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.

Piezo2 is not an indispensable mechanosensor in murine cardiomyocytes.

A short-term increase in ventricular filling leads to an immediate (Frank-Starling mechanism) and a slower (Anrep effect) rise in cardiac contractility, while long-term increased cardiac load (e.g., in arterial hypertension) decreases contractility. Whether these answers to mechanical tension are mediated by specific sensors in cardiomyocytes remains elusive. In this study, the piezo2 protein was evaluated as a potential mechanosensor. Piezo2 was found to be upregulated in various rat and mouse cardiac tissues upon mechanical or pharmacological stress. To investigate its function, C57BL/6J mice with homozygous cardiomyocyte-specific piezo2 knockout [Piezo2-KO] were created. To this end, α-MHC-Cre mice were crossed with homozygous "floxed" piezo2 mice. α-MHC-Cre mice crossed with wildtype mice served as controls [WT-Cre+]. In cardiomyocytes of Piezo2-KO mice, piezo2 mRNA was reduced by > 90% and piezo2 protein was not detectable. Piezo2-KO mice displayed no morphological abnormalities or altered cardiac function under nonstressed conditions. In a subsequent step, hearts of Piezo2-KO or WT-Cre+-mice were stressed by either three weeks of increased afterload (angiotensin II, 2.5 mg/kg/day) or one week of hypercontractility (isoprenaline, 30 mg/kg/day). As expected, angiotensin II treatment in WT-Cre+-mice resulted in higher heart and lung weight (per body weight, + 38%, + 42%), lower ejection fraction and cardiac output (- 30%, - 39%) and higher left ventricular anterior and posterior wall thickness (+ 34%, + 37%), while isoprenaline led to higher heart weight (per body weight, + 25%) and higher heart rate and cardiac output (+ 24%, + 54%). The Piezo2-KO mice reacted similarly with the exception that the angiotensin II-induced increases in wall thickness were blunted and the isoprenaline-induced increase in cardiac output was slightly less pronounced. As cardiac function was neither severely affected under basal nor under stressed conditions in Piezo2-KO mice, we conclude that piezo2 is not an indispensable mechanosensor in cardiomyocytes.

Recapitulation of dyssynchrony-associated contractile impairment in asymmetrically paced engineered heart tissue.

BACKGROUND: One third of heart failure patients exhibit dyssynchronized electromechanical activity of the heart (evidenced by a broad QRS-complex). Cardiac resynchronization therapy (CRT) in the form of biventricular pacing improves cardiac output and clinical outcome of responding patients. Technically demanding and laborious large animal models have been developed to better predict responders of CRT and to investigate molecular mechanisms of dyssynchrony and CRT. The aim of this study was to establish a first humanized in vitro model of dyssynchrony and CRT. METHODS: Cardiomyocytes were differentiated from human induced pluripotent stem cells and cast into a fibrin matrix to produce engineered heart tissue (EHT). EHTs were either field stimulated in their entirety (symmetrically) or excited locally from one end (asymmetrically) or they were allowed to beat spontaneously. RESULTS: Asymmetrical pacing led to a depolarization wave from one end to the other end, which was visualized in human EHT transduced with a fast genetic Ca2+-sensor (GCaMP6f) arguing for dyssynchronous excitation. Symmetrical pacing in contrast led to an instantaneous (synchronized) Ca2+-signal throughout the EHT. To investigate acute and long-term functional effects, spontaneously beating human EHTs (0.5-0.8 Hz) were divided into a non-paced control group, a symmetrically and an asymmetrically paced group, each stimulated at 1 Hz. Symmetrical pacing was clearly superior to asymmetrical pacing or no pacing regarding contractile force both acutely and even more pronounced after weeks of continuous stimulation. Contractile dysfunction that can be evoked by an increased afterload was aggravated in the asymmetrically paced group. Consistent with reports from paced dogs, p38MAPK and CaMKII-abundance was higher under asymmetrical than under symmetrical pacing while pAKT was considerably lower. CONCLUSIONS: This model allows for long-term pacing experiments mimicking electrical dyssynchrony vs. synchrony in vitro. Combined with force measurement and afterload stimulus manipulation, it provides a robust new tool to gain insight into the biology of dyssynchrony and CRT.

Hypertrophic signaling compensates for contractile and metabolic consequences of DNA methyltransferase 3A loss in human cardiomyocytes.

The role of DNA methylation in cardiomyocyte physiology and cardiac disease remains a matter of controversy. We have recently provided evidence for an important role of DNMT3A in human cardiomyocyte cell homeostasis and metabolism, using engineered heart tissue (EHT) generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes carrying a knockout of the de novo DNA methyltransferase DNMT3A. Unlike isogenic control EHT, knockout EHT displayed morphological abnormalities such as lipid accumulations inside cardiomyocytes associated with impaired mitochondrial metabolism, as well as functional defects and impaired glucose metabolism. Here, we analyzed the role of DNMT3A in the setting of cardiac hypertrophy. We induced hypertrophic signaling by treatment with 50 nM endothelin-1 and 20 µM phenylephrine for one week and assessed EHT contractility, morphology, DNA methylation, and gene expression. While both knockout EHTs and isogenic controls showed the expected activation of the hypertrophic gene program, knockout EHTs were protected from hypertrophy-related functional impairment. Conversely, hypertrophic treatment prevented the metabolic consequences of a loss of DNMT3A, i.e. abolished lipid accumulation in cardiomyocytes likely by partial normalization of mitochondrial metabolism and restored glucose metabolism and metabolism-related gene expression of knockout EHT. Together, these data suggest an important role of DNA methylation not only for cardiomyocyte physiology, but also in the setting of cardiac disease.

Assessment of Cardiotoxicity With Stem Cell-based Strategies.

PURPOSE: Adverse cardiovascular drug effects pose a substantial medical risk and represent a common cause of drug withdrawal from the market. Thus, current in vitro assays and in vivo animal models still have shortcomings in assessing cardiotoxicity. A human model for more accurate preclinical cardiotoxicity assessment is highly desirable. Current differentiation protocols allow for the generation of human pluripotent stem cell-derived cardiomyocytes in basically unlimited numbers and offer the opportunity to study drug effects on human cardiomyocytes. The purpose of this review is to provide a brief overview of the current approaches to translate studies with pluripotent stem cell-derived cardiomyocytes from basic science to preclinical risk assessment. METHODS: A review of the literature was performed to gather data on the pathophysiology of cardiotoxicity, the current cardiotoxicity screening assays, stem cell-derived cardiomyocytes, and their application in cardiotoxicity screening. FINDINGS: There is increasing evidence that stem cell-derived cardiomyocytes predict arrhythmogenicity with high accuracy. Cardiomyocyte immaturity represents the major limitation so far. However, strategies are being developed to overcome this hurdle, such as tissue engineering. In addition, stem cell-based strategies offer the possibility to assess structural drug toxicity (eg, by anticancer drugs) on complex models that more closely mirror the structure of the heart and contain endothelial cells and fibroblasts. IMPLICATIONS: Pluripotent stem cell-derived cardiomyocytes have the potential to substantially change how preclinical cardiotoxicity screening is performed. To which extent they will replace or complement current approaches is being evaluated.

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.

An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility.

BACKGROUND: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell-derived cardiomyocytes. METHODS: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. RESULTS: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. CONCLUSION: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.

Targeting muscle-enriched long non-coding RNA H19 reverses pathological cardiac hypertrophy.

AIMS: Pathological cardiac remodelling and subsequent heart failure represents an unmet clinical need. Long non-coding RNAs (lncRNAs) are emerging as crucial molecular orchestrators of disease processes, including that of heart diseases. Here, we report on the powerful therapeutic potential of the conserved lncRNA H19 in the treatment of pathological cardiac hypertrophy. METHOD AND RESULTS: Pressure overload-induced left ventricular cardiac remodelling revealed an up-regulation of H19 in the early phase but strong sustained repression upon reaching the decompensated phase of heart failure. The translational potential of H19 is highlighted by its repression in a large animal (pig) model of left ventricular hypertrophy, in diseased human heart samples, in human stem cell-derived cardiomyocytes and in human engineered heart tissue in response to afterload enhancement. Pressure overload-induced cardiac hypertrophy in H19 knock-out mice was aggravated compared to wild-type mice. In contrast, vector-based, cardiomyocyte-directed gene therapy using murine and human H19 strongly attenuated heart failure even when cardiac hypertrophy was already established. Mechanistically, using microarray, gene set enrichment analyses and Chromatin ImmunoPrecipitation DNA-Sequencing, we identified a link between H19 and pro-hypertrophic nuclear factor of activated T cells (NFAT) signalling. H19 physically interacts with the polycomb repressive complex 2 to suppress H3K27 tri-methylation of the anti-hypertrophic Tescalcin locus which in turn leads to reduced NFAT expression and activity. CONCLUSION: H19 is highly conserved and down-regulated in failing hearts from mice, pigs and humans. H19 gene therapy prevents and reverses experimental pressure-overload-induced heart failure. H19 acts as an anti-hypertrophic lncRNA and represents a promising therapeutic target to combat pathological cardiac remodelling.

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.

Aging-regulated anti-apoptotic long non-coding RNA Sarrah augments recovery from acute myocardial infarction.

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.

Phosphomimetic cardiac myosin-binding protein C partially rescues a cardiomyopathy phenotype in murine engineered heart tissue.

Phosphorylation of cardiac myosin-binding protein C (cMyBP-C), encoded by MYBPC3, increases the availability of myosin heads for interaction with actin thus enhancing contraction. cMyBP-C phosphorylation level is lower in septal myectomies of patients with hypertrophic cardiomyopathy (HCM) than in non-failing hearts. Here we compared the effect of phosphomimetic (D282) and wild-type (S282) cMyBP-C gene transfer on the HCM phenotype of engineered heart tissues (EHTs) generated from a mouse model carrying a Mybpc3 mutation (KI). KI EHTs showed lower levels of mutant Mybpc3 mRNA and protein, and altered gene expression compared with wild-type (WT) EHTs. Furthermore, KI EHTs exhibited faster spontaneous contractions and higher maximal force and sensitivity to external [Ca2+] under pacing. Adeno-associated virus-mediated gene transfer of D282 and S282 similarly restored Mybpc3 mRNA and protein levels and suppressed mutant Mybpc3 transcripts. Moreover, both exogenous cMyBP-C proteins were properly incorporated in the sarcomere. KI EHTs hypercontractility was similarly prevented by both treatments, but S282 had a stronger effect than D282 to normalize the force-Ca2+-relationship and the expression of dysregulated genes. These findings in an in vitro model indicate that S282 is a better choice than D282 to restore the HCM EHT phenotype. To which extent the results apply to human HCM remains to be seen.

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.

Long Noncoding RNA-Enriched Vesicles Secreted by Hypoxic Cardiomyocytes Drive Cardiac Fibrosis.

Long non-coding RNAs (lncRNAs) have potential as novel therapeutic targets in cardiovascular diseases, but detailed information about the intercellular lncRNA shuttling mechanisms in the heart is lacking. Here, we report an important novel crosstalk between cardiomyocytes and fibroblasts mediated by the transfer of lncRNA-enriched extracellular vesicles (EVs) in the context of cardiac ischemia. lncRNA profiling identified two hypoxia-sensitive lncRNAs: ENSMUST00000122745 was predominantly found in small EVs, whereas lncRNA Neat1 was enriched in large EVs in vitro and in vivo. Vesicles were taken up by fibroblasts, triggering expression of profibrotic genes. In addition, lncRNA Neat1 was transcriptionally regulated by P53 under basal conditions and by HIF2A during hypoxia. The function of Neat1 was further elucidated in vitro and in vivo. Silencing of Neat1 in vitro revealed that Neat1 was indispensable for fibroblast and cardiomyocyte survival and affected fibroblast functions (reduced migration capacity, stalled cell cycle, and decreased expression of fibrotic genes). Of translational importance, genetic loss of Neat1 in vivo resulted in an impaired heart function after myocardial infarction highlighting its translational relevance.

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.

Correction: Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.

[This corrects the article DOI: 10.1371/journal.pone.0145937.].

S100A4 as a Target of the E3-Ligase Asb2ß and Its Effect on Engineered Heart Tissue.

Background: S100A4 has recently emerged as an important player in cardiac disease, affecting phenotype development in animal models of myocardial infarction and pathological cardiac hypertrophy, albeit it is unclear whether S100A4 exerts a detrimental or beneficial function. The goal of the current study was to analyze S100A4 expression in models of cardiac pathology, investigate its degradation by the ubiquitin-proteasome system (UPS), and furthermore examine the functional effects of S100A4 levels in a 3D model of engineered heart tissue (EHT). Methods and Results: S100A4 mRNA and protein levels were analyzed in different models of cardiac pathology via quantitative RT-PCR and Western blot, showing a higher S100A4 steady-state protein concentration in hearts of Mybpc3-knock-in (KI) hypertrophic cardiomyopathy (HCM) mice. COS-7 cells co-transfected with plasmids encoding mutant (MUT) Asb2ß lacking the E3 ligase activity in combination with V5-tagged S100A4 plasmid presented higher S100A4-V5 protein steady-state concentrations than cells co-transfected with the Asb2ß wild type (WT) plasmid. This effect was blunted by treatment with the specific proteasome inhibitor epoxomicin. Adeno-associated virus serotype 6 (AAV6)-mediated S100A4 overexpression in a 3D model of EHT did not affect contractile parameters. Immunofluorescence analysis showed a cytosolic and partly nuclear expression pattern of S100A4. Gene expression analysis in EHTs overexpressing S100A4-V5 showed markedly lower steady-state concentrations of genes involved in cardiac fibrosis and pathological cardiac hypertrophy. Conclusion: We showed that S100A4 protein level is higher in cardiac tissue of Mybpc3-KI HCM mice probably as a result of a lower degradation by the E3 ligase Asb2ß. While an overexpression of S100A4 did not alter contractile parameters in EHTs, downstream gene expression analysis points toward modulation of signaling cascades involved in fibrosis and hypertrophy.

Pharmacological inhibition of DNA methylation attenuates pressure overload-induced cardiac hypertrophy in rats.

BACKGROUND: Heart failure is associated with altered gene expression and DNA methylation. De novo DNA methylation is associated with gene silencing, but its role in cardiac pathology remains incompletely understood. We hypothesized that inhibition of DNA methyltransferases (DNMT) might prevent the deregulation of gene expression and the deterioration of cardiac function under pressure overload (PO). To test this hypothesis, we evaluated a DNMT inhibitor in PO in rats and analysed DNA methylation in cardiomyocytes. METHODS AND RESULTS: Young male Wistar rats were subjected to PO by transverse aortic constriction (TAC) or to sham surgery. Rats from both groups received solvent or 12.5 mg/kg body weight of the non-nucleosidic DNMT inhibitor RG108, initiated on the day of the intervention. After 4 weeks, we analysed cardiac function by MRI, fibrosis with Sirius Red staining, gene expression by RNA sequencing and qPCR, and DNA methylation by reduced representation bisulphite sequencing (RRBS). RG108 attenuated the ~70% increase in heart weight/body weight ratio of TAC over sham to 47% over sham, partially rescued reduced contractility, diminished the fibrotic response and the downregulation of a set of genes including Atp2a2 (SERCA2a) and Adrb1 (beta1-adrenoceptor). RG108 was associated with significantly lower global DNA methylation in cardiomyocytes by ~2%. The differentially methylated pathways were "cardiac hypertrophy", "cell death" and "xenobiotic metabolism signalling". Among these, "cardiac hypertrophy" was associated with significant methylation differences in the group comparison sham vs. TAC, but not significant between sham+RG108 and TAC+RG108 treatment, suggesting that RG108 partially prevented differential methylation. However, when comparing TAC and TAC+RG108, the pathway cardiac hypertrophy was not significantly differentially methylated. CONCLUSIONS: DNMT inhibitor treatment is associated with attenuation of cardiac hypertrophy and moderate changes in cardiomyocyte DNA methylation. The potential mechanistic link between these two effects and the role of non-myocytes need further clarification.

Human iPSC-derived cardiomyocytes cultured in 3D engineered heart tissue show physiological upstroke velocity and sodium current density.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising tool for drug testing and modelling genetic disorders. Abnormally low upstroke velocity is a current limitation. Here we investigated the use of 3D engineered heart tissue (EHT) as a culture method with greater resemblance to human heart tissue in comparison to standard technique of 2D monolayer (ML) format. INa was measured in ML or EHT using the standard patch-clamp technique. INa density was ~1.8 fold larger in EHT (-18.5 ± 1.9 pA/pF; n = 17) than in ML (-10.3 ± 1.2 pA/pF; n = 23; p < 0.001), approaching densities reported for human CM. Inactivation kinetics, voltage dependency of steady-state inactivation and activation of INa did not differ between EHT and ML and were similar to previously reported values for human CM. Action potential recordings with sharp microelectrodes showed similar upstroke velocities in EHT (219 ± 15 V/s, n = 13) and human left ventricle tissue (LV, 253 ± 7 V/s, n = 25). EHT showed a greater resemblance to LV in CM morphology and subcellular NaV1.5 distribution. INa in hiPSC-CM showed similar biophysical properties as in human CM. The EHT format promotes INa density and action potential upstroke velocity of hiPSC-CM towards adult values, indicating its usefulness as a model for excitability of human cardiac tissue.