Mature Cardiomyocytes Research Articles

Cardiomyocyte Maturation-the Road is not Obstructed.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent one of the most promising ways to treat cardiovascular diseases. High-purity cardiomyocytes (CM) from different cell sources could be obtained at present. However, the immature nature of these cardiomyocytes hinders its further clinical application. From immature to mature state, it involves structural, functional,and metabolic changes in cardiomyocytes. Generally, two types of culturing (2D and 3D) systemshave been reported to induce cardiomyocyte maturation. 2D culture mainly achieves the maturation of cardiomyocytes through long-term culture, co-culture, supplementation of small molecule compounds, and the application of biophysical cues. The combined use of biomaterial's surface topography and biophysical cues also facilitates the maturation of cardiomyocytes. Cardiomyocyte maturation is a complex process involving many signaling pathways, and current methods fail to fully reproduce this process. Therefore, analyzing the signaling pathway network related to the maturation and producing hPSC-CMs with adult-like phenotype is a challenge. In this review, we summarized the structural and functional differences between hPSC-CMs and mature cardiomyocytes, and introduced various methods to induce cardiomyocyte maturation.

Protein Kinase C Regulates Expression and Function of the Cav3.2 T-Type Ca2+ Channel during Maturation of Neonatal Rat Cardiomyocyte.

Two distinct isoforms of the T-type Ca2+ channel, Cav3.1 and Cav3.2, play a pivotal role in the generation of pacemaker potentials in nodal cells in the heart, although the isoform switches from Cav3.2 to Cav3.1 during the early neonatal period with an unknown mechanism. The present study was designed to investigate the molecular system of the parts that are responsible for the changes of T-type Ca2+ channel isoforms in neonatal cardiomyocytes using the whole-cell patch-clamp technique and mRNA quantification. The present study demonstrates that PKC activation accelerates the Ni2+-sensitive beating rate and upregulates the Ni2+-sensitive T-type Ca2+ channel current in neonatal cardiomyocytes as a long-term effect, whereas PKC inhibition delays the Ni2+-sensitive beating rate and downregulates the Ni2+-sensitive T-type Ca2+ channel current. Because the Ni2+-sensitive T-type Ca2+ channel current is largely composed of the Cav3.2-T-type Ca2+ channel, it is accordingly assumed that PKC activity plays a crucial role in the maintenance of the Cav3.2 channel. The expression of Cav3.2 mRNA was highly positively correlated with PKC activity. The expression of a transcription factor Nkx2.5 mRNA, possibly corresponding to the Cav3.2 channel gene, was decreased by an inhibition of PKCβII. These results suggest that PKC activation, presumably by PKCβII, is responsible for the upregulation of CaV3.2 T-type Ca2+ channel expression that interacts with a cardiac-specific transcription factor, Nkx2.5, in neonatal cardiomyocytes.

In vitro maturation of human pluripotent stem cell-derived cardiomyocyte: A promising approach for cell therapy

<i>In vitro</i> maturation of human pluripotent stem cell-derived cardiomyocyte: A promising approach for cell therapy

PLK inhibitors identified by high content phenotypic screening promote maturation of human PSC-derived cardiomyocytes

Human pluripotent stem cells-derived cardiomyocytes (hPSC-CMs) provide an unlimited source of human cardiomyocytes for disease modeling, cell therapies, and other biomedical applications. However, hPSC-CMs remain developmentally immature which limits their suitability in translational applications. High Content Screening (HCS) is a powerful tool for identifying novel molecules and pathways regulating complex biological processes, but no HCS assay for hPSC-CM maturation has yet been reported. PCM1, a centriole satellite protein, is specifically restricted on nuclear envelope in mature cardiomyocytes. We developed a High Content Screen (HCS) based on PCM1 subcellular localization in hPSC-CMs to identify novel molecules promoting cardiomyocyte maturation, which identified 93 from 1693 compounds that enhance maturation of hPSC-CMs, including multiple PLK inhibitors. Volasertib and Centrinone, two PLK inhibitors, can enhance binucleation, and promote metabolic and electrophysiological maturation in hPSC-CMs. Furthermore, PI3K-AKT signaling pathway was found to be suppressed by PLK inhibitors, and VO-Ohpic, a PTEN inhibitor that activates AKT pathway, blunted the effect of PLK inhibitors on hPSC-CM maturation. In summary, our HCS assay found that PLK inhibitors can promote maturation of hPSC-CMs through suppressing AKT signaling pathway.

Direct reprogramming of adult adipose-derived regenerative cells toward cardiomyocytes using six transcriptional factors

SummaryIt is widely accepted that adipose-derived regenerative cells (ADRCs) can differentiate into mesodermal lineage cells. However, reprogramming adult ADRCs into mature cardiomyocytes is challenging. We investigated the induction of myocardial differentiation in ADRCs via direct reprogramming using lentiviral gene transfer. First, we identified candidate transcriptional factors by performing RNA sequencing and ultimately confirmed that the combination of six unique factors (Baf60c, Gata4, Gata6, Klf15, Mef2a, and Myocd) could efficiently express enhanced green fluorescent protein (GFP) in ADRCs isolated from adult alpha-myosin heavy chain promoter-driven GFP transgenic mice. The GFP-positive ADRCs induced by six factors (6F-ADRCs) expressed multiple cardiac genes and revealed cardiac differentiation in bioinformatic analysis. Moreover, injection of 6F-ADRCs into acute myocardial infarcted tissues in vivo resulted in the improvement of survival rate, fractional shortening, and reduction of infarction scar area. This study provides an alternative method for direct reprogramming of adult ADRCs into cardiomyocytes.

Glucocorticoid receptor antagonization propels endogenous cardiomyocyte proliferation and cardiac regeneration.

In mammals, the physiological activation of the glucocorticoid receptor (GR) by glucocorticoids (GCs) promotes the maturation of cardiomyocytes during late gestation, but the effect on postnatal cardiac growth and regenerative plasticity is unclear. Here we demonstrate that the GC-GR axis restrains cardiomyocyte proliferation during postnatal development. Cardiomyocyte-specific GR ablation in conditional knockout (cKO) mice delayed the postnatal cardiomyocyte cell cycle exit, hypertrophic growth and cytoarchitectural maturation. GR-cKO hearts showed increased expression of genes involved in glucose catabolism and reduced expression of genes promoting fatty acid oxidation and mitochondrial respiration. Accordingly, oxygen consumption in GR-cKO cardiomyocytes was less dependent on fatty acid oxidation, and glycolysis inhibition reverted GR-cKO effects on cardiomyocyte proliferation. GR ablation or transient pharmacological inhibition after myocardial infarction in juvenile and/or adult mice facilitated cardiomyocyte survival, cell cycle re-entry and division, leading to cardiac muscle regeneration along with reduced scar formation. Thus, GR restrains heart regeneration and may represent a therapeutic target.

Differentiation of induced pluripotent stem cells to Cardiomyocytes on Cellulose Nanofibril substrate

In vitro differentiation of cardiomyocytes from human induced pluripotent stem cells (hiPSCs) holds great potential in regenerative medicine. Currently the differentiation of iPSCs to cardiomyocytes is mostly done on vitronectin substrate, which is very expensive and difficult to produce on a large scale. For potential future clinical application, an appropriate natural and non-xenogenic substrate for the differentiation of iPS cells is very important. In this study a natural, innocuous, tissue compatible and biodegradable polymer Cellulose Nanofibril (CNF) was used as a fine coat substrate for differentiating iPSCs to cardiomyocytes. We checked the expression of NKX2–5, an early cardiac mesoderm marker and TNNT2, a mature cardiomyocyte marker to confirm the process of becoming iPSCs to cardiomyocytes on both vitronectin and CNF substrates. Our preliminary results suggest that the stage specific markers were expressed on the differentiated cardiomyocytes from both the substrates and were analogous. Thus, we established a natural biomaterial, CNF, that may be used as a potential substrate in tissue engineering applications for the process by which iPSCs become cardiomyocytes.

Expression of Extracellular Vesicle PIWI-Interacting RNAs Throughout hiPSC-Cardiomyocyte Differentiation.

Extracellular Vesicles (EV) play a critical role in the regulation of regenerative processes in wounded tissues by mediating cell-to-cell communication. Multiple RNA species have been identified in EV, although their function still lacks understanding. We previously characterized the miRNA content of EV secreted over hiPSC-cardiomyocyte differentiation and found a distinct miRNA expression in hiPSC-EV driving its in vitro bioactivity. In this work, we investigated the piRNA profiles of EV derived from key stages of the hiPSC-CM differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors (CPC-EV), immature (CMi-EV), and mature (CMm-EV) cardiomyocytes, demonstrating that EV-piRNA expression differs greatly from the miRNA profiles we previously identified. Only four piRNA were significantly deregulated in EV, one in hiPSC-EV, and three in CPC-EV, as determined by differential expression analysis on small RNA-seq data. Our results provide a valuable source of information for further studies aiming at defining the role of piRNA in the bioactivity and therapeutic potential of EV.

Single-cell atlas of multilineage cardiac organoids derived from human induced pluripotent stem cells

Abstract Human induced pluripotent stem cell (hiPSC)-derived cardiac organoids can be used to model human heart development and cardiovascular disease, and provide therapeutic cells to repair the heart. We used single-cell transcriptome analysis to dissect the development of 3D mini-cardiac organoids (MCOs) consisting of hiPSC-derived cardiomyocytes, and endothelial and smooth muscle cells. We found that the 3D matrix-rich microenvironment significantly promoted the maturation of cardiomyocytes, and mixing endothelial and smooth muscle cells with cardiomyocytes led to the formation of cardiac fibroblast highly expressing DLK1. Modulation of DLK1 signaling affected immunomodulatory gene expression in 2D cultured cardiomyocytes. Transplantation of multilineage MCO into a rat model of myocardial infarction significantly improved cardiac function and reduced fibrosis in the infarcted area. Our single-cell analysis of MCO provided rich information about cell state and fate dynamics in the 3D multilineage microenvironment and brought new insight into the molecular mechanism that promotes cardiomyocyte maturation and heart repair.

Maturation of hiPSC-derived cardiomyocytes in cardiac microtissues promotes adult alternative splicing of sodium channel revealing mutation effects associated with cardiac arrhythmia

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Marie Skłodowska-Curie Actions - Individual Fellowship Background The cardiac sodium channel encoded by SCN5A gene undergoes fetal-to-adult isoform switch postnatally, regulated by alternative splicing. Various mutations in SCN5A associated with severe cardiac arrhythmia have been previously studied using cardiomyocytes derived from human induced pluripotent stem-cells (hiPSC-CMs), demonstrating the importance of a human cardiac cellular context for such analysis. Mutations in the adult isoform of SCN5A are however of difficult evaluation in hiPSC-CMs, because of their typical immature/fetal-like characteristics. Combining hiPSC-CMs in 3-dimensional microtissues together with hiPSC-derived cardiac fibroblasts and endothelial cells induced maturation in gene expression and functional properties, including electrophysiology. Purpose We investigated whether the maturation promoted by the microtissue system could reveal the functional phenotype of a mutation in the adult isoform of SCN5A. Methods We derived hiPSC-CMs from a cardiac arrhythmia patient carrying two compound mutations in SCN5A: p.W156X in exon 4 and p.R225W in the adult splicing variant of exon 6 (exon 6B). Using CRISPR/Cas9, we corrected exon 4 mutation to investigate specific effects of exon 6B mutation on sodium current by single-cell patch clamp. HiPSC-CMs were matured in tricell-type cardiac microtissues and analysed after dissociation. The mechanism underlying exon 6B expression was investigated by overexpressing or knocking-out the alternative splicing regulator MBNL1 in hiPSC-CMs. Results ddPCR analysis showed low expression of exon 6B-containing transcripts in hiPSC-CMs, accompanied by no effect of exon 6B mutation on the sodium current. Instead, maturing hiPSC-CMs in cardiac microtissues promoted SCN5A exon 6B expression and revealed the contribution of both SCN5A mutations to the diseased cellular electrophysiology. MBNL1 was upregulated in microtissues and indeed its overexpression was sufficient to increase exon 6B levels in hiPSC-CMs, while lack of MBNL1 prevented the switch of SCN5A splicing isoforms. Conclusions Our data demonstrate that a maturation of hiPSC-CMs is required to express the adult SCN5A isoform. This was achieved in the microtissue system via upregulation of MBNL1, and allowed to reveal SCN5A mutation contributions, by dissecting changes in ionic current that cause adult arrhythmic disease phenotypes in humans.

Small molecule-mediated rapid maturation of human induced pluripotent stem cell derived cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Gravitation Program “Materials Driven Regeneration” by the Netherlands Organization for Scientific Research (RegmedXB #024.003.013) and the Marie Skłodowska-Curie Actions (Grant agreement RESCUE #801540). The EU-funded project BRAV3 (H2020, ID:874827) Background Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature human primary cardiomyocytes: the ability to use fatty acids as an energy source, high mitochondrial mass, increased nuclei polyploidism, synchronized electrical conduction, and forceful contractions. Instead, their phenotype is similar to immature cardiomyocytes in the late fetal stage. This immaturity represents a bottleneck to their application in 1) disease modeling – as most cardiac (genetic) diseases have a middle-age onset – and 2) clinical use, where integration and functional coupling are key. So far, the mainly used methods to enhance iPSC-CM maturation include prolonged time-in-culture, 3D culture, cyclic mechanical stretch, and electrical stimulation with specialized media. However, these protocols are laborious, costly, and not easily scalable. Methods In this study, we developed a simple, low cost, and rapid protocol using two peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A/PGC-1α) activating small molecules: Asiatic Acid (AA) and GW501516 (GW) to promote cardiomyocyte maturity by inducing a metabolic switch to fatty acid utilization and increased mitochondrial biogenesis. Results Monolayers of iPSC-CMs were incubated with AA and GW every other day for 10 days resulting in increased expression of fatty acid-metabolism-related genes (5 and 10-fold increase in CPT1B gene expression, respectively), mitochondria biogenesis (protein expression of ATP5A) and fusion (50 and 100-fold increase in OPA1 gene expression, respectively). In addition, AA treated iPSC-CMs responded in the seahorse mitochondria stress test more rapidly to an artificial increase in mitochondrial activity and showed a higher flexibility in substrate utilization in the seahorse stress test. A more mature electrophysiological functionality was shown by increased ion channel gene expression (KCNA4, SCN5A, GJA1, CACNA1C, and SCN1B) and enhanced synchronous contraction in treated samples. Moreover, maturation was further shown by increased sarcomeric gene expression (5 and 7-fold increase in TNNI3 in AA and GW respectively) and nuclear polyploidism (&amp;gt;4N fold 2.16 and 1.48-fold increase in AA and GW respectively). Conclusions Collectively, these findings show that AA and GW trigger a metabolic switch and induce extensive maturation of iPSC-CMs, providing a rapid and cost-effective method to obtain iPSC-CMs that more closely resemble their adult counterparts.

Characterization of cardiomyocyte membrane microdomains setup during postnatal development

MAGUKs are one of the largest families of scaffolding proteins associated with intercellular junctions. Deletion of CASK is lethal, with full-term mice dying within hours of birth. The role of CASK was first established in neural development, but postnatal malformations have also been reported in the lungs and intestines. Previous results showed that CASK, in the adult cardiomyocyte, has an exclusive localization at the lateral membrane (LM), within the focal adhesion complexes. Preliminary data also suggest that CASK, in neonatal cardiomyocytes, plays a major role in the establishment and maintenance of intercellular junctions. To investigate the expression and organization of CASK and cardiomyocyte membrane microdomains during postnatal development of the Rat heart. Kinetics of CASK and polarity marker expression and localization during the developmental process from neonatal (P0) to adult (P60 days postnatal) were performed by western blot and immunofluorescence. CASK expression is maximal during early postnatal development (P0 to P5) and decreases from P20, to be strongly repressed at the adult stage. Structural polarity markers such as adherens (N-cadherin) and tight (ZO-1) junctions, as well as electrical polarity markers (NaV1.5, Kir2.1) follow the same kinetics. Only Cx43, a marker of electrical junctions, peaks at P20. CASK is very intense in the epicardial and subepicardial layers at P0 and P5 but weak in the midcardium. From P20 onward, as the fibers become oriented, CASK distributes throughout the myocardium and begins to become membranous at P20 to be fully localized to LM in a striated pattern at P60. Localization of N-cadherin to the intercalated disc (ID) is observed as early as P20, while Cx43 is still lateralized, as is ZO-1. At P60, Cx43 is restricted to the ID while ZO-1 retains a lateral localization but is also distributed to the ID. Like CASK, NaV1.5 is highly expressed in the epicardial and subepicardial layers at P0 and P5. From P20 onwards, both NaV1.5 and Kir2.1 adopt a cytosolic localization in the midcardium and concentrates at the ID, where CASK is excluded. This study shows that the expression level of polarity markers is inversely correlated with terminal cardiomyocyte maturation. In addition, the localization of CASK and NaV1.5 in the epicardial and subepicardial layers suggests that these proteins may have a role in early cardiac morphogenesis, before myocardial differentiation.

Therapeutic Ultrasound Effects on Human Induced Pluripotent Stem Cell Cardiomyocytes Measured Optically and with Spectral Ultrasound

To the best of our knowledge, therapeutic ultrasound (TUS) is thus far an unexplored means of delivering mechanical stimulation to cardiomyocyte cultures, which is necessary to engineer a more mature cardiomyocyte phenotype in vitro. Spectral ultrasound (SUS) may provide a way to non-invasively, non-disruptively and inexpensively monitor growth and change in cell cultures over long periods. Compared with other measurement methods, SUS as an acoustic measurement tool will not be affected by an acoustic therapy, unlike electrical measurement methods, in which motion caused by acoustic therapy can affect measurements. Further SUS has the potential to provide functional as well as morphological information in cell cultures. Human induced pluripotent stem cell cardiomyocytes (iPS-CMs) were imaged with calcium fluorescence microscopy while TUS was being applied. TUS was applied at 600 kHz and 1, 3.4 and 6 W/cm2 for a continuous 1 s pulse. Measures of the instantaneous beat frequency, repolarization rate and calcium spike amplitude were calculated from the fluorescence data. At 600 kHz, TUS at 1 and 6 W/cm2 had significant effects on the shortening of both the repolarization rate and instantaneous beat rate of the iPS-CMs (p < 0.05), while TUS at 3.4 and 6 W/cm2 had significant effects on the shortening of the calcium spike amplitude (p < 0.05). Three SUS measures and one gray-level measure were captured from the iPS-CM monolayers while they were simultaneously being imaged with calcium-labeled confocal microscopy. The gray-level measure performed the best of all SUS measures; however, it was not reliable enough to produce a consistent determination of the beat rate of the cell. Finally, SUS measures were captured using three different transducers while simultaneously applying TUS. A center-of-mass (COM) measure calculated from the wavelet transform scalogram of the time-averaged radiofrequency data revealed that SUS was able to detect a change in the frequency content of the reflected ultrasound at 1 and 6 W/cm2 before and after ultrasound application (p < 0.05), showing promise for the ability of SUS to measure changes in the beating behavior of iPS-CMs. Overall, SUS is promising as a method for constant monitoring of dynamic cell and tissue culture and growth.

AP-1 activation mediates post-natal cardiomyocyte maturation.

Post-natal maturation of mammalian cardiomyocytes proceeds rapidly after birth, with most of the myocytes exiting cell cycle, becoming binucleated, and adopting oxidative phosphorylation as the primary metabolic route. The triggers and transcriptional programmes regulating cardiomyocyte maturation have not been fully understood yet. We performed single-cell RNA-Seq in post-natal rat hearts in order to identify the important factors for this process. Single-cell RNA-Seq profiling was performed of post-natal Day 1 and Day 7 rat hearts, and we found that members of the activating protein 1 (AP-1) transcription factors showed a transient up-regulation in the maturing cardiomyocytes, suggesting their functional involvement in the process. Activating members of the AP-1 family by palmitate or adrenergic stimulation inhibited cardiomyocyte cytokinesis and promoted cardiomyocyte maturation. In contrast, knocking down AP-1 members Atf3 and Jun promoted cardiomyocyte cytokinesis, reduced polyploidy, and inhibited maturation. Mechanistically, RNA-Seq results and rescue experiments indicated that AP-1 members activate the expression of fatty acid metabolic genes to promote cardiomyocyte maturation. Finally, intraperitoneal injection of AP-1 inhibitor T-5224 in neonatal mice inhibits cardiomyocyte maturation in vivo. Our results are the first evidence implicating AP-1 transcription factors in post-natal cardiomyocyte maturation both in vitro and in vivo, which expand our understanding of the molecular mechanism of cardiomyocyte maturation, and may lead to novel therapies to treat congenital heart diseases.

Metabolic Determinants in Cardiomyocyte Function and Heart Regenerative Strategies.

Heart disease is the leading cause of mortality in developed countries. The associated pathology is characterized by a loss of cardiomyocytes that leads, eventually, to heart failure. In this context, several cardiac regenerative strategies have been developed, but they still lack clinical effectiveness. The mammalian neonatal heart is capable of substantial regeneration following injury, but this capacity is lost at postnatal stages when cardiomyocytes become terminally differentiated and transit to the fetal metabolic switch. Cardiomyocytes are metabolically versatile cells capable of using an array of fuel sources, and the metabolism of cardiomyocytes suffers extended reprogramming after injury. Apart from energetic sources, metabolites are emerging regulators of epigenetic programs driving cell pluripotency and differentiation. Thus, understanding the metabolic determinants that regulate cardiomyocyte maturation and function is key for unlocking future metabolic interventions for cardiac regeneration. In this review, we will discuss the emerging role of metabolism and nutrient signaling in cardiomyocyte function and repair, as well as whether exploiting this axis could potentiate current cellular regenerative strategies for the mammalian heart.

Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different age-dependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56–2.2 × 106 cells/heart) and viability (~70–100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell type-specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR < 0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rod-shaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

Three-dimensional cardiac organoid formation accelerates the functional maturation of human induced pluripotent stem cell-derived cardiomyocytes

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) offer a promising source for heart regeneration, disease modeling, and drug screening. Recent developments in organoid technology have made it possible to study how hiPSC-derived CMs interact together, and this culture system mimics the tissue environment and behavior of the cardiac cells in our body. However, the similarities and differences between conventional 2-dimensional (2D) culture and 3-dimensional (3D) organoid culture systems for CM differentiation have been incompletely elucidated. To study how the individual microenvironment formed by each culture system affects the properties of CMs differentiated from hiPSCs, we conducted a comparative study between 2D monolayer and direct 3D cardiac organoid (hiCO) differentiation from hiPSCs throughout the sequential differentiation stages. Although identical differentiation cues were applied to hiPSCs, the 3D differentiation system strongly exhibited higher mesoderm commitment and cardiac induction than 2D monolayer differentiation. In the late stage of differentiation, the 3D hiCOs showed a higher frequency of a mature myofibrillar isoform switching in sarcomere structure of differentiated CMs than was observed in monolayer culture, although over 94% of cardiac troponin T-positive cells resulted at the end point of differentiation in both systems. Furthermore, the accelerated structural maturation in 3D hiCOs resulted in increased expression of cardiac-specific ion channel genes and Ca2+ transient properties, with a high signal amplitude and rapid contractility. The present study provides details surrounding the 2D and 3D culture methods for CM differentiation from iPSCs, and focuses on 3D cell culture as an improved strategy for approaching and applying cardiac maturation.

Ryanodine receptor 2 (RYR2) dysfunction activates the unfolded protein response and perturbs cardiomyocyte maturation.

Calcium-handling capacity is a major gauge of cardiomyocyte maturity. Ryanodine receptor 2 (RYR2) is the pre-dominant calcium channel that releases calcium from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) to activate cardiomyocyte contraction. Although RYR2 was previously implied as a key regulator of cardiomyocyte maturation, the mechanisms remain unclear. The aim of this study is to solve this problem. We performed Cas9/AAV9-mediated somatic mutagenesis to knockout RYR2 specifically in cardiomyocytes in mice. We conducted a genetic mosaic analysis to dissect the cell-autonomous function of RYR2 during cardiomyocyte maturation. We found that RYR2 depletion triggered ultrastructural and transcriptomic defects relevant to cardiomyocyte maturation. These phenotypes were associated with the drastic activation of ER stress pathways. The ER stress alleviator tauroursodeoxycholic acid partially rescued the defects in RYR2-depleted cardiomyocytes. Overexpression of ATF4, a key ER stress transcription factor, recapitulated defects in RYR2-depleted cells. Integrative analysis of RNA-Seq and bioChIP-Seq data revealed that protein biosynthesis-related genes are the major direct downstream targets of ATF4. RYR2-regulated ER homeostasis is essential for cardiomyocyte maturation. Severe ER stress perturbs cardiomyocyte maturation primarily through ATF4 activation. The major downstream effector genes of ATF4 are related to protein biosynthesis.

Regulation of extracellular matrix composition by fibroblasts during perinatal cardiac maturation

BackgroundCardiac fibroblasts are the main non-myocyte population responsible for extracellular matrix (ECM) production. During perinatal development, fibroblast expansion coincides with the transition from hyperplastic to hypertrophic myocardial growth. Therefore, we investigated the consequences of fibroblast loss at the time of cardiomyocyte maturation by depleting fibroblasts in the perinatal mouse. Methods and resultsWe evaluated the microenvironment of the perinatal heart in the absence of fibroblasts and the potential functional impact of fibroblast loss in regulation of cardiomyocyte cell cycle arrest and binucleation. Cre-mediated expression of diphtheria toxin A in PDGFRα expressing cells immediately after birth eliminated 70–80% of the cardiac fibroblasts. At postnatal day 5, hearts lacking fibroblasts appeared similar to controls with normal morphology and comparable numbers of endothelial and smooth muscle cells, despite a pronounced reduction in fibrillar collagen. Immunoblotting and proteomic analysis of control and fibroblast-deficient hearts identified differential abundance of several ECM proteins. In addition, fibroblast loss decreased tissue stiffness and resulted in increased cardiomyocyte mitotic index, DNA synthesis, and cytokinesis. Moreover, decellularized matrix from fibroblast-deficient hearts promoted cardiomyocyte DNA replication. While cardiac architecture was not overtly affected by fibroblast reduction, few pups survived past postnatal day 11, suggesting an overall requirement for PDGFRα expressing fibroblasts. ConclusionsThese studies demonstrate the key role of fibroblasts in matrix production and cardiomyocyte cross-talk during mouse perinatal heart maturation and revealed that fibroblast-derived ECM may modulate cardiomyocyte maturation in vivo. Neonatal depletion of fibroblasts demonstrated that although hearts can tolerate reduced ECM composition, fibroblast loss eventually leads to perinatal death as the approach simultaneously reduced fibroblast populations in other organs.

Hair-follicle-associated pluripotent (HAP) stem cells differentiate into mature beating cardiomyocyte sheets on flexible substrates in vitro.

Cardiomyocytes have been differentiated from various stem cells such as human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), but it is difficult to produce mature cardiomyocytes. We showed rat hair-follicle-associated pluripotent (HAP) stem cells have pluripotency and produced mature beating cardiomyocyte sheets differentiated from rat HAP stem cells. The upper parts of rat vibrissa hair follicles were cultured in 10% FBS DMEM and stained with antibodies of the ectoderm, mesoderm, endoderm system to show the differentiation of multiple cell types. Moreover, HAP stem cells were cultured under three different conditions to decide the most suitable culture conditions for making beating cardiomyocyte sheets. The beating cardiomyocyte sheets were shown to be mature by staining sarcomere structures. Isoproterenol alone and the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) effectively induced beating long-fiber cardiomyocytes, which formed beating sheets, only in the presence of all four agents. Flexible substrates were essential for the differentiation of sheets of mature beating cardiomyocytes for HAP stem cells. The features of the cardiomyocytes differentiated from HAP stem cells demonstrate they have clinical potential for heart regeneration.