Human metapneumovirus infection in the cardiac paediatric ICU before and during COVID-19 pandemic: a retrospective cohort analysis.

INTRODUCTION: This study investigates the hygiene standards in the context of the COVID-19 pandemic and their impact on the perioperative incidence of human metapneumovirus as well as the typical symptom burden of human metapneumovirus-infected children with CHDs. MATERIALS AND METHODS: Between March 2018 and July 2021, all patients of a cardiac paediatric ICU of a German university hospital were included in this retrospective cohort analysis. RESULTS: A total of 589 patients with CHD were included in the analysis. Three hundred and fifty-two patients (148 females and 204 males) were admitted before the introduction of social distancing and face masks between March 2018 and 15 April 2020 (cohort A). Two hundred and thirty-seven patients (118 females and 119 males) were admitted after the introduction between April 16 and July 2021 (cohort B). In cohort A, human metapneumovirus was detected in 11 out of 352 patients (3.1%) during their stay at cardiac paediatric ICU. In cohort B, one patient out of 237 (0.4%) tested positive for human metapneumovirus. Patients who tested positive for human metapneumovirus stayed in cardiac paediatric ICU for a median of 17.5 days (range, 2-45 days). Patients without a detected human metapneumovirus infection stayed in the cardiac paediatric ICU for a median of 4 days (range, 0.5-114 days). Nine out of 12 (75%) human metapneumovirus-positive patients showed atelectasis. CONCLUSION: Perioperative human metapneumovirus infections prolong cardiac paediatric ICU stay in children with CHD. In affected patients, pulmonary impairment with typical symptoms appears. Under certain circumstances, a complication-rich perioperative infection with human metapneumovirus could be prevented in paediatric cardiac high-risk patients by prophylactic hygiene intervention.

[SARS-CoV-2 positive child-What to do if inhalation induction of anesthesia is unavoidable?] / SARS-CoV-2-positives Kind ­ Was tun bei unvermeidbarer inhalativer Narkoseeinleitung?

Induction of anesthesia by inhalation is very common in children due to difficult venous conditions and especially in uncooperative children. During the study on complications in the pediatric anesthesia in Europe (APRICOT study) including almost 30,000 patients, 48% of the children were induced by inhalation.Under the conditions of the corona pandemic, however, induction of anesthesia by inhalation represents an increased risk of infection due to the potential release of aerosols. Rapid sequence induction is recommended for anesthesia induction and definitive airway management for adults and children in the current pandemic situation.The present case demonstrates that there can be situations in children in which induction of anesthesia by inhalation is unavoidable and shows a potential procedure for reducing the risk of infection for the anesthesia personnel.

Severe Hyponatraemia Associated with the Use of Arginine-Vasopressin in Two Neonates with Complex Congenital Heart Disease.

We report two cases of neonates with complex congenital heart disease and volume, catecholamine, and corticosteroid refractory shock treated with arginine-vasopressin. Arginine-vasopressin was able to reverse critical hypotension, but both patients developed severe hyponatremia, which recovered after discontinuation of arginine-vasopressin. Close control and prompt substitution of serum sodium is required in neonates with advanced heart failure on high-dose vasopressin therapy.

Severe heart failure and the need for mechanical circulatory support and heart transplantation in pediatric patients with myocarditis: Results from the prospective multicenter registry "MYKKE".

Myocarditis represents an important cause for acute heart failure. MYKKE, a prospective multicenter registry of pediatric patients with myocarditis, aims to gain knowledge on courses, diagnostics, and therapy of pediatric myocarditis. The role of mechanical circulatory support (MCS) in children with severe heart failure and myocarditis is unclear. The aim of this study was to determine characteristics and outcome of patients with severe heart failure requiring MCS and/or heart transplantation. The MYKKE cohort between September 2013 and 2016 was analyzed. A total of 195 patients were prospectively enrolled by 17 German hospitals. Twenty-eight patients (14%) received MCS (median 1.5 years), more frequently in the youngest age group (0-2 years) than in the older groups (P < 0.001; 2-12 and 13-18 years). In the MCS group, 50% received a VAD, 36% ECMO, and 14% both, with a survival rate of 79%. The weaning rate was 43% (12/28). Nine (32%) patients were transplanted, one had ongoing support, and six (21%) died. Histology was positive for myocarditis in 63% of the MCS group. Patients within the whole cohort with age <2 years and/or ejection fraction <30% had a significantly worse survival with high risk for MCS, transplantation, and death (P < 0.001). Myocarditis represents a life-threatening disease with an overall mortality of 4.6% in this cohort. The fulminant form more often affected the youngest, leading to significantly higher rate of MCS, transplantation, and mortality. MCS represents an important and life-saving therapeutic option in children with myocarditis with a weaning rate of 43%.

Different Responses to Drug Safety Screening Targets between Human Neonatal and Infantile Heart Tissue and Cardiac Bodies Derived from Human-Induced Pluripotent Stem Cells.

AIMS: Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) have become a promising tool in cardiovascular safety pharmacology. Immaturity of iPS-CMs remains an ongoing concern. We compared electrophysiological and contractile features of cardiac bodies (hiPS-CBs) derived from human-induced pluripotent stem cells and human neonatal and infantile myocardial slices relevant for drug screening. METHODS AND RESULTS: Myocardial tissue slices were prepared from biopsies obtained from patients undergoing surgery for hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot (TOF). Electrophysiological features and response to Ik,r blockade as well as contractile properties were investigated using microelectrodes and isometric force measurements and were compared to hiPS-CBs. Both native myocardial tissue slices as well as hiPS-CBs showed action potential prolongation after Ik,r blockade, but early afterdepolarisations could be observed in native myocardial tissue slices only. The force-frequency relationship (FFR) varied at lower frequencies and was negative throughout at higher frequencies in hiPS-CBs. In contrast, native myocardial tissue slices exhibited positive, negative, and biphasic FFRs. In contrast to native myocardial tissue slices, hiPS-CBs failed to show an inotropic response to ß-adrenergic stimulation. Although all groups showed ß-adrenergic induced positive lusitropy, the effect was more pronounced in myocardial tissue slices. CONCLUSION: hiPS-CBs were able to reproduce AP prolongation after Ik,r blockade, but to a lesser extent compared to human neonatal and infantile myocardial tissue slices. Early afterdepolarisations could not be induced in hiPS-CBs. Contractile force was differently regulated by ß-adrenergic stimulation in hiPS-CBs and the native myocardium. If used for cardiotoxicity screening, caution is warranted as hiPS-CBs might be less sensitive to pharmacologic targets compared to the native myocardium of neonates and infants.

Stenting the Fontan pathway in paediatric patients with obstructed extracardiac conduits.

OBJECTIVES: An unobstructed extracardiac conduit (ECC) is essential for optimal Fontan haemodynamics. We aimed to evaluate the feasibility and results of percutaneous transcatheter stenting of the ECC in paediatric patients with a significant Fontan pathway obstruction. METHODS: Our institutional database was searched to identify all Fontan patients who had a stent placed in their ECC. Medical records, cardiac catheterisation data and echocardiographic investigations were reviewed. Vessel diameters were normalised to account for differences in body surface area. RESULTS: Nineteen Fontan patients (age 6.5±3.2 years; male 78.9%) with a significant stenosis of their Dacron ECC graft were identified. Seven patients presented with protein-losing enteropathy (36.8%). An ECC obstruction was suspected on echocardiography in only 6/19 patients (31.6%). The mean minimum diameter of the ECC was 8.3±2.4 mm. A stenosis of >45% was seen in the majority of patients (n=12, 63.1%). Significant correlations between the severity of the ECC obstruction and Fontan pathway vessel diameters were found (all p<0.05). Stenting was successful in all children. The ECC diameter increased significantly after stenting (p<0.0001). An acute clinical benefit of ECC stenting was observed in 18/19 (94.7%) patients. ECC patency was good during a mean follow-up of 1.8±0.9 years. CONCLUSIONS: The feasibility and acute results of percutaneous transcatheter ECC stenting are promising and may provide a good alternative to postpone surgery to a later age. The mechanisms contributing to the development of ECC stenoses are likely multifactorial.

Towards a proposal for a universal diagnostic definition of protein-losing enteropathy in Fontan patients: a systematic review.

OBJECTIVE: A standardised diagnostic definition of protein-losing enteropathy (PLE) in Fontan patients serves both patient care and research. The present study determined whether a diagnostic definition of PLE was routinely used in published clinical Fontan studies, and to identify potentially relevant diagnostic criteria for composing a uniform PLE definition. METHODS: A systematic review was conducted in adherence to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) recommendations. Published clinical Fontan studies that were written in English and included at least four patients with PLE were selected. PLE definitions were quantitatively analysed using a lateral thinking tool in which definitions were fractionated into constituent pieces of information (building blocks or diagnostic criteria). RESULTS: We identified 364 papers. In the final analysis, data from 62 published articles were extracted. A diagnostic definition of PLE was used in only 27/62 (43.5%) of selected studies, and definitions were very heterogeneous. We identified eight major diagnostic criteria. Hypoalbuminaemia (n=23 studies, 85.2%), clinical presentation (n=18, 66.7%), documentation of enteric protein loss (n=16, 59.3%) and exclusion of other causes of hypoproteinaemia (n=17, 63.0%), were the most frequently used diagnostic criteria. Most studies used three diagnostic variables (n=13/27, 48.1%). Cut-off values for laboratory parameters (serum albumin, protein or faecal α-1-antitrypsin) were frequently incorporated in the PLE definition (n=16, 59.3%). CONCLUSIONS: Establishment of a universally accepted PLE definition for routine use in clinical research and daily practice is required. The diagnostic criteria may help constitute a diagnostic PLE definition.

The Interaction between Adult Cardiac Fibroblasts and Embryonic Stem Cell-Derived Cardiomyocytes Leads to Proarrhythmic Changes in In Vitro Cocultures.

Transplantation of stem cell-derived cardiomyocytes is one of the most promising therapeutic approaches after myocardial infarction, as loss of cardiomyocytes is virtually irreversible by endogenous repair mechanisms. In myocardial scars, transplanted cardiomyocytes will be in immediate contact with cardiac fibroblasts. While it is well documented how the electrophysiology of neonatal cardiomyocytes is modulated by cardiac fibroblasts of the same developmental stage, it is unknown how adult cardiac fibroblasts (aCFs) affect the function of embryonic stem cell-derived cardiomyocytes (ESC-CMs). To investigate the effects of aCFs on ESC-CM electrophysiology, we performed extra- and intracellular recordings of murine aCF-ESC-CM cocultures. We observed that spontaneous beating behaviour was highly irregular in aCF-ESC-CM cocultures compared to cocultures with mesenchymal stem cells (coefficient of variation of the interspike interval: 40.5 ± 15.2% versus 9.3 ± 2.0%, p = 0.008) and that action potential amplitude and maximal upstroke velocity (V max) were reduced (amplitude: 52.3 ± 1.7 mV versus 65.1 ± 1.5 mV, V max: 7.0 ± 1.0 V/s versus 36.5 ± 5.3 V/s), while action potential duration (APD) was prolonged (APD50: 25.6 ± 1.0 ms versus 16.8 ± 1.9 ms, p < 0.001; APD90: 52.2 ± 1.5 ms versus 43.3 ± 3.3 ms, p < 0.01) compared to controls. Similar changes could be induced by aCF-conditioned medium. We conclude that the presence of aCFs changes automaticity and induces potentially proarrhythmic changes of ESC-CM electrophysiology.

Low serum 25-hydroxyvitamin D levels and secondary hyperparathyroidism in Fontan patients.

BACKGROUND: Limited data exist on the vitamin D status in Fontan patients. We determined the prevalence and potential risk factors of vitamin D deficiency in this patient subset. Methods and results Data were collected from 27 Fontan patients (55.6% male, mean age 8.1±5.3 years). Protein-losing enteropathy was diagnosed in six patients (22.2%). Vitamin D deficiency was defined as a serum 25-hydroxyvitamin D level of <20 ng/ml. The neutrophil-to-lymphocyte ratio, a marker of systemic inflammation, was calculated. Associations between laboratory measurements and patient characteristics were explored. Mean serum 25-hydroxyvitamin D level was 14.1±10.4 ng/ml. Vitamin D deficiency was found in 19/27 patients (70.3%). Only skin type was associated with vitamin D deficiency (p=0.04). Hyperparathyroidism was present in 5/21 (23.8%) patients, and was more prevalent in patients with protein-losing enteropathy (p<0.001). Parathyroid hormone levels correlated with parameters of systemic inflammation (neutrophil-to-lymphocyte ratio: r=0.484, p=0.026; relative lymphocyte count: r=-0.635, p=0.002). Vitamin D supplementation significantly increased serum 25-hydroxyvitamin D levels (p<0.0001), and was accompanied by a reduction in parathyroid hormone concentrations (p=0.032). CONCLUSIONS: A high prevalence of vitamin D deficiency was found among Fontan patients, independent of age, time after Fontan procedure, ventricular morphology, and presence of protein-losing enteropathy. A potentially important link between parathyroid hormone levels and systemic inflammation is suggested.

Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and ß-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 µM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05). Ni(2+) was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca(2+)-release decreased force development to 54 ± 3% in ventricular (n=13, p<0.05) and to 52 ± 8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca(2+)-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169 ± 7% force compared to baseline, n=8, p<0.05) and 10 s (198 ± 9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after ß-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

Electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes are cell line-dependent.

BACKGROUND: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs) requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs) of different murine ESC lines. METHODS: Two wild-type (D3 and R1) and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7) were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP) and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC) promoter. Action potentials (APs) were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. RESULTS: Spontaneous AP frequency and AP duration (APD) as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. CONCLUSION: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts - MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3 ± 2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment. The implications of these findings are critically discussed.

Mesenchymal stem cells and their conditioned medium improve integration of purified induced pluripotent stem cell-derived cardiomyocyte clusters into myocardial tissue.

Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) might become therapeutically relevant to regenerate myocardial damage. Purified iPS-CMs exhibit poor functional integration into myocardial tissue. The aim of this study was to investigate whether murine mesenchymal stem cells (MSCs) or their conditioned medium (MScond) improves the integration of murine iPS-CMs into myocardial tissue. Vital or nonvital embryonic murine ventricular tissue slices were cocultured with purified clusters of iPS-CMs in combination with murine embryonic fibroblasts (MEFs), MSCs, or MScond. Morphological integration was assessed by visual scoring and functional integration by isometric force and field potential measurements. We observed a moderate morphological integration of iPS-CM clusters into vital, but a poor integration into nonvital, slices. MEFs and MSCs but not MScond improved morphological integration of CMs into nonvital slices and enabled purified iPS-CMs to confer force. Coculture of vital slices with iPS-CMs and MEFs or MSCs resulted in an improved electrical integration. A comparable improvement of electrical coupling was achieved with the cell-free MScond, indicating that soluble factors secreted by MSCs were involved in electrical coupling. We conclude that cells such as MSCs support the engraftment and adhesion of CMs, and confer force to noncontractile tissue. Furthermore, soluble factors secreted by MSCs mediate electrical coupling of purified iPS-CM clusters to myocardial tissue. These data suggest that MSCs may increase the functional engraftment and therapeutic efficacy of transplanted iPS-CMs into infarcted myocardium.

Time-course of the electrophysiological maturation and integration of transplanted cardiomyocytes.

Electrophysiological maturation and integration of transplanted cardiomyocytes are essential to enhance safety and efficiency of cell replacement therapy. Yet, little is known about these important processes. The aim of our study was to perform a detailed analysis of electrophysiological maturation and integration of transplanted cardiomyocytes. Fetal cardiomyocytes expressing enhanced green fluorescent protein were transplanted into cryoinjured mouse hearts. At 6, 9 and 12 days after transplantation, viable slices of recipient hearts were prepared and action potentials of transplanted and host cardiomyocytes within the slices were recorded by microelectrodes. In transplanted cells embedded in healthy host myocardium, action potential duration at 50% repolarization (APD50) decreased from 32.2 ± 3.3 ms at day 6 to 27.9 ± 2.6 ms at day 9 and 19.6 ± 1.6 ms at day 12. The latter value matched the APD50 of host cells (20.5 ± 3.2 ms, P=0.78). Integration improved in the course of time: 26% of cells at day 6 and 53% at day 12 revealed no conduction blocks up to a stimulation frequency of 10 Hz. APD50 was inversely correlated to the quality of electrical integration. In transplanted cells embedded into the cryoinjury, which showed no electrical integration, APD50 was 49.2 ± 4.3 ms at day 12. Fetal cardiomyocytes transplanted into healthy myocardium integrate electrically and mature after transplantation, their action potential properties after 12 days are comparable to those of host cardiomyocytes. Quality of electrical integration improves over time, but conduction blocks still occur at day 12 after transplantation. The pace of maturation correlates with the quality of electrical integration. Transplanted cells embedded in cryoinjured tissue still possess immature electrophysiological properties after 12 days.

Fibroblasts support functional integration of purified embryonic stem cell-derived cardiomyocytes into avital myocardial tissue.

Transplantation of purified pluripotent stem cell-derived cardiomyocytes into damaged myocardium might become a therapy to improve contractile function after myocardial infarction. However, engraftment remains problematic. Aim of this study was to investigate whether murine embryonic fibroblasts (MEFs) support the functional integration of purified embryonic stem cell-derived cardiomyocytes (ES-CMs). Neonatal murine ventricular tissue slices were subjected to oxygen and glucose deprivation to simulate irreversible ischemia. Vital tissue slices served as control. Vital and avital tissue slices were cultured with or without MEFs before coculturing with clusters of puromycin-selected ES-CMs. Integration of ES-CM clusters was assessed morphologically, motility by long-term microscopy, and functional integration by isometric force measurements. We observed a good morphological integration into vital but a poor integration into avital slices. Adding MEFs improved morphological integration into irreversibly damaged slices and enabled purified ES-CMs to migrate and to confer force. We conclude that noncardiomyocytes like MEFs support morphological integration and force transmission of purified ES-CMs by enabling adhesion and migration.

Induced pluripotent stem cells: a new approach for physiological research.

The generation of induced pluripotent stem (iPS) cells by controlled delivery of reprogramming factors enables the derivation of pluripotent cells from a variety of somatic cell types. Patient-tailored iPS cells remove the major roadblock of immune rejection for clinical applications associated with the use of human embryonic stem (hES) cells. Beside therapeutic issues, iPS cell technology opens the door for broader research on human pluripotent cells because ethical limitations are lifted with iPS cells compared to hES cells. Scientists are now able to generate iPS cells for disease modelling and use them in basic research of physiological and pathophysiological models. In this concise review, we discuss the state of the art in the field of iPS cell induction by cell fusion or defined factors. Techniques to derive pluripotent cells from somatic sources are introduced and discussed, as well as some biological factors that influence the generation of iPS cells. We compare ES and iPS cells to answer the question whether these cells are identical, and we finish with an outlook on clinical research with iPS cells with a focus on cardiovascular medicine.

Electrophysiological basis of the first heart beats.

AIMS: Earlier studies on cardiac pacemaker activity were inconclusive regarding the electrophysiological mechanism(s) of first electrical activity generation during cardiac development. We therefore aimed to investigate the role of ion channels in action potential (AP) formation and pacemaker activity during very early murine heart development (E8.5). METHODS AND RESULTS: The voltage clamp mode of the whole-cell patch clamp technique was used to study whole cell ion currents at the single cell level. Extracellular field potentials (FPs) of whole mount hearts were recorded by the use of multi-electrodes arrays (MEAs). RT-PCR was performed to study mRNA expression of the tested ion channels. E8.5 cardiomyocytes displayed membrane potential (MP) oscillations, primitive APs but also more mature-like APs. Cells with primitive APs expressed higher levels of Ca(v)1.2, HCN4, Ca(v)3.2 and NCX1 than cells with MP oscillations and mature-like cells expressed higher levels of K(ir)3.1 (P-like) or K(ir)2.1 (V- and A- like) than cells with primitive APs. Blockers of I(CaL) (verapamil+nifedipine), I(NCX) (KB-R7943 and SEA0400), and I(CaT) (Ni(2+)) led to a complete halt of cardiac APs and FPs even when applied alone, while blocker of I(f) (Cs(+)) only suppressed AP/FP frequency. CONCLUSIONS: Our data imply that i) The high expression of Ca(v)1.2, HCN4, Ca(v)3.2, and NCX1 contributed to the shift from MP oscillations to primitive APs at E8.5. ii) Spontaneous AP generation may rely on the sum of transsarcolemmal Ca(2+) influx through I(CaL), I(NCX) and I(CaT) since blockage of each individual ion channel/exchanger led to an immediate halt of beating at E8.5.

Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells.

Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC-CMs) and iPS cells (iPS-CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5-7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force-frequency relationship (FFR), effects of Ca(2+), Ni(2+), nifedipine, ryanodine, beta-adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC-CMs and iPS-CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca(2+) and decreased with nifedipine. Force was reduced by Ni(2+). Isoproterenol (1 microM) increased the force most pronounced in embryonic tissue (207+/-31%, n=7; ESC-CMs: 123+/-5%, n=4; iPS-CMs: 120+/-4%, n=8). EC(50) values were similar. Contractile properties of iPS-CMs and ESC-CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the beta-adrenergic response of iPS-CMs and ESC-CMs.

Fibroblasts facilitate the engraftment of embryonic stem cell-derived cardiomyocytes on three-dimensional collagen matrices and aggregation in hanging drops.

There is growing interest in the use of cardiomyocytes purified from embryonic stem (ES) cells for tissue engineering and cardiomyoplasty. However, most transplanted cells are lost shortly after transplantation due to the lack of integration into the host tissue and subsequent apoptosis. Here we examine whether murine embryonic fibroblasts (MEFs) can support the integration of purified murine ES cell-derived cardiomyocytes in a 3-dimensional tissue culture model based on a freezed-dryed collagen matrix with tubular structure. Collagen matrix was seeded either with cardiomyocytes alone or in combination with MEFs. The collagen sponges that were transplanted with cardiomyocytes alone showed neither morphological nor functional integration of viable cells. Cardiomyocytes also did not appear to be capable of attaching quantitatively to any of 16 different 2-dimensional biomaterials. However, cardiomyocytes co-cultured with MEFs formed fiber-like structures of rod-shaped cells with organized sarcomeric structure that contracted spontaneously. Electrical coupling between cardiomyocytes was suggested by strong expression of connexin 43. In addition, MEFs as well as cardiac fibroblasts supported re-aggregation of dissociated cardiomyocytes in hanging drops in the absence of collagen matrix. We conclude that fibroblasts promote cardiomyocyte engraftment and formation of functional 3-dimensional tissue in vitro. Elucidation of the mechanism of this phenomenon may help improve the integration of cardiomyocytes in vivo.

Cardiac myocytes derived from murine reprogrammed fibroblasts: intact hormonal regulation, cardiac ion channel expression and development of contractility.

AIMS: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells. METHODS AND RESULTS: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to beta-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of I(Na), I(f), I(NCX), and I(CaL), respectively, and suggested their involvement in cardiac pacemaking, with I(CaL) being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to beta-adrenergic stimulation. CONCLUSIONS: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty.