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.

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.

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.

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.

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.

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.

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.

[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.

[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.

Selective site pacing in paediatric patients: how to recreate a new loop in the right atrium to follow body growth.

In children, intracardiac loops are created to avoid a lead's fracture due to rapid body growth. Selective site pacing needs the insertion of two 4.1 Fr lumenless leads that do not allow direct manoeuvrability and should be extracted when tension on the lead occurs. We describe an easy, safe, and inexpensive technique to reposition the leads and generate new intracardiac loops.

Impact of corticosteroids on late growth of radiofrequency lesions in infant pigs: histopathological and electroanatomical findings.

AIMS: Corticosteroids attenuate late growth of radiofrequency (RF) lesions in the thigh muscle of infant rats. We sought to assess the impact of these drugs on the late growth of RF lesions in immature swine myocardium and to determine the electroanatomical mapping (EAM) characteristics of these lesions. METHODS AND RESULTS: Radiofrequency (60°C; 60 s) lesions were created in the right atrium (n = 2) and ventricle (n = 2) of 14 piglets (age 65 days; weight 5 kg) and 3 adults. Piglets were divided into: controls (n = 7) and treated (n = 7), receiving hydrocortisone (10 mg/kg iv after RF) and prednisone (1 mg/kg/day) for 29 days. After 8 months, animals were sacrificed for histological analysis. In four piglets, endocardial and epicardial voltage EAM were performed. In infant groups, the dimensions of atrial (11 ± 5 vs. 13 ± 7 mm) and ventricular (12 ± 3 vs. 11 ± 3 mm) lesions were similar. In adults, atrial (6 ± 1 mm) and ventricular (6 ± 1 mm) lesions were smaller. In controls, ventricular lesions depicted dense fibrosis and multiple strands of fibrous tissue extending from the lesion into normal muscle. Treated piglets revealed scars exhibiting less dense fibrosis with predominance of fibroadipose tissue and less collagen proliferation. Large atrial and ventricular low-voltage areas corresponding to the macroscopic lesions were identified in all animals. CONCLUSION: Radiofrequency lesions in infant pigs reveal late growth and invasion of normal muscle by intense collagen proliferation. Corticosteroids do not prevent late enlargement of the lesions but modulate the fibrotic proliferation. The expressive growth of the lesion may generate low-voltage areas detectable by EAM.

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.

Requirement of transcription factor NFAT in developing atrial myocardium.

Nuclear factor of activated T cell (NFAT) is a ubiquitous regulator involved in multiple biological processes. Here, we demonstrate that NFAT is temporally required in the developing atrial myocardium between embryonic day 14 and P0 (birth). Inhibition of NFAT activity by conditional expression of dominant-negative NFAT causes thinning of the atrial myocardium. The thin myocardium exhibits severe sarcomere disorganization and reduced expression of cardiac troponin-I (cTnI) and cardiac troponin-T (cTnT). Promoter analysis indicates that NFAT binds to and regulates transcription of the cTnI and the cTnT genes. Thus, regulation of cytoskeletal protein gene expression by NFAT may be important for the structural architecture of the developing atrial myocardium.

Derivation of a size-independent variable for scaling of cardiac dimensions in a normal paediatric population.

AIMS: It is general practice to correct cardiac chamber size for body size by the process of scaling or normalization. Normalization is most commonly performed using simple linear or isometric correction; however, there is increasing evidence that this approach may be flawed. Likewise, there is little agreement concerning the appropriate scaling variable (measure of body size) for normalization. Therefore, we aimed to establish the optimal method for correcting the differences in body size in a large population of echocardiographically normal paediatric subjects. METHODS AND RESULTS: We compared the relative ability of standard size variables including height (HT), body weight (BW), body mass index (BMI), and body surface area (BSA), in both isometric and allometric models, to remove the effect of body size in 4109 consecutive echocardiographically normal subjects<18 years of age, using the left atrial dimension (LAD) as a reference standard. Simple linear normalization resulted in significant residual correlations (r=-0.57 to -0.92) of the indexed value with the body size variable, the correlations with weight (WT) and BSA actually increasing. In contrast, correction by the optimal allometric exponent (AE) removed the effects of the indexed variable (residual correlations -0.01 to 0.01), with BW and BSA best removing the effects of all the measures of body size. CONCLUSION: Conventional linear correction for body size is inaccurate in children and paradoxically increases the relationship of the indexed parameter with WT and BSA. Conversely, correction using the optimal AE removes the effect of that variable, with WT best correction for all measures of body size.

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.

Regulation of cardiac innervation and function via the p75 neurotrophin receptor.

Homeostatic regulation of cardiac function is dependent on the balance of inputs from the sympathetic and parasympathetic nervous systems. We investigated whether the p75 neurotrophin receptor plays a developmental role in cardiac innervation by analyzing sympathetic and parasympathetic fibers in the atria of p75 knockout and wildtype mice at several stages of postnatal development, and examining the effect on control of heart rate. We found that parasympathetic innervation of the atria in p75-/- mice was similar to wildtype at all time points, but that the density of sympathetic innervation was dynamically regulated. Compared to wildtype mice, the p75-/- mice had less innervation at postnatal day 4, an increase at day 28, and decreased innervation in adult mice. These changes reflect defects in initial fiber in-growth and the timing of the normal developmental decrease in sympathetic innervation density in the atria. Thus, p75 regulates both the growth and stability of cardiac sympathetic fibers. The distribution of sympathetic fibers was also altered, so that many regions lacked innervation. Basal heart rate was depressed in adult p75-/- mice, and these mice exhibited a diminished heart rate response to restraint stress. This resulted from the lack of sympathetic innervation rather than increased parasympathetic transmission or a direct effect of p75 in cardiac cells. Norepinephrine was elevated in p75-/- atria, but stimulating norepinephrine release with tyramine produced less tachycardia in p75-/- mice than wild type mice. This suggests that altered density and distribution of sympathetic fibers in p75-/- atria impairs the control of heart rate.

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.

Molecular cloning and characterization of human cardiac alpha- and beta-form myosin heavy chain complementary DNA clones. Regulation of expression during development and pressure overload in human atrium.

We have constructed and characterized two types of myosin heavy chain (MHC) cDNA clones (pHMHC2, pHMHC5) from a fetal human heart cDNA library. Comparison of the nucleotide and deduced amino acid sequences between pHMHC2 and pHMHC5 shows 95.1 and 96.2% homology, respectively. The carboxyl-terminal peptide and 3'-untranslated (3'-UT) regions are highly divergent and specific for these cDNA clones. By using the synthetic oligonucleotide probes that are complementary to the unique 3'-UT regions of these cDNA clones, we demonstrate that pHMHC2 is exclusively transcribed in the atrium, whereas the mRNA for pHMHC5 is predominantly expressed in the ventricle. This result indicates that pHMHC2 and pHMHC5 code for alpha- and beta-form MHCs, respectively. Furthermore, we show that beta-form MHC mRNA is expressed in adult atrium at a low level but scarcely expressed in fetal atrium. Finally, we demonstrate that MHC isozymic transition in pressure-overloaded atrium is, at least in part, regulated at a pretranslational level.

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.