First experience with real-time magnetic resonance imaging-based investigation of respiratory influence on cardiac function in pediatric congenital heart disease patients with chronic right ventricular volume overload.

BACKGROUND: Congenital heart disease (CHD) is often associated with chronic right ventricular (RV) volume overload. Real-time magnetic resonance imaging (MRI) enables the analysis of cardiac function during free breathing. OBJECTIVE: To evaluate the influence of respiration in pediatric patients with CHD and chronic RV volume overload. METHODS AND MATERIALS: RV volume overload patients (n=6) and controls (n=6) were recruited for cardiac real-time MRI at 1.5 tesla during free breathing. Breathing curves from regions of interest reflecting the position of the diaphragm served for binning images in four different tidal volume classes, each in inspiration and expiration. Tidal volumes were estimated from these curves by data previously obtained by magnetic resonance-compatible spirometry. Ventricular volumes indexed to body surface area and Frank-Starling relationships referenced to the typical tidal volume indexed to body height (TTVi) were compared. RESULTS: Indexed RV end-diastolic volume (RV-EDVi) and indexed RV stroke volume (RV-SVi) increased during inspiration (RV-EDVi/TTVi: RV load: + 16 ± 4%; controls: + 22 ± 13%; RV-SVi/TTVi: RV load: + 21 ± 6%; controls: + 35 ± 17%; non-significant for comparison). The increase in RV ejection fraction during inspiration was significantly lower in RV load patients (RV load: + 1.1 ± 2.2%; controls: + 6.1 ± 1.5%; P=0.01). The Frank-Starling relationship of the RV provided a significantly reduced slope estimate in RV load patients (inspiration: RV load: 0.75 ± 0.11; controls: 0.92 ± 0.02; P=0.02). CONCLUSION: In pediatric patients with CHD and chronic RV volume overload, cardiac real-time MRI during free breathing in combination with respiratory-based binning indicates an impaired Frank-Starling relationship of the RV.

Hyperinsulinemic Hypoglycemia Associated with a CaV1.2 Variant with Mixed Gain- and Loss-of-Function Effects.

The voltage-dependent L-type calcium channel isoform CaV1.2 is critically involved in many physiological processes, e.g., in cardiac action potential formation, electromechanical coupling and regulation of insulin secretion by beta cells. Gain-of-function mutations in the calcium voltage-gated channel subunit alpha 1 C (CACNA1C) gene, encoding the CaV1.2 α1-subunit, cause Timothy syndrome (TS), a multisystemic disorder that includes autism spectrum disorders and long QT (LQT) syndrome. Strikingly, TS patients frequently suffer from hypoglycemia of yet unproven origin. Using next-generation sequencing, we identified a novel heterozygous CACNA1C mutation in a patient with congenital hyperinsulinism (CHI) and associated hypoglycemic episodes. We characterized the electrophysiological phenotype of the mutated channel using voltage-clamp recordings and in silico action potential modeling experiments. The identified CaV1.2L566P mutation causes a mixed electrophysiological phenotype of gain- and loss-of-function effects. In silico action potential modeling supports that this mixed electrophysiological phenotype leads to a tissue-specific impact on beta cells compared to cardiomyocytes. Thus, CACNA1C variants may be associated with non-syndromic hyperinsulinemic hypoglycemia without long-QT syndrome, explained by very specific electrophysiological properties of the mutated channel. We discuss different biochemical characteristics and clinical impacts of hypoglycemia in the context of CACNA1C variants and show that these may be associated with significant morbidity for Timothy Syndrome patients. Our findings underline that the potential of hypoglycemia warrants careful attention in patients with CACNA1C variants, and such variants should be included in the differential diagnosis of non-syndromic congenital hyperinsulinism.

Comparison of cardiac volumetry using real-time MRI during free-breathing with standard cine MRI during breath-hold in children.

BACKGROUND: Cardiac real-time magnetic resonance imaging (RT-MRI) provides high-quality images even during free-breathing. Difficulties in post-processing impede its use in clinical routine. OBJECTIVE: To demonstrate the feasibility of quantitative analysis of cardiac free-breathing RT-MRI and to compare image quality and volumetry during free-breathing RT-MRI in pediatric patients to standard breath-hold cine MRI. MATERIALS AND METHODS: Pediatric patients (n = 22) received cardiac RT-MRI volumetry during free breathing (1.5 T; short axis; 30 frames per s) in addition to standard breath-hold cine imaging in end-expiration. Real-time images were binned retrospectively based on electrocardiography and respiratory bellows. Image quality and volumetry were compared using the European Cardiovascular Magnetic Resonance registry score, structure visibility rating, linear regression and Bland-Altman analyses. RESULTS: Additional time for binning of real-time images was 2 min. For both techniques, image quality was rated good to excellent. RT-MRI was significantly more robust against artifacts (P < 0.01). Linear regression revealed good correlations for the ventricular volumes. Bland-Altman plots showed a good limit of agreement (LoA) for end-diastolic volume (left ventricle [LV]: LoA -0.1 ± 2.7 ml/m2, right ventricle [RV]: LoA -1.9 ± 3.4 ml/m2), end-systolic volume (LV: LoA 0.4 ± 1.9 ml/m2, RV: LoA 0.6 ± 2.0 ml/m2), stroke volume (LV: LoA -0.5 ± 2.3 ml/m2, RV: LoA -2.6 ± 3.3 ml/m2) and ejection fraction (LV: LoA -0.5 ± 1.6%, RV: LoA -2.1 ± 2.8%). CONCLUSION: Compared to standard cine MRI with breath hold, RT-MRI during free breathing with retrospective respiratory binning offers good image quality, reduced image artifacts enabling fast quantitative evaluations of ventricular volumes in clinical practice under physiological conditions.

Spirometry-based reconstruction of real-time cardiac MRI: Motion control and quantification of heart-lung interactions.

PURPOSE: To test the feasibility of cardiac real-time MRI in combination with retrospective gating by MR-compatible spirometry, to improve motion control, and to allow quantification of respiratory-induced changes during free-breathing. METHODS: Cross-sectional real-time MRI (1.5T; 30 frames/s) using steady-state free precession contrast during free-breathing was combined with MR-compatible spirometry in healthy adult volunteers (n = 4). Retrospective binning assigned images to classes that were defined by electrocardiogram and spirometry. Left ventricular eccentricity index as an indicator of septal position and ventricular volumes in different respiratory phases were calculated to assess heart-lung interactions. RESULTS: Real-time MRI with MR-compatible spirometry is feasible and well tolerated. Spirometry-based binning improved motion control significantly. The end-diastolic epicardial eccentricity index increased significantly during inspiration (1.04 ± 0.04 to 1.19 ± 0.05; P < .05). During inspiration, right ventricular end-diastolic volume (79 ± 17 mL/m2 to 98 ± 18 mL/m2 ), stroke volume (41 ± 8 mL/m2 to 59 ± 11 mL/m2 ) and ejection fraction (53 ± 3% to 60 ± 1%) increased significantly, whereas the end-systolic volume remained almost unchanged. Left ventricular end-diastolic volume, left ventricular stroke volume, and left ventricular ejection fraction decreased during inspiration, whereas the left ventricular end-systolic volume increased. The relationship between stroke volume and end-diastolic volume (Frank-Starling relationship) based on changes induced by respiration allowed for a slope estimate of the Frank-Starling curve to be 0.9 to 1.1. CONCLUSION: Real-time MRI during free-breathing combined with MR-compatible spirometry and retrospective binning improves image stabilization, allows quantitative image analysis, and importantly, offers unique opportunities to judge heart-lung interactions.

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.

A new model to perform electrophysiological studies in the early embryonic mouse heart.

BACKGROUND: The first electrocardiograms (ECGs) have been recorded with a capillary electrometer in the late 19(th) century by John Burdon Sanderson and Augustus Waller. In 1903 Willem Einthoven used the much more sensitive string galvanometer and was awarded Nobel Price in Medicine for this discovery. Though the physical principles of that era are still in use, there have been many advances but also challenges in cardiac electrophysiology over the last decades. One challenge is to record electrocardiograms of rather small animals such as mice and even smaller organisms such as their embryos. As mice belong to the most routinely used laboratory animals it is important to better understand their physiology and specific diseases. We therefore aimed to study whether it is feasible to measure electrical activities of embryonic mouse hearts. METHODS AND RESULTS: For our studies we used substrate-integrated Microelectrode Arrays combined with newly developed stimulation electrodes to perform electrophysiological studies in these hearts. The system enabled us to perform ECG-like recordings with atrio-ventricular (anterograde) and ventriculo-atrial (retrograde) stimulation. The functional separation of atria and ventricles, indicated by a stable atrio-ventricular conduction time, occurred clearly earlier than the morphological separation. Electrical stimulation induced a reversible prolongation of the anterograde and retrograde conduction up to atrio-ventricular conduction blocks at higher frequencies. CONCLUSION: These results yield new insight into functional aspects of murine cardiac development, and may help as a new diagnostic tool to uncover the functional and electrophysiological background of embryonic cardiac phenotypes of genetically altered mice.

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.

Brown-Vialetto-Van Laere syndrome: a riboflavin-unresponsive patient with a novel mutation in the C20orf54 gene.

Brown-Vialetto-Van Laere syndrome (Online Mendelian Inheritance in Man number 211530) is a neurodegenerative disorder characterized by pontobulbar palsy affecting cranial nerves (mainly VII-XII). Sensorineural deafness is often the leading sign, followed by other neurologic signs. Inheritance is often autosomal recessive, with mutations in the C20orf54 gene (Online Mendelian Inheritance in Man number 613350). Three previous patients with mutations in the C20orf54 gene and clinical signs of Brown-Vialetto-Van Laere or Fazio-Londe syndrome revealed a metabolic profile suggesting a multiple acyl-coenzyme A dehydrogenase defect. They benefited from riboflavin. We describe a 3-year-old girl with early-onset Brown-Vialetto-Van Laere syndrome and a novel mutation in the C20orf54 gene (c.989G>T). On T(2)-weighted imaging, increased signal intensity of the vestibular nuclei bilaterally, the pedunculus cerebellaris superior and the central tegmental tract were observed during acute clinical deterioration. Her metabolic profile was normal. Trials with steroids, immunoglobulins, and riboflavin produced no effect. The patient recovered slowly during subsequent months, with residual deficits. Brown-Vialetto-Van Laere syndrome should be considered in patients with sensorineural hearing loss and pontobulbar palsy. Patients should be screened for riboflavin deficiency and a therapy with riboflavin may provide effective treatment in some affected patients.

Urinary NT-proBNP, NGAL, and H-FABP may predict hemodynamic relevance of patent ductus arteriosus in very low birth weight infants.

BACKGROUND: Hemodynamically significant patent ductus arteriosus (hsPDA) is the most common functional cardiovascular disease in preterm infants. The necessity to treat hsPDA can neither be derived solely from clinical nor from echocardiographic criteria. OBJECTIVE: The aim of this study was to establish non-invasive parameters which can differentiate hsPDA from non-hsPDA. METHODS: Urinary protein levels of NT-proBNP, NGAL, and H-FABP were measured and correlated with the necessity of therapy for PDA. In 37 neonates (<1,500 g), urinary protein concentrations were tested on days 0, 2, and 7 by ELISA methodology. Of 37 infants, 12 required therapeutic interventions according to current treatment standards. RESULTS: Infants receiving an intervention for PDA showed significantly higher levels of pro-BNP, NGAL, and H-FABP at all time points except for NT-proBNP on day 0. Infants requiring a second or third course of ibuprofen had significantly higher levels of H-FABP and NGAL. In all samples, the concentration of the three proteins correlated positively with each other. CONCLUSIONS: The present study shows that measurement of urinary proteins is a powerful and non-invasive method to quantify the effect of PDA on systemic perfusion in preterm infants. Furthermore, NGAL and H-FABP may be used to indicate the necessity of pharmacological or surgical treatment of PDA.

Contractile properties of early human embryonic stem cell-derived cardiomyocytes: beta-adrenergic stimulation induces positive chronotropy and lusitropy but not inotropy.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide the unique opportunity to study the very early development of the human heart. The aim of this study was to investigate the effect of calcium and beta-adrenergic stimulation on the contractile properties of early hESC-CMs. Beating clusters containing hESC-CMs were co-cultured in vitro with noncontractile slices of neonatal murine ventricles. After 5-7 days, when beating clusters had integrated morphologically into the damaged tissue, isometric force measurements were performed during spontaneous beating as well as during electrical field stimulation. Spontaneous beating stopped when extracellular calcium ([Ca²âº](ec)) was removed or after administration of the Ca²âº channel blocker nifedipine. During field stimulation at a constant rate, the developed force increased with incremental concentrations of [Ca²âº](ec). During spontaneous beating, rising [Ca²âº](ec) increased beating rate and developed force up to a [Ca²âº](ec) of 2.5 mM. When [Ca²âº](ec) was increased further, spontaneous beating rate decreased, whereas the developed force continued to increase. The beta-adrenergic agonist isoproterenol induced a dose-dependent increase of the frequency of spontaneous beating; however, it did not significantly change the developed force during spontaneous contractions or during electrical stimulation at a constant rate. Force developed by early hESC-CMs depends on [Ca²âº](ec) and on the L-type Ca²âº channel. The lack of an inotropic reaction despite a pronounced chronotropic response after beta-adrenergic stimulation most likely indicates immaturity of the sarcoplasmic reticulum. For cell-replacement strategies, further maturation of cardiac cells has to be achieved either in vitro before or in vivo after transplantation.

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.

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.

Physiological differences between transplanted and host tissue cause functional decoupling after in vitro transplantation of human embryonic stem cell-derived cardiomyocytes.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) might provide cells to repopulate injured myocardium. Electrical coupling of these cells to the host myocardium is a prerequisite for improved functionality. The aim of this study was to investigate electrical interaction of hESC-CMs with myocardial tissue and to identify factors challenging functional integration. Beating clusters containing hESC-CMs were cocultured in vitro with viable slices of late-stage embryonic murine ventricles. Field potentials recorded with micro-electrode arrays and video data were analyzed. The effects of heptanol, electrical pacing, beta-adrenergic, and muscarinic stimulation on coupling were studied. Beating clusters integrated morphologically and functionally resulting in a synchronized beating pattern after two to four days of coculture. Heptanol-induced conduction block between transplanted cells and host tissue and immunoreactivity for connexin43 suggested electrical coupling via gap junctions. Beta-adrenergic or muscarinic stimulation induced uncoupling and arrhythmias probably due to genetically determined differences of hormonal modulation of spontaneous beating rates of transplanted cells and host tissue. HESC-CMs can integrate functionally and develop synchronized beating. Interventions unraveling the different electrophysiological properties of transplanted and host tissue induce functional disintegration. Successful cellular replacement has to improve coupling but should also aim to transplant cardiomyocytes with similar electrophysiological properties as the host tissue.

Biological pacemakers: characterization in an in vitro coculture model.

BACKGROUND: Biological pacemakers could be an alternative or complement to electronic pacemakers. Embryonic stem cells (ESCs) can be differentiated in vitro to spontaneously active cells. Although numerous studies show that ESC-derived cardiomyocytes (ESC-CMs) and other cell types are capable to exert pacemaker function in vivo, detailed analyses of pattern and safety of conduction on a tissue level are rare. METHODS: Murine ESCs (mESCs) expressing enhanced green fluorescent protein and puromycin resistance under control of the promoter of alpha-myosin (heavy chain) were differentiated to cardiomyocytes (mESC-CMs) and purified by negative antibiotic selection. Ventricles of mouse embryonic hearts (embryonic day 16.5) were embedded in agarose and sliced along the short axis. Clusters of mESC-CMs and the murine, vital heart slices were cocultured on multielectrode arrays for 4 days. Field potentials and videos were recorded daily to investigate beating behavior and excitation spreading within the slice. RESULTS: On the first day of coculture, the mean beating rate of the tissue slices cocultured with mESC-CMs (n = 19) did not differ significantly from the beating rate of control slices (n = 19) (37 +/- 10 versus 19 +/- 7 bpm, P = .133). After 4 days of coculture, beating rates were significantly higher in cocultures than in control slices (154 +/- 22 versus 49 +/- 8 bpm, P < .001). On day 4, 1:1 coupling could be found in 1 of 19 preparations; 2:1, 3:1, or 4:1 coupling in another 4 of 19 preparations; 14 of 19 propagation patterns were irregular. CONCLUSION: In this in vitro model, the increase of the beating rate suggests that purified mESC-CMs can pace native heart tissue, albeit with low efficiency.

Neonatal murine heart slices. A robust model to study ventricular isometric contractions.

BACKGROUND/AIMS: Cardiac function is increasingly studied using murine models. However, current multicellular preparations to investigate contractile properties have substantial technical and biological limitations and are especially difficult to apply to the developing murine heart. METHODS: Newborn murine hearts were cut with a vibratome into viable tissue slices. The structural and functional integrity of the tissue was shown by histology, ATP content and sharp electrode recordings. RESULTS: Within the first 48 hours after slicing structure remained intact without induction of apoptosis. ATP concentrations and action potential parameters were comparable to those of physiological tissue. Isometric force measurements demonstrated a physiological force-frequency relationship with a ;primary-phase' negative force-frequency relationship up to 1-2 Hz and a ;secondary-phase' positive force-frequency relationship up to 8 Hz. (-)-Isoproterenol (10(-6) mol/l) increased active force to 251 +/- 35% (n=15) of baseline values and shortened relaxation times indicating a preserved beta-adrenergic regulation of contraction. Changes of the force-frequency relationship after application of ryanodine and nifedipine indicated functionality of calcium release from the sarcoplasmic reticulum and of L-type calcium channels. CONCLUSION: Generation of viable, physiological intact ventricular slices from neonatal hearts is feasible and provides a robust model to study loaded contractions.

Electrophysiological maturation and integration of murine fetal cardiomyocytes after transplantation.

In the present study, we investigated the electrophysiological maturation and integration of immature cardiomyocytes after transplantation; maturation and integration are essential to achieve the cardiac regeneration. Murine fetal cardiomyocytes (FCMs) (d12.5-d15.5) expressing enhanced green fluorescent protein under the control of the alpha-actin promoter were injected into cryoinjured areas and adjacent myocardium of cryoinjured mouse ventricles. Viable short axis tissue slices (thickness, 150 microm) of the ventricles were prepared 5 to 6 days after transplantation. Glass microelectrodes were used for measurements of action potentials in transplanted FCMs and host cardiomyocytes within the slices. Stimulation at frequencies of up to 10 Hz was performed via a unipolar electrode placed in viable host tissue. Transplanted FCMs could be distinguished clearly from host tissue by their green fluorescence and their electrophysiological properties: maximal upstroke velocity (V(max)) was significantly lower and action potential duration at 50% repolarization (APD(50)) was significantly longer compared with values of adult cardiomyocytes. Transplanted FCMs surrounded by cryoinjured tissue showed spontaneous electrical and contractile activity, which was in no case synchronous with host tissue. V(max) and APD(50) of these nonintegrated cells matched values of cultivated dissociated FCMs. In contrast, 82% of transplanted FCMs surrounded by viable host tissue were electrically integrated; ie, electrical and contractile activity was synchronous with host tissue and these cells had more mature action potential parameters (significantly higher V(max) and shorter APD(50)) compared with nonintegrated FCMs. In conclusion, electrophysiological maturation and integration of transplanted FCMs depend on an embedment in viable host myocardium. FCMs surrounded by cryoinjured tissue maintain physiological but immature AP properties.

Regulation of Na+/Ca2+ exchange current in the normal and failing heart.

Cardiac NCX is modulated by diverse regulatory elements. Although there is consensus about the regulatory function of Na+ and Ca2+ and other elements, for example, ATP, there is still a controversial debate about the functional role of cyclic nucleotides and protein kinases. Future studies should focus on that topic since disturbances of cAMP/cGMP concentration and kinase activity may lead to severe functional disorders in the diseased heart. S100A1 is presumably a novel regulator of NCX.