous infiltration of the lungs, heart, and other organs thought to be caused by a dysregulated immune system, influenced by genetic factors. Here, we present a case where an individual initially diagnosed with HCM was experiencing frequent episodes of ventricular tachycardia (VT). Further workup, including advanced imaging along with whole genome screening (WGS), revealed an unlikely diagnosis of concurrent cardiac sarcoidosis (CS). Clinical History: A 62-year-old man had ventricular fibrillation with workup notable for an ECG with deeply inverted T-waves, normal coronary angiography, and an echocardiogram and MRI showing apical HCM. An ICD was placed. Six years later, he had VT requiring a shock and was started on sotalol. He has now developed recurrent exercise-induced VT. While VT is seen HCM, the increasing frequency prompted further evaluation. A CT coronary angiogram showed normal coronaries and mediastinal lymphadenopathy. Lymph node biopsy revealed noncaseating granulomas and a PET/CT revealed hypermetabolic basal myocardium meeting HRS criteria for CS. Given minimal apical FDG uptake and a low likelihood that he had CS for 9 years without significant fibrosis, he likely has both HCM and CS. Using WGS, four genetic variants previously described in CS and HCM were identified confirming this diagnosis, HLA-DRB1, HLA-DQA1, ALPK3, and TTN. Discussion: HLA-DRB1 and HLA-DQA1 are both major histocompatibility complexes associated with CS. ALPK3 encodes alpha kinase 3 while TTN encodes titin. Truncating variants of ALPK3 are associated with HCM while pathogenic TTN variants have been described to cause HCM, DCM, and ARVC. This case reflects the potential of WGS in aiding clinicians in confirming an unlikely diagnosis.

Introduction: Troponin inhibitor3 ( TNNI3 ) is a thin-filament protein that regulates contraction of thick filaments. The role of the switch domain of TNNI3 (aa147-163), which interacts with the calcium-binding pocket of troponin C, is poorly defined. Pathogenic mutations to the switch domain cause restrictive cardiomyopathy in humans, but no therapies exist that address the underlying problem of this mutation at the sarcomeric level. Further, few models of genetic restrictive cardiomyopathy exist to aid in development of new therapies. Hypothesis: Substitution of alanine with valine at position 157 (A157V) in the switch domain of TNNI3 causes restrictive cardiomyopathy by blunting response to adrenergic stimulus. Methods: A known pathogenic mutation to the TNNI3 switch domain (A157V) was identified in a family of patients with cardiomyopathy and restrictive features. A mutant knock-in mouse homozygous for this mutation (A157V) was generated using CRISPR-Cas9 and used to elucidate the function of the switch domain. Results: Compared to wild type controls (WT), mutant A157V mice demonstrate significant restrictive features on invasive hemodynamics that worsen with age but do not show evidence of systolic dysfunction or hypertrophy on echocardiography. Heart size and myocyte cross-sectional area were significantly smaller in mutant A157V mice compared to WT controls. Molecular dynamics simulations revealed reduced TNNI3 activation in response to PKA-mediated phosphorylation at serine23/24. Isolated myocytes from A157V mice demonstrated impaired relaxation, lower peak systolic calcium and delayed reuptake of calcium into the sarcoplasmic reticulum compared to WT controls. Conclusions: The A157V mutation to the switch domain of TNNI3 , a critical regulatory domain that interacts with the calcium binding pocket of troponin C, causes diastolic dysfunction by impairing responsiveness to PKA-mediated phosphorylation of S23/24. This mouse model recapitulates the key restrictive features of human disease and could be used as a platform to study future targeted therapeutics for thin filament cardiomyopathy.

Abstract Danon disease is a fatal X-linked recessive disease caused by a lack of expression of the lysosomal associated membrane protein type 2 (LAMP2), leading to severe vacuolar cardiomyopathy. Most patients with Danon progress to end-stage heart failure or death without advanced therapies. In this study, we investigated the therapeutic efficacy of systemic transplantation of ex vivo gene-modified Lamp2-/- (Lamp2 KO) hematopoietic stem and progenitor cells (HSPCs) using a lentiviral vector containing the human LAMP2B transgene, pCCL-LAMP2B, in the mouse model of Danon disease, Lamp2 KO mice. Transplanted pCCL-LAMP2B-HSPCs efficiently engrafted and differentiated into macrophages in heart. LAMP2B was found in cardiomyocytes and improved cardiac systolic as well as locomotor functions were observed in pCCL-LAMP2B-HSPCs recipient mice compared to non-treated or Lamp2 KO mice receiving Lamp2 KO HSPCs. In addition, we also demonstrated that pCCL-LAMP2B-HSPCs rescued autophagic flux and activity in the heart. In vitro, we cocultured WT macrophages with Lamp2 KO fibroblasts and observed transfer of LAMP2B and rescue of the autophagic flux in the diseased cells confirming cross-correction despite LAMP2B being a lysosomal transmembrane protein.

Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through β-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.

Introduction: Previous studies describe a morbid restrictive cardiomyopathy in patients with substitution of alanine with valine at amino acid 157 (A157V) within the calcium-binding domain of the inhibitory subunit of cardiac troponin (TNNI3); yet the precise mechanism of disease remains unknown. Hypothesis: A murine model of A157V TNNI3 would recapitulate key features of disease and serve as a model of cardiac restriction. Methods: TNNI3 A157V mice were generated using a cloning-free CRISPR/Cas-9 system to introduce a point mutation (c.470c>T) resulting in missense substitution of valine for alanine at amino acid 157 in Exon 7 of TNNI3 . Heterozygous and homozygous mice were generated in a C57BL/6 background. At one year of age animals had echocardiography and electrocardiogram (ECG) performed, followed by invasive hemodynamics. Mice were then sacrificed for cardiac morphometry and hearts were fixed and embedded for histology. TNNI3 A157V adeno-associated virus-9 (AAV-9) construct was generated and injected retro-orbitally into 8-12 week old wild-type mice. Results: Serial echocardiography at 2, 4, 6 and 12 months of age demonstrated normal wall thickness, ejection fraction and strain. No differences were noted in cardiac arrhythmias assessed using ECG. Hemodynamics performed at 15 months of age demonstrated significantly reduced minimum derivative of pressure/time (dP/dT) and Tau in response to dobutamine in TNNI3 A157V mice v. controls. No significant differences between genotypes were detected in cardiac morphometry (heart weight and heart weight normalized to body weight or tibia length), myocyte cross sectional area measured on trichrome-stained heart sections, or myocardial oxygen consumption rate measured by oroboros. Mice injected with TNNI3 A157V AAV-9 demonstrated expression of the mutant TNNI3 construct on western analysis and immunohistochemistry. Conclusions: We have developed a novel mouse model in which substitution of valine for alanine at amino acid 157 in TNNI3 induces diastolic dysfunction despite the absence of cardiac hypertrophy that recapitulates key features of human disease and can serve as a platform for further mechanistic and translational studies.

Danon disease (DD) is a rare X-linked autophagic vacuolar myopathy associated with multiorgan dysfunction, including the heart, skeletal muscle, and liver. There are no specific treatments, and most male patients die from advanced heart failure during the second or third decade of life. DD is caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene, a key mediator of autophagy. LAMP2 has three isoforms: LAMP2A, LAMP2B, and LAMP2C. LAMP2B is the predominant isoform expressed in cardiomyocytes. This study evaluates the efficacy of human LAMP2B gene transfer using a recombinant adeno-associated virus 9 carrying human LAMP2B (AAV9.LAMP2B) in a Lamp2 knockout (KO) mouse, a DD model. AAV9.LAMP2B was intravenously injected into 2- and 6-month-old Lamp2 KO male mice to assess efficacy in adolescent and adult phenotypes. Lamp2 KO mice receiving AAV9.LAMP2B demonstrated dose-dependent restoration of human LAMP2B protein in the heart, liver, and skeletal muscle tissue. Impaired autophagic flux, evidenced by increased LC3-II, was abrogated by LAMP2B gene transfer in all tissues in both cohorts. Cardiac function was also improved, and transaminases were reduced in AAV9.LAMP2B-treated KO mice, indicating favorable effects on the heart and liver. Survival was also higher in the older cohort receiving high vector doses. No anti-LAMP2 antibodies were detected in mice that received AAV9.LAMP2B. In summary, LAMP2B gene transfer improves metabolic and physiologic function in a DD murine model, suggesting that a similar therapeutic approach may be effective for treating patients with this highly morbid disease.

Chronic heart failure and cardiac arrhythmias have high morbidity and mortality, and drugs for the prevention and management of these diseases are a large part of the pharmaceutical market. Among these drugs are plant-derived cardiac glycosides, which have been used by various cultures over millennia as both medicines and poisons. We report that digoxin and related compounds activate the NLRP3 inflammasome in macrophages and cardiomyocytes at concentrations achievable during clinical use. Inflammasome activation initiates the maturation and release of the inflammatory cytokine IL-1β and the programmed cell death pathway pyroptosis in a caspase-1-dependent manner. Notably, the same fluxes of potassium and calcium cations that affect heart contraction also induce inflammasome activation in human but not murine cells. Pharmaceuticals that antagonize these fluxes, including glyburide and verapamil, also inhibit inflammasome activation by cardiac glycosides. Cardiac glycoside-induced cellular cytotoxicity and IL-1β signaling are likewise antagonized by inhibitors of the NLRP3 inflammasome or the IL-1 receptor-targeting biological agent anakinra. Our results inform on the molecular mechanism by which the inflammasome integrates the diverse signals that activate it through secondary signals like cation flux. Furthermore, this mechanism suggests a contribution of the inflammasome to the toxicity and adverse events associated with cardiac glycosides use in humans and that targeted anti-inflammatories could provide an additional adjunct therapeutic countermeasure.

Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD.

ABSTRACTHuman genetic variants are usually represented by four values with variable length: chromosome, position, reference and alternate alleles. Thereis no guarantee that these components are represented in a consistent way across different data sources, and processing variant-based data can be inefficient because four different comparison operations are needed for each variant, three of which are string comparisons. Working with strings, in contrast to numbers, poses extra challenges on computer memory allocation and data-representation. Existing variant identifiers do not typicallyrepresent every possible variant we may be interested in, nor they are directly reversible. To overcome these limitations,VariantKey, a novel reversible numerical encoding schema for human genetic variants, is presented here alongside a multi-language open-source software implementation (http://github.com/genomicspls/variantkey). VariantKey represents variants as single 64 bit numeric entities, while preserving the ability to be searched and sorted by chromosome and position. The individual components of short variants can be directly read back from the VariantKey, while long variants are supported with a fast lookup table.Highlights~100 compounds identified by high-content screen inhibit SGs in HEK293, NPCs and iPS-MNs.ALS-associated RBPs are recruited to SGs in an RNA-dependent mannerMolecules with planar moieties prevent recruitment of ALS-associated RBPs to SGsCompounds inhibit TDP-43 accumulation in SGs and inTARDBPmutant iPS-MNs.

Understanding the mechanisms that control human cardiomyocyte proliferation might be applicable to regenerative medicine. We screened a whole genome collection of human miRNAs, identifying 96 to be capable of increasing proliferation (DNA synthesis and cytokinesis) of human iPSC-derived cardiomyo-cytes. Chemical screening and computational approaches indicated that most of these miRNAs (67) target different components of the Hippo pathway and that their activity depends on the nuclear translocation of the Hippo transcriptional effector YAP. 53 of the 67 miRNAs are present in human iPSC cardio-myocytes, yet anti-miRNA screening revealed that none are individually essential for basal proliferation of hiPSC cardiomyocytes despite the importance of YAP for proliferation. We propose a model in which multiple endogenous miRNAs redundantly suppress Hippo signaling to sustain the cell cycle of immature cardiomyocytes.

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

Increasing angiogenesis has long been considered a therapeutic target for improving heart function after injury such as acute myocardial infarction. However, gene, protein and cell therapies to increase microvascularization have not been successful, most likely because the studies failed to achieve regulated and concerted expression of pro-angiogenic and angiostatic factors needed to produce functional microvasculature. Here, we report that the transcription factor RBPJ is a homoeostatic repressor of multiple pro-angiogenic and angiostatic factor genes in cardiomyocytes. RBPJ controls angiogenic factor gene expression independently of Notch by antagonizing the activity of hypoxia-inducible factors (HIFs). In contrast to previous strategies, the cardiomyocyte-specific deletion of Rbpj increased microvascularization of the heart without adversely affecting cardiac structure or function even into old age. Furthermore, the loss of RBPJ in cardiomyocytes increased hypoxia tolerance, improved heart function and decreased pathological remodelling after myocardial infarction, suggesting that inhibiting RBPJ might be therapeutic for ischaemic injury.