Accelerating therapeutic development and clinical trial readiness for STXBP1 and SYNGAP1 disorders.

Gene-targeted therapies for genetic neurodevelopmental disorders (NDDs) are becoming a reality. The Center for Epilepsy and Neurodevelopmental Disorders (ENDD) is currently focused on the development of therapeutics for STXBP1 and SYNGAP1 disorders. Here we review the known clinical features of these disorders, highlight the biological role of STXBP1 and SYNGAP1, and discuss our current understanding of pathogenic mechanisms and therapeutic development. Finally, we provide our perspective as scientists and parents of children with NDDs, and comment on the current challenges for both clinical and basic science endeavors.

Integrated Stress Response Potentiates Ponatinib-Induced Cardiotoxicity.

BACKGROUND: Mitochondrial dysfunction is a primary driver of cardiac contractile failure; yet, the cross talk between mitochondrial energetics and signaling regulation remains obscure. Ponatinib, a tyrosine kinase inhibitor used to treat chronic myeloid leukemia, is among the most cardiotoxic tyrosine kinase inhibitors and causes mitochondrial dysfunction. Whether ponatinib-induced mitochondrial dysfunction triggers the integrated stress response (ISR) to induce ponatinib-induced cardiotoxicity remains to be determined. METHODS: Using human induced pluripotent stem cells-derived cardiomyocytes and a recently developed mouse model of ponatinib-induced cardiotoxicity, we performed proteomic analysis, molecular and biochemical assays to investigate the relationship between ponatinib-induced mitochondrial stress and ISR and their role in promoting ponatinib-induced cardiotoxicity. RESULTS: Proteomic analysis revealed that ponatinib activated the ISR in cardiac cells. We identified GCN2 (general control nonderepressible 2) as the eIF2α (eukaryotic translation initiation factor 2α) kinase responsible for relaying mitochondrial stress signals to trigger the primary ISR effector-ATF4 (activating transcription factor 4), upon ponatinib exposure. Mechanistically, ponatinib treatment exerted inhibitory effects on ATP synthase activity and reduced its expression levels resulting in ATP deficits. Perturbed mitochondrial function resulting in ATP deficits then acts as a trigger of GCN2-mediated ISR activation, effects that were negated by nicotinamide mononucleotide, an NAD+ precursor, supplementation. Genetic inhibition of ATP synthase also activated GCN2. Interestingly, we showed that the decreased abundance of ATP also facilitated direct binding of ponatinib to GCN2, unexpectedly causing its activation most likely because of a conformational change in its structure. Importantly, administering an ISR inhibitor protected human induced pluripotent stem cell-derived cardiomyocytes against ponatinib. Ponatinib-treated mice also exhibited reduced cardiac function, effects that were attenuated upon systemic ISRIB administration. Importantly, ISRIB does not affect the antitumor effects of ponatinib in vitro. CONCLUSIONS: Neutralizing ISR hyperactivation could prevent or reverse ponatinib-induced cardiotoxicity. The findings that compromised ATP production potentiates GCN2-mediated ISR activation have broad implications across various cardiac diseases. Our results also highlight an unanticipated role of ponatinib in causing direct activation of a kinase target despite its role as an ATP-competitive kinase inhibitor.

Chronic activation of tubulin tyrosination in HCM mice and human iPSC-engineered heart tissues improves heart function.

Rationale: Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and associated with left ventricular (LV) hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with the findings that -α-tubulin detyrosination (dTyr-tub) is markedly elevated in heart failure. Acute reduction of dTyr-tub by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, and thus poses a new perspective for HCM treatment. Objective: In this study, we tested the impact of chronic tubulin tyrosination in a HCM mouse model ( Mybpc3 -knock-in; KI), in human HCM cardiomyocytes and in SVBP-deficient human engineered heart tissues (EHTs). Methods and Results: AAV9-mediated TTL transfer was applied in neonatal wild-type (WT) rodents and 3-week-old KI mice and in HCM human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We show that i) TTL for 6 weeks dose-dependently reduced dTyr-tub and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes; ii) TTL for 12 weeks improved diastolic filling, cardiac output and stroke volume and reduced stiffness in KI mice; iii) TTL for 10 days normalized cell hypertrophy in HCM hiPSC-cardiomyocytes; iv) TTL induced a marked transcription and translation of several tubulins and modulated mRNA or protein levels of components of mitochondria, Z-disc, ribosome, intercalated disc, lysosome and cytoskeleton in KI mice; v) SVBP-deficient EHTs exhibited reduced dTyr-tub levels, higher force and faster relaxation than TTL-deficient and WT EHTs. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs. Conclusion: This study provides the first proof-of-concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.

An Unbiased Screen Identified the Hsp70-BAG3 Complex as a Regulator of Myosin-Binding Protein C3.

Variants in the gene myosin-binding protein C3 (MYBPC3) account for approximately 50% of familial hypertrophic cardiomyopathy (HCM), leading to reduced levels of myosin-binding protein C3 (MyBP-C), the protein product made by gene MYBPC3. Elucidation of the pathways that regulate MyBP-C protein homeostasis could uncover new therapeutic strategies. Toward this goal, we screened a library of 2,426 bioactive compounds and identified JG98, an allosteric modulator of heat shock protein 70 that inhibits interaction with Bcl-2-associated athanogene (BAG) domain co-chaperones. JG98 reduces MyBP-C protein levels. Furthermore, genetic reduction of BAG3 phenocopies treatment with JG-98 by reducing MYBP-C protein levels.. Thus, an unbiased compound screen identified the heat shock protein 70-BAG3 complex as a regulator of MyBP-C stability.

Adult human cardiomyocyte mechanics in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is an extracellular matrix disorder characterized by defects in collagen-1 transport or synthesis, resulting in bone abnormalities. Although reduced collagen in OI hearts has been associated with reduced myocardial stiffness and left ventricular remodeling, its impact on cardiomyocyte (CM) function has not been studied. Here, we explore the tissue-level and CM-level properties of a heart from a deceased organ donor with OI type I. Proteomics and histology confirmed strikingly low expression of collagen 1. Trabecular stretch confirmed low stiffness on the tissue level. However, CMs retained normal viscoelastic properties as revealed by nanoindentation. Interestingly, OI CMs were hypercontractile relative to nonfailing controls after 24 h of culture. In response to 48 h of culture on surfaces with physiological (10 kPa) and pathological (50 kPa) stiffness, OI CMs demonstrated a greater reduction in contractility than nonfailing CMs, suggesting that OI CMs may have an impaired stress response. Levels of detyrosinated α-tubulin, known to be responsive to extracellular stiffness, were reduced in OI CMs. Together these data confirm multiple CM-level adaptations to low stiffness that extend our understanding of OI in the heart and how CMs respond to extracellular stiffness.NEW & NOTEWORTHY In a rare donation of a heart from an individual with osteogenesis imperfecta (OI), we explored cardiomyocyte (CM) adaptations to low stiffness. This represents the first assessment of cardiomyocyte mechanics in OI. The data reveal the hypercontractility of OI CMs with rapid rundown when exposed to acute stiffness challenges, extending our understanding of OI. These data demonstrate that the impact of OI on myocardial mechanics includes cardiomyocyte adaptations beyond known direct effects on the extracellular matrix.

Mapping PTBP2 binding in human brain identifies SYNGAP1 as a target for therapeutic splice switching.

Alternative splicing of neuronal genes is controlled partly by the coordinated action of polypyrimidine tract binding proteins (PTBPs). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal. Here, we define the PTBP2 footprint in the human transcriptome using brain tissue and human induced pluripotent stem cell-derived neurons (iPSC-neurons). We map PTBP2 binding sites, characterize PTBP2-dependent alternative splicing events, and identify novel PTBP2 targets including SYNGAP1, a synaptic gene whose loss-of-function leads to a complex neurodevelopmental disorder. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and nonsense-mediated decay, and that antisense oligonucleotides (ASOs) that disrupt PTBP binding redirect splicing and increase SYNGAP1 mRNA and protein expression. In SYNGAP1 haploinsufficient iPSC-neurons generated from two patients, we show that PTBP2-targeting ASOs partially restore SYNGAP1 expression. Our data comprehensively map PTBP2-dependent alternative splicing in human neurons and cerebral cortex, guiding development of novel therapeutic tools to benefit neurodevelopmental disorders.

Desmin intermediate filaments and tubulin detyrosination stabilize growing microtubules in the cardiomyocyte.

In heart failure, an increased abundance of post-translationally detyrosinated microtubules stiffens the cardiomyocyte and impedes its contractile function. Detyrosination promotes interactions between microtubules, desmin intermediate filaments, and the sarcomere to increase cytoskeletal stiffness, yet the mechanism by which this occurs is unknown. We hypothesized that detyrosination may regulate the growth and shrinkage of dynamic microtubules to facilitate interactions with desmin and the sarcomere. Through a combination of biochemical assays and direct observation of growing microtubule plus-ends in adult cardiomyocytes, we find that desmin is required to stabilize growing microtubules at the level of the sarcomere Z-disk, where desmin also rescues shrinking microtubules from continued depolymerization. Further, reducing detyrosination (i.e. tyrosination) below basal levels promotes frequent depolymerization and less efficient growth of microtubules. This is concomitant with tyrosination promoting the interaction of microtubules with the depolymerizing protein complex of end-binding protein 1 (EB1) and CAP-Gly domain-containing linker protein 1 (CLIP1/CLIP170). The dynamic growth and shrinkage of tyrosinated microtubules reduce their opportunity for stabilizing interactions at the Z-disk region, coincident with tyrosination globally reducing microtubule stability. These data provide a model for how intermediate filaments and tubulin detyrosination establish long-lived and physically reinforced microtubules that stiffen the cardiomyocyte and inform both the mechanism of action and therapeutic index for strategies aimed at restoring tyrosination for the treatment of cardiac disease.

Extracellular stiffness induces contractile dysfunction in adult cardiomyocytes via cell-autonomous and microtubule-dependent mechanisms.

The mechanical environment of the myocardium has a potent effect on cardiomyocyte form and function, yet an understanding of the cardiomyocyte responses to extracellular stiffening remains incomplete. We therefore employed a cell culture substrate with tunable stiffness to define the cardiomyocyte responses to clinically relevant stiffness increments in the absence of cell-cell interactions. When cultured on substrates magnetically actuated to mimic the stiffness of diseased myocardium, isolated rat adult cardiomyocytes exhibited a time-dependent reduction of sarcomere shortening, characterized by slowed contraction and relaxation velocity, and alterations of the calcium transient. Cardiomyocytes cultured on stiff substrates developed increases in viscoelasticity and microtubule detyrosination in association with early increases in the α-tubulin detyrosinating enzyme vasohibin-2 (Vash2). We found that knockdown of Vash2 was sufficient to preserve contractile performance as well as calcium transient properties in the presence of extracellular substrate stiffening. Orthogonal prevention of detyrosination by overexpression of tubulin tyrosine ligase (TTL) was also able to preserve contractility and calcium homeostasis. These data demonstrate that a pathologic increment of extracellular stiffness induces early, cell-autonomous remodeling of adult cardiomyocytes that is dependent on detyrosination of α-tubulin.

Transcriptional, Post-Transcriptional, and Post-Translational Mechanisms Rewrite the Tubulin Code During Cardiac Hypertrophy and Failure.

A proliferated and post-translationally modified microtubule network underlies cellular growth in cardiac hypertrophy and contributes to contractile dysfunction in heart failure. Yet how the heart achieves this modified network is poorly understood. Determining how the "tubulin code"-the permutations of tubulin isoforms and post-translational modifications-is rewritten upon cardiac stress may provide new targets to modulate cardiac remodeling. Further, while tubulin can autoregulate its own expression, it is unknown if autoregulation is operant in the heart or tuned in response to stress. Here we use heart failure patient samples and murine models of cardiac remodeling to interrogate transcriptional, autoregulatory, and post-translational mechanisms that contribute to microtubule network remodeling at different stages of heart disease. We find that autoregulation is operant across tubulin isoforms in the heart and leads to an apparent disconnect in tubulin mRNA and protein levels in heart failure. We also find that within 4 h of a hypertrophic stimulus and prior to cardiac growth, microtubule detyrosination is rapidly induced to help stabilize the network. This occurs concomitant with rapid transcriptional and autoregulatory activation of specific tubulin isoforms and microtubule motors. Upon continued hypertrophic stimulation, there is an increase in post-translationally modified microtubule tracks and anterograde motors to support cardiac growth, while total tubulin content increases through progressive transcriptional and autoregulatory induction of tubulin isoforms. Our work provides a new model for how the tubulin code is rapidly rewritten to establish a proliferated, stable microtubule network that drives cardiac remodeling, and provides the first evidence of tunable tubulin autoregulation during pathological progression.

The microtubule cytoskeleton in cardiac mechanics and heart failure.

The microtubule network of cardiac muscle cells has unique architectural and biophysical features to accommodate the demands of the working heart. Advances in live-cell imaging and in deciphering the 'tubulin code' have shone new light on this cytoskeletal network and its role in heart failure. Microtubule-based transport orchestrates the growth and maintenance of the contractile apparatus through spatiotemporal control of translation, while also organizing the specialized membrane systems required for excitation-contraction coupling. To withstand the high mechanical loads of the working heart, microtubules are post-translationally modified and physically reinforced. In response to stress to the myocardium, the microtubule network remodels, typically through densification, post-translational modification and stabilization. Under these conditions, physically reinforced microtubules resist the motion of the cardiomyocyte and increase myocardial stiffness. Accordingly, modified microtubules have emerged as a therapeutic target for reducing stiffness in heart failure. In this Review, we discuss the latest evidence on the contribution of microtubules to cardiac mechanics, the drivers of microtubule network remodelling in cardiac pathologies and the therapeutic potential of targeting cardiac microtubules in acquired heart diseases.

Cardiomyocyte Microtubules: Control of Mechanics, Transport, and Remodeling.

Microtubules are essential cytoskeletal elements found in all eukaryotic cells. The structure and composition of microtubules regulate their function, and the dynamic remodeling of the network by posttranslational modifications and microtubule-associated proteins generates diverse populations of microtubules adapted for various contexts. In the cardiomyocyte, the microtubules must accommodate the unique challenges faced by a highly contractile, rigidly structured, and long-lasting cell. Through their canonical trafficking role and positioning of mRNA, proteins, and organelles, microtubules regulate essential cardiomyocyte functions such as electrical activity, calcium handling, protein translation, and growth. In a more specialized role, posttranslationally modified microtubules form load-bearing structures that regulate myocyte mechanics and mechanotransduction. Modified microtubules proliferate in cardiovascular diseases, creating stabilized resistive elements that impede cardiomyocyte contractility and contribute to contractile dysfunction. In this review, we highlight the most exciting new concepts emerging from recent studies into canonical and noncanonical roles of cardiomyocyte microtubules.

Integrated landscape of cardiac metabolism in end-stage human nonischemic dilated cardiomyopathy.

Heart failure (HF) is a leading cause of mortality. Failing hearts undergo profound metabolic changes, but a comprehensive evaluation in humans is lacking. We integrate plasma and cardiac tissue metabolomics of 678 metabolites, genome-wide RNA-sequencing, and proteomic studies to examine metabolic status in 87 explanted human hearts from 39 patients with end-stage HF compared with 48 nonfailing donors. We confirm bioenergetic defects in human HF and reveal selective depletion of adenylate purines required for maintaining ATP levels. We observe substantial reductions in fatty acids and acylcarnitines in failing tissue, despite plasma elevations, suggesting defective import of fatty acids into cardiomyocytes. Glucose levels, in contrast, are elevated. Pyruvate dehydrogenase, which gates carbohydrate oxidation, is de-repressed, allowing increased lactate and pyruvate burning. Tricarboxylic acid cycle intermediates are significantly reduced. Finally, bioactive lipids are profoundly reprogrammed, with marked reductions in ceramides and elevations in lysoglycerophospholipids. These data unveil profound metabolic abnormalities in human failing hearts.

Need for Speed: The Importance of Physiological Strain Rates in Determining Myocardial Stiffness.

The heart is viscoelastic, meaning its compliance is inversely proportional to the speed at which it stretches. During diastolic filling, the left ventricle rapidly expands at rates where viscoelastic forces impact ventricular compliance. In heart disease, myocardial viscoelasticity is often increased and can directly impede diastolic filling to reduce cardiac output. Thus, treatments that reduce myocardial viscoelasticity may provide benefit in heart failure, particularly for patients with diastolic heart failure. Yet, many experimental techniques either cannot or do not characterize myocardial viscoelasticity, and our understanding of the molecular regulators of viscoelasticity and its impact on cardiac performance is lacking. Much of this may stem from a reliance on techniques that either do not interrogate viscoelasticity (i.e., use non-physiological rates of strain) or techniques that compromise elements that contribute to viscoelasticity (i.e., skinned or permeabilized muscle preparations that compromise cytoskeletal integrity). Clinically, cardiac viscoelastic characterization is challenging, requiring the addition of strain-rate modulation during invasive hemodynamics. Despite these challenges, data continues to emerge demonstrating a meaningful contribution of viscoelasticity to cardiac physiology and pathology, and thus innovative approaches to characterize viscoelasticity stand to illuminate fundamental properties of myocardial mechanics and facilitate the development of novel therapeutic strategies.

X-ROS Signaling Depends on Length-Dependent Calcium Buffering by Troponin.

The stretching of a cardiomyocyte leads to the increased production of reactive oxygen species that increases ryanodine receptor open probability through a process termed X-ROS signaling. The stretching of the myocyte also increases the calcium affinity of myofilament Troponin C, which increases its calcium buffering capacity. Here, an integrative experimental and modeling study is pursued to explain the interplay of length-dependent changes in calcium buffering by troponin and stretch-activated X-ROS calcium signaling. Using this combination, we show that the troponin C-dependent increase in myoplasmic calcium buffering during myocyte stretching largely offsets the X-ROS-dependent increase in calcium release from the sarcoplasmic reticulum. The combination of modeling and experiment are further informed by the elimination of length-dependent changes to troponin C calcium binding in the presence of blebbistatin. Here, the model suggests that it is the X-ROS signaling-dependent Ca2+ release increase that serves to maintain free myoplasmic calcium concentrations during a change in myocyte length. Together, our experimental and modeling approaches have further defined the relative contributions of X-ROS signaling and the length-dependent calcium buffering by troponin in shaping the myoplasmic calcium transient.

Cochlear supporting cells require GAS2 for cytoskeletal architecture and hearing.

In mammals, sound is detected by mechanosensory hair cells that are activated in response to vibrations at frequency-dependent positions along the cochlear duct. We demonstrate that inner ear supporting cells provide a structural framework for transmitting sound energy through the cochlear partition. Humans and mice with mutations in GAS2, encoding a cytoskeletal regulatory protein, exhibit hearing loss due to disorganization and destabilization of microtubule bundles in pillar and Deiters' cells, two types of inner ear supporting cells with unique cytoskeletal specializations. Failure to maintain microtubule bundle integrity reduced supporting cell stiffness, which in turn altered cochlear micromechanics in Gas2 mutants. Vibratory responses to sound were measured in cochleae from live mice, revealing defects in the propagation and amplification of the traveling wave in Gas2 mutants. We propose that the microtubule bundling activity of GAS2 imparts supporting cells with mechanical properties for transmitting sound energy through the cochlea.

Microtubules orchestrate local translation to enable cardiac growth.

Hypertension, exercise, and pregnancy are common triggers of cardiac remodeling, which occurs primarily through the hypertrophy of individual cardiomyocytes. During hypertrophy, stress-induced signal transduction increases cardiomyocyte transcription and translation, which promotes the addition of new contractile units through poorly understood mechanisms. The cardiomyocyte microtubule network is also implicated in hypertrophy, but via an unknown role. Here, we show that microtubules are indispensable for cardiac growth via spatiotemporal control of the translational machinery. We find that the microtubule motor Kinesin-1 distributes mRNAs and ribosomes along microtubule tracks to discrete domains within the cardiomyocyte. Upon hypertrophic stimulation, microtubules redistribute mRNAs and new protein synthesis to sites of growth at the cell periphery. If the microtubule network is disrupted, mRNAs and ribosomes collapse around the nucleus, which results in mislocalized protein synthesis, the rapid degradation of new proteins, and a failure of growth, despite normally increased translation rates. Together, these data indicate that mRNAs and ribosomes are actively transported to specific sites to facilitate local translation and assembly of contractile units, and suggest that properly localized translation - and not simply translation rate - is a critical determinant of cardiac hypertrophy. In this work, we find that microtubule based-transport is essential to couple augmented transcription and translation to productive cardiomyocyte growth during cardiac stress.

Pathogenic LMNA variants disrupt cardiac lamina-chromatin interactions and de-repress alternative fate genes.

Pathogenic mutations in LAMIN A/C (LMNA) cause abnormal nuclear structure and laminopathies. These diseases have myriad tissue-specific phenotypes, including dilated cardiomyopathy (DCM), but how LMNA mutations result in tissue-restricted disease phenotypes remains unclear. We introduced LMNA mutations from individuals with DCM into human induced pluripotent stem cells (hiPSCs) and found that hiPSC-derived cardiomyocytes, in contrast to hepatocytes or adipocytes, exhibit aberrant nuclear morphology and specific disruptions in peripheral chromatin. Disrupted regions were enriched for transcriptionally active genes and regions with lower LAMIN B1 contact frequency. The lamina-chromatin interactions disrupted in mutant cardiomyocytes were enriched for genes associated with non-myocyte lineages and correlated with higher expression of those genes. Myocardium from individuals with LMNA variants similarly showed aberrant expression of non-myocyte pathways. We propose that the lamina network safeguards cellular identity and that pathogenic LMNA variants disrupt peripheral chromatin with specific epigenetic and molecular characteristics, causing misexpression of genes normally expressed in other cell types.