Background Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modelling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) towards an adult phenotype under defined conditions. Methods We systematically investigated cell composition, matrix and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We employed morphological, functional, and transcriptome analyses to benchmark maturation of EHM. Results EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M-bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency-response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β1- and β2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and NT-proBNP release; all are classical hallmarks of heart failure. Additionally, we demonstrate scalability of EHM according to anticipated clinical demands for cardiac repair. Conclusions We provide proof-of-concept for a universally applicable technology for the engineering of macro-scale human myocardium for disease modelling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.

The ability to generate patient-specific induced pluripotent stem cells (iPSCs) provides a unique opportunity for modeling heart disease in vitro. In this study, we generated iPSCs from a patient with dilated cardiomyopathy (DCM) caused by a missense mutation S635A in RNA-binding motif protein 20 (RBM20) and investigated the functionality and cell biology of cardiomyocytes (CMs) derived from patient-specific iPSCs (RBM20-iPSCs). The RBM20-iPSC-CMs showed abnormal distribution of sarcomeric α-actinin and defective calcium handling compared to control-iPSC-CMs, suggesting disorganized myofilament structure and altered calcium machinery in CMs of the RBM20 patient. Engineered heart muscles (EHMs) from RBM20-iPSC-CMs showed that not only active force generation was impaired in RBM20-EHMs but also passive stress of the tissue was decreased, suggesting a higher visco-elasticity of RBM20-EHMs. Furthermore, we observed a reduced titin (TTN) N2B-isoform expression in RBM20-iPSC-CMs by demonstrating a reduction of exon skipping in the PEVK region of TTN and an inhibition of TTN isoform switch. In contrast, in control-iPSC-CMs both TTN isoforms N2B and N2BA were expressed, indicating that the TTN isoform switch occurs already during early cardiogenesis. Using next generation RNA sequencing, we mapped transcriptome and splicing target profiles of RBM20-iPSC-CMs and identified different cardiac gene networks in response to the analyzed RBM20 mutation in cardiac-specific processes. These findings shed the first light on molecular mechanisms of RBM20-dependent pathological cardiac remodeling leading to DCM. Our data demonstrate that iPSC-CMs coupled with EHMs provide a powerful tool for evaluating disease-relevant functional defects and for a deeper mechanistic understanding of alternative splicing-related cardiac diseases.

Evolution often results in morphologically similar solutions in different organisms, a phenomenon known as convergence. However, there is little knowledge of the processes that lead to convergence at the genetic level. The genes of the Hox cluster control morphology in animals. They may also be central to the convergence of morphological traits, but whether morphological similarities also require similar changes in Hox gene function is disputed. In arthropods, body subdivision into a region with locomotory appendages (“thorax”) and a region with reduced appendages (“abdomen”) has evolved convergently in several groups, e.g., spiders and insects. In insects, legs develop in the expression domain of the Hox gene Antennapedia ( Antp ), whereas the Hox genes Ultrabithorax ( Ubx ) and abdominal-A mediate leg repression in the abdomen. Here, we show that, unlike Antp in insects, the Antp gene in the spider Achaearanea tepidariorum represses legs in the first segment of the abdomen (opisthosoma), and that Antp and Ubx are redundant in the following segment. The down-regulation of Antp in A. tepidariorum leads to a striking 10-legged phenotype. We present evidence from ectopic expression of the spider Antp gene in Drosophila embryos and imaginal tissue that this unique function of Antp is not due to changes in the Antp protein, but likely due to divergent evolution of cofactors, Hox collaborators or target genes in spiders and flies. Our results illustrate an interesting example of convergent evolution of abdominal leg repression in arthropods by altering the role of distinct Hox genes at different levels of their action.

AimWe have previously reported that early phase (1 week) of experimental volume overload (VO) has an adaptive phenotype while wall stress‐matched pressure overload (PO) is maladaptive. Here we investigate the transition from adaptation to heart failure (HF) in long‐term VO.Methods and results FVB/N wild‐type mice were subjected to VO induced by aortocaval shunt, and were followed by serial echocardiography until in vivo left ventricular ejection fraction was below <50% (135 ± 35 days). Heart failure was evident from increased lung and liver weight and increased mortality compared with sham. Maladaptive remodelling resulted in significantly reduced sarcomeric titin phosphorylation (causing increased sarcomeric stiffness), whereas interstitial fibrosis was not increased. This was paralleled by re‐expression of the fetal gene program, activation of calcium/calmodulin‐dependent protein kinase II (CaMKII), decreased protein kinase B (Akt) phosphorylation, high oxidative stress, and increased apoptosis. Consistently, development of HF and mortality were significantly aggravated in Akt‐deficient mice.ConclusionTransition to HF in VO is associated with decreased Akt and increased CaMKII signalling pathways together with increased oxidative stress and apoptosis. Lack of interstitial fibrosis together with sarcomeric titin hypophosphorylation indicates an increased stiffness at the sarcomeric but not matrix level in VO‐induced HF (in contrast to PO). Transition to HF may result from myocyte loss and myocyte dysfunction owing to increased stiffness.

Despite many aspects of the regulation of segmentation being conserved among arthropods, the evolution of novel gene functions has played an important role in the evolution of developmental regulation and the emergence of new segmental structures. Moreover the study of such novel gene functions can be informative with respect to the patterns and direction of evolutionary changes in developmental programs. The homeobox gene Distal-less (Dll) is known for its conserved function in appendage development in metazoans. In arthropods, Dll is required for the specification of distal appendage structures. Here we describe a novel and unexpected role of Dll in the spider Achaearanea tepidariorum. We detect At-Dll transcripts not only in the appendages, but unexpectedly also in an anterior domain during early development, prior to the specification of the limb primordia. A similar early Dll domain is present in the distantly related spider Pholcus phalangioides. In A. tepidariorum this early At-Dll expression is required for head segmentation. RNA interference results in spiders that lack either the first or the first and the second walking leg segments. The early At-Dll expression is also required for the activation of the segment polarity genes engrailed and hedgehog in this region. Our work identifies the Distal-less gene as a novel factor in anterior spider segmentation with a gap gene-like function. This novel role of Dll is interesting because Dll expression is reduced in this region in crustaceans and the homologous insect segment, the mandible segment, does not express Dll and does not require this gene for patterning. We therefore discuss the possible implications of our results for understanding the evolution and diversification of the mandible segment.

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Chromatin remodelling precedes transcriptional and structural changes in heart failure. A body of work suggests roles for the developmental Wnt signalling pathway in cardiac remodelling. Hitherto, there is no evidence supporting a direct role of Wnt nuclear components in regulating chromatin landscapes in this process. We show that transcriptionally active, nuclear, phosphorylated(p)Ser675-β-catenin and TCF7L2 are upregulated in diseased murine and human cardiac ventricles. We report that inducible cardiomyocytes (CM)-specific pSer675-β-catenin accumulation mimics the disease situation by triggering TCF7L2 expression. This enhances active chromatin, characterized by increased H3K27ac and TCF7L2 occupancies to cardiac developmental and remodelling genes in vivo. Accordingly, transcriptomic analysis of β-catenin stabilized hearts shows a strong recapitulation of cardiac developmental processes like cell cycling and cytoskeletal remodelling. Mechanistically, TCF7L2 co-occupies distal genomic regions with cardiac transcription factors NKX2–5 and GATA4 in stabilized-β-catenin hearts. Validation assays revealed a previously unrecognized function of GATA4 as a cardiac repressor of the TCF7L2/β-catenin complex in vivo, thereby defining a transcriptional switch controlling disease progression. Conversely, preventing β-catenin activation post-pressure-overload results in a downregulation of these novel TCF7L2-targets and rescues cardiac function. Thus, we present a novel role for TCF7L2/β-catenin in CMs-specific chromatin modulation, which could be exploited for manipulating the ubiquitous Wnt pathway.

BackgroundGene duplications provide genetic material for the evolution of new morphological and physiological features. One copy can preserve the original gene functions while the second copy may evolve new functions (neofunctionalisation). Gene duplications may thus provide new genes involved in evolutionary novelties.ResultsWe have studied the duplicated homeobox gene homothorax (hth) in the spider species Parasteatoda tepidariorum and Pholcus phalangioides and have compared these data with previously published data from additional spider species. We show that the expression pattern of hth1 is highly conserved among spiders, consistent with the notion that this gene copy preserves the original hth functions. By contrast, hth2 has a markedly different expression profile especially in the prosomal appendages. The pattern in the pedipalps and legs consists of several segmental rings, suggesting a possible role of hth2 in limb joint development. Intriguingly, however, the hth2 pattern is much less conserved between the species than hth1 and shows a species specific pattern in each species investigated so far.ConclusionsWe hypothesise that the hth2 gene has gained a new patterning function after gene duplication, but has then undergone a second phase of diversification of its new role in the spider clade. The evolution of hth2 may thus provide an interesting example for a duplicated gene that has not only contributed to genetic diversity through neofunctionalisation, but beyond that has been able to escape evolutionary conservation after neofunctionalisation thus forming the basis for further genetic diversification.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-1013-0) contains supplementary material, which is available to authorized users.