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.

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.

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.

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.