Serine biosynthesis as a novel therapeutic target for dilated cardiomyopathy.

AIMS: Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro. METHODS AND RESULTS: Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions. CONCLUSIONS: A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.

Collagen type I and hyaluronic acid based hybrid scaffolds for heart valve tissue engineering.

Tissue engineers have achieved limited success so far in designing an ideal scaffold for aortic valve; scaffolds lack in mechanical compatibility, appropriate degradation rate, and microstructural similarity. This paper, therefore, has demonstrated a carbodiimide-based sequential crosslinking technique to prepare aortic valve extracellular matrix mimicking (ECM) hybrid scaffolds from collagen type I and hyaluronic acid (HA), the building blocks of heart valve ECM, with tailorable crosslinking densities. Swelling studies revealed that crosslinking densities of parent networks increased with increasing the concentration of the crosslinking agents whereas crosslinking densities of hybrid scaffolds averaged from those of parent collagen and HA networks. Hybrid scaffolds also offered a wide range of pore size (66-126 µm) which fulfilled the criteria for valvular tissue regeneration. Scanning electron microscopy and images of Alcian blue-Periodic acid Schiff stained samples suggested that our crosslinking technique yielded an ECM mimicking microstructure with interlaced bands of collagen and HA in the hybrid scaffolds. The mutually reinforcing networks of collagen and HA also resulted in increased bending moduli up to 1660 kPa which spanned the range of natural aortic valves. Cardio sphere-derived cells (CDCs) from rat hearts showed that crosslinking density affected the available cell attachment sites on the surface of the scaffold. Increased bending moduli of CDCs seeded scaffolds up to two folds (2-6 kPa) as compared to the non-seeded scaffolds (1 kPa) suggested that an increase in crosslinking density of the scaffolds could not only increase the in vitro bending modulus but also prevented its disintegration in the cell culture medium.

Using iPSC Models to Probe Regulation of Cardiac Ion Channel Function.

PURPOSE OF REVIEW: Cardiovascular disease is the leading contributor to mortality and morbidity. Many deaths of heart failure patients can be attributed to sudden cardiac death due primarily to ventricular arrhythmia. Currently, most anti-arrhythmics modulate ion channel conductivity or ß-adrenergic signaling, but these drugs have limited efficacy for some indications, and can potentially be proarrhythmic. RECENT FINDINGS: Recent studies have shown that mutations in proteins other than cardiac ion channels may confer susceptibility to congenital as well as acquired arrhythmias. Additionally, ion channels themselves are subject to regulation at the levels of channel expression, trafficking and post-translational modification; thus, research into the regulation of ion channels may elucidate disease mechanisms and potential therapeutic targets for future drug development. This review summarizes the current knowledge of the molecular mechanisms of arrhythmia susceptibility and discusses technological advances such as induced pluripotent stem cell-derived cardiomyocytes, gene editing, functional genomics, and physiological screening platforms that provide a new paradigm for discovery of new therapeutic targets to treat congenital and acquired diseases of the heart rhythm.

Ambiguity in the Presentation of Decellularized Tissue Composition: The Need for Standardized Approaches.

Decellularization offers great potential to the field of tissue engineering, as this method gives rise to scaffold material with the native organ architecture by removing all cellular material and leaving much of the extracellular matrix (ECM) intact. However, many parameters may affect decellularization efficacy and ECM retention and, therefore, decellularization protocols need to be optimized for specific needs. This requires robust methods for comparison of decellularized tissue composition. Various representation methods are used in literature to express tissue composition (DNA, glycosaminoglycans, collagen, other ECM proteins, and growth factors). Here, we present and compare the various methods used and demonstrate that normalization to either dry or wet decellularized weight might be misleading and may overestimate true component retention. Moreover, the magnitude of the confounding effect is likely to be decellularization treatment dependent. As a result, we propose alternative comparison strategies: normalization to whole organ or to a unit of whole initial organ weight. We believe proper assessment of decellularized tissue composition is paramount for the successful comparison of different decellularization protocols and clinical translation.

A deep learning platform to assess drug proarrhythmia risk.

Drug safety initiatives have endorsed human iPSC-derived cardiomyocytes (hiPSC-CMs) as an in vitro model for predicting drug-induced cardiac arrhythmia. However, the extent to which human-defined features of in vitro arrhythmia predict actual clinical risk has been much debated. Here, we trained a convolutional neural network classifier (CNN) to learn features of in vitro action potential recordings of hiPSC-CMs that are associated with lethal Torsade de Pointes arrhythmia. The CNN classifier accurately predicted the risk of drug-induced arrhythmia in people. The risk profile of the test drugs was similar across hiPSC-CMs derived from different healthy donors. In contrast, pathogenic mutations that cause arrhythmogenic cardiomyopathies in patients significantly increased the proarrhythmic propensity to certain intermediate and high-risk drugs in the hiPSC-CMs. Thus, deep learning can identify in vitro arrhythmic features that correlate with clinical arrhythmia and discern the influence of patient genetics on the risk of drug-induced arrhythmia.

Designing Novel BCR-ABL Inhibitors for Chronic Myeloid Leukemia with Improved Cardiac Safety.

Development of tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL oncogene constitutes an effective approach for the treatment of chronic myeloid leukemia (CML) and/or acute lymphoblastic leukemia. However, currently available inhibitors are limited by drug resistance and toxicity. Ponatinib, a third-generation inhibitor, has demonstrated excellent efficacy against both wild type and mutant BCR-ABL kinase, including the "gatekeeper" T315I mutation that is resistant to all other currently available TKIs. However, it is one of the most cardiotoxic of the FDA-approved TKIs. Herein, we report the structure-guided design of a novel series of potent BCR-ABL inhibitors, particularly for the T315I mutation. Our drug design paradigm was coupled to iPSC-cardiomyocyte models. Systematic structure-activity relationship studies identified two compounds, 33a and 36a, that significantly inhibit the kinase activity of both native BCR-ABL and the T315I mutant. We have identified the most cardiac-safe TKIs reported to date, and they may be used to effectively treat CML patients with the T315I mutation.

Reengineering Ponatinib to Minimize Cardiovascular Toxicity.

Small molecule tyrosine kinase inhibitors (TKI) have revolutionized cancer treatment and greatly improved patient survival. However, life-threatening cardiotoxicity of many TKIs has become a major concern. Ponatinib (ICLUSIG) was developed as an inhibitor of the BCR-ABL oncogene and is among the most cardiotoxic of TKIs. Consequently, use of ponatinib is restricted to the treatment of tumors carrying T315I-mutated BCR-ABL, which occurs in chronic myeloid leukemia (CML) and confers resistance to first- and second-generation inhibitors such as imatinib and nilotinib. Through parallel screening of cardiovascular toxicity and antitumor efficacy assays, we engineered safer analogs of ponatinib that retained potency against T315I BCR-ABL kinase activity and suppressed T315I mutant CML tumor growth. The new compounds were substantially less toxic in human cardiac vasculogenesis and cardiomyocyte contractility assays in vitro. The compounds showed a larger therapeutic window in vivo, leading to regression of human T315I mutant CML xenografts without cardiotoxicity. Comparison of the kinase inhibition profiles of ponatinib and the new compounds suggested that ponatinib cardiotoxicity is mediated by a few kinases, some of which were previously unassociated with cardiovascular disease. Overall, the study develops an approach using complex phenotypic assays to reduce the high risk of cardiovascular toxicity that is prevalent among small molecule oncology therapeutics. SIGNIFICANCE: Newly developed ponatinib analogs retain antitumor efficacy but elicit significantly decreased cardiotoxicity, representing a therapeutic opportunity for safer CML treatment.

Mechanical and Degradation Properties of Hybrid Scaffolds for Tissue Engineered Heart Valve (TEHV).

In addition to biocompatibility, an ideal scaffold for the regeneration of valvular tissue should also replicate the natural heart valve extracellular matrix (ECM) in terms of biomechanical properties and structural stability. In our previous paper, we demonstrated the development of collagen type I and hyaluronic acid (HA)-based scaffolds with interlaced microstructure. Such hybrid scaffolds were found to be compatible with cardiosphere-derived cells (CDCs) to potentially regenerate the diseased aortic heart valve. This paper focused on the quantification of the effect of crosslinking density on the mechanical properties under dry and wet conditions as well as degradation resistance. Elastic moduli increased with increasing crosslinking densities, in the dry and wet state, for parent networks, whereas those of interlaced scaffolds were higher than either network alone. Compressive and storage moduli ranged from 35 ± 5 to 95 ± 5 kPa and 16 ± 2 kPa to 113 ± 6 kPa, respectively, in the dry state. Storage moduli, in the dry state, matched and exceeded those of human aortic valve leaflets (HAVL). Similarly, degradation resistance increased with increasing the crosslinking densities for collagen-only and HA-only scaffolds. Interlaced scaffolds showed partial degradation in the presence of either collagenase or hyaluronidase as compared to when exposed to both enzymes together. These results agree with our previous findings that interlaced scaffolds were composed of independent collagen and HA networks without crosslinking between them. Thus, collagen/HA interlaced scaffolds have the potential to fill in the niche for designing an ideal tissue engineered heart valve (TEHV).

Physiological and pharmacological stimulation for in vitro maturation of substrate metabolism in human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable human cardiac cells to be studied in vitro, although they use glucose as their primary metabolic substrate and do not recapitulate the properties of adult cardiomyocytes. Here, we have explored the interplay between maturation by stimulation of fatty acid oxidation and by culture in 3D. We have investigated substrate metabolism in hiPSC-CMs grown as a monolayer and in 3D, in porous collagen-derived scaffolds and in engineered heart tissue (EHT), by measuring rates of glycolysis and glucose and fatty acid oxidation (FAO), and changes in gene expression and mitochondrial oxygen consumption. FAO was stimulated by activation of peroxisome proliferator-activated receptor alpha (PPARα), using oleate and the agonist WY-14643, which induced an increase in FAO in monolayer hiPSC-CMs. hiPSC-CMs grown in 3D on collagen-derived scaffolds showed reduced glycolysis and increased FAO compared with monolayer cells. Activation of PPARα further increased FAO in cells on collagen/elastin scaffolds but not collagen or collagen/chondroitin-4-sulphate scaffolds. In EHT, FAO was significantly higher than in monolayer cells or those on static scaffolds and could be further increased by culture with oleate and WY-14643. In conclusion, a more mature metabolic phenotype can be induced by culture in 3D and FAO can be incremented by pharmacological stimulation.

Small-molecule probe reveals a kinase cascade that links stress signaling to TCF/LEF and Wnt responsiveness.

Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through ß-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.

Trilayer scaffold with cardiosphere-derived cells for heart valve tissue engineering.

Natural polymers collagen, glycosaminoglycans, and elastin are promising candidate materials for heart valve tissue engineering scaffolds. This work produced trilayer scaffolds that resembled the layered structures of the extracellular matrices of native heart valves. The scaffolds showed anisotropic bending moduli (in both dry and hydrated statuses) depending on the loading directions (lower in the With Curvature direction than in the Against Curvature direction), which mimicked the characteristic behavior of the native heart valves. The interactions between cardiosphere-derived cells and the scaffolds were characterized by multiphoton microscopy, and relatively similar cell distributions were observed on different layers (a cell density of 3,000-4,000 mm-3 and a migration depth of 0.3-0.4 mm). The trilayer scaffold has represented a forwarding step from the previous studies, in attempting to better replicate a native heart valve structurally, mechanically, and biologically.

Reengineering an Antiarrhythmic Drug Using Patient hiPSC Cardiomyocytes to Improve Therapeutic Potential and Reduce Toxicity.

Modeling cardiac disorders with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes is a new paradigm for preclinical testing of candidate therapeutics. However, disease-relevant physiological assays can be complex, and the use of hiPSC-cardiomyocyte models of congenital disease phenotypes for guiding large-scale screening and medicinal chemistry have not been shown. We report chemical refinement of the antiarrhythmic drug mexiletine via high-throughput screening of hiPSC-CMs derived from patients with the cardiac rhythm disorder long QT syndrome 3 (LQT3) carrying SCN5A sodium channel variants. Using iterative cycles of medicinal chemistry synthesis and testing, we identified drug analogs with increased potency and selectivity for inhibiting late sodium current across a panel of 7 LQT3 sodium channel variants and suppressing arrhythmic activity across multiple genetic and pharmacological hiPSC-CM models of LQT3 with diverse backgrounds. These mexiletine analogs can be exploited as mechanistic probes and for clinical development.

Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na+) channels and sarcoplasmic reticulum calcium (Ca2+) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na+ channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.

AlleleProfileR: A versatile tool to identify and profile sequence variants in edited genomes.

Gene editing strategies, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9), are revolutionizing biology. However, quantitative and sensitive detection of targeted mutations are required to evaluate and quantify the genome editing outcomes. Here we present AlleleProfileR, a new analysis tool, written in a combination of R and C++, with the ability to batch process the sequence analysis of large and complex genome editing experiments, including the recently developed base editing technologies.

Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ.

NOTCH plays a pivotal role during normal development and in congenital disorders and cancer. γ-secretase inhibitors are commonly used to probe NOTCH function, but also block processing of numerous other proteins. We discovered a new class of small molecule inhibitor that disrupts the interaction between NOTCH and RBPJ, which is the main transcriptional effector of NOTCH signaling. RBPJ Inhibitor-1 (RIN1) also blocked the functional interaction of RBPJ with SHARP, a scaffold protein that forms a transcriptional repressor complex with RBPJ in the absence of NOTCH signaling. RIN1 induced changes in gene expression that resembled siRNA silencing of RBPJ rather than inhibition at the level of NOTCH itself. Consistent with disruption of NOTCH signaling, RIN1 inhibited the proliferation of hematologic cancer cell lines and promoted skeletal muscle differentiation from C2C12 myoblasts. Thus, RIN1 inhibits RBPJ in its repressing and activating contexts, and can be exploited for chemical biology and therapeutic applications.

Will iPSC-cardiomyocytes revolutionize the discovery of drugs for heart disease?

Cardiovascular disease remains the largest single cause of mortality in the Western world, despite significant advances in clinical management over the years. Unfortunately, the development of new cardiovascular medicines is stagnating and can in part be attributed to the difficulty of screening for novel therapeutic strategies due to a lack of suitable models. The advent of human induced pluripotent stem cells and the ability to make limitless numbers of cardiomyocytes could revolutionize heart disease modeling and drug discovery. This review summarizes the state of the art in the field, describes the strengths and weaknesses of the technology, and applications where the model system would be most appropriate.

An Automated Platform for Assessment of Congenital and Drug-Induced Arrhythmia with hiPSC-Derived Cardiomyocytes.

The ability to produce unlimited numbers of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) harboring disease and patient-specific gene variants creates a new paradigm for modeling congenital heart diseases (CHDs) and predicting proarrhythmic liabilities of drug candidates. However, a major roadblock to implementing hiPSC-CM technology in drug discovery is that conventional methods for monitoring action potential (AP) kinetics and arrhythmia phenotypes in vitro have been too costly or technically challenging to execute in high throughput. Herein, we describe the first large-scale, fully automated and statistically robust analysis of AP kinetics and drug-induced proarrhythmia in hiPSC-CMs. The platform combines the optical recording of a small molecule fluorescent voltage sensing probe (VoltageFluor2.1.Cl), an automated high throughput microscope and automated image analysis to rapidly generate physiological measurements of cardiomyocytes (CMs). The technique can be readily adapted on any high content imager to study hiPSC-CM physiology and predict the proarrhythmic effects of drug candidates.

Stem Cell Therapy for the Heart: Blind Alley or Magic Bullet?

When stressed by ageing or disease, the adult human heart is unable to regenerate, leading to scarring and hypertrophy and eventually heart failure. As a result, stem cell therapy has been proposed as an ultimate therapeutic strategy, as stem cells could limit adverse remodelling and give rise to new cardiomyocytes and vasculature. Unfortunately, the results from clinical trials to date have been largely disappointing. In this review, we discuss the current status of the field and describe various limitations and how future work may attempt to resolve these to make way to successful clinical translation.

Chronic High-Fat Feeding Affects the Mesenchymal Cell Population Expanded From Adipose Tissue but Not Cardiac Atria.

UNLABELLED: Mesenchymal stem cells offer a promising approach to the treatment of myocardial infarction and prevention of heart failure. However, in the clinic, cells will be isolated from patients who may be suffering from comorbidities such as obesity and diabetes, which are known to adversely affect progenitor cells. Here we determined the effect of a high-fat diet (HFD) on mesenchymal stem cells from cardiac and adipose tissues. Mice were fed a HFD for 4 months, after which cardiosphere-derived cells (CDCs) were cultured from atrial tissue and adipose-derived mesenchymal cells (ADMSCs) were isolated from epididymal fat depots. HFD raised body weight, fasted plasma glucose, lactate, and insulin. Ventricle and liver tissue of HFD-fed mice showed protein changes associated with an early type 2 diabetic phenotype. At early passages, more ADMSCs were obtained from HFD-fed mice than from chow-fed mice, whereas CDC number was not affected by HFD. Migratory and clonogenic capacity and release of vascular endothelial growth factor did not differ between cells from HFD- and chow-fed animals. CDCs from chow-fed and HFD-fed mice showed no differences in surface marker expression, whereas ADMSCs from HFD-fed mice contained more cells positive for CD105, DDR2, and CD45, suggesting a high component of endothelial, fibroblast, and hematopoietic cells. Both Noggin and transforming growth factor ß-supplemented medium induced an early stage of differentiation in CDCs toward the cardiomyocyte phenotype. Thus, although chronic high-fat feeding increased the number of fibroblasts and hematopoietic cells within the ADMSC population, it left cardiac progenitor cells largely unaffected. SIGNIFICANCE: Mesenchymal cells are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. In the clinic, cells are isolated from patients who may be suffering from comorbidities such as obesity and diabetes. This study examined the effect of a high-fat diet on mesenchymal cells from cardiac and adipose tissues. It was demonstrated that a high-fat diet did not affect cardiac progenitor cells but increased the number of fibroblasts and hematopoietic cells within the adipose-derived mesenchymal cell population.