Advancing Commotio cordis Safety Standards Using the Total Human Models for Safety (THUMS).

Commotio cordis is one of the leading causes of sudden cardiac death in youth baseball. Currently, there are chest protector regulations regarding the prevention of Commotio cordis in baseball and lacrosse; however, they are not fully optimized. For the advancement of Commotio cordis safety, it is vital to include various age groups and a variety of impact angles in the testing process. This study employed finite element models and simulated Commotio cordis-inducing baseball collisions for different velocities, impact angles, and age groups. Commotio cordis risk response was characterized in terms of left ventricular strain and pressure, chest band and rib deformation, and force from impact. Normalized rib and chest band deformation when correlated with left ventricular strain resulted in R2 = 0.72, and R2 = 0.76, while left ventricular pressure resulted in R2 = 0.77, R2 = 0.68 across all velocities and impact angles in the child models. By contrast, the resultant reaction force risk metric as used by the National Operating Committee on Standards for Athletic Equipment (NOCSAE) demonstrated a correlation of R2 = 0.20 in the child models to ventricular strain, while illustrating a correlation to pressure of R2 = 0.74. When exploring future revisions to Commotio cordis safety requirements, the inclusion of deformation-related risk metrics at the level of the left ventricle should be considered.

Enabling comprehensive optogenetic studies of mouse hearts by simultaneous opto-electrical panoramic mapping and stimulation.

During the last decade, cardiac optogenetics has turned into an essential tool for investigating cardiac function in general and for assessing functional interactions between different myocardial cell types in particular. To advance exploitation of the unique research opportunities offered by this method, we develop a panoramic opto-electrical measurement and stimulation (POEMS) system for mouse hearts. The core of the experimental platform is composed of 294 optical fibers and 64 electrodes that form a cup which embraces the entire ventricular surface of mouse hearts and enables straightforward 'drop&go' experimentation. The flexible assignment of fibers and electrodes to recording or stimulation tasks permits a precise tailoring of experiments to the specific requirements of individual optogenetic constructs thereby avoiding spectral congestion. Validation experiments with hearts from transgenic animals expressing the optogenetic voltage reporters ASAP1 and ArcLight-Q239 demonstrate concordance of simultaneously recorded panoramic optical and electrical activation maps. The feasibility of single fiber optical stimulation is proven with hearts expressing the optogenetic voltage actuator ReaChR. Adaptation of the POEMS system to larger hearts and incorporation of additional sensors can be achieved by redesigning the system-core accordingly.

Advancing mechanobiology by performing whole-cell patch clamp recording on mechanosensitive cells subjected simultaneously to dynamic stretch events.

A comprehensive understanding of mechano-electrical coupling requires continuous intracellular electrical recordings being performed on cells undergoing simultaneously in vivo like strain events. Here, we introduce a linear strain single-cell electrophysiology (LSSE) system that meets these requirements by delivering highly reproducible unidirectional strain events with magnitudes up to 12% and strain rates exceeding 200%s-1 to adherent cells kept simultaneously in whole-cell patch-clamp recording configuration. Proof-of-concept measurements with NIH3T3 cells demonstrate that stable recording conditions are maintained over tens of strain cycles at maximal amplitudes and strain rates thereby permitting a full electrophysiological characterization of mechanically activated ion currents. Because mechano-electrical responses to predefined strain patterns can be investigated using any adherent wild-type or genetically modified cell type of interest, the LSSE system offers the perspective of providing advanced insights into mechanosensitive ion channel function that can finally be compared quantitatively among different types of channels and cells.

The Role of Membrane Capacitance in Cardiac Impulse Conduction: An Optogenetic Study With Non-excitable Cells Coupled to Cardiomyocytes.

Non-excitable cells (NECs) such as cardiac myofibroblasts that are electrotonically coupled to cardiomyocytes affect conduction velocity (θ) by representing a capacitive load (CL: increased membrane to be charged) and a resistive load (RL: partial depolarization of coupled cardiomyocytes). In this study, we untangled the relative contributions of both loading modalities to NEC-dependent arrhythmogenic conduction slowing. Discrimination between CL and RL was achieved by reversibly removing the RL component by light activation of the halorhodopsin-based hyperpolarizing membrane voltage actuator eNpHR3.0-eYFP (enhanced yellow fluorescent protein) expressed in communication-competent fibroblast-like NIH3T3 cells (3T3 HR cells) that served as a model of coupled NECs. Experiments were conducted with strands of neonatal rat ventricular cardiomyocytes coated at increasing densities with 3T3 HR cells. Impulse conduction along preparations stimulated at 2.5 Hz was assessed with multielectrode arrays. The relative density of 3T3 HR cells was determined by dividing the area showing eYFP fluorescence by the area covered with cardiomyocytes [coverage factor (CF)]. Compared to cardiomyocytes, 3T3 HR cells exhibited a depolarized membrane potential (-34 mV) that was shifted to -104 mV during activation of halorhodopsin. Without illumination, 3T3 HR cells slowed θ along the preparations from ∼330 mm/s (control cardiomyocyte strands) to ∼100 mm/s (CF = ∼0.6). Illumination of the preparation increased the electrogram amplitudes and induced partial recovery of θ at CF > 0.3. Computer simulations demonstrated that the θ deficit observed during illumination was attributable in full to the CL represented by coupled 3T3 HR cells with θ showing a power-law relationship to capacitance with an exponent of -0.78 (simulations) and -0.99 (experiments). The relative contribution of CL and RL to conduction slowing changed as a function of CF with CL dominating at CF ≤ ∼0.3, both mechanisms being equally important at CF = ∼0.5, and RL dominating over CL at CF > 0.5. The finding that RL did not affect θ at CFs ≤ 0.3 is explained by the circumstance that, at the respective moderate levels of cardiomyocyte depolarization, supernormal conduction stabilized propagation. The findings provide experimental estimates for the dependence of θ on membrane capacitance in general and suggest that the myocardium can absorb moderate numbers of electrotonically coupled NECs without showing substantial alterations of θ.

Nanovolcano microelectrode arrays: toward long-term on-demand registration of transmembrane action potentials by controlled electroporation.

Volcano-shaped microelectrodes (nanovolcanoes) functionalized with nanopatterned self-assembled monolayers have recently been demonstrated to report cardiomyocyte action potentials after gaining spontaneous intracellular access. These nanovolcanoes exhibit recording characteristics similar to those of state-of-the-art micro-nanoelectrode arrays that use electroporation as an insertion mechanism. In this study, we investigated whether the use of electroporation improves the performance of nanovolcano arrays in terms of action potential amplitudes, recording durations, and yield. Experiments with neonatal rat cardiomyocyte monolayers grown on nanovolcano arrays demonstrated that electroporation pulses with characteristics derived from analytical models increased the efficiency of nanovolcano recordings, as they enabled multiple on-demand registration of intracellular action potentials with amplitudes as high as 62 mV and parallel recordings in up to ~76% of the available channels. The performance of nanovolcanoes showed no dependence on the presence of functionalized nanopatterns, indicating that the tip geometry itself is instrumental for establishing a tight seal at the cell-electrode interface, which ultimately determines the quality of recordings. Importantly, the use of electroporation permitted the recording of attenuated cardiomyocyte action potentials during consecutive days at identical sites, indicating that nanovolcano recordings are nondestructive and permit long-term on-demand recordings from excitable cardiac tissues. Apart from demonstrating that less complex manufacturing processes can be used for next-generation nanovolcano arrays, the finding that the devices are suitable for performing on-demand recordings of electrical activity from multiple sites of excitable cardiac tissues over extended periods of time opens the possibility of using the devices not only in basic research but also in the context of comprehensive drug testing.

High-speed mechano-active multielectrode array for investigating rapid stretch effects on cardiac tissue.

Systematic investigations of the effects of mechano-electric coupling (MEC) on cellular cardiac electrophysiology lack experimental systems suitable to subject tissues to in-vivo like strain patterns while simultaneously reporting changes in electrical activation. Here, we describe a self-contained motor-less device (mechano-active multielectrode-array, MaMEA) that permits the assessment of impulse conduction along bioengineered strands of cardiac tissue in response to dynamic strain cycles. The device is based on polydimethylsiloxane (PDMS) cell culture substrates patterned with dielectric actuators (DEAs) and compliant gold ion-implanted extracellular electrodes. The DEAs induce uniaxial stretch and compression in defined regions of the PDMS substrate at selectable amplitudes and with rates up to 18 s-1. Conduction along cardiomyocyte strands was found to depend linearly on static strain according to cable theory while, unexpectedly, being completely independent on strain rates. Parallel operation of multiple MaMEAs provides for systematic high-throughput investigations of MEC during spatially patterned mechanical perturbations mimicking in-vivo conditions.

TGF-ß1 (Transforming Growth Factor-ß1) Plays a Pivotal Role in Cardiac Myofibroblast Arrhythmogenicity.

BACKGROUND: TGF-ß1 (transforming growth factor-ß1) importantly contributes to cardiac fibrosis by controlling differentiation, migration, and collagen secretion of cardiac myofibroblasts. It is still elusive, however, to which extent TGF-ß1 alters the electrophysiological phenotype of myofibroblasts and cardiomyocytes and whether it affects proarrhythmic myofibroblast-cardiomyocyte crosstalk observed in vitro. METHODS AND RESULTS: Patch-clamp recordings of cultured neonatal rat ventricular myofibroblasts revealed that TGF-ß1, applied for 24 to 48 hours at clinically relevant concentrations (≤2.5 ng/mL), causes substantial membrane depolarization concomitant with a several-fold increase of transmembrane currents. Transcriptome analysis revealed TGF-ß1-dependent changes in 29 of 63 ion channel/pump/connexin transcripts, indicating a pleiotropic effect on the electrical phenotype of myofibroblasts. Whereas not affecting cardiomyocyte membrane potentials and cardiomyocyte-cardiomyocyte gap junctional coupling, TGF-ß1 depolarized cardiomyocytes coupled to myofibroblasts by ≈20 mV and increased gap junctional coupling between myofibroblasts and cardiomyocytes >5-fold as reflected by elevated connexin 43 and consortin transcripts. TGF-ß1-dependent cardiomyocyte depolarization resulted from electrotonic crosstalk with myofibroblasts as demonstrated by immediate normalization of cardiomyocyte electrophysiology after targeted disruption of coupled myofibroblasts and by cessation of ectopic activity of cardiomyocytes coupled to myofibroblasts during pharmacological gap junctional uncoupling. In cardiac fibrosis models exhibiting slow conduction and ectopic activity, block of TGF-ß1 signaling completely abolished both arrhythmogenic conditions. CONCLUSIONS: TGF-ß1 profoundly alters the electrophysiological phenotype of cardiac myofibroblasts. Apart from possibly contributing to the control of cell function in general, the changes proved to be pivotal for proarrhythmic myofibroblast-cardiomyocyte crosstalk in vitro, which suggests that TGF-ß1 may play a potentially important role in arrhythmogenesis of the fibrotic heart.

Myofibroblasts Electrotonically Coupled to Cardiomyocytes Alter Conduction: Insights at the Cellular Level from a Detailed In silico Tissue Structure Model.

Fibrotic myocardial remodeling is typically accompanied by the appearance of myofibroblasts (MFBs). In vitro, MFBs were shown to slow conduction and precipitate ectopic activity following gap junctional coupling to cardiomyocytes (CMCs). To gain further mechanistic insights into this arrhythmogenic MFB-CMC crosstalk, we performed numerical simulations in cell-based high-resolution two-dimensional tissue models that replicated experimental conditions. Cell dimensions were determined using confocal microscopy of single and co-cultured neonatal rat ventricular CMCs and MFBs. Conduction was investigated as a function of MFB density in three distinct cellular tissue architectures: CMC strands with endogenous MFBs, CMC strands with coating MFBs of two different sizes, and CMC strands with MFB inserts. Simulations were performed to identify individual contributions of heterocellular gap junctional coupling and of the specific electrical phenotype of MFBs. With increasing MFB density, both endogenous and coating MFBs slowed conduction. At MFB densities of 5-30%, conduction slowing was most pronounced in strands with endogenous MFBs due to the MFB-dependent increase in axial resistance. At MFB densities >40%, very slow conduction and spontaneous activity was primarily due to MFB-induced CMC depolarization. Coating MFBs caused non-uniformities of resting membrane potential, which were more prominent with large than with small MFBs. In simulations of MFB inserts connecting two CMC strands, conduction delays increased with increasing insert lengths and block appeared for inserts >1.2 mm. Thus, electrophysiological properties of engineered CMC-MFB co-cultures depend on MFB density, MFB size and their specific positioning in respect to CMCs. These factors may influence conduction characteristics in the heterocellular myocardium.

PITX2 Modulates Atrial Membrane Potential and the Antiarrhythmic Effects of Sodium-Channel Blockers.

BACKGROUND: Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 transcription factor (PITX2) might play an important role in regulating gene expression and electrical function of the adult left atrium (LA). OBJECTIVES: After determining LA PITX2 expression in AF patients requiring rhythm control therapy, the authors assessed the effects of Pitx2c on LA electrophysiology and the effect of antiarrhythmic drugs. METHODS: LA PITX2 messenger ribonucleic acid (mRNA) levels were measured in 95 patients undergoing thoracoscopic AF ablation. The effects of flecainide, a sodium (Na+)-channel blocker, and d,l-sotalol, a potassium channel blocker, were studied in littermate mice with normal and reduced Pitx2c mRNA by electrophysiological study, optical mapping, and patch clamp studies. PITX2-dependent mechanisms of antiarrhythmic drug action were studied in human embryonic kidney (HEK) cells expressing human Na channels and by modeling human action potentials. RESULTS: Flecainide 1 µmol/l was more effective in suppressing atrial arrhythmias in atria with reduced Pitx2c mRNA levels (Pitx2c+/-). Resting membrane potential was more depolarized in Pitx2c+/- atria, and TWIK-related acid-sensitive K+ channel 2 (TASK-2) gene and protein expression were decreased. This resulted in enhanced post-repolarization refractoriness and more effective Na-channel inhibition. Defined holding potentials eliminated differences in flecainide's effects between wild-type and Pitx2c+/- atrial cardiomyocytes. More positive holding potentials replicated the increased effectiveness of flecainide in blocking human Nav1.5 channels in HEK293 cells. Computer modeling reproduced an enhanced effectiveness of Na-channel block when resting membrane potential was slightly depolarized. CONCLUSIONS: PITX2 mRNA modulates atrial resting membrane potential and thereby alters the effectiveness of Na-channel blockers. PITX2 and ion channels regulating the resting membrane potential may provide novel targets for antiarrhythmic drug development and companion therapeutics in AF.

Pharmacological Modulation of Hemodynamics in Adult Zebrafish In Vivo.

INTRODUCTION: Hemodynamic parameters in zebrafish receive increasing attention because of their important role in cardiovascular processes such as atherosclerosis, hematopoiesis, sprouting and intussusceptive angiogenesis. To study underlying mechanisms, the precise modulation of parameters like blood flow velocity or shear stress is centrally important. Questions related to blood flow have been addressed in the past in either embryonic or ex vivo-zebrafish models but little information is available for adult animals. Here we describe a pharmacological approach to modulate cardiac and hemodynamic parameters in adult zebrafish in vivo. MATERIALS AND METHODS: Adult zebrafish were paralyzed and orally perfused with salt water. The drugs isoprenaline and sodium nitroprusside were directly applied with the perfusate, thus closely resembling the preferred method for drug delivery in zebrafish, namely within the water. Drug effects on the heart and on blood flow in the submental vein were studied using electrocardiograms, in vivo-microscopy and mathematical flow simulations. RESULTS: Under control conditions, heart rate, blood flow velocity and shear stress varied less than ± 5%. Maximal chronotropic effects of isoprenaline were achieved at a concentration of 50 µmol/L, where it increased the heart rate by 22.6 ± 1.3% (n = 4; p < 0.0001). Blood flow velocity and shear stress in the submental vein were not significantly increased. Sodium nitroprusside at 1 mmol/L did not alter the heart rate but increased blood flow velocity by 110.46 ± 19.64% (p = 0.01) and shear stress by 117.96 ± 23.65% (n = 9; p = 0.03). DISCUSSION: In this study, we demonstrate that cardiac and hemodynamic parameters in adult zebrafish can be efficiently modulated by isoprenaline and sodium nitroprusside. Together with the suitability of the zebrafish for in vivo-microscopy and genetic modifications, the methodology described permits studying biological processes that are dependent on hemodynamic alterations.

The European Network for Translational Research in Atrial Fibrillation (EUTRAF): objectives and initial results.

Atrial fibrillation (AF) is the most common sustained arrhythmia in the general population. As an age-related arrhythmia AF is becoming a huge socio-economic burden for European healthcare systems. Despite significant progress in our understanding of the pathophysiology of AF, therapeutic strategies for AF have not changed substantially and the major challenges in the management of AF are still unmet. This lack of progress may be related to the multifactorial pathogenesis of atrial remodelling and AF that hampers the identification of causative pathophysiological alterations in individual patients. Also, again new mechanisms have been identified and the relative contribution of these mechanisms still has to be established. In November 2010, the European Union launched the large collaborative project EUTRAF (European Network of Translational Research in Atrial Fibrillation) to address these challenges. The main aims of EUTRAF are to study the main mechanisms of initiation and perpetuation of AF, to identify the molecular alterations underlying atrial remodelling, to develop markers allowing to monitor this processes, and suggest strategies to treat AF based on insights in newly defined disease mechanisms. This article reports on the objectives, the structure, and initial results of this network.

Optical recording of calcium currents during impulse conduction in cardiac tissue.

We explore the feasibility of obtaining a spatially resolved picture of [Formula: see text] inward currents ([Formula: see text]) in multicellular cardiac tissue by differentiating optically recorded [Formula: see text] transients that accompany propagating action potentials. Patterned growth strands of neonatal rat ventricular cardiomyocytes were stained with the [Formula: see text] indicators Fluo-4 or Fluo-4FF. Preparations were stimulated at 1 Hz, and [Formula: see text] transients were recorded with high spatiotemporal resolution ([Formula: see text], 2 kHz analog bandwidth) with a photodiode array. Signals were differentiated after appropriate digital filtering. Differentiation of [Formula: see text] transients resulted in optically recorded calcium currents (ORCCs) that carried the temporal and pharmacological signatures of L-type [Formula: see text] inward currents: the time to peak amounted to [Formula: see text] (Fluo-4FF) and [Formula: see text] (Fluo-4), full-width at half-maximum was [Formula: see text], and ORCCs were completely suppressed by [Formula: see text][Formula: see text]. Also, and as reported before from patch-clamp studies, caffeine reversibly depressed the amplitude of ORCCs. The results demonstrate that the differentiation of [Formula: see text] transients can be used to obtain a spatially resolved picture of the initial phase of [Formula: see text] in cardiac tissue and to assess relative changes of activation/fast inactivation of [Formula: see text] following pharmacological interventions.

Aggravation of cardiac myofibroblast arrhythmogeneicity by mechanical stress.

AIMS: Myofibroblasts (MFBs) as appearing in the myocardium during fibrotic remodelling induce slow conduction following heterocellular gap junctional coupling with cardiomyocytes (CMCs) in bioengineered tissue preparations kept under isometric conditions. In this study, we investigated the hypothesis that strain as developed during diastolic filling of the heart chambers may modulate MFB-dependent slow conduction. METHODS AND RESULTS: Effects of defined levels of strain on single-cell electrophysiology (patch clamp) and impulse conduction in patterned growth cell strands (optical mapping) were investigated in neonatal rat ventricular cell cultures (Wistar) grown on flexible substrates. While 10.5% strain only minimally affected conduction times in control CMC strands (+3.2%, n.s.), it caused a significant slowing of conduction in the fibrosis model consisting of CMC strands coated with MFBs (conduction times +26.3%). Increased sensitivity to strain of the fibrosis model was due to activation of mechanosensitive channels (MSCs) in both CMCs and MFBs that aggravated the MFB-dependent baseline depolarization of CMCs. As found in non-strained preparations, baseline depolarization of CMCs was partly due to the presence of constitutively active MSCs in coupled MFBs. Constitutive activity of MSCs was not dependent on the contractile state of MFBs, because neither stimulation (thrombin) nor suppression (blebbistatin) thereof significantly affected conduction velocities in the non-strained fibrosis model. CONCLUSIONS: The findings demonstrate that both constitutive and strain-induced activity of MSCs in MFBs significantly enhance their depolarizing effect on electrotonically coupled CMCs. Ensuing aggravation of slow conduction may contribute to the precipitation of strain-related arrhythmias in fibrotically remodelled hearts.

The natural cardioprotective particle HDL modulates connexin43 gap junction channels.

AIMS: High-density lipoprotein (HDL) is known for its cardioprotective properties independent from its cholesterol transport activity. These properties are mediated by activation of kinases such as protein kinase C (PKC). Connexin43 (Cx43) is a gap junction protein present in ventricular cardiomyocytes. PKC-dependent phosphorylation modifies Cx43 gap junction channel properties and is involved in cardioprotection. We hypothesized that cardioprotective properties of HDL may be mediated in part by affecting Cx43 gap junction channels. METHODS AND RESULTS: Neonatal rat cardiomyocytes were treated with HDL and Cx43 phosphorylation was evaluated by western blotting and immunofluorescence. We found that HDL promoted phosphorylation of Cx43 with a maximal induction at 5 min, which was inhibited by pre-treatment with various PKC inhibitors. Sphingosine-1-phosphate (S1P), a component of HDL, induced effects that were similar to those of HDL. These compounds significantly reduced diffusion of fluorescent dye among cardiomyocytes (∼50%) which could be prevented by PKC inhibition. As observed during optical recordings of transmembrane voltage, HDL and S1P depressed impulse conduction only minimally (<5%). Moreover, 5 min of HDL and S1P treatment at the onset of reperfusion significantly reduced infarct size (∼50%) in response to 30 min ischaemia in ex vivo experiments. CONCLUSION: Short-term treatment with HDL or S1P induces phosphorylation of Cx43 by a PKC-dependent pathway. HDL-induced phosphorylation of Cx43 reduced the diffusion of large tracer molecules between cells, whereas impulse conduction was maintained. Moreover, 5 min treatment with HDL confers cardioprotection against ischaemia/reperfusion injury. These results link Cx43 for the first time to the short-term cardioprotective effects of HDL.

Abolishing myofibroblast arrhythmogeneicity by pharmacological ablation of α-smooth muscle actin containing stress fibers.

RATIONALE: Myofibroblasts typically appear in the myocardium after insults to the heart like mechanical overload and infarction. Apart from contributing to fibrotic remodeling, myofibroblasts induce arrhythmogenic slow conduction and ectopic activity in cardiomyocytes after establishment of heterocellular electrotonic coupling in vitro. So far, it is not known whether α-smooth muscle actin (α-SMA) containing stress fibers, the cytoskeletal components that set myofibroblasts apart from resident fibroblasts, are essential for myofibroblasts to develop arrhythmogenic interactions with cardiomyocytes. OBJECTIVE: We investigated whether pharmacological ablation of α-SMA containing stress fibers by actin-targeting drugs affects arrhythmogenic myofibroblast-cardiomyocyte cross-talk. METHODS AND RESULTS: Experiments were performed with patterned growth cell cultures of neonatal rat ventricular cardiomyocytes coated with cardiac myofibroblasts. The preparations exhibited slow conduction and ectopic activity under control conditions. Exposure to actin-targeting drugs (Cytochalasin D, Latrunculin B, Jasplakinolide) for 24 hours led to disruption of α-SMA containing stress fibers. In parallel, conduction velocities increased dose-dependently to values indistinguishable from cardiomyocyte-only preparations and ectopic activity measured continuously over 24 hours was completely suppressed. Mechanistically, antiarrhythmic effects were due to myofibroblast hyperpolarization (Cytochalasin D, Latrunculin B) and disruption of heterocellular gap junctional coupling (Jasplakinolide), which caused normalization of membrane polarization of adjacent cardiomyocytes. CONCLUSIONS: The results suggest that α-SMA containing stress fibers importantly contribute to myofibroblast arrhythmogeneicity. After ablation of this cytoskeletal component, cells lose their arrhythmic effects on cardiomyocytes, even if heterocellular electrotonic coupling is sustained. The findings identify α-SMA containing stress fibers as a potential future target of antiarrhythmic therapy in hearts undergoing structural remodeling.

A protective antiarrhythmic role of ursodeoxycholic acid in an in vitro rat model of the cholestatic fetal heart.

UNLABELLED: Intrahepatic cholestasis of pregnancy may be complicated by fetal arrhythmia, fetal hypoxia, preterm labor, and, in severe cases, intrauterine death. The precise etiology of fetal death is not known. However, taurocholate has been demonstrated to cause arrhythmia and abnormal calcium dynamics in cardiomyocytes. To identify the underlying reason for increased susceptibility of fetal cardiomyocytes to arrhythmia, we studied myofibroblasts (MFBs), which appear during structural remodeling of the adult diseased heart. In vitro, they depolarize rat cardiomyocytes via heterocellular gap junctional coupling. Recently, it has been hypothesized that ventricular MFBs might appear in the developing human heart, triggered by physiological fetal hypoxia. However, their presence in the fetal heart (FH) and their proarrhythmogenic effects have not been systematically characterized. Immunohistochemistry demonstrated that ventricular MFBs transiently appear in the human FH during gestation. We established two in vitro models of the maternal heart (MH) and FH, both exposed to increasing doses of taurocholate. The MH model consisted of confluent strands of rat cardiomyocytes, whereas for the FH model, we added cardiac MFBs on top of cardiomyocytes. Taurocholate in the FH model, but not in the MH model, slowed conduction velocity from 19 to 9 cm/s, induced early after depolarizations, and resulted in sustained re-entrant arrhythmias. These arrhythmic events were prevented by ursodeoxycholic acid, which hyperpolarized MFB membrane potential by modulating potassium conductance. CONCLUSION: These results illustrate that the appearance of MFBs in the FH may contribute to arrhythmias. The above-described mechanism represents a new therapeutic approach for cardiac arrhythmias at the level of MFB.

Cardiac fibroblasts in cell culture systems: myofibroblasts all along?

The cytoarchitecture of the working myocardium is characterized by densely packed cardiomyocytes that are embedded in a three-dimensional network of numerous fibroblasts. Although the importance of cardiac fibroblasts in maintaining an orderly structured extracellular matrix is well recognized, less is known about their potential paracrine and electrotonic interactions with cardiomyocytes. This is partly the result of the complex intermingling of both cell types in vivo that tends to preclude a direct investigation of heterocellular crosstalk. It is for that reason that most of our present knowledge regarding stromal-parenchymal cell interactions is based on culture systems that permit direct access to either cell type. An often disregarded feature of such studies is that cardiac fibroblasts in standard two-dimensional cell culture have a pronounced tendency to undergo a phenotype switch to myofibroblasts. This cell type typically appears in injured hearts where it contributes importantly to fibrotic remodeling. The present review focuses on recent insights into electrical and paracrine crosstalk between myofibroblasts and cardiomyocytes while acknowledging that a comprehensive understanding of stromal-parenchymal cell interactions will depend on future methodological developments that permit retaining the fibroblast phenotype in cell culture systems and that will, most importantly, allow direct investigations of heterocellular crosstalk in intact tissue.