Glycoproteomics Reveals Decorin Peptides With Anti-Myostatin Activity in Human Atrial Fibrillation.

BACKGROUND: Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). METHODS: Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. RESULTS: ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. On enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were identified consistently in the flowthrough. Of all ECM proteins identified, decorin was found to be the most fragmented. Within its protein core, 18 different cleavage sites were identified. In contrast, less cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was detected predominantly in ventricles in comparison with atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had greater levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. CONCLUSIONS: This proteomics study is the first to analyze the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages, can regulate the local bioavailability of antihypertrophic and profibrotic growth factors.

DNA methylation in an engineered heart tissue model of cardiac hypertrophy: common signatures and effects of DNA methylation inhibitors.

DNA methylation affects transcriptional regulation and constitutes a drug target in cancer biology. In cardiac hypertrophy, DNA methylation may control the fetal gene program. We therefore investigated DNA methylation signatures and their dynamics in an in vitro model of cardiac hypertrophy based on engineered heart tissue (EHT). We exposed EHTs from neonatal rat cardiomyocytes to a 12-fold increased afterload (AE) or to phenylephrine (PE 20 µM) and compared DNA methylation signatures to control EHT by pull-down assay and DNA methylation microarray. A 7-day intervention sufficed to induce contractile dysfunction and significantly decrease promoter methylation of hypertrophy-associated upregulated genes such as Nppa (encoding ANP) and Acta1 (α-skeletal actin) in both intervention groups. To evaluate whether pathological consequences of AE are affected by inhibiting de novo DNA methylation we applied AE in the absence and presence of DNA methyltransferase (DNMT) inhibitors: 5-aza-2'-deoxycytidine (aza, 100 µM, nucleosidic inhibitor), RG108 (60 µM, non-nucleosidic) or methylene disalicylic acid (MDSA, 25 µM, non-nucleosidic). Aza had no effect on EHT function, but RG108 and MDSA partially prevented the detrimental consequences of AE on force, contraction and relaxation velocity. RG108 reduced AE-induced Atp2a2 (SERCA2a) promoter methylation. The results provide evidence for dynamic DNA methylation in cardiac hypertrophy and warrant further investigation of the potential of DNA methylation in the treatment of cardiac hypertrophy.

Deciphering the microRNA signature of pathological cardiac hypertrophy by engineered heart tissue- and sequencing-technology.

Pathological cardiac hypertrophy and fibrosis are modulated by a set of microRNAs, most of which have been detected in biologically complex animal models of hypertrophy by arrays with moderate sensitivity and disregard of passenger strand (previously "star") microRNAs. Here, we aimed at precisely analyzing the microRNA signature of cardiac hypertrophy and fibrosis by RNA sequencing in a standardized in vitro hypertrophy model based on engineered heart tissue (EHT). Spontaneously beating, force-generating fibrin EHTs from neonatal rat heart cells were subjected to afterload enhancement for 7days (AE-EHT), and EHTs without intervention served as controls. AE resulted in reduced contractile force and relaxation velocity, fibrotic changes and reactivation of the fetal gene program. Small RNAs were extracted from control and AE-EHTs and sequencing yielded almost 750 different mature microRNAs, many of which have never been described before in rats. The detection of both arms of the precursor stem-loop (pre-miRNA), namely -3p and -5p miRs, was frequent. 22 abundantly sequenced microRNAs were >1.3× upregulated and 15 abundantly sequenced microRNAs downregulated to <0.77×. Among the upregulated microRNAs were 3 pairs of guide and passenger strand microRNAs (miR-21-5p/-3p, miR-322-5p/-3p, miR-210-3p/-5p) and one single passenger strand microRNA (miR-140-3p). Among downregulated microRNAs were 3 pairs (miR-133a-3p/-5p, miR-30e-5p/3p, miR-30c-5p/-3p). Preincubating EHTs with anti-miR-21-5p markedly attenuated the AE-induced contractile failure, cardiomyocyte hypertrophy and fibrotic response, recapitulating prior results in whole animals. Taken together, AE-induced pathological hypertrophy in EHTs is associated with 37 differentially regulated microRNAs, including many passenger strands. Antagonizing miR-21-5p ameliorates dysfunction in this model.

Identification of hypertrophy-modulating Cullin-RING ubiquitin ligases in primary cardiomyocytes.

Cullin-RING ubiquitin ligases (CRL) regulate numerous biological processes in the heart and have been implicated in regulating cardiac hypertrophy. This study aimed to identify novel hypertrophy-modulating CRLs in cardiomyocytes (CM). A functional genomic approach using siRNA-mediated depletion and automated microscopy was employed to screen for cell size-modulating CRLs in neonatal rat CM. Screening hits were confirmed by 3H-isoleucine incorporation. Of 43 targets screened, siRNA-mediated depletion of Fbxo6, Fbxo45, and Fbxl14 resulted in decreased cell size, whereas depletion of Fbxo9, Fbxo25, Fbxo30, Fbxo32, Fbxo33, Cullin1, Roc1, Ddb1, Fbxw4, and Fbxw5 led to a markedly increased cell size under basal conditions. In CM stimulated with phenylephrine (PE), depletion of Fbxo6, Fbxo25, Fbxo33, Fbxo45, and Fbxw4 further augmented PE-induced hypertrophy. As a proof-of-concept, the CRLFbox25 was analysed by transverse aortic constriction (TAC) resulting in a 4.5-fold increase in Fbxo25 protein concentrations compared to control animals. In cell culture, siRNA-mediated depletion of Fbxo25 resulted in a ∼ 37% increase in CM cell size and ∼41% increase in 3H-isoleucine incorporation. Depleting Fbxo25 resulted in upregulation of Anp and Bnp. In summary, we identified 13 novel CRLs as positive or negative regulators of CM hypertrophy. Of these, CRLFbox25 was further characterized, as a potential modulator of cardiac hypertrophy.

Integrating single-cell genomics pipelines to discover mechanisms of stem cell differentiation.

Pluripotent stem cells underpin a growing sector that leverages their differentiation potential for research, industry, and clinical applications. This review evaluates the landscape of methods in single-cell transcriptomics that are enabling accelerated discovery in stem cell science. We focus on strategies for scaling stem cell differentiation through multiplexed single-cell analyses, for evaluating molecular regulation of cell differentiation using new analysis algorithms, and methods for integration and projection analysis to classify and benchmark stem cell derivatives against in vivo cell types. By discussing the available methods, comparing their strengths, and illustrating strategies for developing integrated analysis pipelines, we provide user considerations to inform their implementation and interpretation.

Effects of the Delta Opioid Receptor Agonist DADLE in a Novel Hypoxia-Reoxygenation Model on Human and Rat-Engineered Heart Tissue: A Pilot Study.

Intermittent hypoxia and various pharmacological compounds protect the heart from ischemia reperfusion injury in experimental approaches, but the translation into clinical trials has largely failed. One reason may lie in species differences and the lack of suitable human in vitro models to test for ischemia/reperfusion. We aimed to develop a novel hypoxia-reoxygenation model based on three-dimensional, spontaneously beating and work performing engineered heart tissue (EHT) from rat and human cardiomyocytes. Contractile force, the most important cardiac performance parameter, served as an integrated outcome measure. EHTs from neonatal rat cardiomyocytes were subjected to 90 min of hypoxia which led to cardiomyocyte apoptosis as revealed by caspase 3-staining, increased troponin I release (time control vs. 24 h after hypoxia: cTnI 2.7 vs. 6.3 ng/mL, ** p = 0.002) and decreased contractile force (64 ± 6% of baseline) in the long-term follow-up. The detrimental effects were attenuated by preceding the long-term hypoxia with three cycles of 10 min hypoxia (i.e., hypoxic preconditioning). Similarly, [d-Ala2, d-Leu5]-enkephalin (DADLE) reduced the effect of hypoxia on force (recovery to 78 ± 5% of baseline with DADLE preconditioning vs. 57 ± 5% without, p = 0.012), apoptosis and cardiomyocyte stress. Human EHTs presented a comparable hypoxia-induced reduction in force (55 ± 5% of baseline), but DADLE failed to precondition them, likely due to the absence of δ-opioid receptors. In summary, this hypoxia-reoxygenation in vitro model displays cellular damage and the decline of contractile function after hypoxia allows the investigation of preconditioning strategies and will therefore help us to understand the discrepancy between successful conditioning in vitro experiments and its failure in clinical trials.

Magnetic Adjustment of Afterload in Engineered Heart Tissues.

Afterload is known to drive the development of both physiological and pathological cardiac states. As such, studying the outcomes of altered afterload states could yield important insights into the mechanisms controlling these critical processes. However, an experimental technique for precisely fine-tuning afterload in heart tissue over time is currently lacking. Here, a newly developed magnetics-based technique for achieving this control in engineered heart tissues (EHTs) is described. In order to produce magnetically responsive EHTs (MR-EHTs), the tissues are mounted on hollow silicone posts, some of which contain small permanent magnets. A second set of permanent magnets is press-fit into an acrylic plate such that they are oriented with the same polarity and are axially-aligned with the post magnets. To adjust afterload, this plate of magnets is translated toward (higher afterload) or away (lower afterload) from the post magnets using a piezoelectric stage fitted with an encoder. The motion control software used to adjust stage positioning allows for the development of user-defined afterload regimens while the encoder ensures that the stage corrects for any inconsistencies in its location. This work describes the fabrication, calibration, and implementation of this system to enable the development of similar platforms in other labs around the world. Representative results from two separate experiments are included to exemplify the range of different studies that can be performed using this system.

Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research.

The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR Support Protocol 4: Identity: Short tandem repeat analysis.

A magnetics-based approach for fine-tuning afterload in engineered heart tissues.

Afterload plays important roles during heart development and disease progression, however, studying these effects in a laboratory setting is challenging. Current techniques lack the ability to precisely and reversibly alter afterload over time. Here, we describe a magnetics-based approach for achieving this control and present results from experiments in which this device was employed to sequentially increase afterload applied to rat engineered heart tissues (rEHTs) over a 7-day period. The contractile properties of rEHTs grown on control posts marginally increased over the observation period. The average post deflection, fractional shortening, and twitch velocities measured for afterload-affected tissues initially followed this same trend, but fell below control tissue values at high magnitudes of afterload. However, the average force, force production rate, and force relaxation rate for these rEHTs were consistently up to 3-fold higher than in control tissues. Transcript levels of hypertrophic or fibrotic markers and cell size remained unaffected by afterload, suggesting that the increased force output was not accompanied by pathological remodeling. Accordingly, the increased force output was fully reversed to control levels during a stepwise decrease in afterload over 4 hours. Afterload application did not affect systolic or diastolic tissue lengths, indicating that the afterload system was likely not a source of changes in preload strain. In summary, the afterload system developed herein is capable of fine-tuning EHT afterload while simultaneously allowing optical force measurements. Using this system, we found that small daily alterations in afterload can enhance the contractile properties of rEHTs, while larger increases can have temporary undesirable effects. Overall, these findings demonstrate the significant role that afterload plays in cardiac force regulation. Future studies with this system may allow for novel insights into the mechanisms that underlie afterload-induced adaptations in cardiac force development.

Blockade of miR-140-3p prevents functional deterioration in afterload-enhanced engineered heart tissue.

Afterload enhancement (AE) of rat engineered heart tissue (EHT) in vitro leads to a multitude of changes that in vivo are referred to as pathological cardiac hypertrophy: e.g., cardiomyocyte hypertrophy, contractile dysfunction, reactivation of fetal genes and fibrotic changes. Moreover AE induced the upregulation of 22 abundantly expressed microRNAs. Here, we aimed at evaluating the functional effect of inhibiting 7 promising microRNAs (miR-21-5p, miR-146b-5p, miR-31a-5p, miR-322-5p, miR-450a-5p, miR-140-3p and miR-132-3p) in a small-range screen. Singular transfection of locked nucleic acid (LNA)-based anti-miRs at 100 nM (before the one week AE-procedure) led to a powerful reduction of the targeted microRNAs. Pretreatment with anti-miR-146b-5p, anti-miR-322-5p or anti-miR-450a-5p did not alter the AE-induced contractile decline, while anti-miR-31a-5p-pretreatment even worsened it. Anti-miR-21-5p and anti-miR-132-3p partially attenuated the AE-effect, confirming previous reports. LNA-anti-miR against miR-140-3p, a microRNA recently identified as a prognostic biomarker of cardiovascular disease, also attenuated the AE-effect. To simplify future in vitro experiments and to create an inhibitor for in vivo applications, we designed shorter miR-140-3p-inhibitors and encountered variable efficiency. Only the inhibitor that effectively repressed miR-140-3p was also protective against the AE-induced contractile decline. In summary, in a small-range functional screen, miR-140-3p evolved as a possible new target for the attenuation of afterload-induced pathological cardiac hypertrophy.

Toward Second-Generation Cardiomyogenic and Anti-cardiofibrotic 1,4-Dihydropyridine-Class TGFß Inhibitors.

Innovative therapeutic modalities for pharmacological intervention of transforming growth factorâ€…ß (TGFß)-dependent diseases are of great value. b-Annelated 1,4-dihydropyridines (DHPs) might be such a class, as they induce TGFß receptor type II degradation. However, intrinsic drawbacks are associated with this compound class and were systematically addressed in the presented study. It was possible to install polar functionalities and bioisosteric moieties at distinct sites of the molecules while maintaining TGFß-inhibitory activities. The introduction of a 2-amino group or 7-N-alkyl modification proved to be successful strategies. Aqueous solubility was improved by up to seven-fold at pH 7.4 and 200-fold at pH 3 relative to the parent ethyl 4-(biphenyl-4-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate. The therapeutic potential of the presented DHPs was further underscored in view of a potential dual mode of action: The differentiation of committed human iPSC-derived cardiac progenitor cells (CPCs) was potently stimulated, and the rescue of cardiac fibrosis phenotypes was observed in engineered heart tissue (EHT) constructs.

Analysis of fibrosis in control or pressure overloaded rat hearts after mechanical unloading by heterotopic heart transplantation.

Mechanical unloading (MU) by implantation of left ventricular assist devices (LVAD) has become clinical routine. This procedure has been shown to reverse cardiac pathological remodeling, with the underlying molecular mechanisms incompletely understood. Most studies thus far were performed in non-standardized human specimens or MU of healthy animal hearts. Our study investigates cardiac remodeling processes in sham-operated healthy rat hearts and in hearts subjected to standardized pathological pressure overload by transverse aortic constriction (TAC) prior to MU by heterotopic heart transplantation (hHTx/MU). Rats underwent sham or TAC surgery. Disease progression was monitored by echocardiography prior to MU by hHTx/MU. Hearts after TAC or TAC combined with hHTx/MU were removed and analyzed by histology, western immunoblot and gene expression analysis. TAC surgery resulted in cardiac hypertrophy and impaired cardiac function. TAC hearts revealed significantly increased cardiac myocyte diameter and mild fibrosis. Expression of hypertrophy associated genes after TAC was higher compared to hearts after hHTx/MU. While cardiac myocyte cell diameter regressed to the level of sham-operated controls in all hearts subjected to hHTx/MU, fibrotic remodeling was significantly exacerbated. Transcription of pro-fibrotic and apoptosis-related genes was markedly augmented in all hearts after hHTx/MU. Sarcomeric proteins involved in excitation-contraction coupling displayed significantly lower phosphorylation levels after TAC and significantly reduced total protein levels after hHTx/MU. Development of myocardial fibrosis, cardiac myocyte atrophy and loss of sarcomeric proteins was observed in all hearts that underwent hHTX/MU regardless of the disease state. These results may help to explain the clinical experience with low rates of LVAD removal due to lack of myocardial recovery.

Author Correction: Differentiation of cardiomyocytes and generation of human engineered heart tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Differentiation of cardiomyocytes and generation of human engineered heart tissue.

Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)-in a strip format-that generate force under auxotonic stretch conditions. 10-15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.

Human Engineered Heart Tissue: Analysis of Contractile Force.

Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating engineered heart tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane- and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human heart tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling.

Ataxin-10 is part of a cachexokine cocktail triggering cardiac metabolic dysfunction in cancer cachexia.

OBJECTIVES: Cancer cachexia affects the majority of tumor patients and significantly contributes to high mortality rates in these subjects. Despite its clinical importance, the identity of tumor-borne signals and their impact on specific peripheral organ systems, particularly the heart, remain mostly unknown. METHODS AND RESULTS: By combining differential colon cancer cell secretome profiling with large-scale cardiomyocyte phenotyping, we identified a signature panel of seven "cachexokines", including Bridging integrator 1, Syntaxin 7, Multiple inositol-polyphosphate phosphatase 1, Glucosidase alpha acid, Chemokine ligand 2, Adamts like 4, and Ataxin-10, which were both sufficient and necessary to trigger cardiac atrophy and aberrant fatty acid metabolism in cardiomyocytes. As a prototypical example, engineered secretion of Ataxin-10 from non-cachexia-inducing cells was sufficient to induce cachexia phenotypes in cardiomyocytes, correlating with elevated Ataxin-10 serum levels in murine and human cancer cachexia models. CONCLUSIONS: As Ataxin-10 serum levels were also found to be elevated in human cachectic cancer patients, the identification of Ataxin-10 as part of a cachexokine cocktail now provides a rational approach towards personalized predictive, diagnostic and therapeutic measures in cancer cachexia.