CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes.

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.

Generating ring-shaped engineered heart tissues from ventricular and atrial human pluripotent stem cell-derived cardiomyocytes.

The functions of the heart are achieved through coordination of different cardiac cell subtypes (e.g., ventricular, atrial, conduction-tissue cardiomyocytes). Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer unique opportunities for cardiac research. Traditional studies using these cells focused on single-cells and utilized mixed cell populations. Our goal was to develop clinically-relevant engineered heart tissues (EHTs) comprised of chamber-specific hPSC-CMs. Here we show that such EHTs can be generated by directing hPSCs to differentiate into ventricular or atrial cardiomyocytes, and then embedding these cardiomyocytes in a collagen-hydrogel to create chamber-specific, ring-shaped, EHTs. The chamber-specific EHTs display distinct atrial versus ventricular phenotypes as revealed by immunostaining, gene-expression, optical assessment of action-potentials and conduction velocity, pharmacology, and mechanical force measurements. We also establish an atrial EHT-based arrhythmia model and confirm its usefulness by applying relevant pharmacological interventions. Thus, our chamber-specific EHT models can be used for cardiac disease modeling, pathophysiological studies and drug testing.

Left ventricular and left atrial volume ratio assessed by three-dimensional echocardiography: Novel indices for evaluating age-related change in left heart chamber size.

We hypothesized that left ventricular and left atrial volume ratio (LVLAVR) assessed by three-dimensional echocardiography (3DE) reflects age- and gender-related change in left chamber size. We aimed to (1) determine the reference values of LVLAVR, (2) investigate their age and gender dependency, and (3) clarify which anthropometric and echocardiography parameters are closely associated with these indices. Both left ventricular (LV) and left atrial (LA) volume curves were obtained using 3DE speckle tracking analytical software, and the LVLAVR curve throughout one cardiac cycle was created, from which LVLAVR at ventricular end-diastole and at ventricular end-systole were determined in 313 healthy subjects (age, 20-85 years; 51% men). The mean values of LVLAVR at ventricular end-diastole and ventricular end-systole in male subjects were 5.74 ± 1.54 and 1.37 ± 0.35, respectively. Corresponding values in female subjects were significantly lower (5.20 ± 1.47, p = .003 and 1.13 ± 0.29, p < .001) than the values in male subjects. LVLAVR at ventricular end-diastole step wisely decreased to advanced aging, and had a highest F ratio compared with other left chamber volumetric parameters in both genders. LV mass and LA ejection fraction were significantly associated with LVLAVR at ventricular end-diastole. In contrast, LV mass and LV ejection fraction were significantly coupled with LVLAVR at ventricular end-systole. This study provides the reference values for LVLAVR from a relatively large number of healthy subjects. LVLAVR may be a sensitive parameter to reflect age- and gender-related change in LV and LA volumes. Further studies should be required to determine its clinical usefulness over traditional echocardiography parameters in various cardiovascular diseases.

Effect of Neuropeptide Y on Action Potential of Working Right Atrial Cardiomyocytes in Early Postnatal Ontogeny.

The effects of neuropeptide Y (10-9-10-6 M) on electrical activity of right atrial cardiomyocytes of rats aging 7, 21, and 100 days were examined in vitro. Neuropeptide Y affected the amplitude-temporal parameters of the action potential in these cells. It decreased the duration of repolarization phase in 7-day-old rats in concentrations of 10-8 and 10-7 M, in 21-day-old rats at 10-8 and 10-6 M, and in 100-day-old at 10-6 M. The data indicate elevation of total membrane potassium current under the action of neuropeptide Y.

Hippo signaling determines the number of venous pole cells that originate from the anterior lateral plate mesoderm in zebrafish.

The differentiation of the lateral plate mesoderm cells into heart field cells constitutes a critical step in the development of cardiac tissue and the genesis of functional cardiomyocytes. Hippo signaling controls cardiomyocyte proliferation, but the role of Hippo signaling during early cardiogenesis remains unclear. Here, we show that Hippo signaling regulates atrial cell number by specifying the developmental potential of cells within the anterior lateral plate mesoderm (ALPM), which are incorporated into the venous pole of the heart tube and ultimately into the atrium of the heart. We demonstrate that Hippo signaling acts through large tumor suppressor kinase 1/2 to modulate BMP signaling and the expression of hand2, a key transcription factor that is involved in the differentiation of atrial cardiomyocytes. Collectively, these results demonstrate that Hippo signaling defines venous pole cardiomyocyte number by modulating both the number and the identity of the ALPM cells that will populate the atrium of the heart.

A COUP-TFII Human Embryonic Stem Cell Reporter Line to Identify and Select Atrial Cardiomyocytes.

Reporter cell lines have already proven valuable in identifying, tracking, and purifying cardiac subtypes and progenitors during differentiation of human pluripotent stem cells (hPSCs). We previously showed that chick ovalbumin upstream promoter transcription factor II (COUP-TFII) is highly enriched in human atrial cardiomyocytes (CMs), but not ventricular. Here, we targeted mCherry to the COUP-TFII genomic locus in hPSCs expressing GFP from the NKX2.5 locus. This dual atrial NKX2.5EGFP/+-COUP-TFIImCherry/+ reporter line allowed identification and selection of GFP+ (G+)/mCherry+ (M+) CMs following cardiac differentiation. These cells exhibited transcriptional and functional properties of atrial CMs, whereas G+/M- CMs displayed ventricular characteristics. Via CRISPR/Cas9-mediated knockout, we demonstrated that COUP-TFII is not required for atrial specification in hPSCs. This new tool allowed selection of human atrial and ventricular CMs from mixed populations, of relevance for studying cardiac specification, developing human atrial disease models, and examining distinct effects of drugs on the atrium versus ventricle.

Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes.

KEY POINTS: Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart. Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur. We report alterations to the atrial systolic Ca2+ transient that have implications for the function of the atrial in the elderly. We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca2+ in the old atria. The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops. ABSTRACT: Many cardiovascular diseases, including those affecting the atria, are associated with advancing age. Arrhythmias, including those in the atria, can arise as a result of electrical remodelling or alterations in Ca2+ homeostasis. In the atria, age-associated changes in the action potential have been documented. However, little is known about remodelling of intracellular Ca2+ homeostasis in the healthy aged atria. Using single atrial myocytes from young and old Welsh Mountain sheep, we show the free Ca2+ transient amplitude and rate of decay of systolic Ca2+ decrease with age, whereas sarcoplasmic reticulum (SR) Ca content increases. An increase in intracellular Ca buffering explains both the decrease in Ca2+ transient amplitude and decay kinetics in the absence of any change in sarcoendoplasmic reticulum calcium transport ATPase function. Ageing maintained the integrated Ca2+ influx via ICa-L but decreased peak ICa-L . Decreased peak ICa-L was found to be responsible for the age-associated increase in SR Ca content but not the decrease in Ca2+ transient amplitude. Instead, decreased peak ICa-L offsets increased SR load such that Ca2+ release from the SR was maintained during ageing. The results of the present study highlight a novel mechanism by which increased Ca buffering decreases systolic Ca2+ in old atria. Furthermore, for the first time, we have shown that SR Ca content is increased in old atrial myocytes.

Sfrp5 identifies murine cardiac progenitors for all myocardial structures except for the right ventricle.

Upon acquirement of pulmonary circulation, the ancestral heart may have been remodelled coincidently with, or accompanied by, the production and rearrangement of progenitor cells. However, the progenitor populations that give rise to the left ventricle (LV) and sinus venosus (SV) are still ambiguous. Here we show that the expression of Secreted frizzled-related protein Sfrp5 in the mouse identifies common progenitors for the outflow tract (OFT), LV, atrium and SV but not the right ventricle (RV). Sfrp5 expression begins at the lateral sides of the cardiac crescent, excluding early differentiating regions, and continues in the venous pole, which gives rise to the SV. Lineage-tracing analysis revealed that descendants of Sfrp5-expressing cells at E7.5 contribute not only to the SV but also to the LV, atria and OFT and are found also in the dorsal splanchnic mesoderm accompanied by the expression of the secondary heart field marker, Islet1. These findings provide insight into the arrangement of cardiac progenitors for systemic circulation.

Foxa2 identifies a cardiac progenitor population with ventricular differentiation potential.

The recent identification of progenitor populations that contribute to the developing heart in a distinct spatial and temporal manner has fundamentally improved our understanding of cardiac development. However, the mechanisms that direct atrial versus ventricular specification remain largely unknown. Here we report the identification of a progenitor population that gives rise primarily to cardiovascular cells of the ventricles and only to few atrial cells (<5%) of the differentiated heart. These progenitors are specified during gastrulation, when they transiently express Foxa2, a gene not previously implicated in cardiac development. Importantly, Foxa2+ cells contribute to previously identified progenitor populations in a defined pattern and ratio. Lastly, we describe an analogous Foxa2+ population during differentiation of embryonic stem cells. Together, these findings provide insight into the developmental origin of ventricular and atrial cells, and may lead to the establishment of new strategies for generating chamber-specific cell types from pluripotent stem cells.

Evolution and Distribution of Teleost myomiRNAs: Functionally Diversified myomiRs in Teleosts.

Myosin heavy chain (MYH) genes belong to a multigene family, and the regulated expression of each member determines the physiological and contractile muscle properties. Among these, MYH6, MYH7, and MYH14 occupy unique positions in the mammalian MYH gene family because of their specific expression in slow/cardiac muscles and the existence of intronic micro(mi) RNAs. MYH6, MYH7, and MYH14 encode miR-208a, miR-208b, and miR-499, respectively. These MYH encoded miRNAs are designated as myomiRs because of their muscle-specific expression and functions. In mammals, myomiRs and host MYHs form a transcription network involved in muscle fiber-type specification; thus, genomic positions and expression patterns of them are well conserved. However, our previous studies revealed divergent distribution and expression of MYH14/miR-499 among teleosts, suggesting the unique evolution of myomiRs and host MYHs in teleosts. Here, we examined distribution and expression of myomiRs and host MYHs in various teleost species. The major cardiac MYH isoforms in teleosts are an intronless gene, atrial myosin heavy chain (amhc), and ventricular myosin heavy chain (vmhc) gene that encodes an intronic miRNA, miR-736. Phylogenetic analysis revealed that vmhc/miR-736 is a teleost-specific myomiR that differed from tetrapoda MYH6/MYH7/miR-208s. Teleost genomes also contain species-specific orthologs in addition to vmhc and amhc, indicating complex gene duplication and gene loss events during teleost evolution. In medaka and torafugu, miR-499 was highly expressed in slow/cardiac muscles whereas the expression of miR-736 was quite low and not muscle specific. These results suggest functional diversification of myomiRs in teleost with the diversification of host MYHs.

Multicolor mapping of the cardiomyocyte proliferation dynamics that construct the atrium.

The orchestrated division of cardiomyocytes assembles heart chambers of distinct morphology. To understand the structural divergence of the cardiac chambers, we determined the contributions of individual embryonic cardiomyocytes to the atrium in zebrafish by multicolor fate-mapping and we compare our analysis to the established proliferation dynamics of ventricular cardiomyocytes. We find that most atrial cardiomyocytes become rod-shaped in the second week of life, generating a single-muscle-cell-thick myocardial wall with a striking webbed morphology. Inner pectinate myofibers form mainly by direct branching, unlike delamination events that create ventricular trabeculae. Thus, muscle clones assembling the atrial chamber can extend from wall to lumen. As zebrafish mature, atrial wall cardiomyocytes proliferate laterally to generate cohesive patches of diverse shapes and sizes, frequently with dominant clones that comprise 20-30% of the wall area. A subpopulation of cardiomyocytes that transiently express atrial myosin heavy chain (amhc) contributes substantially to specific regions of the ventricle, suggesting an unappreciated level of plasticity during chamber formation. Our findings reveal proliferation dynamics and fate decisions of cardiomyocytes that produce the distinct architecture of the atrium.

Chamber Specific Gene Expression Landscape of the Zebrafish Heart.

The organization of structure and function of cardiac chambers in vertebrates is defined by chamber-specific distinct gene expression. This peculiarity and uniqueness of the genetic signatures demonstrates functional resolution attributed to the different chambers of the heart. Altered expression of the cardiac chamber genes can lead to individual chamber related dysfunctions and disease patho-physiologies. Information on transcriptional repertoire of cardiac compartments is important to understand the spectrum of chamber specific anomalies. We have carried out a genome wide transcriptome profiling study of the three cardiac chambers in the zebrafish heart using RNA sequencing. We have captured the gene expression patterns of 13,396 protein coding genes in the three cardiac chambers-atrium, ventricle and bulbus arteriosus. Of these, 7,260 known protein coding genes are highly expressed (≥10 FPKM) in the zebrafish heart. Thus, this study represents nearly an all-inclusive information on the zebrafish cardiac transcriptome. In this study, a total of 96 differentially expressed genes across the three cardiac chambers in zebrafish were identified. The atrium, ventricle and bulbus arteriosus displayed 20, 32 and 44 uniquely expressing genes respectively. We validated the expression of predicted chamber-restricted genes using independent semi-quantitative and qualitative experimental techniques. In addition, we identified 23 putative novel protein coding genes that are specifically restricted to the ventricle and not in the atrium or bulbus arteriosus. In our knowledge, these 23 novel genes have either not been investigated in detail or are sparsely studied. The transcriptome identified in this study includes 68 differentially expressing zebrafish cardiac chamber genes that have a human ortholog. We also carried out spatiotemporal gene expression profiling of the 96 differentially expressed genes throughout the three cardiac chambers in 11 developmental stages and 6 tissue types of zebrafish. We hypothesize that clustering the differentially expressed genes with both known and unknown functions will deliver detailed insights on fundamental gene networks that are important for the development and specification of the cardiac chambers. It is also postulated that this transcriptome atlas will help utilize zebrafish in a better way as a model for studying cardiac development and to explore functional role of gene networks in cardiac disease pathogenesis.

Right Atrial Flap Repair for Left Superior Vena Cava Draining into Left Atrium.

There are different surgical methods for the repair of persistent left superior vena cava that connects directly to the left atrium. We describe an extracardiac surgical technique that includes direct anastomosis of persistent left superior vena cava to the right atrium with right atrial flap and autologous pericardium. We have performed this procedure in four cases and there is no obstruction at postoperative control studies. Right atrial flap repair is a feasible extracardiac technique that offers growth potential.

Different Myocardial Sensitivity in Newborn and Mature Rats to Selective Stimulation of M3 Cholinoreceptors.

The effects of selective activation of subtype 3 muscarinic (M3) receptors on electrical activity of isolated preparations of the atrial and ventricular myocardium of the newborn and 4-month-old rats were examined. Application of muscarinic receptor agonist pilocarpine (10(-5) M) in preparations with M2 cholinoreceptors blocked by selective antagonist methoctramine (10(-7) M) decreased the duration of action potentials in the atrial and ventricular myocardium. Selective blocker of M3 cholinoreceptors 4-DAMP (10(-8) M) prevented this effect. While stimulation of ventricular M3 cholinoreceptors with pilocarpine was significantly stronger in newborn pups than in mature rats, similar stimulation of atrial receptors revealed no significant difference in both groups.

[Morphological and molecular bases of cardiac development]. / Morfologiczne i molekularne podstawyrozwoju serca.

The heart is a mesoderm-derived organ, whose formation is regulated by various genes. Initially, the most important is expression of Nkx2.5, CR1, pitx2, anf and mhc2a, which are responsible for differentiation of cardiomyocytes. In a later phase activation of mhc2b, pitx2c, mesp1, pcmf1, vmhc, xin, mcl2v, mlc2a, mlc2a, mef2, hand1 and hand2 was revealed. Their expression is regulated by various molecules, including transcription (XIN, GATA, MEF, Tbx5, Baf60c, PECAM, tie-2, MEF2) and growth (VEGF, FGF, PDGF) factors, as well as proteins (i.e., dickkopf-1, cerberus, cytotactin, fibrillin, nodal, thrombomodulin, Wnt, bone morphometric ones - BMP2, BMP 4, BMP5, BMP7) and other substances, such as retinoid and folic acid. Crucial steps in cardiac organogenesis are development of the ventricle and atrial formation, as well as septation and valve formation. Any disturbances of such processes may lead to various congenital heart diseases and defects that could be initiated by various genetic, epigenetic or environmental factors. The most common heart malformations are: stenosis (coarctation) of the aorta and pulmonary trunk, bicuspid aortic valve, atrial and/or ventricular septal defect, persistent truncus arteriosus (Botallo duct), transposition of the great vessels, tricuspid atresia, hypoplastic left and right heart, as well as syndrome of Lutembachera, Cantrell, Ebstein, Eisenmenger and Shone and trilogy, tetralogy, pentalogy of Fallot.

Atrial identity is determined by a COUP-TFII regulatory network.

Atria and ventricles exhibit distinct molecular profiles that produce structural and functional differences between the two cardiac compartments. However, the factors that determine these differences remain largely undefined. Cardiomyocyte-specific COUP-TFII ablation produces ventricularized atria that exhibit ventricle-like action potentials, increased cardiomyocyte size, and development of extensive T tubules. Changes in atrial characteristics are accompanied by alterations of 2,584 genes, of which 81% were differentially expressed between atria and ventricles, suggesting that a major function of myocardial COUP-TFII is to determine atrial identity. Chromatin immunoprecipitation assays using E13.5 atria identified classic atrial-ventricular identity genes Tbx5, Hey2, Irx4, MLC2v, MLC2a, and MLC1a, among many other cardiac genes, as potential COUP-TFII direct targets. Collectively, our results reveal that COUP-TFII confers atrial identity through direct binding and by modulating expression of a broad spectrum of genes that have an impact on atrial development and function.

Development of the hearts of lizards and snakes and perspectives to cardiac evolution.

Birds and mammals both developed high performance hearts from a heart that must have been reptile-like and the hearts of extant reptiles have an unmatched variability in design. Yet, studies on cardiac development in reptiles are largely old and further studies are much needed as reptiles are starting to become used in molecular studies. We studied the growth of cardiac compartments and changes in morphology principally in the model organism corn snake (Pantherophis guttatus), but also in the genotyped anole (Anolis carolinenis and A. sagrei) and the Philippine sailfin lizard (Hydrosaurus pustulatus). Structures and chambers of the formed heart were traced back in development and annotated in interactive 3D pdfs. In the corn snake, we found that the ventricle and atria grow exponentially, whereas the myocardial volumes of the atrioventricular canal and the muscular outflow tract are stable. Ventricular development occurs, as in other amniotes, by an early growth at the outer curvature and later, and in parallel, by incorporation of the muscular outflow tract. With the exception of the late completion of the atrial septum, the adult design of the squamate heart is essentially reached halfway through development. This design strongly resembles the developing hearts of human, mouse and chicken around the time of initial ventricular septation. Subsequent to this stage, and in contrast to the squamates, hearts of endothermic vertebrates completely septate their ventricles, develop an insulating atrioventricular plane, shift and expand their atrioventricular canal toward the right and incorporate the systemic and pulmonary venous myocardium into the atria.

Evaluation of cardiac systolic and diastolic functions in small for gestational age babies during the first months of life: a prospective follow-up study.

OBJECTIVE: The purpose of this study was to evaluate the cardiac functions and age-related changes of these functions in full-term small for gestational age infants during the first 3 months of life. METHODS: Cardiac functions of 20 term small for gestational age and 20 term appropriate for gestational age infants were studied using conventional and tissue Doppler echocardiography on postnatal day 5 and at 1 and 3 months. RESULTS: Ventricular diameters, ventricular wall thicknesses, and left ventricular mass significantly increased with age in both groups. All these parameters were significantly lower in small for gestational age infants. No differences were detected by conventional echocardiography between the groups in systolic and diastolic functions. Systolic velocity, early diastolic and atrial contraction velocities, and the ratio between early diastolic and atrial contraction velocities determined by tissue Doppler echocardiography increased with age. Systolic velocity was lower in the small for gestational age babies for all myocardial regions on the 5th day. Peak early diastolic velocity was decreased in the small for gestational age babies at the first and second evaluations for all myocardial regions. The ratio between early diastolic and atrial contraction velocities was significantly lower in the small for gestational age babies for the interventricular septum and right ventricle. Significant positive correlations were detected between the Ponderal index and systolic and early diastolic velocities. CONCLUSION: The present findings suggest that systolic and diastolic function indices including tissue Doppler measures are significantly affected in small for gestational age babies during the first 3 months of life.

[Ultrastructural estimation facilities of atrial cardiomyocyte secretory activity].

The wide experience in the ultrastructural study of myoendocrine cells of different animal species in normal and experimental conditions allows us to choose the optimal methodology that gives the most complete information about the state of intracellular secretory apparatus. It is revealed that the combined set of atrial myoendocrine cell qualitative and quantitative parameters allows defining the natriuretic regulatory system status, as well as it's acute and chronic responses to hemodynamic changes. The information value of such approach is illustrated by examples of the ontogenetic investigation in two rat lines: with normal arterial blood pressure and with inherited stress-induced arterial hypertension. The proposed methodology is quite sensitive and descriptive; so it is of high importance due to insufficiency of other universal, specific, and accurate methods for cardiac natriuretic peptides investigation.

Retinoic acid-driven Hox1 is required in the epidermis for forming the otic/atrial placodes during ascidian metamorphosis.

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.